# Patent

## Title
Mapkap kinase-2 as a specific target for blocking proliferation of P53-defective cells

## Abstract

The present invention relates to compounds and pharmaceutical compositions for treating cellular proliferative disorders, e.g., in patients having one or more p53-deficient cells, screening assays for identifying such compounds, and methods for treating such disorders.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Ser. No. 11/273,567, filed Nov. 14, 2005, which claims benefit of U.S. Provisional Application No. 60/627,352, filed Nov. 12, 2004, each of which is hereby incorporated by reference.
This application also claims benefit of U.S. Provisional Application Nos. 60/794,451, filed Apr. 24, 2006; 60/800,298, filed May 12, 2006; and 60/873,904, filed Dec. 8, 2006, each of which is hereby incorporated by reference.


STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
The present research was supported by grants from the National Institutes of Health (grant numbers GM60594 and CA112967) and from the National Institute of Environmental Health Sciences (grant number ES015339). The U.S. government has certain rights to this invention.


BACKGROUND OF THE INVENTION
The maintenance of genomic integrity is essential for the health of multi-cellular organisms. DNA damage checkpoints constitute a mechanism where cell division is delayed to allow repair of damaged DNA, or if the extent of DNA damage is beyond repair, induce apoptosis. The three major DNA damage-responsive cell cycle checkpoints are the G1/S checkpoint, intra S-phase checkpoint, and the G2/M checkpoint.
In response to DNA damage, eukaryotic cells activate a complex signaling network to arrest the cell cycle and facilitate DNA repair. This signaling network has traditionally been divided into two major protein kinase pathways, one mediated by Ataxia-Telangiectasia mutated (ATM) through Chk2, and the other mediated by Ataxia-Telangiectasia and Rad-3 related (ATR) through Chk1. Some cross-talk exists between the ATM/Chk2 and ATR/Chk1 kinase pathways, particularly when signaling through one pathway is partially or totally deficient. Normally, however the pathways show only partial functional overlap in response to particular forms of DNA damage. The ATM/Chk2 pathway responds primarily to DNA double strand breaks (DSBs), while the ATR/Chk1 pathway is activated by bulky DNA lesions, and following replication fork collapse during S-phase. The tumor suppressor protein p53 is a major downstream effector of these DNA damage kinase pathways. In normal cells, p53-dependent signaling results in G1 arrest, mainly mediated by transcriptional upregulation of p21. In addition, p21 also appears to play a role in sustaining the G2 checkpoint after γ-irradiation. If the DNA damage is extensive, however, then p53-dependent pathways target the damaged cell for apoptotic cell death through both the intrinsic and extrinsic pathways. Most tumor cells show specific disruptions in the p53 pathway, leading to selective loss of the G1 checkpoint. These cells are then entirely dependent on intra-S and G2/M checkpoints to maintain their genomic integrity in response to DNA damage.
In contrast to the DNA damage-specific activation of Chk1 and Chk2, the p38MAPK pathway is a general stress-activated kinase pathway that responds to various cellular stimuli, including cytokines, hyperosmolarity, and UV irradiation. Activity of p38MAPK is important for G2/M checkpoint function in immortalized fibroblasts and HeLa cells following UV exposure. Furthermore, MAPKAP Kinase-2 (MK2) is the critical downstream effector kinase of p38MAPK required for UV-induced cell cycle checkpoints in U2OS cells.
Whether the observed activation of p38 MAPK/MK2 is a direct result of UV-induced DNA lesions, or results instead from other non-genotoxic effects of UV radiation has been unclear. Similarly, whether the p38MAPK/MK2 pathway is an important part of a general cellular response to genotoxic stress has been unclear. There exists a need to better understand this checkpoint and to develop methods and therapies for disease treatment based on this improved understanding.
SUMMARY OF THE INVENTION
We now report that MAPKAP kinase-2 is specifically activated in response to DNA damage caused by chemotherapeutic agents in an ATR and/or ATM-dependent manner, and that MAPKAP kinase-2 is critical for the activation of G1, S-phase and G2/M checkpoints after exposure to these drugs. Down-regulation of MAPKAP kinase-2 using RNA interference profoundly increases the anti-proliferative and cytotoxic effects of cisplatin and doxorubucin on tumor cells in vitro, and in a murine tumor model in vivo. MAPKAP kinase-2 depletion is especially effective in increasing the chemosensitivity of p53-deficient cells, suggesting that compounds that target MAPKAP kinase-2 can be used as specific therapeutics that can sensitize p53-deficient tumor cells without sensitizing normal cells. At the systems level, in response to DNA damage, Chk1 and MAPKAP kinase-2 appear to function in parallel independent pathways that converge to phosphorylate similar molecular targets, such that checkpoint abrogation following MAPKAP kinase-2 depletion can be rescued by overexpression of Chk1.
Based on these results, we have invented novel methods of treating cellular proliferative disorders by inhibiting MAPKAP kinase-2 expression. We have also discovered MAPKAP kinase-2 inhibitors, pharmaceutical compounds containing such inhibitors that are useful for treating cellular proliferative disorders, and screening methods for identifying additional inhibitors. The methods and compounds of the invention may be used, for example, to treat cancer or to aid in the development of other anti-cancer therapies.
Accordingly, in one aspect, the invention features a method for treating a cellular proliferative disorder in a patient that includes the steps of: (a) determining whether the patient has a p53-deficient cell; and (b) if the patient has a p53-deficient cell, administering to the patient a compound, e.g., UCN-01, that is capable of inhibiting an activity of a MAPKAP kinase-2 polypeptide. Any method can be used to determine whether the patient has a p53-deficient cell, e.g., an antibody assay of a cell sample, e.g., from a tumor biopsy. The MAPKAP kinase-2 inhibition can be either specific or non-specific. The activity being inhibited may include, for example, MAPKAP kinase-2 polypeptide expression or substrate-binding. The method may also include the step of administering an additional treatment to the patient, such as a chemotherapeutic agent or radiation therapy, such that the compound and the chemotherapeutic agent or the radiation therapy are administered in amounts sufficient to treat the patient's cellular proliferative disorder. The additional treatment may be administered simultaneously or nonsimultaneously, e.g., up to twenty-eight days apart, in relation to the administration of the inhibitory compound. Any chemotherapeutic agent or radiation therapy known in the art may be useful in the methods of the invention. Exemplary chemotherapeutic agents are antimicrotubule drugs, e.g., nocodazole; compounds that create double-strand DNA breakage, e.g., doxorubicin and daunorubicin; compounds that induce single-strand DNA breaks, e.g., camptothecin; and cross-linking agents, e.g., cisplatin. Exemplary cellular proliferative disorders include neoplasms, e.g., any known form of cancer. In one embodiment, a solid tumor may be treated by injecting a MAPKAP kinase-2 inhibitor, alone or in combination with an additional therapeutic agent, directly into the tumor or by systemic administration. If given as a monotherapy, the compound is administered in an amount sufficient to treat the patient's cellular proliferative disorder; alternatively, in the case of combination therapy, the combination of compounds is collectively administered in an amount sufficient to treat the patient's cellular proliferative disorder.
An inhibitory compound used in the foregoing method may include a covalently-linked moiety capable of translocating across a biological membrane, such as a penetratin or TAT peptide. Alternatively, such a compound may be administered in the form of a prodrug. Suitable compounds include small molecule inhibitors of MAPKAP kinase-2 biological activity, RNA molecules useful in RNA interference therapy, RNA molecules useful in antisense therapy, and peptides capable of inhibiting a MAPKAP kinase-2 polypeptide. For example, an RNA molecule useful in the methods of the invention includes a double-stranded small interfering nucleic acid (siNA) molecule that is capable of directing cleavage of a MAPKAP kinase-2 RNA via RNA interference, wherein each strand of the siNA molecule is about 18 to 23 nucleotides in length, and one strand of the siNA molecule includes a nucleotide sequence that is substantially identical to the sequence of the MAPKAP kinase-2 RNA. In one embodiment, the siNA molecule includes RNA, the sequence of such RNA including, for example, any one of SEQ ID NOs: 29-32. Small hairpin nucleic acid (shNA) molecules may also be used in the methods of the invention. Alternatively, antisense therapy may be performed by administering a nucleobase oligomer, wherein the sequence of the oligomer is complementary to at least 10 consecutive residues of a nucleotide sequence encoding a MAPKAP kinase-2 polypeptide. Therapy may also be performed by utilizing a compound that includes a peptide or peptidomimetic, e.g., containing the amino acid sequence [L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17), wherein X represents any amino acid and the peptide or peptidomimetic includes no more than 50 amino acids. Hydrophobic amino acids are selected from the group consisting of alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. In one embodiment, the peptide or peptidomimetic includes the amino acid sequence LQRQLSI (SEQ ID NO: 16).
In one aspect of the invention, administering a compound that is capable of inhibiting an activity of a MAPKAP kinase-2 polypeptide for treating a cellular proliferative disorder in a patient sensitizes p53-deficient cells to chemotherapeutic challenge. This can result in increased likelihood of death of aberrantly proliferating cells in a patient, in comparison to p53-deficient cells in a patient not receiving a MAPKAP kinase-2 inhibitor. The chemotherapeutics can be administered subsequently or concomitantly to administration of a MAPKAP kinase-2 inhibitor. An exemplary chemotherapeutic used in this regimen is UCN-01. Increased chemosensitivity of p53-deficient cells to a MAPKAP kinase-2 treatment may alter a DNA damage-responsive cell cycle checkpoint, in comparison to control p-53 deficient cells in a control patient not receiving the described therapy. Alteration of a DNA damage-responsive cell cycle checkpoint may occur at G1/S phase arrest, where Cdc25a degradation impaired. Alteration of a DNA damage-responsive cell cycle checkpoint may occur at G2/M phase arrest, where, the interaction between Cdc25b and a 14-3-3 protein is reduced. The above-mentioned alterations of cell cycle checkpoints may increase the likelihood of cell death, including cell death by apoptosis.
The invention further features kits that include: (a) a means of detecting the level of p53 polypeptide expression or activity in a cellular sample, e.g., by using an anti-p53 antibody; and (b) a compound that is capable of inhibiting an activity of a MAPKAP kinase-2 polypeptide, e.g., UCN-01. In some instances, the kit can also contain one or more chemotherapeutic agents.
The invention additionally features a method for treating a cellular proliferative disorder in a patient including administering to the patient a compound that is capable of inhibiting an activity of a MAPKAP kinase-2 polypeptide. In some instances, the cellular proliferative disorder includes the presence of one or more p53-deficient cells, e.g., tumor cells, in the patient. Administration of a MAPKAP kinase-2 polypeptide together with a chemotherapeutic compound has the desired effect of reducing tumor size. Administration of the described therapy can be, e.g., by direct injection into the tumor.
The invention further features a method for identifying a compound that may be an inhibitor of substrate binding to a MAPKAP kinase-2 polypeptide, the method including the steps of: contacting the MAPKAP kinase-2 polypeptide and a compound capable of binding the MAPKAP kinase-2 polypeptide under conditions allowing the formation of a complex between the compound and the MAPKAP kinase-2 polypeptide; contacting the complex with a candidate compound; and measuring the displacement of the compound capable of binding the MAPKAP kinase-2 polypeptide from the MAPKAP kinase-2 polypeptide. The displacement of the compound capable of binding identifies the candidate compound as a compound that may be an inhibitor of substrate binding to a MAPKAP kinase-2 polypeptide. In one embodiment, the compound capable of binding the MAPKAP kinase-2 polypeptide includes a peptide or peptidomimetic, e.g., containing the amino acid sequence [L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17), wherein the peptide or peptidomimetic includes no more than 50 amino acids. For example, the peptide or peptidomimetic may include the amino acid sequence LQRQLSI (SEQ ID NO: 16). In the foregoing method, a substrate-binding fragment of a MAPKAP kinase-2 polypeptide may be utilized in place of a full-length MAPKAP kinase-2 polypeptide.
Variations of the foregoing aspect are also possible in the methods of the invention. The MAPKAP kinase-2 polypeptide, or substrate-binding fragment thereof, and compound capable of binding the polypeptide may be contacted in the presence of a candidate compound, and any means of measuring the binding of the MAPKAP kinase-2 polypeptide and the compound capable of binding may be used in the methods of the invention. In general, if the amount of binding of the MAPKAP kinase-2 polypeptide and the compound capable of binding is decreased in the presence of the candidate compound in comparison to the amount of binding measured in the absence of the candidate compound, then the candidate compound is determined to be an inhibitor of substrate binding using the methods of the invention.
In another aspect, the invention features a method for identifying a compound that may be an inhibitor of substrate binding to a MAPKAP kinase-2 polypeptide or substrate-binding fragment thereof, the method including the steps of: providing a three-dimensional model of the MAPKAP kinase-2 polypeptide having at least one atomic coordinate, or surrogate thereof, from Table 1 for at least three of the residues Ile74, Glu145, Lys188, Glu190, Phe210, Cys224, Tyr225, Thr226, Pro227, Tyr228, Tyr229, and Asp345, or atomic coordinates that have a root mean square deviation of the coordinates of less than 3 Å; and producing a structure for a candidate compound, the structure defining a molecule having sufficient surface complementary to the MAPKAP kinase-2 polypeptide to bind the MAPKAP kinase-2 polypeptide in an aqueous solution.
The invention further features a compound that includes a peptide or peptidomimetic, e.g., containing the amino acid sequence [L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17), wherein the peptide or peptidomimetic includes no more than 50 amino acids. In one embodiment, the peptide or peptidomimetic includes the amino acid sequence LQRQLSI (SEQ ID NO: 16). An inhibitory compound of the invention may include a covalently-linked moiety capable of translocating across a biological membrane, such as a penetratin or TAT peptide. Alternatively, such a compound may be administered in the form of a prodrug.
In another aspect, the invention features a pharmaceutical composition for treating a cellular proliferative disorder in a patient, the composition including: a compound that is capable of inhibiting an activity of a MAPKAP kinase-2 polypeptide; and a chemotherapeutic agent, wherein the composition is formulated in an amount sufficient to treat the cellular proliferative disorder. Any chemotherapeutic agent known in the art may be useful in the compositions of the invention. An inhibitory compound useful in the pharmaceutical composition may include a covalently-linked moiety capable of translocating across a biological membrane, such as a penetratin or TAT peptide. Alternatively, such a compound may be administered in the form of a prodrug. Any compounds described in any of the foregoing aspects, including small molecule inhibitors, compounds containing siNA molecules, antisense RNA molecules, or peptides, may be useful in the pharmaceutical compositions of the invention.
In any of the foregoing aspects of the invention, it is desirable that the inhibitory compounds be specific inhibitors of MAPKAP kinase-2, e.g., compounds that inhibit a MAPKAP kinase-2 polypeptide without also substantially inhibiting related kinases such as Chk1, Chk2, and p38 SAPK, although compounds that inhibit a MAPKAP kinase-2 polypeptide in a less selective or non-selective manner are also useful in the methods of the invention.
As used throughout this specification and the appended claims, the following terms have the meanings specified.
By an “amino acid fragment” is meant an amino acid residue that has been incorporated into a peptide chain via its alpha carboxyl, its alpha nitrogen, or both. A terminal amino acid is any natural or unnatural amino acid residue at the amino-terminus or the carboxy-terminus. An internal amino acid is any natural or unnatural amino acid residue that is not a terminal amino acid.
By “analog” is meant a molecule that is not identical but has analogous features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
By “antisense,” as used herein in reference to nucleic acids, is meant a nucleic acid sequence, regardless of length, that is complementary to the coding strand of a gene.
By “atomic coordinates” is meant those three-dimensional coordinates of the atoms in a crystalline material derived from mathematical equations related to the patterns obtained on diffraction of x-rays by the atoms (x-ray scattering centers) of the crystalline material. The diffraction data are used to calculate an electron density map of the unit cell of the crystal. These electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal. Atomic coordinates can be transformed, as is known to those skilled in the art, to different coordinate systems (i.e., surrogate systems) without affecting the relative positions of the atoms.
By “binding to” a molecule is meant having a physicochemical affinity for that molecule. Binding may be measured by any of the methods of the invention, e.g., using an in vitro translation binding assay.
By “biological activity” of a polypeptide or other compound is meant having structural, regulatory, or biochemical functions of a naturally occurring molecule. For example, one biological activity of a MAPKAP kinase-2 polypeptide is substrate binding, e.g., peptide binding, which may be measured using in vivo or in vitro binding assays.
By “caged compound” is meant a biologically active molecule coupled to a cleavable moiety such that the resulting coupled compound lacks biological activity as long as the moiety remains attached. Such a moiety prevents bioaction by sterically shielding one or more chemical groups of the molecule. The moiety may be removed by any means, including enzymatic, chemical, or photolytic; removal of the moiety results in restoration of the molecule's biological activity.
By “candidate compound” is meant any nucleic acid molecule, polypeptide, or other small molecule that is assayed for its ability to alter gene or protein expression levels, or the biological activity of a gene or protein by employing one of the assay methods described herein. Candidate compounds include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof.
By “cellular proliferative disorder” is meant any pathological condition in which there is an abnormal increase or decrease in cell proliferation. Exemplary cellular proliferative disorders include cancer or neoplasms, inflammatory diseases, or hyperplasias (e.g., some forms of hypertension, prostatic hyperplasia).
By “chemotherapeutic agent” is meant one or more chemical agents used in the treatment or control of proliferative diseases, including cancer. Chemotherapeutic agents include cytotoxic and cytostatic agents.
By “complex” is meant a chemical association of two or more molecules. Complexes may include a network of weak electrostatic bonds that maintain the association of the molecules. Other types of interactions, such as covalent, ionic, hydrogen bond, hydrophobic, or van der Waals interactions, may be present instead of or in addition to electrostatic bonds between members of a complex.
By “computer modeling” is meant the application of a computational program to determine one or more of the following: the location and binding proximity of a ligand to a binding moiety, the occupied space of a bound ligand, the amount of complementary contact surface between a binding moiety and a ligand, the deformation energy of binding of a given ligand to a binding moiety, and some estimate of hydrogen bonding strength, van der Waals interaction, hydrophobic interaction, and/or electrostatic interaction energies between ligand and binding moiety. Computer modeling can also provide comparisons between the features of a model system and a candidate compound. For example, a computer modeling experiment can compare a pharmacophore model of the invention with a candidate compound to assess the fit of the candidate compound with the model. Examples of techniques useful in the above evaluations include: quantum mechanics, molecular mechanics, molecular dynamics, Monte Carlo sampling, systematic searches and distance geometry methods. Further descriptions of computer modeling programs are provided elsewhere herein.
By “detectably-labeled” is meant any means for marking and identifying the presence of a molecule, e.g., a peptide or a peptidomimetic small molecule that interacts with a MAPKAP kinase-2 domain. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radionuclides (e.g., with an isotope such as 32P, 33P, 125I, or 35S), nonradioactive labeling (e.g., chemiluminescent labeling or fluorescein labeling), and epitope tags.
If required, molecules can be differentially labeled using markers that can distinguish the presence of multiply distinct molecules. For example, a peptide that interacts with a MAPKAP kinase-2 domain can be labeled with fluorescein and a MAPKAP kinase-2 domain can be labeled with Texas Red. The presence of the peptide can be monitored simultaneously with the presence of the MAPKAP kinase-2 domain.
By “fragment” is meant a portion of a polypeptide or nucleic acid having a region that is substantially identical to a portion of a reference protein or nucleic acid and retains at least 50%, 75%, 80%, 90%, 95%, or even 99% of at least one biological activity of the reference protein or nucleic acid.
By “hydrophobic” in the context of amino acids is meant any of the following amino acids: alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, or valine.
By “inhibit an activity of a MAPKAP kinase-2 polypeptide” is meant to reduce one or more biological activities of MAPKAP kinase-2 polypeptide. Desirably, the inhibition is a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in biological activity, relative to a control activity, for example the expression or substrate-binding capability of a naturally occurring MAPKAP kinase-2 polypeptide. An example of a compound that inhibits a MAPKAP kinase-2 polypeptide is UCN-01.
By “MAPKAP kinase-2 biological activity” is meant any activity known to be caused in vivo or in vitro by a MAPKAP kinase-2 polypeptide. For example, such activity could be caused by at least one of the following: function in a DNA damage response pathway, cell cycle control, transcriptional regulation, chromatin remodeling, or substrate binding. In one assay for MAPKAP kinase-2 biological activity, the ability of MAPKAP kinase-2, or a fragment or mutant thereof comprising a substrate-binding domain, to bind a substrate is measured.
By “MAPKAP kinase-2 nucleic acid” is meant a nucleic acid that encodes all or a portion of a MAPKAP kinase-2 polypeptide or is substantially identical to all or a portion of the nucleic acid sequence of Genbank Accession Nos. NM—004759 (SEQ ID NO: 1) or NM—032960 (SEQ ID NO: 2), or analog thereof.
By “MAPKAP kinase-2 polypeptide” and “MK2” are used interchangeably herein, and denote a polypeptide substantially identical to all or a portion of the polypeptide sequence of Genbank Accession Nos. NP—004750 (SEQ ID NO: 3) or P49137 (SEQ ID NO: 4), or analog thereof, and having MAPKAP kinase-2 biological activity.
By “neoplasia” or “neoplasm” is meant a disease characterized by the pathological proliferation of a cell or tissue and its subsequent migration to or invasion of other tissues or organs. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasias can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof. Neoplasias include cancers, such as acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, acute erythroleukemia, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, colon cancer, colon carcinoma, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing's tumor, glioma, heavy chain disease, hemangioblastoma, hepatoma, Hodgkin's disease, large cell carcinoma, leiomyosarcoma, liposarcoma, lung cancer, lung carcinoma, lymphangioendotheliosarcoma, lymphangiosarcoma, macroglobulinemia, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, neuroblastoma, non-Hodgkin's disease, oligodendroglioma, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rhabdomyosarcoma, renal cell carcinoma, retinoblastoma, schwannoma, sebaceous gland carcinoma, seminoma, small cell lung carcinoma, squamous cell carcinoma, sweat gland carcinoma, synovioma, testicular cancer, uterine cancer, Waldenstrom's fibrosarcoma, and Wilm's tumor.
By “nucleic acid” is meant an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid, or analog thereof. This term includes oligomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
Specific examples of some preferred nucleic acids may contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH2—NH_CH2, CH2—N(CH3)—O—CH2, CH2—O—N(CH3)—CH2, CH2—N(CH3)—N(CH3)—CH2 and O—N(CH3)—CH2—CH2 backbones (where phosphodiester is O—P—CH2). Also preferred are oligonucleotides having morpholino backbone structures (Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506). In other preferred embodiments, such as the protein-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (P. E. Nielsen et al. Science 199: 254, 1997). Other preferred oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2′ position: OH, SH, SCH3, F, OCN, O(CH2)nNH2 or O(CH2)n CH3, where n is from 1 to about 10; C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
Other preferred embodiments may include at least one modified base form. Some specific examples of such modified bases include 2-(amino)adenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine, or other heterosubstituted alkyladenines.
By “p53-deficient cell” is meant a cell expressing substantially less p53 polypeptide, or exhibiting substantially less p53 polypeptide activity, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% less p53 expression or activity, than a corresponding wild-type cell. For example, p53-deficient cells, e.g., tumor cells, are present in some neoplastic disorders. p53-deficient cells include cells with one or more p53 gene mutations, e.g., point mutations or null mutations, that reduce or eliminate expression or activity.
By a “peptidomimetic” is meant a compound that is capable of mimicking or antagonizing the biological actions of a natural parent peptide. A peptidomimetic may include non-peptidic structural elements, unnatural peptides, synthesized organic molecules, naturally occurring organic molecules, nucleic acid molecules, and components thereof. Identification of a peptidomimetic can be accomplished by screening methods incorporating a binding pair and identifying compounds that displace the binding pair. Alternatively, a peptidomimetic can be designed in silico, by molecular modeling of a known protein-protein interaction, for example, the interaction of a peptide of the invention and a MAPKAP kinase-2 domain. In one embodiment, the peptidomimetic will displace one member of a binding pair by occupying the same binding interface. It is desirable that the peptidomimetic have a higher binding affinity to the binding interface.
By “pharmaceutically acceptable excipient” is meant a carrier that is physiologically acceptable to the subject to which it is administered and that preserves the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable excipient is physiological saline. Other physiologically acceptable excipients and their formulations are known to one skilled in the art and described, for example, in “Remington: The Science and Practice of Pharmacy,” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins).
By “phosphopeptide” is meant a peptide in which one or more phosphate moieties are covalently linked to serine, threonine, tyrosine, aspartic acid, histidine amino acid residues, or amino acid analogs. A peptide can be phosphorylated to the extent of the number of serine, threonine, tyrosine, or histidine amino acid residues that is present. A phosphopeptide may be phosphorylated at four independent Ser/Thr/Tyr residues, at three independent Ser/Thr/Tyr residues, or at two independent Ser/Thr/Tyr residues. Desirably, a phosphopeptide is phosphorylated at one Ser/Thr/Tyr residue regardless of the presence of multiple Ser, Thr, or Tyr residues.
Typically, a phosphopeptide is produced by expression in a prokaryotic or eukaryotic cell under appropriate conditions or in translation extracts where the peptide is subsequently isolated, and phosphorylated using an appropriate kinase. Alternatively, a phosphopeptide may be synthesized by standard chemical methods, for example, using N-α-FMOC-protected amino acids (including appropriate phosphoamino acids). In a desired embodiment, the use of non-hydrolysable phosphate analogs can be incorporated to produce non-hydrolysable phosphopeptides (Jenkins et al., J. Am. Chem. Soc., 124:6584-6593, 2002; herein incorporated by reference). Such methods of protein synthesis are commonly used and practiced by standard methods in molecular biology and protein biochemistry (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1994, J. Sambrook and D. Russel, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Woodbury N.Y., 2000). In one embodiment, a phosphopeptide is generally not longer than 100 amino acid residues in length. Shorter phosphopeptides, e.g., less than 50, 25, 20, or 15 residues, are also possible. Phosphopeptides may be as short as 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues long.
By “protein” or “polypeptide” or “peptide” is meant any chain of more than two natural or unnatural amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring or non-naturally occurring polypeptide or peptide, as is described herein. As used herein, a natural amino acid is a natural α-amino acid having the L-configuration, such as those normally occurring in natural proteins. Unnatural amino acid refers to an amino acid, which normally does not occur in proteins, e.g., an epimer of a natural α-amino acid having the L configuration, that is to say an amino acid having the unnatural D-configuration; or a (D,L)-isomeric mixture thereof; or a homologue of such an amino acid, for example, a β-amino acid, an α,α-disubstituted amino acid, or an α-amino acid wherein the amino acid side chain has been shortened by one or two methylene groups or lengthened to up to 10 carbon atoms, such as an α-amino alkanoic acid with 5 up to and including 10 carbon atoms in a linear chain, an unsubstituted or substituted aromatic (α-aryl or α-aryl lower alkyl), for example, a substituted phenylalanine or phenylglycine. Other amino acids that may also be incorporated into a polypeptide include ornithine (O or Orn) and hydroxyproline (Hyp).
Polypeptides or derivatives thereof may be fused or attached to another protein or peptide, for example, as a Glutathione-S-Transferase (GST) fusion polypeptide. Other commonly employed fusion polypeptides include, but are not limited to, maltose-binding protein, Staphylococcus aureus protein A, Flag-Tag, HA-tag, green fluorescent proteins (e.g., eGFP, eYFP, eCFP, GFP, YFP, CFP), red fluorescent protein, polyhistidine (6×His), and cellulose-binding protein.
By “prodrug” is meant a compound that is modified in vivo, resulting in formation of a biologically active drug compound, for example by hydrolysis in blood. A thorough discussion of prodrug modifications is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and Judkins et al., Synthetic Communications 26(23):4351-4367, 1996, each of which is incorporated herein by reference.
By “purified” is meant separated from other components that naturally accompany it. Typically, a factor is substantially pure when it is at least 50%, by weight, free from proteins, antibodies, and naturally-occurring organic molecules with which it is naturally associated. The factor may be at least 75%, 90%, or even 99%, by weight, pure. A substantially pure factor may be obtained by chemical synthesis, separation of the factor from natural sources, or production of the factor in a recombinant host cell that does not naturally produce the factor. Proteins, vesicles, and organelles may be purified by one skilled in the art using standard techniques such as those described by Coligan et al. (Current Protocols in Protein Science, John Wiley & Sons, New York, 2000). The factor is desirably at least 2, 5, or 10 times as pure as the starting material, as measured using polyacrylamide gel electrophoresis or column chromatography (including HPLC) analysis (Coligan et al., supra). Exemplary methods of purification include (i) salting-out, i.e., (NH4)2SO4 precipitation; (ii) conventional chromatography, e.g., ion exchange, size exclusion, hydrophobic interaction, or reverse-phase; (iii) affinity chromatography, e.g., immunoaffinity, active site affinity, dye affinity, or immobilized-metal affinity; and (iv) preparative electrophoresis, e.g., isoelectric focusing or native PAGE.
By “RNA interference” (RNAi) is meant a phenomenon where double-stranded RNA homologous to a target mRNA leads to degradation of the targeted mRNA. More broadly defined as degradation of target mRNAs by homologous siRNAs.
By “sensitivity” or “sensitivity to an agent” is meant an increased likelihood of cell death in response to genotoxic stress. An exemplary means of sensitivity occurs when a patient having p53-deficient tumor cell is administered a composition including MAPKAP kinase-2 polypeptide inhibitor and a chemotherapeutic agent, resulting in reduction of tumor size. A reduction in tumor size in the described patient receiving the described therapy is determined in comparison to a control p53-deficient tumor cell in a control patient not receiving the described therapy. Desirably, tumors are reduced is size by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in comparison to the described control.
By “siNA” is meant small interfering nucleic acids. One exemplary siNA is composed of ribonucleic acid (siRNA). siRNAs can be 21-25 nt RNAs derived from processing of linear double-stranded RNA. siRNAs assemble in complexes termed RISC(RNA-induced silencing complex) and target homologous RNA sequences for endonucleolytic cleavage. Synthetic siRNAs also recruit RISCs and are capable of cleaving homologous RNA sequences
By “specifically inhibit an activity of a MAPKAP kinase-2 polypeptide” is meant to reduce one or more biological activities of MAPKAP kinase-2 polypeptide, without substantially inhibiting related kinases, e.g., Chk1, Chk2, and p38 SAPK. Desirably, the specific inhibition is a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in biological activity, relative to a control activity, for example the expression or substrate-binding capability of a naturally occurring MAPKAP kinase-2 polypeptide. An exemplary means of specific inhibition occurs through use of RNA interference. An example of a compound that inhibits a MAPKAP kinase-2 polypeptide, but does not do so specifically, is UCN-01.
By “substantially identical” is meant a polypeptide or nucleic acid exhibiting at least 75%, 85%, 90%, 95%, or even 99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 35 amino acids, 45 amino acids, 55 amino acids, or even 70 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 60 nucleotides, 90 nucleotides, or even 120 nucleotides.
Sequence identity is typically measured using publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12: 387, 1984), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990). The well-known Smith Waterman algorithm may also be used to determine identity. The BLAST program is publicly available from NCBI and other sources (e.g., BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, Md. 20894). These software programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions for amino acid comparisons typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By “substantially inhibit” is meant to reduce one or more activities of the molecule being inhibited by at least 50%, 60%, 70%, 80%, 90%, 95%, or even 98% compared to a control activity value.
By “substrate-binding fragment” in reference to a MAPKAP kinase-2 polypeptide is meant a portion of the polypeptide that is capable of binding a peptide or peptidomimetic substrate. For example, fragments of MAPKAP kinase-2 polypeptide that include the region Phe46-Asp345 (with reference to SEQ ID NO: 3) are substrate-binding fragments.
By “surrogate,” in the context of atomic coordinates, is meant any modification (e.g., mathematical modification or scaling) of the coordinates that preserves the relative relationships among the coordinates.
By “three-dimensional model” is meant a three-dimensional representation of a molecule's structure. Computer modeling may be used to generate such a model in conjunction with structural data. These data could include x-ray crystallographic data, nuclear magnetic resonance data, electron microscopy data, or any other source of experimental or theoretical data useful for generating a model of a molecule or complex of molecules.
By “treating” a disease, disorder, or condition is meant preventing or delaying an initial or subsequent occurrence of a disease, disorder, or condition; increasing the disease-free survival time between the disappearance of a condition and its reoccurrence; stabilizing or reducing an adverse symptom associated with a condition; or inhibiting, slowing, or stabilizing the progression of a condition. Desirably, at least 20, 40, 60, 80, 90, or 95% of the treated subjects have a complete remission in which all evidence of the disease disappears. In another desirable embodiment, the length of time a patient survives after being diagnosed with a condition and treated with a compound of the invention is at least 20, 40, 60, 80, 100, 200, or even 500% greater than (i) the average amount of time an untreated patient survives or (ii) the average amount of time a patient treated with another therapy survives.
By “unnatural amino acid” is meant an organic compound that has a structure similar to a natural amino acid, where it mimics the structure and reactivity of a natural amino acid. The unnatural amino acid as defined herein generally increases or enhances the properties of a peptide (e.g., selectivity, stability, binding affinity) when the unnatural amino acid is either substituted for a natural amino acid or incorporated into a peptide. Unnatural amino acids and peptides including such amino acids are described, e.g., in U.S. Pat. Nos. 6,566,330 and 6,555,522.
Other features and advantages of the invention will be apparent from the following description of the desirable embodiments thereof, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1E depict the substrate specificity and kinetic analysis of substrate phosphorylation by p38α SAPK. FIG. 1A is a table showing p38 substrate specificity determined using oriented peptide library screening. Residues displaying the highest selectivity are shown; those with selection values >1.7 in bold. Abbreviations: MEF2A, Myocyte Enhancer Factor 2; ATF2, Activating Transcription Factor 2; 3PK1, MAP Kinase-activated Protein Kinase-3. FIG. 1B is a graph showing the kinetics of in vitro phosphorylation of an optimal p38 peptide (p38tide) and a peptide from p47phox (p47tide) by p38α kinase. FIG. 1C is a graph showing the kinetics of in vitro phosphorylation of wild-type GST-p47phox, the Ser345→Ala mutant, and the Ser348→Ala mutant. Typical data from n=3 experiments is shown. FIG. 1D shows the in vitro phosphorylation of full-length wild-type or mutant p47phox proteins. Samples were analyzed by SDS-PAGE/autoradiography. FIG. 1E is a table of kinetic parameters for the reactions shown in FIG. 1C.
FIGS. 2A-2C depict the involvement of MAPKAP kinase-2 in the phosphorylation of Cdc25B and Cdc25C after DNA damage. FIG. 2A shows the phosphorylation of full-length wild type Cdc25B or a Ser−323→Ala mutant by p38α SAPK or MAPKAP kinase-2. After phosphorylation, generation of a 14-3-3 binding site on Cdc25B was determined by a 14-3-3-MBP pulldown followed by SDSPAGE/autoradiography. In FIG. 2B, the kinetics of MAPKAP kinase-2 phosphorylation and generation of a 14-3-3-binding site on Cdc25B were measured in U2OS cells following 20 J/m2 UV-irradiation. In FIG. 2C, signaling events in the G2/M, G1, and S-phase checkpoint response were analyzed in GFP siRNA- or MAPKAP kinase-2 siRNA-treated U2OS cells before and two hours after UV-induced DNA damage. Equal loading was determined by western blotting for β-actin.
FIGS. 3A-3C depict the substrate specificity and kinetic analysis of substrate phosphorylation by MAPKAP kinase-2. FIG. 3A is a table showing MAPKAP kinase-2 substrate specificity determined by oriented peptide library screening. Abbreviations: HSP27, Heat Shock protein 27; 5-LO, 5-Lipoxygenase; LSP1, lymphocyte-specific protein; SRF, Serum Response Factor; GS, Glycogen Synthase; TH, Tyrosine Hydroxylase. FIG. 3B is a graph showing the kinetics of in vitro phosphorylation of the optimal MAPKAP kinase-2 peptide (MK2tide) by MAPKAP kinase-2. FIG. 3C is a table of kinetic parameters for MAPKAP kinase-2 phosphorylation of wild-type and mutant MK2tides.
FIGS. 4A-4K show that MAPKAP kinase-2 is required for G2/M arrest following DNA damage. FIG. 4A shows that UV-C irradiation induces DNA damage as revealed by nuclear foci formation. U2OS cells were mock irradiated or exposed to 20 J/m2 of UV-C radiation and immunostained two hours later using an anti-phospho(Ser/Thr) ATM/ATR substrate antibody. FIG. 4B is a graph depicting a FACS analysis of GFP siRNA-treated non-irradiated U2OS cells placed in 50 ng/ml nocodazole-containing media for 16 hours. Cells were analyzed for DNA content by PI staining. FIG. 4C is a graph depicting a FACS analysis of GFP siRNA-treated non-irradiated U2OS cells placed in 50 ng/ml nocodazole-containing media for 16 hours. Cells were analyzed for phospho-histone H3 staining as a marker of mitotic entry. FIG. 4D is a graph depicting a FACS analysis of GFP siRNA-treated U2OS cells irradiated as described for FIG. 4A and then placed in 50 ng/ml nocodazole-containing media for an additional 16 hours. Cells were analyzed for DNA content by PI staining. FIG. 4E is a graph depicting a FACS analysis of GFP siRNA-treated U2OS cells irradiated as described for FIG. 4A and then placed in 50 ng/ml nocodazole-containing media for an additional 16 hours. Cells were analyzed for phospho-histone H3 staining as a marker of mitotic entry. FIG. 4F is a graph depicting a FACS analysis of MAPKAP kinase-2 siRNA-treated non-irradiated U2OS cells placed in 50 ng/ml nocodazole-containing media for 16 hours. Cells were analyzed for DNA content by PI staining. FIG. 4G is a graph depicting a FACS analysis of MAPKAP kinase-2 siRNA-treated non-irradiated U2OS cells placed in 50 ng/ml nocodazole-containing media for 16 hours. Cells were analyzed for phospho-histone H3 staining as a marker of mitotic entry. FIG. 4H is a graph depicting a FACS analysis of MAPKAP kinase-2 siRNA-treated U2OS cells irradiated as described for FIG. 4A and then placed in 50 ng/ml nocodazole-containing media for 16 hours. Cells were analyzed for DNA content by PI staining. FIG. 4I is a graph depicting a FACS analysis of MAPKAP kinase-2 siRNA-treated U2OS cells irradiated as described for FIG. 4A and then placed in 50 ng/ml nocodazole-containing media for 16 hours. Cells were analyzed for phospho-histone H3 staining as a marker of mitotic entry. FIG. 4J is a graph depicting the results of an experiment in which GFP siRNA- or MAPKAP kinase-2-siRNA treated U2OS cells were either mock treated or exposed to 20 J/m2 of UV-C irradiation, and analyzed as described for FIGS. 4B-4I. Representative results of each experiment are shown. FIG. 4K is a graph depicting the results of an experiment in which GFP siRNA- or MAPKAP kinase-2-siRNA treated U2OS cells were either mock treated or exposed to 10 Gy of ionizing radiation, and analyzed as described for FIGS. 4B-4I. Representative results of each experiment are shown.
FIGS. 5A-5E show that MAPKAP kinase-2 is required for S-phase arrest and cell survival following DNA damage. In FIG. 5A, GFP siRNA- or MAPKAP kinase-2-siRNA-treated U2OS cells were mock treated or UV-irradiated and allowed to recover for 30 min. BrdU was added and cells were fixed and analyzed by FACS for DNA content and BrdU incorporation twelve hours later. FIG. 5B is a graph showing the percentage of cells in FIG. 5A showing BrdU incorporation at two and twelve hours following BrdU addition. In FIG. 5C, GFP siRNA- or MAPKAP kinase-2-siRNA-treated U2OS cells were either mock treated or UV-irradiated, allowed to recover for 30 min, then pulse-labeled with BrdU for 30 minutes. At the indicated times after irradiation the distribution of DNA content was analyzed in the BrdU-positive population. In FIG. 5D, GFP siRNA- or MAPKAP kinase-2-siRNA treated U2OS cells were either mock treated or irradiated at the indicated UV-C dose. Cells were stained with Crystal Violet forty-eight hours later and visualized. Insets show a magnified view. FIG. 5E is a graph showing the results of quantitative colony forming assays performed by plating cells at a density of  ̃100 cells per 35 mm2 dish. Cells were treated as in FIG. 5D, and assays were performed in triplicate for each condition.
FIGS. 6A-6E show a comparison of active site electrostatic potentials and hydrophobicity for the substrate binding cleft of MAPKAP kinase-2, Akt and Chk1. FIG. 6A is a table showing the optimal substrate phosphorylation motifs for Akt/PKB, Chk1, Chk2 and MAPKAP kinase-2. FIG. 6B is a ribbons representation of the MAPKAP kinase-2 kinase domain in a similar orientation as that shown in FIGS. 6C-6E (upper), and in an orthogonal orientation (lower) with stick representations of the substrate peptide in the active site. The figure was created using Molscript and Raster3D. FIG. 6C shows molecular surface representations of the Akt/PKB active site (PDB code 106K) using GRASP. Electrostatic potentials (left) and hydrophobicity (right) are indicated by shading. The GSK3 substrate peptide GRPRTTSFAE (SEQ ID NO: 5), with the phospho-acceptor indicated in bold, is shown in stick representation. FIG. 6D shows molecular surface representations of the MAPKAP kinase-2 active site (PDB code 1NY3). Electrostatic and hydrophobic potentials are shaded as in FIG. 6C. The optimal substrate peptide LQRQLSIA (SEQ ID NO: 6) is shown in stick representation. FIG. 6E shows molecular surface representations of the Chk1 active site (PDB code 1IA8). Electrostatic and hydrophobic potentials are shaded as in FIG. 6C. Stick representation of the modeled Cdc25C substrate peptide (LYRSPSMPL) (SEQ ID NO: 7) is shown. The region corresponding to the Ser−5, Ser−3 and Ser+1 positions of the substrate peptides in FIGS. 6C, 6D, and 6E is indicated by dashed circles.
FIG. 7 is a representation of a unified model of the kinase-dependent DNA damage checkpoint. In this model, parallel pathways in the DNA damage checkpoint signal transduction network converge on common substrates by signaling to downstream kinases with similar phosphorylation motif specificities. φ indicates hydrophobic residues. The dashed line from Chk1 to Cdc25B/C indicates that this phosphorylation event remains controversial in response to ionizing radiation.
In FIG. 8A, HeLa cells were incubated with different chemotherapeutic agents for various times as indicated. Lysates were probed for MAPKAP kinase-2, phospho T334 MAPKAP kinase-2 and β-actin as indicated. In FIG. 8B, U2OS cells were incubated with different chemotherapeutic agents for various times as indicated. Lysates were probed for MAPKAP kinase-2, phospho T334 MAPKAP kinase-2 and β-actin as indicated. In FIG. 8C, U87MG cells were incubated with different chemotherapeutic agents for various times as indicated. Lysates were probed for MAPKAP kinase-2, phospho T334 MAPKAP kinase-2 and b-actin as indicated.
In FIG. 9A, U2OS cells were stably transfected with a lentiviral transfection system. Cells were then treated with cisplatin and nocodazole as indicated. After the incubation, cells were harvested and labeled with propidium iodide and phospho histone H3 antibodies for subsequent FACS analysis. In FIG. 9B, U2OS cells were stably transfected with a lentiviral transfection system. Cells were then treated with doxorubicin and nocodazole as indicated. After the incubation, cells were harvested and labeled with propidium iodide and phospho histone H3 antibodies for subsequent FACS analysis.
In FIG. 10, U2OS cells were transiently transfected with siRNA targeting GFP or MAPKAP kinase-2 as indicated. After incubation with cisplatin for the indicated times, cells were harvested and lysates were blotted for CDC25A, MAPKAP kinase-2 and β-actin as indicated.
FIG. 11A is a graph summarizing the results of two independent plating assays. Transiently transfected cells were treated with cisplatin for 8 hours. After the incubation, cells were split 1:20 and re-seeded into new dishes. After a week, cells were methanol-fixed and stained with modified GiemsaTM stain for 2 min. Colonies were then counted. FIG. 11B is a graph summarizing the results of two independent plating assays. Transiently transfected cells were treated with doxorubicin for 8 hours. After the incubation, cells were split 1:20 and re-seeded into new dishes. After a week, cells were methanol-fixed and stained with modified GiemsaTM stain for 2 min. Colonies were then counted. FIG. 11C shows photographs of the original plates for FIG. 11A. Insets show higher magnifications of single colonies. FIG. 11D shows photographs of the original plates for FIG. 11B. Insets show higher magnifications of single colonies.
FIGS. 12A-12F show that the MAPKAP kinase-2 pathway is activated after DNA-damaging chemotherapy. In FIGS. 12A-12D, the kinetics of MAPKAP kinase-2 and p38 MAPK activation are shown. U2OS cells were treated with 10 μM cisplatin (FIG. 12A), 10 μM camptothecin (FIG. 12B), 10 μM doxorubicin (FIG. 12C), or DMSO control (FIG. 12D) for the indicated times. Cell lysates were probed for total and phosphorylated/activated forms of MAPKAP kinase-2 (MK-2) and p38 MAPK by western blotting. β-actin staining served as a loading control. FIG. 12E shows that MAPKAP kinase-2 activation is p38 MAPK-dependent. U2OS cells were treated with the p38 MAPK specific inhibitor SB203580 (10 μM) or DMSO vehicle for 30 min prior to exposure to chemotherapeutic drugs as in FIGS. 12A-12D. Total and phosphorylated/activated p38 was determined by immunoblotting as above. FIG. 12F shows that activation of MAPKAP kinase-2 parallels the formation of γH2AX nuclear foci. U2OS cells were either mock treated or incubated with cisplatin (10 μM), camptothecin (10 μM) or doxorubicin (10 μM). Cells were immunostained one and four hours later using an antibody against γ-H2AX, and counterstained with DAPI (FIG. 12 F). FIG. 12 G is a summary of the requirement for ATM and/or ATR for the activation of MK2.
FIGS. 13A-13C show that ATM/ATR are required for activation of MAPKAP kinase-2 by DNA damaging drugs. FIG. 13A shows that MAPKAP kinase-2 activation by doxorubicin, but not by cisplatin or UV, involves ATM. GM05849 A-T fibroblasts and corresponding control GM00637 fibroblasts were treated with cisplatin (10 μM), doxorubicin (10 μM), UV irradiation (20J/m2) or DMSO (control) for two or eight hours. Cell lysates were probed for total and activated MAPKAP kinase-2 by immunoblotting as in FIG. 12, with β-actin as a loading control. FIG. 13B shows that MAPKAP kinase-2 activation by both cisplatin and doxorubicin is ATR-dependent. GM18366 ATR-defective cells from a patient with Seckel syndrome or the corresponding control GM00023 fibroblasts were treated and analyzed as in FIG. 13A. In contrast to cisplatin and doxorubicin, MAPKAP kinase-2 activation after UV did not require ATR. FIG. 13C shows that caffeine inhibits MAPKAP kinase-2 activation by cisplatin and doxorubicin but not UV. U2OS cells were treated with 20 mM caffeine or vehicle alone for 30 min prior to exposure to the DNA damaging agents. in FIG. 13A. Cell lysates were analyzed for MAPKAP kinase-2 activation as above.
FIGS. 14A-14C show that MAPKAP Kinase 2 mediates a G2/M arrest following doxorubicin treatment. FIG. 14A shows that RNAi down-regulation of MAPKAP kinase-2 ablates the doxorubicin-induced G2/M checkpoint. U2OS cells stably expressing control luciferase shRNA or MAPKAP kinase-2 shRNA were cultured in the absence or presence of 10 μM doxorubicin and cell cycle profiles analyzed thirty hours later by FACS using PI for DNA content and phosphohistone H3 staining as an indicator of mitosis. In the lower set of panels, nocodazole (100 nM) was added three hours following doxorubicin addition. Note that in addition to loss of the prominent G2/M checkpoint, the G1 and S phase components are also eliminated in MK2-depleted cells following doxorubicin+nocodazole treatment. The efficiency of MK2 depletion was analyzed by immunoblotting total cell lysates (lower left). FIG. 14B shows that down-regulation of MAPKAP kinase-2 does not impair Chk1 activation. Luciferase shRNA- or MAPKAP kinase-2 shRNA expressing U2OS cells were mock treated or exposed to 10 μM doxorubicin for thirty hours. Total cell lysates were immunoblotted for the presence of MAPKAP kinase-2 and total and activated forms of Chk1. FIG. 14C shows that doxorubicin and camptothecin-induced binding of Cdc25B to 14-3-3 is lost in MAPKAP kinase-2 depleted cells. U2OS cells were mock treated or treated with 10 M cisplatin, 10 μM camptothecin, or 10 μM doxorubicin for eight hours. The presence of 14-3-3 binding sites on Cdc25B was monitored by incubating the lysates with bead-bound GST-14-3-3 followed by immunoblotting of the pulled-down material.
FIGS. 15A-15C show that MAPKAP kinase-2 controls the G1/S checkpoint in response to cisplatin treatment. FIG. 15A shows that RNAi down-regulation of MAPKAP kinase-2 ablates the cisplatin-induced G1/S checkpoint. U2OS cells stably expressing control luciferase shRNA or MAPKAP kinase-2 shRNA were cultured in the absence or presence of 10 μM cisplatin and cell cycle profiles analyzed thirty hours later by FACS using PI for DNA content and phosphohistone H3 staining as an indicator of mitosis. In the lower set of panels, nocodazole (100 nM) was added three hours following cisplatin addition. FIG. 15B shows that cisplatin-induced reduction of Cdc25A levels is impaired in MAPKAP kinase-2 depleted cells despite activation of Chk1. Luciferase shRNA- or MAPKAP kinase-2 shRNA expressing U2OS cells were mock treated or exposed to 10 μM cisplatin for eight and twelve hours. Total cell lysates were immunoblotted for Cdc25A, total MAPKAP kinase-2 and activated Chk1. α-actin was used as a loading control. FIG. 15C shows that MAPKAP kinase-2 phosphorylates Cdc25A in vitro. GST-tagged Cdc25A was phosphorylated in 30 μl kinase reactions containing either 0.3 μM Chk1 or 0.1 μM MAPKAP kinase-2 for 20 min at 30° C. Samples were analyzed by SDS-PAGE autoradiography. Equal substrate loading was assessed by immunoblotting for GST.
FIGS. 16A-16D show that MAPKAP kinase-2 depletion sensitizes U2OS cells to the antiproliferative effects of cisplatin and doxorubicin. Luciferase shRNA- or MAPKAP Kinase 2 shRNA-expressing U2OS cells were mock treated or treated for eight hours with increasing doses of cisplatin (FIG. 16A) or doxorubicin (FIG. 16B) in a clonogenic survival assay. Cells were washed, trypsinized, and replated at a density of 5000 cells/10 cm2 dish. Eight days later, colonies were visualized using Crystal Violet staining and counted. Insets show magnified views. FIGS. 16C and 16D are graphs showing the quantitation of the results shown in FIGS. 16A and 16B. Assays were performed in triplicate for each condition and normalized to mock treated cells.
FIGS. 17A-17D show that MAPKAP kinase-2 depletion suppresses tumor formation in vivo after implantation of chemotherapy-treated cancer cells. H-Ras-V12 transformed p53−/− MEFs were transfected with siRNA oligonucleotides against GFP or MAPKAP kinase-2. Forty-eight hours following transfection, cells were treated for eight hours with either cisplatin (1 μm), doxorubicin (0.1 μm), or vehicle alone. Two individual injections of 106 cells each were performed into the subcutaneous tissues of each flank of NCR nude outbred mice. FIG. 17A is a dorsal view of the resulting tumors fifteen days after each of the indicated treatments. Control siRNA-transfected cells are in the right flank and MAPKAP kinase-2 siRNA-treated cells are in the left flank. FIG. 17B is a close-up view of the resulting tumors that formed in the absence of DNA damaging chemotherapy pre-treatment. In FIG. 17C, the efficiency of siRNA-mediated knockdown on murine MAPKAP kinase-2 was assessed by immunoblotting lysates from the MEFs prior to tumor implantation. FIG. 17D is a graph showing an analysis of tumor weight at the fifteen-day endpoint.
FIGS. 18A-18D show that MAPKAP kinase-2 depletion enhances regression of established tumors after DNA damaging chemotherapy in a murine model. In FIG. 18A, H-Ras-V12 transformed p53−/− MEFs were infected with lentiviruses encoding U6 promoter-driven luciferase shRNA or MAPKAP kinase-2 shRNA, and CMV promoter-driven GFP. Three days post-infection, GFP expressing cells were selected by FACS and cultured for an additional 7 days. Efficiency of MAPKAP kinase-2 knockdown in the entire GFP-positive population was then assessed by immunoblotting of total cell lysates. In FIG. 18B, following subcutaneous injection of 106 cells into the flanks of NCR nude outbred mice as in FIG. 17, tumor growth was measured every two days. The arrow indicates the start of intraperitoneal administration of DMSO, cisplatin, or doxorubicin on day twelve. In the absence of DNA damaging chemotherapy, the MAPKAP kinase-2 depleted tumors were statistically significantly larger than the control tumors at each time point beginning on day thirteen (Student's t-test, 2-tailed, p<0.02). In contrast, after cisplatin or doxorubicin treatment the MAPKAP kinase-2 depleted tumors were statistically smaller than the control tumors beginning on days twenty-one and twenty-three, respectively (p<0.02). In FIG. 18C, the upper panels are dorsal views of the tumors in situ fourteen days after initiation of the indicated treatments, corresponding to twenty-six days after tumor cell implantation. Middle panels are corresponding fluorescence images. Lower panels are close-up views of the excised tumors. FIG. 18D is a graph showing an analysis of tumor weight at the twenty-six-day endpoint.
FIG. 19 shows that cisplatin and doxorubicin activate MAPKAP kinase-2 independently of Chk1. U2OS cells were transfected with siRNA oligonucleotides targeting GFP or Chk1. Forty eight hours following transfection, cells were treated with 10 μM cisplatin or 10 μM doxorubicin for twelve hours, lysed, and probed for levels of total and phosphorylated/activated MAPKAP kinase-2. The efficiency of the knockdown was assessed by immunoblotting for Chk1.
FIGS. 20A-20E show that overexpression of Chk1 rescues the loss of G1/S and G2/M checkpoints and enhances resistance to genotoxic stress in MAPKAP kinase-2-depleted cells. In FIGS. 20A-20B, luciferase and MAPKAP kinase-2 shRNA expressing U2OS cells were transiently transfected with human Chk1 or empty vector alone. Cells were then exposed to 10 μM cisplatin (FIG. 20A) or 10 μM doxorubicin (FIG. 20B). To arrest cycling cells in mitosis, 100 nM nocodazole was added to the media after three hours of treatment where indicated. The cell cycle profile was analyzed after thirty hours of treatment by FACS using PI for DNA content and phospho-histone H3 staining. In FIGS. 20C-20E, the cell types in FIGS. 20A and 20B above were used in clonogenic survival assays as in FIG. 17. Cells were treated with increasing doses of cisplatin (FIG. 20C) or doxorubicin (FIG. 20D) for eight hours, or mock irradiated or UV irradiated (20J/m2) (FIG. 20E), washed, trypsinized and seeded at a density of 5000 cells/10 cm2 dish. The number of surviving colonies was quantitated eight days later. Assays were performed in triplicate for each condition and normalized to mock treated cells.
FIGS. 21A-21D show that UCN-01 potently inhibits MAPKAP kinase-2. In FIG. 21A, in vitro kinase assays in the presence of increasing doses of UCN-01 were performed with Chk1 and MAPKAP kinase-2 using the MK-2tide as a substrate. FIG. 21B shows the structural basis for UCN-01 inhibition of MAPKAP kinase-2. Ribbon diagrams and molecular surfaces of the Chk1:UCN-01 complex (panels 1, 4) and the MAPKAP kinase-2:staurosporine complex with UCN-01 are modeled onto staurosporine (panels 2, 3, 5). Panel 3 is rotated 90° from the view in panel 2. The arrow points to the unique 7-hydroxy group of UCN-01 with Van der Waals radii indicated by dots. FIG. 21C shows that UCN-01 inhibits MAPKAP kinase-2 in U2OS cells. Luciferase shRNA- or MAPKAP Kinase 2 shRNA-expressing cells were incubated at 37° C. or 42° C. for two hours in the absence or presence of 200 nM UCN-01. Cells were lysed and probed for total hsp-27, hsp-27 pS82, and MAPKAP kinase-2 by immunoblotting. FIG. 21D shows that UCN-01 inhibition of hsp-27 is independent of Chk1. GFP or Chk1 siRNA-transfected cells were incubated at 42° C. or 37° C. for two hours in the absence or presence of 200 nM UCN-01. Cells were then lysed and probed for total hsp-27, hsp-27 pS82, and Chk1 by immunoblotting.
FIG. 22 is a representation of a model for MAPKAP kinase-2 checkpoint signaling in response to DNA damaging chemotherapy. Checkpoint function in response to DNA damaging agents normally requires the combined action of both the Chk1 and MAPKAP kinase-2 pathways, and both pathways are simultaneously inhibited by the indolocarbazole drug UCN-01.
FIGS. 23A-23B show that camptothecin activates MAPKAP kinase-2 in an ATR-dependent and ATM-independent manner. In FIG. 23A, GM18366 ATR-defective cells from a patient with Seckel syndrome or the corresponding control GM00023 fibroblasts were treated with camptothecin (10 μM) for two or eight hours. Cell lysates were probed for total and activated MAPKAP kinase-2 by immunoblotting, and with anti-α-actin as a loading control. In FIG. 23B, GM05849 A-T fibroblasts and corresponding control GM00637 fibroblasts were treated and analyzed as in FIG. 23A.
FIGS. 24A-24D show that treatment with cisplatin and doxorubicin preferentially induce different cell cycle checkpoints. In FIG. 24A, the cell cycle profile of asynchronous control U2OS cells was analyzed by FACS using PI for DNA content and phospho-histone H3 staining. FIG. 24B shows that cells treated with doxorubicin (10 μM) for eighteen hours preferentially accumulate at the G2/M boundary, with a smaller component in G1 and S. FIG. 24C shows that cells treated with cisplatin (10 μM) for eighteen hours preferentially accumulate in G1/S with a small component at G2/M. FIG. 24D shows that cells treated with nocodazole (100 nM) for eighteen hours accumulate in M with  ̃42% staining strongly for phospho-histone H3.
FIG. 25 shows the effect of overexpression of Chk1 in control and MAPKAP kinase-2 knockdown U2OS cells. U2OS cells stably expressing luciferase or MAPKAP kinase-2 shRNA were transiently transfected with FLAG-tagged Chk1 in the expression vector pHURRA, or with vector alone. Cell lysates were probed for total MAPKAP kinase-2 and Chk1 levels, with β-actin as a loading control.
FIG. 26 shows that overexpression of Chk1 rescues the loss of the UV-induced G1/S checkpoint. Luciferase and MAPKAP kinase-2 shRNA expressing U2OS cells were transiently transfected with human Chk1 or empty vector alone. Cells were then irradiated with 20 J/m2 of UV light. To arrest cycling cells in mitosis, 100 nM nocodazole was added to the media after three hours of treatment where indicated. The cell cycle profile was analyzed after thirty hours of treatment by FACS using PI for DNA content and phospho-histone H3 staining. In addition to the prominent G1/S checkpoint, a minor G2/M checkpoint was also observed in the control cells, but not the MAPKAP kinase-2 depleted cells, after irradiation, and this checkpoint was also restored upon overexpression of Chk1.
FIG. 27 shows that Chk1 overexpression rescues clonogenic survival in MAPKAP kinase-2 depleted cells treated with cisplatin. U2OS cells stably expressing luciferase or MAPKAP kinase-2 shRNA were transiently transfected with FLAG-tagged Chk1 in the expression vector pHURRA, or with vector alone. Cells were treated with increasing doses of cisplatin for eight hours, washed, trypsinized and seeded at a density of 5000 cells/10 cm2 dish. Surviving colonies were stained with crystal violet and counted eight days later.
FIG. 28 shows that Chk1 overexpression rescues clonogenic survival in MAPKAP kinase-2 depleted cells treated with doxorubicin. U2OS cells as in FIG. 27 were treated with increasing doses of doxorubicin and analyzed as described above.
FIGS. 29A-29B are graphs showing the results of a colony survival assay in which mouse embryonic fibroblasts (MEFs) were treated with increasing doses of doxorubicin or cisplatin for eight hours, rinsed twice with PBS and once with media, and re-plated at an initial density of 5,000 cells/10 cm2 dish. After eleven days, the number of colonies on the plate was counted and normalized to the number of colonies formed by the same cell type in the absence of treatment with chemotherapeutic drugs. RNA interference using short hairpin RNAs was used with both the p53 wild-type MEFs and the p53−/− MEFs to knock down the levels of MAPKAP kinase-2 (MK2 shRNA). Short hairpin RNAs against luciferase (luciferase shRNA) were used as a control. Loss of MAPKAP kinase-2 activity resulted in increased sensitivity to both doxorubicin and cisplatin (i.e. decreased survival after treatment) only in the MEFs that lacked p53. FIG. 29C is a series of Western blots using the indicated antibodies. These blots verify that p53 polypeptide, and the known p53-responsive gene product p21, is induced by DNA damage only in the p53 wild-type cells (lanes 2, 3, 5, and 6), but not in the p53−/− cells (lanes 7-12). The blot also verifies that the short hairpin RNA against MAPKAP kinase-2 knocked down the levels of MAPKAP kinase-2 in the MK-2 shRNA treated cells (lanes 4-6 and 10-12). This also prevented MAPKAP kinase-2 activation in the MAPKAP kinase-2 shRNA-treated cells after DNA-damaging chemotherapy, as shown by the lack of phospho-MAPKAP kinase-2 in lanes 5-6 and 11-12, compared with the presence of phospho-MAPKAP kinase-2 in the luciferase shRNA treated cells after DNA-damaging chemotherapy (lanes 2-3 and 8-9).
FIGS. 30A-30D represent pS3WT/WT and p53−/− MEFs stably expressing RNAi hairpins against luciferase (FIGS. 30A and 30C) or MK2 (FIGS. 30B, and 30D). Cells were then treated with low dose cisplatin (1.01M) or doxorubicin (0.1 μM) for thirty hours, fixed and stained with antibodies against phosphohistone H3, γ-H2AX and cleaved caspase-3. Positive staining for γ-H2AX in combination with phospho-histone H3 and cleaved caspase-3 labeling is indicative of mitotic catastrophe, was only observed in MK2 depleted p53−/− cells (FIG. 30D, arrows). Arrowhead in that panel shows a γ-H2AX-positive cell that does not stain for either phospho-histone H3 or cleaved caspase-3. Scale bar, 5 μm.
FIGS. 31A-31C are histograms and western blot images demonstrating MAPKAP Kinase 2 mediates a G2/M arrest following doxorubicin treatment. RNAi down-regulation of MK2 ablates the doxorubicin-induced G2/M checkpoint (FIG. 31A). p53−/− MEFs stably expressing control luciferase shRNA (FIG. 31A left panels) or MK2 shRNA (FIG. 31A right panels) were cultured in the absence or presence of 10 μM doxorubicin and cell cycle profiles analyzed thirty hours later by FACS using PI for DNA content (blue) and phospho-histone H3 staining as an indicator of mitosis (red). In the lower set of panels, nocodazole (100 ng/ml) was added three hours following doxorubicin addition. Note that in addition to loss of the prominent G2/M checkpoint, the G1 and S phase components are also eliminated in MK2-depleted cells following doxorubicin+nocodazole treatment. Down-regulation of MK2 does not impair Chk1 activation (FIG. 31B). Luciferase shRNA or MK2 shRNA expressing p53−/− MEFs were mock treated or exposed to 10 μM doxorubicin or 10 μM camptothecin for thirty hours. Total cell lysates were immunoblotted for the presence of MK2 and total and activated forms of Chk1. Doxorubicin and camptothecin-induced binding of Cdc25B to 14-3-3 is lost in MK2 depleted cells (FIG. 31C). p53−/− MEFs cells either expressing a luciferase hairpin (upper panel) or a MK2 specific hairpin (lower panel) were mock treated or treated with 10M camptothecin (cam) or 10 μM doxorubicin (dox) for eight hour. The presence of 14-3-3-binding sites on Cdc25B was monitored by incubating the lysates with bead-bound GST-14-3-3 β/ζ followed by immunoblotting of the pulled-down material.
FIGS. 32A-32B are histograms and western blot images demonstrating MK2 controls the S-phase checkpoint in response to cisplatin treatment. RNAi down-regulation of MK2 ablates the cisplatin-induced S-phase checkpoint (FIG. 32A). p53−/− MEFs stably expressing control luciferase shRNA (FIG. 32A, left panels) or MK2shRNA (FIG. 32A, right panels) were cultured in the absence or presence of 10 μM cisplatin and cell cycle profiles analyzed thirty hours later by FACS using PI for DNA content (blue) and phospho-histone H3 staining as an indicator of mitosis (red). In the lower set of panels, nocodazole (100 ng/ml) was added three hours following cisplatin addition. Cisplatin-induced degradation of Cdc25A is impaired in MK2 depleted cells despite activation of Chk1 (FIG. 32B). Luciferase shRNA- or MK2 shRNA expressing p53−/− MEFs were mock treated or exposed to 10M cisplatin for eight and twelve hours. Total cell lysates were immunoblotted for Cdc25A, total MK2 and activated Chk1. β-actin was used as a loading control.
FIGS. 33A-33B are models for re-wiring of cell cycle checkpoint pathways in p53-proficient and deficient cells. Checkpoint function in p53-proficient cells is mediated primarily through a robust, sustained p53 response downstream of ATM, together with Chk1 (FIG. 33A). Although not shown explicitly in the diagram, Chk1 also directly phosphorylates p53 (Shieh et al., 2000). Under these conditions the presence of MK2 is not required for cell survival after exposure to DNA damaging agents. In p53-deficient cancer cells (FIG. 33B), checkpoint signaling following exposure to DNA damaging agents is mediated through the combined action of both the Chk1 and the p38 MAPK/MK2pathways. In this situation the p38MAPK/MK2 branch of checkpoint signaling becomes essential for cell survival after DNA damage. Both pathways are simultaneously inhibited by the indolocarbazole drug UCN-01.



DETAILED DESCRIPTION OF THE INVENTION
The invention features methods and compounds that are useful in treating cellular proliferative disorders. The methods of treatment feature administration of a compound that is capable of inhibiting an activity of a MAPKAP kinase-2 polypeptide, or a substrate-binding fragment thereof. Such compounds include, without limitation, compounds that contain peptides, peptidomimetics, or nucleic acid molecules. The invention further features screening assays for identifying MAPKAP kinase-2 inhibitors. In addition, the invention includes pharmaceutical compositions and compounds, e.g., peptides and peptidomimetics, that target the substrate-binding site of MAPKAP kinase-2, thereby inhibiting it.
It was recently shown that, in addition to the ATR-Chk1 pathway, the p38 SAPK pathway is also required for full activation of the DNA damage response following UV irradiation. We now demonstrate that MAPKAP kinase-2, a direct downstream target of p38 SAPK, is directly responsible for phosphorylating Cdc25B and C and maintaining the G1, S, and G2/M checkpoints in response to UV-induced DNA damage. Thus, MAPKAP kinase-2 constitutes a third checkpoint kinase, in addition to Chk1 and Chk2, involved in coordinating the DNA damage response of higher eukaryotic cells.
A number of important questions regarding this third DNA damage response pathway have not been previously answered. Is p38 MAPK/MAPKAP kinase-2 activation after DNA damage dependent on ATR or ATM? Is p38 MAPK/MAPKAP kinase-2 cascade important for DNA damage checkpoints in response to other types of genotoxic stress besides UV? How are signals from the Chk1 pathway and the MAPKAP kinase-2 pathway integrated together at the systems level? We were particularly interested in investigating whether MAPKAP kinase-2/Chk3 participates in the genotoxic stress response of cells exposed to conventional anti-cancer chemotherapeutic agents. A demonstration that MAPKAP kinase-2 has an important role in preventing cells with chemotherapy-induced DNA damage from progressing through the cell cycle would implicate MAPKAP kinase-2 as a clinically important target for anti-cancer drug design.
Defining the Optimal Phosphorylation Motif for p38 SAPK
To identify substrates and targets of the p38 SAPK signaling pathway involved in DNA damage responses, we determined the optimal substrate phosphorylation motif for p38ax SAPK using oriented peptide library screening. Efficient peptide phosphorylation by p38 SAPK required a fixed Pro residue in the Ser+1 position, consistent with the known identification of p38 SAPK as a Pro-directed MAP kinase. Screening performed with a library containing the degenerate sequence XXXXSPXXXX (SEQ ID NO: 8) (X denotes all amino acids except Cys, Ser, Thr, and Tyr) displayed strongest selection for Pro in the Ser−2 position with weaker selection for other aliphatic residues (FIG. 1A). Additional selection was also observed at the Ser−3, Ser−1, and Ser+2 positions.
To further refine the optimal phosphorylation motif, a secondary screen was performed based on results from the initial screen by using a library with Pro fixed in both the Ser−2 and Ser+1 positions, and Ser, Thr, and Tyr included in the X positions. This revealed selection for Gln, Met, and Gly in the Ser−1 position, along with slightly weaker selection for Pro, Ser and Thr (FIG. 1A). Gly was the preferred residue in the Ser−3 position, along with Ile, Val, and Tyr. Hydrophobic residues, particularly aromatic and β-branched amino acids, were selected at the Ser+2 position. The resulting optimal motif for p38α SAPK determined by oriented peptide library screening closely matches the sequence of mapped p38 MAPK phosphorylation sites on most, though not all, known substrates (FIG. 1A).
A peptide containing the optimal p38 SAPK consensus phosphorylation motif GPQSPI (SEQ ID NO: 9), “p38tide,” was synthesized for kinetic analysis. This peptide was readily phosphorylated by p38 SAPK in vitro; however, it failed to display saturable Michaelis-Menton-type kinetics (FIG. 1B). Instead, the initial velocity increased linearly with increasing p38tide concentration up to 1400 μM. This finding suggests that additional interactions besides an optimal phosphorylation motif are likely to be involved in optimizing p38 SAPK-substrate binding, such as MAP kinase docking sites.
To search for potential p38 SAPK substrates, particularly those relevant to DNA damage signaling, the Swiss-Prot database was queried with the p38 SAPK consensus phosphorylation motif using Scansite. Other than GADD153, a known p38 SAPK substrate, we were unable to identify any DNA damage response proteins in the top 250 hits. Database searching did, however, reveal two tandem near-optimal p38 SAPK phosphorylation sites (Ser345 and Ser348) in p47phox, a cytosolic component of the NADPH oxidase enzyme. A peptide containing this sequence, PGPQSPGSPL (SEQ ID NO: 10), “p47tide,” was strongly phosphorylated by p38 SAPK, but like p38tide, the isolated peptide displayed linear non-saturable kinetics (FIG. 1B).
Wild-type and mutant versions of GST-tagged full-length p47phox protein, rather than isolated peptides, were then used as substrates for in vitro phosphorylation reactions. The wild-type full-length p47phox protein was rapidly phosphorylated by p38α SAPK (FIGS. 1C and 1D). Mutation of Ser345→Ala had a more pronounced effect on p47phox phosphorylation than mutation of Ser348→Ala, in excellent agreement with the observation that the Ser345 site is a better match for the optimal p38 SAPK consensus motif than the Ser348 site. Simultaneous mutation of both Ser345 and Ser348 to Ala eliminated phosphorylation of p47phox by p38 SAPK altogether. Kinetic analysis revealed classical Michaelis-Menton behavior for p38 SAPK phosphorylation of the wild-type p47phox with a Km of 6.0 μM and a Vmax of 36.6 nmol/min/μg. Mutation of Ser345 to Ala both increased the Km and reduced the Vmax, while mutation of Ser348 to Ala primarily increased the Km (FIG. 1E).
These data from isolated peptides and intact proteins argue that efficient substrate phosphorylation by p38 SAPK requires sequences with reasonable matches to the optimal substrate motif determined by oriented peptide library screening, and that additional interactions involving MAPK docking sites are likely to be critical for strong kinase-substrate interactions. Several docking motifs have been identified for p38 SAPK, particularly a short cluster of positively charged amino acid residues often flanked by hydrophobic amino acids. Two sequences corresponding to this type of docking motif are present near the p38SAPK phosphorylation sites in p47 phox, 1HQRSRKRLSQ (SEQ ID NO: 11) and VRFLQQRRRQA (SEQ ID NO: 12). Mutation of RRR to LLL in the latter motif decreased the rate of p38α SAPK phosphorylation of p47phox by over 70%.
Bulavin et al. (Nature, 411:102-107, 2001) implicated p38 SAPK in the DNA damage response pathway and reported that p38 SAPK was directly responsible for generating a 14-3-3-binding site on Cdc25B (Ser323 in Cdc25B2; Ser309 in Cdc25B1) in response to UV-C-induced DNA damage. Like p47phox, Cdc25B contains a potential p38 SAPK docking motif, PVQNKRRRSV (SEQ ID NO: 13); however, the sequence flanking Ser323, LXRSPSMP (SEQ ID NO: 14), lacks a Pro in the Ser+1 position and does not resemble the optimal p38 SAPK motif shown in FIG. 1A. As shown in FIG. 2A, recombinant p38 SAPK readily phosphorylated bacterially produced Cdc25B in vitro. However, this phosphorylation did not induce 14-3-3-binding, and a Ser323→Ala mutant form of Cdc25B was phosphorylated by p38 SAPK equivalently to the wild-type Cdc25B protein. These data argue that, while Cdc25B may be a p38 SAPK substrate, this phosphorylation event is not responsible for the 14-3-3-binding event that results in a G2/M checkpoint.
Defining the Optimal Phosphorylation Motif for MAPKAP Kinase-2
A number of Ser/Thr kinases are activated downstream of p38 SAPK, including MAPKAP Kinases-2 and -3, MNK1 and MNK2, MSK1 and MSK2, and PRAK. In response to UV-B-induced DNA damage, She et al. (Oncogene, 21:1580-1589, 2002) reported that MAPKAP kinase-2 could phosphorylate p53 on Ser20, the same site that is phosphorylated by two well-established checkpoint kinases, Chk1 and Chk2. Both Chk1 and Chk2 can also phosphorylate Cdc25 family members to create 14-3-3 binding sites, suggesting that MAPKAP kinase-2 might share a similar motif. The optimal substrate phosphorylation motif for MAPKAP kinase-2 was therefore investigated using oriented peptide library screening.
Efficient peptide phosphorylation by MAPKAP kinase-2 was only observed with a library containing a fixed Arg in the Ser−3 position (XXXXRXXSXXXX (SEQ ID NO: 15), where X denotes all amino acids except Cys, Ser, Thr, or Tyr). A critical step in determining protein kinase phosphorylation motifs by peptide library screening involves purification of the phosphorylated peptides from the non-phosphorylated peptide background. In the case of MAPKAP kinase-2, this was dramatically improved by conversion of all Glu and Asp residues to their methyl esters prior to metal-affinity chromatography and sequencing. This resulting motif revealed clear amino acid selection at almost all degenerate positions (FIG. 3A). MAPKAP kinase-2 displayed strong selection for the hydrophobic residues Leu, Phe, Ile, and Val in the Ser−5 position and the Ser+1 position. Strong selection was also observed for Gln in the Ser−2 position, and modest selection for Leu in the Ser−1 position. The motif determined for MAPKAP kinase-2 using oriented peptide library screening is in remarkably good agreement with the sequence of mapped MAPKAP kinase-2 phosphorylation sites on known substrates (FIG. 3A, bottom), which primarily contain Leu, Ile or Phe in the Ser−5 position; Arg in the Ser−3 position; Gln, Ser, or Thr in the Ser−2 position; Leu, Val or Pro in the Ser−1 position; and hydrophobic residues along with Glu in the Ser+1 position. The preference for polar residues Ser and Thr in addition to Gln in the Ser−2 position in known MAPKAP kinase-2 substrates would not have been detected by oriented peptide library screening, since Ser and Thr were not present in the library.
To verify the peptide library screening results, individual peptides (MK2tides) containing the optimal MAPKAP kinase-2 consensus motif LQRQLSI (SEQ ID NO: 16), or mutant versions with Ala substituted at each position in the motif, were synthesized and used for kinetic analysis (FIGS. 3B and 3C). The optimal MK2tide displayed a Km value two-fold lower than the best MAPKAP kinase-2 peptide substrate known to date, a sequence derived from HSP27. Substitution of Ala at each position in the motif affected Km and Vmax differently, with some positions showing primarily a Km effect (i.e., Arg in the Ser−3 position), while others revealed a primary effect on Vmax (i.e., Leu in the Ser−5 position) (FIG. 3C). The rank order of importance of key residues is Arg-3>Leu-5≈Ile+1>Gln-3. The optimal MK2tide had neither the lowest Km nor the highest Vmax, but rather had the highest Vmax/Km ratio, consistent with the fact that the peptide library screening approach selects substrates on the basis of optimal Vmax/Km, rather than low Km or high Vmax alone. Combining the data from oriented peptide library screening, known substrate sequences, and our kinetic studies, the optimal MAPKAP kinase-2 phosphorylation motif is [L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17).
The optimal MAPKAP kinase-2 substrate motif is an excellent match for the known Ser323 phosphorylation/14-3-3 binding motif in Cdc25B, as well as the Ser216 phosphorylation/14-3-3-binding site in Cdc25C (FIG. 3A). Initial experiments focused on Cdc25B, since, unlike Cdc25C, Cdc25B can be produced in modest quantities in bacteria, and the Ser323 site in Cdc25B had been previously reported to be a direct p38 SAPK site. Incubation of recombinant Cdc25B with purified MAPKAP kinase-2 resulted in significant Cdc25B phosphorylation and strong binding of the phosphorylated protein to 14-3-3 (FIG. 2A). Mutation of Ser323→Ala substantially reduced the ability of MAPKAP kinase-2 to phosphorylate Cdc25B, and completely eliminated the ability of Cdc25B to bind to 14-3-3 (FIG. 2A). These in vitro results strongly suggest that MAPKAP kinase-2 is the critical Cdc25/14-3-3 checkpoint kinase downstream of DNA damage signals relayed by the p38 SAPK pathway.
MAPKAP Kinase-2 is Critical for the G2/M Checkpoint following UV-Induced DNA Damage
The importance of MAPKAP kinase-2 in DNA damage checkpoint function was investigated in U2OS cells. Activation of MAPKAP kinase-2 in response to UV-C irradiation-induced DNA damage (FIG. 4A) was monitored by its reduced mobility on SDS-PAGE gels, and by immunoblotting using a phospho-specific antibody against pThr344, a site phosphorylated by p38 and required for MAPKAP kinase-2 activation. As shown in FIG. 2B, MAPKAP kinase-2 was activated within one hour of irradiation, and remained activated for the eight hour duration of the experiment. The kinetics of MAPKAP kinase-2 activation paralleled the ability of Cdc25B from these cells to bind to 14-3-3. Based on these data, a two hour time point was chosen for use in further studies.
RNA interference was used to confirm a direct role for endogenous MAPKAP kinase-2 in the UV-induced DNA damage response. Treatment of U2OS cells with MAPKAP kinase-2-specific siRNA oligonucleotides, but not with control GFP siRNA oligonucleotides, resulted in a substantial reduction of MAPKAP kinase-2 to nearly undetectable levels by forty-eight hours after transfection (FIG. 2C). No reduction in the levels or UV-C-induced activation of p38 SAPK, Chk1 or Chk2 was observed in these cells. Despite the presence of these other active kinases, siRNA-mediated knockdown of MAPKAP kinase-2 caused a loss of both Cdc25B- and Cdc25C-binding to 14-3-3 after UV-C exposure (FIG. 2C).
We studied cell cycle progression in the control GFP and MAPKAP kinase-2 knockdown cells following UV-C-irradiation using FACS (FIGS. 4A-4K). In these experiments, cells were irradiated with 20 J/m2 of UV-C radiation, allowed to recover for two hours, then placed in nocodazole-containing media for an additional sixteen hours to cause any cells progressing through the cell cycle to arrest in mitosis, where they can stained for the mitotic marker phosho-histone H3. Under these conditions, un-irradiated cultures of asynchronous GFP siRNA-transfected cells accumulated in a 4N-DNA-containing peak, with prominent levels of phospho-histone H3 staining (FIGS. 4B and 4C), consistent with a nocodazole-mediated M-phase arrest. In response to UV-irradiation, control cells displayed a prominent G1, S, and G2 distribution, with near-complete loss of phosphohistone H3 staining, indicating intact G1, S, and G2 checkpoints (FIGS. 4D and 4E).
The behavior of the MAPKAP kinase-2 siRNA transfected cells was dramatically different. In the absence of UV irradiation, MAPKAP kinase-2 siRNA transfected cells, like control GFP siRNA-transfected cells, accumulate in a 4N DNA-containing peak with high levels of phospho-histone H3 staining (FIGS. 4F and 4G). Following UV-induced DNA damage, however, the MAPKAP kinase-2 knockdown cells failed to arrest cell cycle progression. Instead, these cells proceeded to enter mitosis to the same extent as unirradiated cells, as shown by a comparable 4N-DNA peak and similar levels of phoshohistone H3 staining as those observed in un-irradiated cells (FIGS. 4H and 41). Together with the Cdc25B/C: 14-3-3 results in FIG. 2C, these FACS data demonstrate that MAPKAP kinase-2 is critical for the UV-induced G2/M checkpoint in response to UV-irradiation. In contrast to the UV response, summarized in FIG. 4J, the G2/M checkpoint response to ionizing radiation in MAPKAP kinase-2 knockdown cells is intact (FIG. 4K).
MAPKAP Kinase-2 is Critical for the S-Phase Checkpoint and G1 Arrest Following UV-Induced DNA Damage
The MAPKAP kinase-2 knockdown cells in FIGS. 4A-4K also showed a loss of the G1 and S-phase checkpoints following DNA damage, since UV-irradiation of asynchronous cultures resulted in accumulation of the cells in a 4N DNA-containing peak when the cells were transferred to nocodazole-containing medium. To investigate the direct role of MAPKAP kinase-2 in S-phase checkpoint function, control or MAPKAP kinase-2 knockdown U2OS cells were UV-irradiated, allowed to recover for 30 min, and then labeled with BrdU for various times. In the absence of irradiation, 42% of the control siRNA-transfected cells showed substantial BrdU incorporation after twelve hours, compared with 53% of the MAPKAP kinase-2-siRNA transfected cells (FIGS. 5A and 5B). When the cells were irradiated with 20 J/m2 of UV light prior to BrdU labeling, only 3.5% of the control siRNA transfected cells showed BrdU incorporation at twelve hours. In marked contrast, 48% of the MAPKAP kinase-2-knockdown cells continued to incorporate substantial amounts of BrdU. A similar difference in BrdU uptake between control siRNA-treated cells and MAPKAP kinase-2-knockdown cells was also seen at shorter times after irradiation (FIG. 5B).
Examination of the FACS profiles twelve hours following UV-irradiation revealed a dramatic decrease in the G1 population in the MAPKAP kinase-2-knockdown cells compared with the control GFP siRNA-transduced cells (FIG. 5A, right-most upper and lower FACS profiles). This loss of the G1 peak, together with the increased percentage of cells showing BrdU incorporation at twelve hours versus two hours of labeling, implies that endogenous MAPKAP kinase-2 plays important roles in both the inhibition of DNA synthesis following damage (S-phase checkpoint function), and in the damage-induced arrest of cells in G1 prior to S-phase entry (G1/S checkpoint function). Loss of the G1/S and S-phase checkpoints in MAPKAP kinase-2 knockdown cells was associated with higher levels of Cdc25A, decreased levels of p53, and reduced phosphorylation of p53 on Ser20 following UV-irradiation compared with control siRNA-treated cells (FIG. 2C).
The fate of S-phase control or MAPKAP kinase-2 siRNA-treated cells in response to UV-C-induced DNA damage was examined by using FACS. In this experiment, asynchronous cells were mock-treated or irradiated with 20 J/m2 of UV-C radiation and then pulse-labeled with BrdU. The cells showing BrdU uptake were subsequently analyzed ten and twenty hours later (FIG. 5C). In both non-irradiated control and MAPKAP kinase-2 knockdown cells, the BrdU pulse-labeled population showed a late S and G2/M distribution at ten hours, and a re-appearance of a G1 peak at twenty hours, indicating full transit through the cell cycle. In response to UV-C irradiation, control siRNA-treated cells failed to show significant BrdU uptake upon which to gate for FACS analysis (FIG. 5C, lower left panel). In contrast, the large population of MAPKAP kinase-2 siRNA treated cells, which had lost the S-phase checkpoint and incorporated BrdU, went on to display a greatly reduced G1 peak at twenty hours, with many cells showing DNA staining >4N (FIG. 5C, bracket in lower right panel), consistent with mitotic death and exit from the cell cycle.
MAPKAP Kinase-2 Depleted Cells are more Sensitive to DNA Damage-Induced Cell Death
The experiments in FIGS. 4 and 5A-5C indicate that MAPKAP kinase-2 is involved in each of the cell cycle checkpoints triggered by UV-induced DNA damage. To determine the effect of MAPKAP kinase-2 depletion on cell survival, we transfected cells with control siRNA or MAPKAP kinase-2 siRNA for forty-eight hours, trypsinized, replated, and analyzed for colony formation in response to various doses of UV-C irradiation twelve hours after re-plating. As shown in FIGS. 5D and 5E, MAPKAP kinase-2 knockdown cells displayed a significant reduction in colony formation when compared to control-treated cells at all doses of UV-C irradiation examined. This difference in survival after UV-C exposure was most pronounced at low to moderate UV doses.
A Structural Model for MAPKAP Kinase-2 Substrate Selectivity.
The optimal phosphorylation motif determined for MAPKAP kinase-2 is strikingly similar to that determined for two other checkpoint kinases, Chk1 and Chk2 (FIG. 6A). All three of these CAMK superfamily members—MAPKAP kinase-2, Chk1, and Chk2—strongly select for aliphatic residues in the Ser−5 position, Arg in the Ser−3 position, and aromatic/aliphatic residues in the Ser+1 position, along with additional less stringent selection for particular amino acids in other positions (FIG. 6A). In contrast, members of the AGC kinase superfamily, such as Akt/PKB and conventional protein kinase C superfamily members, preferentially phosphorylate sequences containing Arg residues in both the Ser−5 and Ser−3 positions, and play important roles in anti-apoptotic signaling and other signaling events unique to differentiated cell function, rather than critical roles in cell cycle control.
To investigate the structural basis for substrate motif selection, we performed molecular modeling studies of activated MAPKAP kinase-2, using the published MAPKAP kinase-2:ADP co-crystal structure (Underwood et al., Structure, 11:627-636, 2003) as a base model. The optimal substrate peptide LQRQLSIA (SEQ ID NO: 6) was modeled into the kinase active site in an extended conformation (FIG. 6D), and the kinase:substrate complex compared with the structures of Akt/PKB:AMP-PNP:GSK3-peptide ternary complex (Yang et al., Nat. Struct. Biol., 9:940-944, 2002) (FIG. 6C) and the Chk1 crystal structure containing a modeled Cdc25C peptide (Chen et al., Cell, 100:681-692, 2000) (FIG. 6E). Strong selection for Arg in the Ser−3 position for MAPKAP kinase-2, Akt/PKB and Chk1 is rationalized by the presence of a conserved glutamate residue at a similar location in all three kinases (Glu145 in MAPKAP kinase-2, Glu236 in Akt/PKB and Glu91 in Chk1), which in Akt/PKB forms a bidentate salt bridge with the Ser−3 arginine guanidino group on GSK3-peptide. Similarly, selection for a hydrophobic residue at the Ser+1 position is explained by a hydrophobic pocket that is conserved at this position in all three kinases. The pocket is lined by Phe310, Pro314, Leu317 and Phe359 in Akt/PKB and by Met167, Leu171, Val174, Leu178 and Leu179 in Chk1. The corresponding Ser+1 pocket in MAPKAP kinase-2 is lined by Pro223, Pro227, Val234 and Leu235. Within this region, Gly312 in Akt/PKB and Gly169 in Chk1 are replaced by Tyr225 in MAPKAP kinase-2, which may reduce the depth of the MAPKAP kinase-2 hydrophobic pocket and explain selection for branched chain aliphatic residues in this position compared with Phe selection by Akt/PKB and Chk1.
The marked contrast between Arg selection at the Ser−5 position in Akt/PKB with the corresponding selection for hydrophobic residues at this position by MAPKAP kinase-2 and Chk1 is accounted for by the presence of Glu342 in Akt/PKB at the base of the Ser−5 pocket. This residue is not conserved in MAPKAP kinase-2 and Chk1, and is instead substituted by Ile255 in MAPKAP kinase-2 and by Ala200 in Chk1. Additional residues, notably Phe147, Pro189, Pro261 and Leu342 in MAPKAP kinase-2, and similarly Phe93, Ile96, Pro98, Pro133 and Leu206 in Chk1, contribute a significant hydrophobic character to this region.
MAPKAP Kinase-2 is Required for the G2/M Checkpoint Following Doxorubicin Treatment.
Treatment of U2OS cells with doxorubicin generates DNA double strand breaks, and induced a prominent G2/M arrest between eighteen and thirty hours following treatment (FIGS. 24A-24B). In addition to this large G2/M population, a minor accumulation of cells in G1 and S phase was also observed. To investigate whether MAPKAP kinase-2 activation was involved in the checkpoint response, RNA interference was used to generate U2OS cells in which MAPKAP kinase-2 protein levels were stably repressed (FIGS. 14A-14C). Introduction of MAPKAP kinase-2-specific shRNA, but not luciferase shRNA, resulted in a robust knockdown of MAPKAP kinase-2 protein when the entire population of transfected cells was analyzed (FIG. 14A).
Asynchronous MAPKAP kinase-2 or luciferase shRNA knockdown cells were mock treated or exposed to doxorubicin for thirty hours, and cell cycle progression was monitored by FACS. In one set of experiments, the spindle poison nocodazole was added to the media three hours after addition of doxorubicin, to cause any cells progressing through the cell cycle to arrest in mitosis. DNA content was monitored by PI staining; phospho-histone-H3 staining was used as an indicator of mitotic entry. As shown in the left panels of FIG. 14A, treatment of control luciferase shRNA knockdown cells with doxorubicin led to the accumulation of cells with 4N DNA content, and a lack of phospho-histone-H3 staining in either the absence or presence of nocodazole. The cells expressing the luciferase shRNAs behaved identically to the untransfected doxorubicin-treated control U2OS cell population (FIG. 24B), with the prominent 4N DNA component and the absence of phospho-histone-H3 staining indicative of an intact G2/M checkpoint. In marked contrast, MAPKAP kinase-2-depleted cells treated with doxorubicin displayed a cell cycle profile essentially identical to that of untreated cells (FIG. 14A, right upper and middle panels). Addition of nocodazole following doxorubicin treatment to the MAPKAP kinase-2 depleted cells caused them to accumulate in a 4N DNA containing peak, with 36.3% of the cells staining positively for phospho-histone H3 (FIG. 14A, right lower panels), a value similar to that of untreated U2OS cells blocked in mitosis with nocodazole (42%) (FIG. 24D). Identical results were obtained using a second unrelated RNAi sequence against MAPKAP kinase-2, indicating that these results did not arise from RNAi off-target effects. MAPKAP kinase-2 depletion did not alter total Chk1 levels or reduce Chk1 activation following DNA damage (FIG. 14B). These findings demonstrate that loss of MAPKAP kinase-2 prevents cells from establishing a functional G2/M checkpoint following doxorubicin-induced DNA damage, despite the presence of activated Chk1.
MAPKAP Kinase-2 Induces Binding of Cdc25B to 14-3-3 in Response to Topoisomerase Inhibitor-Induced DNA Damage.
Two Cdc25 family members, Cdc25B and C, play important roles in initiating and maintaining mitotic entry in normal cells, and are prominent targets of the G2/M checkpoint. Cdc25B is believed to function by activating Cdk1/Cyclin B at the centrosome in late G2 as an initiator of early mitotic events, while Cdc25C functions to further amplify Cdk1/CyclinB activity within a nuclear autoamplification loop once mitosis has begun. In response to γ- or UV-radiation-induced DNA damage, checkpoint kinases phosphorylate Cdc25B and C on Ser323 and Ser216, respectively, to induce their binding to 14-3-3 proteins, which, along with a modest reduction in their catalytic activity, sequesters them in the cytoplasm away from their nuclear cyclin/Cdk substrates. Recent studies suggest that Cdc25B plays a particularly crucial role in initiating and maintaining normal cell cycle G2/M checkpoint responses, since reactivation of Cdc25B is critical for DNA-damaged cells to re-enter the cell cycle. We have shown above that MAPKAP kinase-2 is capable of directly phosphorylating Cdc25B on Ser323 to generate the 14-3-3 binding site. We therefore investigated whether MAPKAP kinase-2 signaling was required for association of Cdc25B with 14-3-3 in response to DNA damage by chemotherapeutic drugs. Control luciferase and MAPKAP kinase-2 knockdown cells were either mock treated or incubated with cisplatin, camptothecin, or doxorubicin. Cell lysates were prepared eight hours later and incubated with recombinant GST-14-3-3β/ζ. Binding of endogenous Cdc25B to 14-3-3 was detected by immunoblotting. As shown in FIG. 14C, both doxorubicin and camptothecin treatment, but not cisplatin exposure, resulted in the generation of stable 14-3-3-binding sites on Cdc25B in the luciferase shRNA control cells. No 14-3-3 binding of Cdc25B, however, was detected in lysates from the MAPKAP kinase-2 depleted cells (FIG. 14C, lower panel). This result is in good agreement with the cell cycle studies in panel A, which showed loss of the G2/M checkpoint in MAPKAP kinase-2 depleted cells after treatment with the topoisomerase inhibitor doxorubicin. These data indicate that loss of the chemotherapy-induced G2/M checkpoint in MAPKAP kinase-2 depleted cells likely arises, at least in part, from loss of Cdc25B binding to 14-3-3 proteins.
MAPKAP Kinase-2 is Required for G1/S Checkpoint Arrest Following Cisplatin Treatment.
In contrast to the G2/M checkpoint response observed in doxorubicin-treated cells, treatment with the DNA intra-strand cross-linker cisplatin caused U2OS cells to predominantly accumulate in the G1 and S phases of the cell cycle over the subsequent thirty hours (FIG. 24C). RNA interference was used to investigate the role of MAPKAP kinase-2 in this process. Control luciferase knockdown cells showed an identical accumulation in G1 and S after cisplatin exposure (FIG. 15A, left panels) as that seen in U2OS cells lacking shRNA. Addition of nocodazole to the luciferase knockdown cells three hours following cisplatin treatment did not reveal the appearance of any mitotic cells over the ensuing twenty-seven hours, as monitored by phospho-histone H3 staining (FIG. 15A, lower left panels), indicating a functionally intact G1/S checkpoint. Depletion of MAPKAP kinase-2 prior to cisplatin exposure resulted in a dramatically different result. As seen in the right panels of FIG. 15A, MAPKAP kinase-2 depleted cells showed a cell cycle profile after cisplatin treatment that was similar to that of untreated cells other than a very slight increase in the total number of cells in S-phase. Strikingly, when nocodazole was added three hours following cisplatin addition, the MAPKAP kinase-2 depleted cells accumulated in a 4N DNA containing peak with  ̃42% of the cells staining strongly for phospho-histone H3. Identical results were obtained in cells treated with a second unrelated siRNA sequence against MAPKAP kinase-2. MAPKAP kinase-2 depletion did not impair activation of Chk1 after cisplatin exposure (FIG. 15B). These data imply that MAPKAP kinase-2 is essential for the cisplatin induced G1/S arrest and that loss of MAPKAP kinase-2 enables U2OS cells to override the cisplatin-induced G1/S checkpoints, despite the presence of activated Chk1, and proceed into mitosis.
MAPKAP Kinase-2 is Required for Cdc25A Degradation in Response to Cisplatin-Induced DNA Damage.
In contrast to the 14-3-3-mediated sequestration of Cdc25B and C involved in the G2/M checkpoint response, the G1 and S phase checkpoints are largely controlled by the phosphorylation-dependent degradation of another Cdc25 isoform, Cdc25A. Based on our observation that depletion of MAPKAP kinase-2 resulted in loss of the G1/S checkpoint response, we investigated whether MAPKAP kinase-2 was required for the degradation of Cdc25A following cisplatin-induced DNA damage. Luciferase shRNA control cells and MAPKAP kinase-2 depleted cells were treated with cisplatin, and cell lysates immunoblotted for Cdc25A at eight and twelve hours following treatment (FIG. 15B). Cdc25A levels decreased dramatically in the control luciferase knockdown cells after treatment with cisplatin. In contrast, in the MAPKAP kinase-2 depleted cells, the level of Cdc25A following cisplatin exposure was only minimally reduced, and remained comparable to that seen in undamaged cells. These data indicate that in the absence of MAPKAP kinase-2, U2OS cells are defective in targeting Cdc25A for degradation in response to cisplatin induced DNA damage. This inability of MAPKAP kinase-2 depleted cells to degrade Cdc25A likely explains the failure of MAPKAP kinase-2 depleted cells to establish a sustained G1/S checkpoint following cisplatin exposure.
The degradation of Cdc25A in response to DNA damage involves the direct phosphorylation of Cdc25A by checkpoint kinases. In response to UV and γ-irradiation, for example, Chk1 phosphorylates Cdc25A at multiple sites facilitating its subsequent ubiquitin-mediated destruction by the proteosome. Chk1, however, is activated normally in the MAPKAP kinase-2 depleted cells after cisplatin treatment (FIG. 15B). Other kinases besides Chk1, such as Chk2, have been recently reported to be able to phosphorylate Cdc25A on at least some of the same sites as Chk1 under certain conditions. Furthermore, we have shown that the optimal amino acid sequence motif on peptides and proteins phosphorylated by MAPKAP kinase-2 is nearly identical to the optimal sequence motif phosphorylated by Chk1 and Chk2. We therefore investigated whether Cdc25A could be a direct MAPKAP kinase-2 substrate. Recombinant Cdc25A was incubated with purified MAPKAP kinase-2 or Chk1 in vitro in the presence of 32P-γ-ATP, and phosphorylation monitored by SDS-PAGE/autoradiography. As shown in FIG. 15C, MAPKAP kinase-2 phosphorylated Cdc25A in vitro as efficiently as Chk1. Together, these findings suggest that degradation of Cdc25A in response to cisplatin treatment either requires direct phosphorylation of Cdc25A by MAPKAP kinase-2, or that MAPKAP kinase-2 activity is required to target Chk1 to Cdc25A in vivo.
Down-Regulation of MAPKAP Kinase-2 Increases the Sensitivity of Tumor Cells to Chemotherapy.
The experiments in FIGS. 15A-15C and 16A-16D indicate that MAPKAP kinase-2 is critical for cisplatin- and doxorubicin-triggered G1/S and G2/M arrest. These checkpoint defects in MAPKAP kinase-2 depleted cells might render them more sensitive to the antiproliferative and cytotoxic effects of chemotherapy. To investigate this, control or MAPKAP kinase-2 knockdown U2OS cells were mock treated or incubated with increasing doses of cisplatin or doxorubicin for eight hours, washed, trypsinized and replated, and assayed for colony formation eight days later (FIGS. 16A-16B). When compared to the control shRNA-treated cells, MAPKAP kinase-2 depleted cells displayed a dramatically increased sensitivity to both cisplatin and doxorubicin treatment, particularly at relatively low drug doses (FIGS. 16C-16D). For example, luciferase shRNA cells treated with either 10 μM cisplatin or 1 μM doxorubicin formed  ̃40% of the number of colonies as those formed by untreated cells, while in MAPKAP kinase-2-depleted cells, these same cisplatin and doxorubicin treatments reduced the number of colonies to only 4% and 2%, respectively, of those seen in the untreated cells.
To establish whether the absence of MAPKAP kinase-2 could also enhance the anti-tumorigenic effect of cisplatin or doxorubicin in vivo, we introduced control or MAPKAP kinase-2 siRNAs into H-Ras-V12 transformed p53−/− MEFs, treated them with either vehicle alone, 1 μM cisplatin or 0.1 μM doxorubicin, and then implanted them into nude mice. Each animal received two injections of MAPKAP kinase-2 siRNA-transfected cells (left flank), and two injections of control siRNA transfected cells (right flank), and tumor formation was assessed at fifteen days. FIG. 17A, left panel, shows that in the absence of treatment with DNA damaging agents, all four injections resulted in formation of solid fibrous tumors after fifteen days. In general, the size of the tumors resulting from injection of MAPKAP kinase-2 depleted cells was larger than that from control siRNA-transfected cells (FIGS. 17A, 17B, and 17D). Pre-treatment of the control siRNA transfected cells with either cisplatin or doxorubicin prior to implantation did not prevent tumor formation. The resulting tumors, however, were reduced to  ̃35% of the size and weight of the tumors formed by untreated cells (FIGS. 17A and 17D). Depletion of MAPKAP kinase-2 prior to treatment with either cisplatin or doxorubicin completely eliminated the formation of tumors (FIGS. 17A, 17C, and 17D), indicating that the enhanced sensitivity of these cells to chemotherapeutic drugs seen in culture was maintained even when the cells were grown within a normal tissue microenvironment.
Taken together with the loss of G1/S and G2/M checkpoints observed by FACS analysis, and the mis-regulation of the mitotic phosphatases Cdc25A and B (FIGS. 14A-14C and 15A-15C), these data provide strong evidence that down-regulation of MAPKAP kinase-2 activity results in enhanced sensitivity of cells to genotoxic stress in vitro and in vivo. These findings have potential therapeutic implications, since they suggest that targeting of MAPKAP kinase-2 with small molecule inhibitors should result in an enhanced sensitivity of tumor cells to conventional chemotherapeutic agents.
MAPKAP Kinase-2 and Chk1 are Activated Independently.
The activation of MAPKAP kinase-2 by cisplatin, camptothecin, doxorubicin, and UV irradiation that we observed is strikingly similar to the activation profile reported for Chk1. Similarly, the impaired G1/S and G2/M checkpoints seen after these DNA damaging stimuli in MAPKAP kinase-2 knockdown cells bears some resemblance to what has been reported for Chk1-deficient cells. These phenotypic similarities prompted us to further investigate whether the activation of Chk1 and MAPKAP kinase-2 was interdependent. As shown in FIGS. 14B and 15B, activation of Chk1 in response to cisplatin and doxorubicin was unimpaired in MAPKAP kinase-2 depleted cells. We therefore investigated the opposite possibility—whether the activation of MAPKAP kinase-2 after DNA damage was dependent on Chk1. U2OS cells were depleted of Chk1 using siRNA, exposed to cisplatin and doxorubicin, and analyzed for activation of MAPKAP kinase-2. As shown in FIG. 19, phosphorylation/activation of MAPKAP kinase-2 occurred normally after treatment with these DNA damaging agents, regardless of the presence or absence of Chk1. Thus, activation of MAPKAP kinase-2 and Chk1 after genotoxic stress appears to occur independently of each other.
The MAPKAP Kinase-2 DNA Damage Checkpoint Phenotype can be Synthetically Rescued by Chk1 Overexpression.
The observation that Chk1 and MAPKAP kinase-2 phosphorylate the same optimal sequence motif, target a set of overlapping substrates, and are activated independently of each other, prompted us to perform a genetic experiment to investigate whether loss of MAPKAP kinase-2 could be rescued by overexpression of Chk1 in mammalian cells (FIGS. 20A-20E). In these experiments, luciferase- or MAPKAP kinase-2 shRNA-expressing cells were transiently transfected with a mammalian Chk1 expression construct, or with an empty vector control (FIG. 25). Cells were exposed to cisplatin, doxorubicin, or UV radiation thirty hours following transfection, harvested after an additional thirty hours, and cell cycle progression analyzed by FACS. In one set of experiments, nocodazole was added to the media three hours following addition of chemotherapy or UV, to cause any cells progressing through the cell cycle to arrest in mitosis.
Consistent with what we observed previously, luciferase shRNA control cells transfected with the empty vector DNA executed a G1/S arrest following exposure to cisplatin (FIG. 20A) and UV irradiation (FIG. 26), and displayed a prominent G2 arrest in response to doxorubicin (FIG. 20B). These cell cycle profiles were unchanged when the luciferase shRNA cells were transfected with Chk1. MAPKAP kinase-2 depleted cells transfected with empty vector DNA broke through both checkpoints and accumulated in mitosis when nocodazole was added to the media (FIGS. 20A-20B). Overexpression of Chk1 in the MAPKAP kinase-2 depleted cells, however, completely restored their ability to establish functional checkpoints following genotoxic stress. The cells now arrested in G1/S in response to cisplatin and UV irradiation, and in G2 following doxorubicin (rightmost panels in FIGS. 20A and 20B, and FIG. 26). Addition of nocodazole to the growth media of these MAPKAP kinase-2 depleted Chk1 over-expressing cells did not increase the number of phosphohistone H3 positive cells. Thus, overexpression of Chk1 prevented MAPKAP kinase-2 depleted cells from progressing through the cell cycle after genotoxic stress.
We investigated whether the synthetic rescue of G1/S and G2/M checkpoints by Chk1 in MAPKAP kinase-2 depleted cells was also sufficient to reduce their susceptibility to chemotherapeutic treatment. Luciferase and MAPKAP kinase-2 knockdown cells transfected with Chk1 or vector alone were mock treated or incubated with increasing doses of cisplatin and doxorubicin for eight hours, or irradiated with 20 J/m2 of UV light. Cells were washed, trypsinized, replated and assayed for colony formation after eight days as described previously (FIGS. 27-28). As summarized in FIGS. 20C-20E, MAPKAP kinase-2 depleted cells, transfected with the empty control vector, showed enhanced sensitivity to the anti-proliferative effects of cisplatin, doxorubicin and UV. Overexpression of Chk1 in these MAPKAP kinase-2 depleted cells restored their clonogenic survival to levels that were indistinguishable from those seen with control cells containing wild-type levels of MAPKAP kinase-2.
UCN-01 is a Potent Inhibitor of both Chk1 and MAPKAP Kinase-2.
The staurosporine derivative 7-hydroxystaurosporin/UCN-01 inhibits Chk1 with an IC50 that is  ̃1000 fold lower than that for Chk2, and hence has been used experimentally as a Chk1-specific inhibitor. Strong circumstantial evidence, however, suggests that UCN-01 inhibits other kinases involved in cell cycle control at similar concentrations as those used for Chk1 inhibition studies. For example, Chk1-depleted cells maintain phosphorylation of Cdc25C on Ser−216 both during asynchronous growth and following γ-irradiation. Phosphorylation at this site is lost when cells are treated with low doses of UCN-01 ( ̃300 nM), indicating that UCN-01 inhibitable kinase(s) other than Chk1 participate in Cdc25C phosphorylation. Based on our finding that MAPKAP kinase-2 is a critical checkpoint regulator, we investigated whether UCN-01 inhibited MAPKAP kinase-2 at doses typically used in Chk1 inhibition experiments. In vitro kinase assays were performed with Chk1 and MAPKAP kinase-2 using an optimal peptide substrate with the core consensus sequence LQRQLSI (SEQ ID NO: 16), similar to the 14-3-3 binding sequence in Cdc25B and C, in the presence of various concentrations of UCN-01. As shown in FIG. 21A, UCN-01 potently inhibited both kinases, with an IC50 value of 35 nM for Chk1 and  ̃95 nM for MAPKAP kinase-2. The IC50 value we measured for Chk1 is in good agreement with previously published data. Importantly, the IC50 value we measured for MAPKAP kinase-2 is significantly below the concentrations of UCN-01 that are used in “Chk1-specific” checkpoint abrogation assays, suggesting that under the conditions used in those studies, both Chk1 and MAPKAP kinase-2 were being inhibited.
To examine the structural basis for UCN-01 inhibition of MAPKAP kinase-2, the structure of the MAPKAP kinase-2:UCN-01 complex was modeled using coordinates from the published MAPKAP kinase-2:staurosporine structure, and compared the results with the co-crystal structure of Chk1:UCN-01 (FIG. 21B). As seen in panels 2, 3 and 5 of FIG. 21B, the 7-hydroxy moiety of UCN-01 can be easily accommodated into the MAPKAP kinase-2:staurosporine structure, where its closest neighboring residues would be Vail 18 (2.8 Å to Cγ2), Leu141 (3.2 Å to Cδ1), and Thr206 (3.6 Å to Cγ2). This lack of steric hindrance, and the overall similarity of the modeled MAPKAP kinase-2:UCN-01 structure to the Chk1:UCN-01 structure (panels 1 and 4 of FIG. 21B), provides a structural rationale for the tight binding observed biochemically.
To verify that MAPKAP kinase-2 is a direct target of UCN-01 in cells, we measured the phosphorylation of the MAPKAP kinase-2-specific substrate hsp-27 after heat shock, a stimulus that activates the p38 MAPK/MAPKAP kinase-2 pathway. Control luciferase shRNA expressing or MAPKAP kinase-2 shRNA expressing U2OS cells were incubated at 42° C. or 37° C. for two hours in the presence or absence of 250 nM UCN-01, and phosphorylation of hsp-27 monitored by immunoblotting with an antibody against pSer82, a well established MAPKAP kinase-2 phosphorylation site. FIG. 21C shows phosphorylation of hsp-27 when the control luciferase shRNA cells were placed at 42° C. (lane 1). This phosphorylation was completely abrogated by treatment with UCN-01 (lane 2). No phosphorylation was observed in MAPKAP kinase-2 knockdown cells placed at 42° C. regardless of the presence or absence of UCN-01 (lanes 3, 4). Likewise, no signal was observed in both the control and MAPKAP kinase-2 knockdown cells that were maintained at 37° C., with or without UCN-01 treatment (lane 5-8). Furthermore, heat shock was equally effective in promoting the phosphorylation of hsp-27 on Ser−82, and UCN-01 was equally effective in blocking Ser−82 phosphorylation in cells that were depleted of Chk1 (FIG. 21D, lanes 1-4). Thus, UCN-01 inhibition of MAPKAP kinase-2 in vivo is independent of Chk1 function. These findings provide strong evidence that UCN-01 is a direct inhibitor of MAPKAP kinase-2 within cells, and suggest that the clinical efficacy of UCN-01 in cancer treatment likely arises from the simultaneous inhibition of two parallel but non-redundant checkpoint pathways involving Chk1 and MAPKAP kinase-2.
Since disruption of the MAPKAP kinase-2 signaling pathway enhances chemotherapeutic responses even in the presence of a functional Chk1 response, and since MAPKAP kinase-2 knock-out mice are viable, in contrast to Chk1 knock-out mice, our results suggest that a MAPKAP kinase-2 specific inhibitor might provide significant clinical benefit with fewer undesirable side-effects. In either case, our current data strongly support the development of clinical MAPKAP kinase-2 inhibitors as viable anti-cancer agents. Given the dependence of p53-defective cells on intra-S and G2/M checkpoint pathways, targeting MAPKAP kinase-2 may be a particularly efficacious approach to treating these types of human cancers. Thus, therapeutic treatments that interfere with MAPKAP kinase-2 function would be expected to preferentially sensitize p53-deficient cells to treatment with DNA-damaging chemotherapeutic drugs without similarly sensitizing wild-type cells. Disorders, e.g., neoplastic disorders, that include p53-deficient cells could be treated effectively and specifically using therapy that combines administration of a MAPKAP kinase-2-interfering compound, e.g., UCN-01, and one or more chemotherapeutic agents, preferably at substantially lower levels than would otherwise be necessary to treat the disorder, thereby largely sparing normal cells from the deleterious effects of chemotherapy.
Model for the Role of MAPKAP Kinase-2
Our data show that a crucial role of p38 SAPK in response to UV-induced DNA damage is the phosphorylation and activation of MAPKAP kinase-2, leading to MAPKAP kinase-2-directed phosphorylation of Cdc25 family members to induce 14-3-3-binding and subsequent cell cycle arrest. In this way, MAPKAP kinase-2 performs similar functions after UV-C induced DNA damage as those performed by Chk1 and Chk2 after exposure of cells to ionizing radiation.
MAPKAP kinase-2 undergoes initial activation in the nucleus with subsequent export of the active kinase to the cytoplasm. Thus, MAPKAP kinase-2 is well-positioned to function as both a nuclear initiator of Cdc25B/C phosphorylation in response to DNA damage, and as a maintenance kinase that keeps Cdc25B/C inhibited in the cytoplasm. A unified model for kinase-dependent DNA damage checkpoints is presented in FIG. 7. In response to ionizing radiation, ATM activation of Chk2 and ATR activation of Chk1 leads to phosphorylation of Cdc25 family members on related sequences corresponding to the checkpoint kinase core “motif” LXRXX[S/T][Hydrophobic] (SEQ ID NO: 18). Similarly, in response to UV-induced DNA damage, ATR activates Chk1 and p38 SAPK activates MAPKAP kinase-2, leading to phosphorylation of the same core motif on Cdc25 family members. The major role of Chk1 appears to involve phosphorylation of Cdc25A after IR, whereas Chk2 appears to phosphorylate all three Cdc25 family members. In the absence of Chk2, Chk1 appears to be able to subsume at least part of this function. Our data now indicate that MAPKAP kinase-2 is the primary effector kinase that targets Cdc25B/C after UV-C exposure. MAPKAP kinase-2 may also be involved in Cdc25A phosphorylation, since we observed that the G1 and S-phase checkpoints were eliminated in the MAPKAP kinase-2 knockdown cells.
The results presented here indicate that the activities of both Chk1 and MAPKAP kinase-2 are required for G1/S and G2/M cell cycle arrest in response to DNA damaging chemotherapy and UV-irradiation (FIG. 22). At a systems level, these observations suggest that the normal DNA damage checkpoint response involves the unified actions of a dedicated DNA damage response pathway (i.e., Chk1) and a potentially more global stress response pathway (MAPKAP kinase-2). Individual kinase activities emerging from each of these pathways appear to be titered to levels that, in combination, are just adequate to arrest the cell cycle after damage, presumably facilitating rapid checkpoint release once the DNA damage has been repaired. In agreement with this hypothesis, overexpression of Chk1 rescued both the G2/M and G1/S cell cycle checkpoint defects observed in MAPKAP kinase-2 depleted cells.
Experimental Procedures
Chemicals, antibodies, and drugs. UCN-01 was the kind gift of R. Schultz, Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (Bethesda, Md.). Cisplatin, doxorubicin and camptothecin, puromycin, and glutathione beads were purchased from Sigma-Aldrich. Propidium iodide was purchased from Calbiochem. Antibodies against total and phosphorylated forms of MAPKAP kinase-2, p38 MAPK, Chk1, Chk2, ATM/ATR substrate, hsp-27, and p53 (pS20) were purchased from Cell Signaling Technology (Beverly, Mass.). Antibodies against β-actin and 5-bromo-2-deoxyuridine (BrdU) were purchased from Sigma-Aldrich; an anti-Cdc25A antibody (MS-640-P1, cocktail) was from NeoMarker (Fremont, Calif.); an anti-Cdc25B antibody was from Transduction Labs, an anti-GST antibody was from Amersham/GE Healthcare, and an anti-phospho histone H3 antibody was from Upstate. Active MAPKAP kinase-2 was purchased from Upstate. Propidium Iodide (PI) was purchased from Calbiochem, amylose beads were purchased from New England Biolabs, Ni-NTA agarose were purchased from QIAGEN, and glutathione beads and BrdU were purchased from Sigma-Aldrich.
Cell culture. U2OS cells, HeLa cells, U87MG cells and H-Ras-V12 transformed p53−/− MEFs were cultured in DMEM supplemented with 10% FCS and penicillin/streptomycin at 37° C. in a humidified incubator supplied with 5% CO2. GM05849 A-T fibroblasts and the corresponding control GM00637 fibroblasts, and GM18366 ATR-defective Seckel syndrome fibroblasts and the corresponding control GM00023 fibroblasts were obtained from the Coriell cell repository and were cultured in MEM supplemented with Eagle's salts, 10% FCS, and penicillin/streptomycin.
Purification of recombinant proteins. Constructs encoding GST- and MBP-fusion proteins were transformed into DH5α or BL21(DE3) strains of E. coli and recombinant proteins obtained by inducing late log-phase cells with 0.4 mM IPTG at 37° C. for three to five hours. Cells were lysed by sonication in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT, 8 μg/mL pepstatin, 8 μg/mL aprotinin, and 8 μg/mL leupeptin. Fusion proteins were purified from cell lysates by using amylose or glutathione beads. Following extensive washing with PBS containing 0.5% NP-40 and a final wash with PBS, fusion proteins were eluted from the beads with HEPES, pH 7.2, containing 40 mM maltose or 20 mM glutathione, followed by exchange into PBS using duplicate Sephadex G-25 columns (NAP-10 columns, Pharmacia). Protein concentrations were determined using the bicinchoninic acid assay (Pierce) as recommended by the manufacturer, using BSA as the standard. Full-length Chk1-GST or full-length Chk2-His6 in pFASTBAC was expressed in Sf9 insect cells. Chk1-expressing cells were lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT, 1.0% NP-40, 8 μg/μL pepstatin, 8 μg/mL aprotinin, 8 μg/mL leupeptin, 2 mM Na3VO4, 10 mM NaF, and 1 μM microcystin, and Chk1 was purified using glutathione beads. Chk1 was eluted from the beads with 10 mM glutathione in 50 mM Tris-HCl, pH 8.0, and dialyzed into kinase buffer. Chk2 expressing cells were lysed in buffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT, 1.0% NP-40, 8 μg/mL pepstatin, 8 μg/mL aprotinin, 8 μg/mL leupeptin, 2 mM Na3VO4, 10 mM NaF, and 1 μM microcystin, and Chk2 was purified using Ni-NTA agrose beads. After washing extensively with lysis buffer containing 40 mM imidazole, Chk2 was eluted from the beads with 100 mM imidazole in 50 mM Tris-HCl, pH 8.0, and dialyzed into kinase buffer.
Point mutations were generated using the Stratagene Quick Change Mutagenesis Kit and confirmed by sequencing the entire coding regions.
Kinase motifscreening with oriented peptide libraries and in vitro kinase assays. Using the methods of the invention, one skilled in the art would be able to utilize a peptide library screen to identify peptides that bind to a p38 SAPK polypeptide, MAPKAP kinase-2 polypeptide, or other biologically relevant target. Peptides identified in such a screen, or related compounds, would have potential therapeutic benefit due to their ability to inhibit the biological activity of, e.g., a MAPKAP kinase-2 polypeptide.
Combinatorial peptide library screening was performed using recombinant purified p38α SAPK, MK2, Chk1 and Chk2 as previously described (Songyang and Cantley, Methods Mol. Biol., 87:87-98, 1998) with minor modifications. Briefly, 5.0 μg of recombinant p38α SAPK, 3.0 μg MK2, 2.0 μg Chk1 and 2.0 μg Chk2 were incubated with 1 mg of each peptide library in 300 μl reaction volumes containing 20 mM HEPES, pH 7.5, 10 mM MgCl2, 3 mM 2-mercaptoethanol, and 100 μM ATP containing 2.5 μCi of 32P-γ-ATP for 120 min at 30° C. Under these conditions, approximately 1% of the peptide mixture was phosphorylated. The reaction mixture was diluted by addition of 300 μl of 30% acetic acid, and the phosphorylated peptides separated from unincorporated 32P-γ-ATP by DEAE column chromatography (1 ml bed volume) using isocratic elution with 30% acetic acid. The peptide mixture (both phosphorylated and unphosphorylated, but free of ATP) eluted within the first 1 ml following the 600 μl void volume of the column. Samples were dried in a Speed-Vac apparatus.
For the p38α SAPK peptide library experiments, the sample was resuspended in 200 μl of 50 mM MES, pH 5.5, containing 1 M NaCl. Separation of phosphorylated from non-phosphorylated peptides was achieved by IMAC using ferric-iminodiacetic acid beads. A 0.5 ml iminodiacetic acid column was charged with 2.5 ml of 20 mM FeCl3 and extensively washed with H2O, then with 3 ml of 500 mM NH4HCO3, pH 8.0, 3 ml of H2O, and 3 ml of 50 mM MES (pH 5.5)/1 M NaCl. The peptide mixture was applied and the column was developed with 3 ml 50 mM MES, pH 5.5, 1 M NaCl, followed by 4 ml of H2O to remove nonphosphorylated peptides. Phosphorylated peptides were then eluted with 2 ml of 500 mM NH4HCO3, pH 8.0, and dried in a Speed-Vac apparatus, and resuspended in 80 μl H2O.
Peptide library screens using basophilic kinase-directed libraries are complicated by a high background of non-phosphorylated Asp/Glu-rich peptides that co-purified with the phosphorylated peptides during the immobilized metal affinity chromatography (IMAC) step prior to peptide sequencing, greatly complicating the analysis. To overcome this problem, we developed a new approach in which peptide libraries are first phosphorylated by the kinase of interest, and then treated with methanolic HCl to convert Asp and Glu residues to their uncharged methyl esters. Using this approach, the background of nonphosphorylated peptides that adhere to the IMAC column was reduced to insignificant levels. Furthermore, the Asp and Glu methyl esters were converted back to their free acids during the sequencing reaction, allowing selection for these residues, if present in the phosphorylation motif, to be accurately measured.
For the MAPKAP kinase-2, Chk1, and Chk2 peptide library experiments, 40 μl of thionyl chloride was added dropwise in a hood to 1 ml of dry methanol. This solution was then used to dissolve each of the dried peptide libraries, followed by stirring at room temperature for one hour. The peptide library was dried down overnight and resuspended in 100 μl of a 1:1:1 mixture of methanol/acetonitrile/water. A 0.5 ml iminodiacetic acid column was charged with 2.5 ml of 20 mM FeCl3 and extensively washed with H2O, then with 3 ml of 500 mM NH4HCO3 (pH 8.0), 3 ml of H2O, and 3 ml of 50 mM MES, pH 5.5, 1 M NaCl. The peptide mixture was applied and the column was developed with 4 ml of H2O followed by 3 ml NH4HCO3, pH 8.0, to remove non-phosphorylated peptides. Phosphorylated peptides were eluted with 2 ml of 500 mM NH4HCO3, pH 11.0, dried in a Speed-Vac apparatus, and resuspended in 40-80 μl H2O.
Following IMAC purification, libraries (0.5-1.5 nmoles) were subjected to automated Edman sequencing using an Applied Biosystems model 477A peptide sequencer. Data analysis was performed by normalizing the abundance (mol-%) of each amino acid in the phosphorylated peptide mixture to that present in the starting libraries. The sums of the final preference ratios were normalized to the total number of amino acids in the degenerate positions within the peptide libraries so that a particular amino acid would have a preference value of 1 in the absence of selectivity at a particular position. The degenerate peptide libraries used for in vitro kinase screening with p38 MAP kinase, MK2, Chk1, and Chk2 consisted of the sequences GAXXXXSPXXXXAKKK [SP library] (SEQ ID NO: 19), where X denotes all amino acids except Cys, Ser, Thr, and Tyr; GAXXXXPXSPXXXXXAKKK [PxSP library] (SEQ ID NO: 20), where X denotes all amino acids except Cys; or GAXXSXXXXAKKK [RxxS library] (SEQ ID NO: 21), where X denotes all amino acids except Cys, Ser, Thr and Tyr. In all libraries, S denotes Ser, P denotes Pro, and R denotes Arg.
Kinase reactions were performed in 30 μl of kinase reaction buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 3 mM 2-mercaptoethanol, 100 μg/ml BSA, 50 μM ATP, 10 μCi 32P-γ-ATP) containing 2.0 μg of recombinant p47 or Cdc25B substrate protein or the specified amount of peptide and 0.10 μg of recombinant p38α SAPK or 0.03 μg of recombinant MAPKAP kinase-2 at 30° C. for the indicated time. The sequences of the p38 optimal peptide and the p47phox peptide were KKAZGPQGPQSPIE (SEQ ID NO: 22) and KKAZGPQSPGSPLE (SEQ ID NO: 23), respectively. For 14-3-3 pulldowns of Cdc25B following in vitro phosphorylation by p38 or MAPKAP kinase-2, 2.0 μg of Cdc25B was incubated with 10-fold excess 14-3-3-MBP and analyzed by autoradiography. For kinetic measurements, the reactions were terminated by the addition of an equal volume of 0.5 percent phosphoric acid, and 5 μl was spotted onto p81 paper. The p81 paper was washed 5× in 0.5 percent phosphoric acid and added to scintillation fluid for scintillation counting. For in vitro phosphorylation reactions, the reactions were terminated by the addition of an equal volume of sample buffer followed by heating at 95° C. for 3 min. Samples were analyzed by SDS-PAGE followed by transfer to nitrocellulose for autoradiography and immunoblotting. The rate of p38α phosphorylation of isolated peptides and full-length p47phox proteins was determined by scintillation counting using peptide concentrations of 100, 400, and 1400 μM, and protein concentrations of 1, 5, 10 and 15 μM, with time points taken at five, ten, and twenty minutes. MAPKAP kinase-2 phosphorylation of MK2tides was performed using peptide concentrations of 5, 10, 20, 40, 80, 160, 320, 500, and 1000 μM, with time points taken at three, six, nine, and twelve minutes. From these enzymatic studies, Km, Vmax and Vmax/Km values were then ascertained. All kinetic experiments were performed a minimum of three times. For each experimental condition in the determination of the Km and Vmax values, we verified that the reaction rates were linear with respect to time for all substrate concentrations and that less than 10% substrate was phosphorylated.
In vitro kinase assays for UCN-01 IC50 determination were performed in 30 μl reactions containing 20 mM HEPES (pH 7.5), 10 mM MgCl2, 3 mM 2-mercaptoethanol, 100 μg/ml BSA, 50 mM ATP, 10 μCi 32P-γ-ATP, and 50 μM. MK2-tide substrate for twenty minutes at 30° C. Chk1 was used at a concentration of 0.3 μM; MAPKAP kinase-2 was used at a concentration of 0.1 μM. Reactions were terminated by adding an equal volume of 0.5% phosphoric acid to the reaction and 5 μl was spotted onto P81 paper. After washing 5× in 0.5% phosphoric acid, sample were subjected to scintillation counting. Cdc25A phosphorylation studies were performed using GST-Cdc25A immunoprecipitated from HEK293T cells transfected with pCMV GST-Cdc25A, a generous gift from Dr. W. Harper (Harvard Medical School). In brief, HEK293T cells were transfected with pCMV GST-Cdc25A construct using the calcium phosphate method described earlier. Cells were harvested thirty-six hours later, lysed in a buffer containing 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1.0% NP-40, 5 mM EDTA, 2 mM DTT, 8 μg/ml pepstatin, 8 μg/ml aprotinin, 8 μg/ml leupeptin, 2 mM Na3VO4, 10 mM NaF, and 1 μM microcystin and cleared by centrifugation. Supernatants were precleared with protein G beads for one hour. GST-Cdc25A was precipitated with 50 μl GSH beads (Sigma-Aldrich). Beads were washed five times in kinase buffer and used in kinase reactions. Kinase reactions were performed in 50 μl of kinase reaction buffer using 0.3 μM Chk1 and 0.1 μM MAPKAP kinase-2. Reactions were performed at 30° C. for twenty minutes and terminated by addition of 50 μl 2× sample buffer. Samples were heated at 95° C. for three minutes, separated on a 12.5% SDS-PAGE, and visualized using a phosphor imager (Molecular Dynamics).
14-3-3 pull-down assays, immunoblotting, and immunofluorescence. U2OS cells were lysed in lysis buffer: 50 mM Tris/HCl, pH7.8, 150 mM NaCl, 1.0% NP-40, 5 mM EDTA, 2 mM DTT, 8 μg/ml pepstatin, 8 μg/ml aprotinin, 8 μg/ml leupeptin, 2 mM Na3VO4, 10 mM NaF, 1 μM microcystin for twenty minutes at 4° C. Clarified lysates (0.5-2 mg protein) were incubated with 20 μL glutathione beads or amylose beads containing 10-20 μg 14-3-3-GST or 14-3-3-MBP, respectively, for 120 minutes at 4° C. Following washing, lysates and bead-bound proteins were analysed by SDS-PAGE, followed by transfer to PVDF membranes and immunoblotted with the indicated antibodies. For immunofluorescence experiments, U2OS cells were seeded onto 18 mm2 coverslips, irradiated or mock-treated, fixed, extracted, and immunostained as described previously (Clapperton et al., Nat. Struct. Mol. Biol., 11:512-518, 2004).
FACS analysis. UV irradiation was performed at 254 nm (UV-C) using a Stratalinker 2400 (Stratagene). U2OS cells were fixed in 70% ethanol overnight at −20° C., permeabilized with PBS containing 0.2% Triton X-100 for twenty minutes at 4° C., blocked with 2% FBS in PBS, and incubated with 1 μg of anti-phospho-histone H3 per 106 cells for sixty minutes on ice. Following washing, cells were incubated with FITC-conjugated goat anti-rabbit antibody (diluted 1:500) for thirty minutes on ice, washed, and resuspended in PBS containing 50 μg/ml PI for twenty minutes immediately prior to FACS analysis. Analysis was performed using a Becton Dickinson FACS machine with CellQuest software.
For BrdU incorporation experiments, cells were incubated with 30 μM BrdU for the indicated times, then fixed and permeabilized as above. Cells were denatured in 2N HCl for twenty minutes at room temperature, neutralized with 0.1M Na2B4O7 (pH 8.5), blocked with 2% FBS in PBS, and incubated with a murine anti-BrdU antibody for sixty minutes on ice. Following washing, cells were incubated with FITC-conjugated goat anti-mouse antibodies and PI as above. Analysis was performed using a Becton Dickinson FACS machine with CellQuest software.
Clonogenic survival assay. Cells were either mock-treated or treated with increasing doses of doxorubicin or cisplatin. After eight hours of treatment, cells were washed three times with growth media and three times with PBS, trypsinized and replated at a concentration of 5000 cells/10 cm2 dish. After eight days, cells were fixed and stained with 0.1% crystal violet (Sigma-Aldrich). Colonies consisting of >50 cells were counted, and surviving fractions were determined by normalization against untreated cells. Experiments were performed in triplicate and are plotted as mean values with standard deviations indicated by the error bars.
Murine tumor models. H-Ras-V 12-transformed p53−/− MEFs were used for in vivo tumor formation assays. Cells were transfected with siRNA oligonucleotides against GFP or murine MAPKAP kinase-2 for forty-eight hours, then mock treated or incubated with 0.1 μM doxorubicin or 1 μM cisplatin for eight hours, washed three times in growth media, three times in PBS, trypsinized, and resuspended at a concentration of 107 cells/ml in PBS. 106 cells were subcutaneously injected into the flanks of nude mice (Ncr nu/nu, Taconic).
For tumor regression assays, H-Ras-V12 transformed p53−/− MEFs were stably transfected with a lentiviral transfer vector encoding for shRNA targeting either MAPKAP kinase-2 or luciferase. 106 cells were injected into the flanks of nude mice as above, and tumors were allowed to form for twelve days. Mice were then treated with either cisplatin (2 mg/kg, intraperitoneal administration 3× per week) or doxorubicin (4 mg/kg, intraperitoneal administration 3× per week), monitored for a total of twenty-six days, and then sacrificed. Tumor diameter was measured periodically during growth and tumors were weighed at the endpoint. Experiments were performed in quadruplicate, and data plotted as sample means with error bars showing standard deviation.
Structural modeling. Activated MAPKAP kinase-2 (phosphorylated on Thr 222) was modeled using the crystal structure of the ADP complex (Underwood et al., Structure, 11:627-636, 2003) with the activation loop (residues 213 to 241) deleted and rebuilt using the corresponding region (residues 299 to 328) from the structure of activated Akt/PKB in complex with AMP-PNP and GSK3-peptide (Yang et al., Nat. Struct. Biol., 9:940-944, 2002) as a template. An optimal peptide, LQRQLSIA (SEQ ID NO: 6), was modeled in the active site based on the GSK3-peptide. Coordinates for the activated MAPKAP kinase-2/peptide complex are listed in Table 1 in standard Protein Data Bank (PDB) format (details about the Protein Data Bank and the associated format for coordinates may be found in Berman et al., Nuc. Acids Res., 28:235-242, 2000). Table 2 lists pairs of atoms in the complex that form the closest protein-peptide contacts and that are useful for designing or identifying additional molecules that bind in the active site. A substrate peptide, LYRSPSMPL (residues 211-219 of human Cdc25C) (SEQ ID NO: 7) in the Chk1 active site was similarly modeled using the GSK3-peptide as a template and manually adjusted to resemble the published model (Chen et al., Cell, 100:681-692, 2000). Structures were superimposed using ALIGN and SUPERIMPOSE. Manual adjustments of the models were made using XFIT from the XtalView suite.
The structure of MAPKAP kinase-2 bound to UCN-01 was modeled using PyMOL with the structure of MAPKAP kinase-2 bound to staurosporine (PDB ID 1NXK) as a base model.







TABLE 1





Coordinates of the activated MAPKAP kinase-2/peptide complex


























ATOM
1
N
PHE
A
46
214.820
109.707
179.069
1.00
118.35
N


ATOM
2
CA
PHE
A
46
214.336
108.388
178.678
1.00
109.39
C


ATOM
3
C
PHE
A
46
215.483
107.382
178.556
1.00
92.52
C


ATOM
4
O
PHE
A
46
216.008
107.116
177.483
1.00
93.11
O


ATOM
5
CB
PHE
A
46
213.617
108.523
177.335
1.00
106.31
C


ATOM
6
CG
PHE
A
46
212.712
107.345
177.122
1.00
100.53
C


ATOM
7
CD1
PHE
A
46
213.246
106.138
176.688
1.00
94.28
C


ATOM
8
CD2
PHE
A
46
211.347
107.468
177.345
1.00
81.06
C


ATOM
9
CE1
PHE
A
46
212.408
105.049
176.480
1.00
71.80
C


ATOM
10
CE2
PHE
A
46
210.515
106.370
177.135
1.00
83.85
C


ATOM
11
CZ
PHE
A
46
211.041
105.159
176.704
1.00
54.26
C


ATOM
12
N
HIS
A
47
215.896
106.847
179.719
1.00
99.95
N


ATOM
13
CA
HIS
A
47
216.976
105.867
179.715
1.00
110.68
C


ATOM
14
C
HIS
A
47
216.467
104.473
179.342
1.00
99.74
C


ATOM
15
O
HIS
A
47
215.591
103.903
179.979
1.00
108.34
O


ATOM
16
CB
HIS
A
47
217.598
105.835
181.111
1.00
128.34
C


ATOM
17
CG
HIS
A
47
217.973
107.234
181.527
1.00
172.75
C


ATOM
18
ND1
HIS
A
47
219.157
107.813
181.211
1.00
209.78
N


ATOM
19
CD2
HIS
A
47
217.216
108.147
182.270
1.00
195.55
C


ATOM
20
CE1
HIS
A
47
219.113
109.047
181.749
1.00
214.52
C


ATOM
21
NE2
HIS
A
47
217.963
109.275
182.389
1.00
216.85
N


ATOM
22
N
VAL
A
48
217.023
103.942
178.238
1.00
93.29
N


ATOM
23
CA
VAL
A
48
216.620
102.613
177.798
1.00
69.76
C


ATOM
24
C
VAL
A
48
217.795
101.841
177.194
1.00
72.39
C


ATOM
25
O
VAL
A
48
218.595
102.370
176.434
1.00
67.87
O


ATOM
26
CB
VAL
A
48
215.510
102.770
176.757
1.00
60.38
C


ATOM
27
CG1
VAL
A
48
215.345
101.470
175.973
1.00
76.52
C


ATOM
28
CG2
VAL
A
48
214.200
103.110
177.443
1.00
87.98
C


ATOM
29
N
LYS
A
49
217.962
100.570
177.519
1.00
63.04
N


ATOM
30
CA
LYS
A
49
219.047
99.811
176.921
1.00
61.31
C


ATOM
31
C
LYS
A
49
218.424
98.945
175.833
1.00
56.33
C


ATOM
32
O
LYS
A
49
217.205
98.904
175.688
1.00
61.95
O


ATOM
33
CB
LYS
A
49
219.741
98.923
177.947
1.00
46.86
C


ATOM
34
CG
LYS
A
49
220.349
99.673
179.103
1.00
52.81
C


ATOM
35
CD
LYS
A
49
221.201
100.796
178.607
1.00
52.94
C


ATOM
36
CE
LYS
A
49
222.255
101.150
179.627
1.00
106.42
C


ATOM
37
NZ
LYS
A
49
223.200
102.166
179.091
1.00
117.49
N


ATOM
38
N
SER
A
50
219.258
98.248
175.073
1.00
67.67
N


ATOM
39
CA
SER
A
50
218.768
97.403
173.994
1.00
61.19
C


ATOM
40
C
SER
A
50
218.240
96.087
174.484
1.00
62.93
C


ATOM
41
O
SER
A
50
218.661
95.592
175.528
1.00
78.88
O


ATOM
42
CB
SER
A
50
219.875
97.119
172.994
1.00
59.19
C


ATOM
43
OG
SER
A
50
220.167
98.279
172.248
1.00
125.35
O


ATOM
44
N
GLY
A
51
217.318
95.516
173.718
1.00
62.13
N


ATOM
45
CA
GLY
A
51
216.773
94.223
174.070
1.00
45.10
C


ATOM
46
C
GLY
A
51
217.728
93.155
173.571
1.00
32.66
C


ATOM
47
O
GLY
A
51
218.558
93.420
172.709
1.00
72.20
O


ATOM
48
N
LEU
A
52
217.634
91.951
174.114
1.00
65.04
N


ATOM
49
CA
LEU
A
52
218.509
90.868
173.671
1.00
48.78
C


ATOM
50
C
LEU
A
52
218.154
90.388
172.252
1.00
54.66
C


ATOM
51
O
LEU
A
52
216.986
90.340
171.866
1.00
70.19
O


ATOM
52
CB
LEU
A
52
218.420
89.677
174.635
1.00
67.84
C


ATOM
53
CG
LEU
A
52
219.214
88.431
174.227
1.00
72.80
C


ATOM
54
CD1
LEU
A
52
220.719
88.700
174.309
1.00
57.35
C


ATOM
55
CD2
LEU
A
52
218.844
87.283
175.128
1.00
66.63
C


ATOM
56
N
GLN
A
53
219.170
90.031
171.477
1.00
68.12
N


ATOM
57
CA
GLN
A
53
218.958
89.546
170.125
1.00
54.88
C


ATOM
58
C
GLN
A
53
219.752
88.291
169.858
1.00
40.76
C


ATOM
59
O
GLN
A
53
220.978
88.278
169.936
1.00
66.36
O


ATOM
60
CB
GLN
A
53
219.372
90.588
169.115
1.00
47.08
C


ATOM
61
CG
GLN
A
53
219.428
90.042
167.731
1.00
60.85
C


ATOM
62
CD
GLN
A
53
220.160
90.974
166.832
1.00
80.61
C


ATOM
63
OE1
GLN
A
53
220.236
90.753
165.627
1.00
99.15
O


ATOM
64
NE2
GLN
A
53
220.723
92.031
167.411
1.00
83.94
N


ATOM
65
N
ILE
A
54
219.050
87.233
169.508
1.00
60.15
N


ATOM
66
CA
ILE
A
54
219.705
85.971
169.238
1.00
52.14
C


ATOM
67
C
ILE
A
54
220.190
85.896
167.802
1.00
51.88
C


ATOM
68
O
ILE
A
54
219.433
85.575
166.893
1.00
58.79
O


ATOM
69
CB
ILE
A
54
218.743
84.814
169.552
1.00
64.06
C


ATOM
70
CG1
ILE
A
54
218.312
84.924
171.010
1.00
33.87
C


ATOM
71
CG2
ILE
A
54
219.412
83.479
169.315
1.00
39.67
C


ATOM
72
CD1
ILE
A
54
217.727
83.680
171.535
1.00
82.78
C


ATOM
73
N
LYS
A
55
221.464
86.197
167.611
1.00
48.72
N


ATOM
74
CA
LYS
A
55
222.064
86.182
166.287
1.00
49.52
C


ATOM
75
C
LYS
A
55
222.019
84.808
165.633
1.00
56.72
C


ATOM
76
O
LYS
A
55
222.270
83.808
166.283
1.00
70.19
O


ATOM
77
CB
LYS
A
55
223.512
86.661
166.383
1.00
51.25
C


ATOM
78
CG
LYS
A
55
223.628
88.111
166.815
1.00
49.44
C


ATOM
79
CD
LYS
A
55
225.059
88.584
166.817
1.00
89.37
C


ATOM
80
CE
LYS
A
55
225.114
90.085
167.040
1.00
73.14
C


ATOM
81
NZ
LYS
A
55
226.521
90.612
167.008
1.00
123.08
N


ATOM
82
N
LYS
A
56
221.710
84.758
164.343
1.00
41.46
N


ATOM
83
CA
LYS
A
56
221.644
83.482
163.643
1.00
66.57
C


ATOM
84
C
LYS
A
56
222.839
83.118
162.767
1.00
55.67
C


ATOM
85
O
LYS
A
56
223.028
81.945
162.455
1.00
66.55
O


ATOM
86
CB
LYS
A
56
220.379
83.413
162.793
1.00
48.95
C


ATOM
87
CG
LYS
A
56
219.242
82.751
163.486
1.00
57.91
C


ATOM
88
CD
LYS
A
56
219.248
83.157
164.927
1.00
78.89
C


ATOM
89
CE
LYS
A
56
217.907
82.899
165.554
1.00
71.88
C


ATOM
90
NZ
LYS
A
56
216.873
83.764
164.935
1.00
82.07
N


ATOM
91
N
ASN
A
57
223.640
84.096
162.361
1.00
48.62
N


ATOM
92
CA
ASN
A
57
224.788
83.802
161.508
1.00
52.30
C


ATOM
93
C
ASN
A
57
225.851
83.039
162.261
1.00
40.32
C


ATOM
94
O
ASN
A
57
225.977
83.197
163.467
1.00
58.36
O


ATOM
95
CB
ASN
A
57
225.380
85.083
161.004
1.00
42.83
C


ATOM
96
CG
ASN
A
57
225.743
86.007
162.123
1.00
73.68
C


ATOM
97
OD1
ASN
A
57
224.859
86.577
162.794
1.00
48.52
O


ATOM
98
ND2
ASN
A
57
227.056
86.182
162.340
1.00
36.35
N


ATOM
99
N
ALA
A
58
226.627
82.223
161.558
1.00
41.49
N


ATOM
100
CA
ALA
A
58
227.661
81.432
162.214
1.00
44.46
C


ATOM
101
C
ALA
A
58
228.613
82.283
163.040
1.00
44.31
C


ATOM
102
O
ALA
A
58
229.267
83.173
162.516
1.00
45.45
O


ATOM
103
CB
ALA
A
58
228.438
80.641
161.194
1.00
32.48
C


ATOM
104
N
ILE
A
59
228.694
81.998
164.334
1.00
46.98
N


ATOM
105
CA
ILE
A
59
229.569
82.753
165.228
1.00
54.79
C


ATOM
106
C
ILE
A
59
231.007
82.870
164.670
1.00
63.43
C


ATOM
107
O
ILE
A
59
231.766
83.777
165.035
1.00
47.27
O


ATOM
108
CB
ILE
A
59
229.605
82.094
166.638
1.00
53.52
C


ATOM
109
CG1
ILE
A
59
230.284
83.012
167.644
1.00
55.42
C


ATOM
110
CG2
ILE
A
59
230.376
80.791
166.588
1.00
40.15
C


ATOM
111
CD1
ILE
A
59
230.311
82.446
169.038
1.00
57.36
C


ATOM
112
N
ILE
A
60
231.375
81.956
163.777
1.00
43.08
N


ATOM
113
CA
ILE
A
60
232.743
81.902
163.274
1.00
57.78
C


ATOM
114
C
ILE
A
60
233.086
83.151
162.458
1.00
60.65
C


ATOM
115
O
ILE
A
60
234.226
83.590
162.377
1.00
72.20
O


ATOM
116
CB
ILE
A
60
232.866
80.659
162.393
1.00
38.44
C


ATOM
117
CG1
ILE
A
60
231.615
80.513
161.518
1.00
50.72
C


ATOM
118
CG2
ILE
A
60
232.965
79.403
163.277
1.00
49.86
C


ATOM
119
CD1
ILE
A
60
231.798
79.479
160.407
1.00
46.70
C


ATOM
120
N
ASP
A
61
232.044
83.704
161.811
1.00
32.79
N


ATOM
121
CA
ASP
A
61
232.254
84.863
160.953
1.00
58.81
C


ATOM
122
C
ASP
A
61
232.607
86.117
161.758
1.00
50.10
C


ATOM
123
O
ASP
A
61
233.397
86.956
161.346
1.00
78.45
O


ATOM
124
CB
ASP
A
61
230.972
85.093
160.149
1.00
36.95
C


ATOM
125
CG
ASP
A
61
230.476
83.758
159.608
1.00
91.42
C


ATOM
126
OD1
ASP
A
61
229.268
83.636
159.400
1.00
76.09
O


ATOM
127
OD2
ASP
A
61
231.293
82.864
159.405
1.00
122.26
O


ATOM
128
N
ASP
A
62
231.954
86.250
162.929
1.00
62.40
N


ATOM
129
CA
ASP
A
62
232.213
87.422
163.761
1.00
63.46
C


ATOM
130
C
ASP
A
62
233.328
87.164
164.778
1.00
43.90
C


ATOM
131
O
ASP
A
62
233.994
88.071
165.261
1.00
74.39
O


ATOM
132
CB
ASP
A
62
230.918
87.786
164.492
1.00
33.21
C


ATOM
133
CG
ASP
A
62
229.843
88.135
163.473
1.00
76.80
C


ATOM
134
OD1
ASP
A
62
230.116
88.979
162.618
1.00
99.65
O


ATOM
135
OD2
ASP
A
62
228.754
87.573
163.546
1.00
84.30
O


ATOM
136
N
TYR
A
63
233.493
85.877
165.132
1.00
44.70
N


ATOM
137
CA
TYR
A
63
234.482
85.539
166.148
1.00
62.53
C


ATOM
138
C
TYR
A
63
235.502
84.519
165.641
1.00
58.43
C


ATOM
139
O
TYR
A
63
235.388
83.956
164.561
1.00
79.42
O


ATOM
140
CB
TYR
A
63
233.742
84.965
167.358
1.00
50.79
C


ATOM
141
CG
TYR
A
63
233.100
86.068
168.120
1.00
44.26
C


ATOM
142
CD1
TYR
A
63
233.802
86.709
169.136
1.00
34.66
C


ATOM
143
CD2
TYR
A
63
231.800
86.477
167.811
1.00
37.87
C


ATOM
144
CE1
TYR
A
63
233.220
87.758
169.831
1.00
27.94
C


ATOM
145
CE2
TYR
A
63
231.215
87.521
168.512
1.00
34.56
C


ATOM
146
CZ
TYR
A
63
231.916
88.158
169.519
1.00
56.82
C


ATOM
147
OH
TYR
A
63
231.353
89.224
170.192
1.00
41.90
O


ATOM
148
N
LYS
A
64
236.554
84.325
166.455
1.00
69.78
N


ATOM
149
CA
LYS
A
64
237.542
83.306
166.129
1.00
60.63
C


ATOM
150
C
LYS
A
64
237.831
82.420
167.340
1.00
67.37
C


ATOM
151
O
LYS
A
64
238.259
82.882
168.389
1.00
66.71
O


ATOM
152
CB
LYS
A
64
238.825
84.004
165.673
1.00
85.98
C


ATOM
153
CG
LYS
A
64
240.084
83.232
166.078
1.00
68.10
C


ATOM
154
CD
LYS
A
64
240.235
81.914
165.315
1.00
96.02
C


ATOM
155
CE
LYS
A
64
241.289
80.993
165.941
1.00
159.06
C


ATOM
156
NZ
LYS
A
64
241.422
79.778
165.141
1.00
151.29
N


ATOM
157
N
VAL
A
65
237.271
81.240
167.130
1.00
54.82
N


ATOM
158
CA
VAL
A
65
237.148
80.288
168.220
1.00
65.09
C


ATOM
159
C
VAL
A
65
238.437
79.492
168.298
1.00
65.21
C


ATOM
160
O
VAL
A
65
238.945
79.029
167.282
1.00
79.23
O


ATOM
161
CB
VAL
A
65
235.960
79.329
168.000
1.00
53.30
C


ATOM
162
CG1
VAL
A
65
235.948
78.263
169.075
1.00
68.94
C


ATOM
163
CG2
VAL
A
65
234.659
80.104
168.039
1.00
50.00
C


ATOM
164
N
THR
A
66
238.971
79.338
169.502
1.00
60.78
N


ATOM
165
CA
THR
A
66
240.214
78.602
169.680
1.00
73.39
C


ATOM
166
C
THR
A
66
240.032
77.346
170.515
1.00
76.31
C


ATOM
167
O
THR
A
66
238.969
77.107
171.080
1.00
77.40
O


ATOM
168
CB
THR
A
66
241.267
79.462
170.376
1.00
65.28
C


ATOM
169
OG1
THR
A
66
241.150
79.289
171.798
1.00
71.85
O


ATOM
170
CG2
THR
A
66
241.067
80.944
170.016
1.00
64.31
C


ATOM
171
N
SER
A
67
241.092
76.554
170.602
1.00
86.86
N


ATOM
172
CA
SER
A
67
241.065
75.318
171.369
1.00
86.84
C


ATOM
173
C
SER
A
67
241.359
75.528
172.838
1.00
78.98
C


ATOM
174
O
SER
A
67
241.140
74.631
173.646
1.00
92.44
O


ATOM
175
CB
SER
A
67
242.082
74.338
170.818
1.00
77.74
C


ATOM
176
OG
SER
A
67
241.681
73.901
169.540
1.00
131.17
O


ATOM
177
N
GLN
A
68
241.868
76.702
173.185
1.00
65.34
N


ATOM
178
CA
GLN
A
68
242.187
76.981
174.571
1.00
79.87
C


ATOM
179
C
GLN
A
68
240.955
76.819
175.448
1.00
75.61
C


ATOM
180
O
GLN
A
68
239.849
77.197
175.067
1.00
70.47
O


ATOM
181
CB
GLN
A
68
242.760
78.387
174.704
1.00
76.67
C


ATOM
182
CG
GLN
A
68
243.238
78.724
176.100
1.00
86.58
C


ATOM
183
CD
GLN
A
68
244.346
79.757
176.087
1.00
151.90
C


ATOM
184
OE1
GLN
A
68
244.745
80.271
177.134
1.00
147.03
O


ATOM
185
NE2
GLN
A
68
244.860
80.060
174.895
1.00
166.06
N


ATOM
186
N
VAL
A
69
241.155
76.244
176.625
1.00
70.23
N


ATOM
187
CA
VAL
A
69
240.064
76.013
177.552
1.00
73.71
C


ATOM
188
C
VAL
A
69
240.157
76.908
178.775
1.00
65.57
C


ATOM
189
O
VAL
A
69
241.009
76.704
179.639
1.00
83.31
O


ATOM
190
CB
VAL
A
69
240.064
74.564
178.023
1.00
69.41
C


ATOM
191
CG1
VAL
A
69
238.896
74.324
178.952
1.00
51.08
C


ATOM
192
CG2
VAL
A
69
240.017
73.648
176.830
1.00
57.48
C


ATOM
193
N
LEU
A
70
239.276
77.899
178.847
1.00
74.02
N


ATOM
194
CA
LEU
A
70
239.259
78.816
179.977
1.00
88.16
C


ATOM
195
C
LEU
A
70
238.865
78.039
181.227
1.00
94.93
C


ATOM
196
O
LEU
A
70
239.171
78.458
182.343
1.00
86.96
O


ATOM
197
CB
LEU
A
70
238.268
79.956
179.718
1.00
80.15
C


ATOM
198
CG
LEU
A
70
238.660
81.015
178.680
1.00
66.85
C


ATOM
199
CD1
LEU
A
70
237.420
81.668
178.097
1.00
90.30
C


ATOM
200
CD2
LEU
A
70
239.553
82.049
179.322
1.00
79.20
C


ATOM
201
N
GLY
A
71
238.194
76.903
181.035
1.00
87.34
N


ATOM
202
CA
GLY
A
71
237.781
76.089
182.166
1.00
87.75
C


ATOM
203
C
GLY
A
71
236.707
75.077
181.825
1.00
83.77
C


ATOM
204
O
GLY
A
71
236.158
75.111
180.730
1.00
84.32
O


ATOM
205
N
LEU
A
72
236.405
74.175
182.757
1.00
88.68
N


ATOM
206
CA
LEU
A
72
235.381
73.155
182.534
1.00
82.29
C


ATOM
207
C
LEU
A
72
234.225
73.239
183.509
1.00
73.28
C


ATOM
208
O
LEU
A
72
234.318
73.871
184.557
1.00
95.04
O


ATOM
209
CB
LEU
A
72
235.976
71.751
182.621
1.00
77.46
C


ATOM
210
CG
LEU
A
72
236.877
71.305
181.473
1.00
94.19
C


ATOM
211
CD1
LEU
A
72
237.217
69.818
181.595
1.00
139.26
C


ATOM
212
CD2
LEU
A
72
236.150
71.568
180.172
1.00
109.03
C


ATOM
213
N
GLY
A
73
233.133
72.577
183.158
1.00
77.98
N


ATOM
214
CA
GLY
A
73
231.963
72.592
184.013
1.00
71.81
C


ATOM
215
C
GLY
A
73
230.762
71.853
183.446
1.00
99.78
C


ATOM
216
O
GLY
A
73
230.843
71.171
182.418
1.00
93.36
O


ATOM
217
N
ILE
A
74
229.635
72.004
184.136
1.00
112.58
N


ATOM
218
CA
ILE
A
74
228.376
71.365
183.765
1.00
114.50
C


ATOM
219
C
ILE
A
74
228.103
71.259
182.270
1.00
110.24
C


ATOM
220
O
ILE
A
74
227.890
70.162
181.750
1.00
94.78
O


ATOM
221
CB
ILE
A
74
227.189
72.085
184.439
1.00
120.25
C


ATOM
222
CG1
ILE
A
74
227.145
71.707
185.921
1.00
127.29
C


ATOM
223
CG2
ILE
A
74
225.889
71.745
183.724
1.00
110.29
C


ATOM
224
CD1
ILE
A
74
225.968
72.285
186.673
1.00
178.93
C


ATOM
225
N
ASN
A
75
228.106
72.393
181.580
1.00
113.84
N


ATOM
226
CA
ASN
A
75
227.844
72.395
180.148
1.00
108.71
C


ATOM
227
C
ASN
A
75
229.044
72.005
179.296
1.00
100.52
C


ATOM
228
O
ASN
A
75
228.935
71.858
178.079
1.00
102.09
O


ATOM
229
CB
ASN
A
75
227.303
73.756
179.736
1.00
95.22
C


ATOM
230
CG
ASN
A
75
225.898
73.967
180.225
1.00
84.98
C


ATOM
231
OD1
ASN
A
75
224.966
73.315
179.749
1.00
88.40
O


ATOM
232
ND2
ASN
A
75
225.730
74.858
181.199
1.00
112.62
N


ATOM
233
N
GLY
A
76
230.186
71.820
179.940
1.00
99.31
N


ATOM
234
CA
GLY
A
76
231.360
71.428
179.198
1.00
93.20
C


ATOM
235
C
GLY
A
76
232.489
72.428
179.282
1.00
96.95
C


ATOM
236
O
GLY
A
76
232.666
73.117
180.285
1.00
101.12
O


ATOM
237
N
LYS
A
77
233.258
72.506
178.205
1.00
82.50
N


ATOM
238
CA
LYS
A
77
234.386
73.409
178.146
1.00
79.18
C


ATOM
239
C
LYS
A
77
233.986
74.820
177.757
1.00
66.93
C


ATOM
240
O
LYS
A
77
233.189
75.025
176.852
1.00
79.35
O


ATOM
241
CB
LYS
A
77
235.410
72.872
177.160
1.00
78.70
C


ATOM
242
N
VAL
A
78
234.523
75.794
178.470
1.00
57.33
N


ATOM
243
CA
VAL
A
78
234.267
77.179
178.150
1.00
49.75
C


ATOM
244
C
VAL
A
78
235.512
77.620
177.410
1.00
79.35
C


ATOM
245
O
VAL
A
78
236.545
77.888
178.028
1.00
64.43
O


ATOM
246
CB
VAL
A
78
234.138
78.020
179.386
1.00
54.95
C


ATOM
247
CG1
VAL
A
78
234.267
79.482
179.023
1.00
45.00
C


ATOM
248
CG2
VAL
A
78
232.810
77.742
180.029
1.00
52.90
C


ATOM
249
N
LEU
A
79
235.414
77.692
176.087
1.00
70.72
N


ATOM
250
CA
LEU
A
79
236.538
78.073
175.245
1.00
61.89
C


ATOM
251
C
LEU
A
79
236.825
79.563
175.185
1.00
64.76
C


ATOM
252
O
LEU
A
79
235.930
80.394
175.360
1.00
63.50
O


ATOM
253
CB
LEU
A
79
236.292
77.599
173.825
1.00
51.24
C


ATOM
254
CG
LEU
A
79
235.907
76.149
173.599
1.00
64.90
C


ATOM
255
CD1
LEU
A
79
235.543
75.983
172.139
1.00
49.75
C


ATOM
256
CD2
LEU
A
79
237.052
75.236
173.978
1.00
80.17
C


ATOM
257
N
GLN
A
80
238.090
79.889
174.919
1.00
72.17
N


ATOM
258
CA
GLN
A
80
238.505
81.274
174.765
1.00
77.62
C


ATOM
259
C
GLN
A
80
238.376
81.604
173.288
1.00
66.11
C


ATOM
260
O
GLN
A
80
238.775
80.813
172.437
1.00
64.63
O


ATOM
261
CB
GLN
A
80
239.955
81.486
175.167
1.00
52.97
C


ATOM
262
CG
GLN
A
80
240.390
82.913
174.863
1.00
93.41
C


ATOM
263
CD
GLN
A
80
241.715
83.310
175.494
1.00
115.14
C


ATOM
264
OE1
GLN
A
80
242.038
84.502
175.567
1.00
116.65
O


ATOM
265
NE2
GLN
A
80
242.490
82.322
175.949
1.00
117.09
N


ATOM
266
N
ILE
A
81
237.792
82.754
172.982
1.00
57.33
N


ATOM
267
CA
ILE
A
81
237.629
83.150
171.596
1.00
57.32
C


ATOM
268
C
ILE
A
81
238.109
84.571
171.400
1.00
54.90
C


ATOM
269
O
ILE
A
81
238.310
85.320
172.362
1.00
61.04
O


ATOM
270
CB
ILE
A
81
236.160
83.072
171.114
1.00
62.17
C


ATOM
271
CG1
ILE
A
81
235.289
84.050
171.897
1.00
51.03
C


ATOM
272
CG2
ILE
A
81
235.628
81.679
171.285
1.00
42.99
C


ATOM
273
CD1
ILE
A
81
233.852
84.088
171.391
1.00
59.46
C


ATOM
274
N
PHE
A
82
238.287
84.930
170.131
1.00
70.25
N


ATOM
275
CA
PHE
A
82
238.753
86.251
169.763
1.00
55.36
C


ATOM
276
C
PHE
A
82
237.815
86.911
168.787
1.00
62.80
C


ATOM
277
O
PHE
A
82
237.287
86.260
167.874
1.00
65.67
O


ATOM
278
CB
PHE
A
82
240.145
86.152
169.152
1.00
42.74
C


ATOM
279
CG
PHE
A
82
241.197
85.862
170.151
1.00
59.25
C


ATOM
280
CD1
PHE
A
82
241.639
86.857
170.999
1.00
44.66
C


ATOM
281
CD2
PHE
A
82
241.687
84.576
170.314
1.00
37.34
C


ATOM
282
CE1
PHE
A
82
242.552
86.576
171.999
1.00
57.81
C


ATOM
283
CE2
PHE
A
82
242.604
84.286
171.315
1.00
54.82
C


ATOM
284
CZ
PHE
A
82
243.032
85.287
172.156
1.00
58.83
C


ATOM
285
N
ASN
A
83
237.592
88.205
168.997
1.00
52.27
N


ATOM
286
CA
ASN
A
83
236.746
88.954
168.104
1.00
66.04
C


ATOM
287
C
ASN
A
83
237.605
89.492
166.971
1.00
53.09
C


ATOM
288
O
ASN
A
83
238.453
90.346
167.171
1.00
64.85
O


ATOM
289
CB
ASN
A
83
236.093
90.099
168.830
1.00
47.48
C


ATOM
290
CG
ASN
A
83
235.214
90.902
167.924
1.00
67.70
C


ATOM
291
OD1
ASN
A
83
235.689
91.744
167.160
1.00
72.99
O


ATOM
292
ND2
ASN
A
83
233.915
90.632
167.977
1.00
70.38
N


ATOM
293
N
LYS
A
84
237.383
88.974
165.774
1.00
63.87
N


ATOM
294
CA
LYS
A
84
238.133
89.376
164.590
1.00
68.20
C


ATOM
295
C
LYS
A
84
238.366
90.876
164.450
1.00
61.51
C


ATOM
296
O
LYS
A
84
239.492
91.340
164.537
1.00
74.81
O


ATOM
297
CB
LYS
A
84
237.412
88.859
163.345
1.00
55.26
C


ATOM
298
CG
LYS
A
84
237.312
87.343
163.283
1.00
33.38
C


ATOM
299
CD
LYS
A
84
236.425
86.918
162.154
1.00
30.89
C


ATOM
300
CE
LYS
A
84
236.759
85.521
161.719
1.00
46.41
C


ATOM
301
NZ
LYS
A
84
236.028
85.182
160.476
1.00
72.55
N


ATOM
302
N
ARG
A
85
237.290
91.622
164.235
1.00
63.12
N


ATOM
303
CA
ARG
A
85
237.359
93.066
164.043
1.00
51.69
C


ATOM
304
C
ARG
A
85
237.905
93.905
165.183
1.00
58.02
C


ATOM
305
O
ARG
A
85
238.197
95.075
164.991
1.00
86.09
O


ATOM
306
CB
ARG
A
85
235.978
93.589
163.647
1.00
60.23
C


ATOM
307
CG
ARG
A
85
235.862
95.086
163.509
1.00
76.93
C


ATOM
308
CD
ARG
A
85
235.516
95.725
164.835
1.00
84.92
C


ATOM
309
NE
ARG
A
85
234.121
96.140
164.907
1.00
93.42
N


ATOM
310
CZ
ARG
A
85
233.541
96.634
165.997
1.00
103.30
C


ATOM
311
NH1
ARG
A
85
234.224
96.777
167.128
1.00
54.02
N


ATOM
312
NH2
ARG
A
85
232.268
96.998
165.949
1.00
119.53
N


ATOM
313
N
THR
A
86
238.050
93.339
166.370
1.00
64.72
N


ATOM
314
CA
THR
A
86
238.565
94.123
167.489
1.00
54.27
C


ATOM
315
C
THR
A
86
239.717
93.398
168.140
1.00
52.40
C


ATOM
316
O
THR
A
86
240.520
93.980
168.856
1.00
57.78
O


ATOM
317
CB
THR
A
86
237.468
94.392
168.536
1.00
58.63
C


ATOM
318
OG1
THR
A
86
236.471
95.246
167.968
1.00
54.24
O


ATOM
319
CG2
THR
A
86
238.045
95.084
169.743
1.00
98.48
C


ATOM
320
N
GLN
A
87
239.772
92.106
167.883
1.00
54.70
N


ATOM
321
CA
GLN
A
87
240.819
91.260
168.397
1.00
49.69
C


ATOM
322
C
GLN
A
87
240.875
91.131
169.927
1.00
63.54
C


ATOM
323
O
GLN
A
87
241.936
90.881
170.491
1.00
78.48
O


ATOM
324
CB
GLN
A
87
242.148
91.761
167.854
1.00
44.04
C


ATOM
325
CG
GLN
A
87
243.145
90.675
167.561
1.00
87.54
C


ATOM
326
CD
GLN
A
87
242.722
89.811
166.413
1.00
70.54
C


ATOM
327
OE1
GLN
A
87
243.394
88.840
166.083
1.00
95.76
O


ATOM
328
NE2
GLN
A
87
241.601
90.157
165.789
1.00
75.13
N


ATOM
329
N
GLU
A
88
239.746
91.291
170.608
1.00
57.33
N


ATOM
330
CA
GLU
A
88
239.751
91.135
172.060
1.00
69.04
C


ATOM
331
C
GLU
A
88
239.352
89.723
172.442
1.00
68.23
C


ATOM
332
O
GLU
A
88
238.622
89.060
171.702
1.00
70.57
O


ATOM
333
CB
GLU
A
88
238.816
92.138
172.708
1.00
73.60
C


ATOM
334
CG
GLU
A
88
239.297
93.538
172.480
1.00
111.93
C


ATOM
335
CD
GLU
A
88
240.559
93.845
173.228
1.00
130.01
C


ATOM
336
OE1
GLU
A
88
240.728
93.298
174.344
1.00
151.01
O


ATOM
337
OE2
GLU
A
88
241.376
94.634
172.698
1.00
169.07
O


ATOM
338
N
LYS
A
89
239.837
89.261
173.592
1.00
80.21
N


ATOM
339
CA
LYS
A
89
239.543
87.904
174.047
1.00
67.45
C


ATOM
340
C
LYS
A
89
238.171
87.815
174.709
1.00
67.74
C


ATOM
341
O
LYS
A
89
237.719
88.752
175.355
1.00
71.95
O


ATOM
342
CB
LYS
A
89
240.632
87.424
175.013
1.00
46.26
C


ATOM
343
N
PHE
A
90
237.504
86.681
174.537
1.00
68.69
N


ATOM
344
CA
PHE
A
90
236.195
86.481
175.128
1.00
63.31
C


ATOM
345
C
PHE
A
90
235.957
85.019
175.477
1.00
70.09
C


ATOM
346
O
PHE
A
90
236.571
84.121
174.892
1.00
76.39
O


ATOM
347
CB
PHE
A
90
235.100
86.935
174.169
1.00
52.49
C


ATOM
348
CG
PHE
A
90
234.975
88.434
174.031
1.00
61.34
C


ATOM
349
CD1
PHE
A
90
235.523
89.091
172.952
1.00
63.36
C


ATOM
350
CD2
PHE
A
90
234.238
89.170
174.942
1.00
60.92
C


ATOM
351
CE1
PHE
A
90
235.330
90.429
172.781
1.00
38.41
C


ATOM
352
CE2
PHE
A
90
234.048
90.529
174.762
1.00
57.58
C


ATOM
353
CZ
PHE
A
90
234.591
91.155
173.683
1.00
50.60
C


ATOM
354
N
ALA
A
91
235.055
84.788
176.431
1.00
67.69
N


ATOM
355
CA
ALA
A
91
234.692
83.444
176.853
1.00
44.28
C


ATOM
356
C
ALA
A
91
233.480
83.000
176.052
1.00
45.25
C


ATOM
357
O
ALA
A
91
232.569
83.788
175.795
1.00
60.93
O


ATOM
358
CB
ALA
A
91
234.360
83.443
178.302
1.00
52.11
C


ATOM
359
N
LEU
A
92
233.477
81.736
175.653
1.00
48.94
N


ATOM
360
CA
LEU
A
92
232.381
81.179
174.882
1.00
50.98
C


ATOM
361
C
LEU
A
92
231.771
79.995
175.610
1.00
66.99
C


ATOM
362
O
LEU
A
92
232.467
79.045
175.939
1.00
82.82
O


ATOM
363
CB
LEU
A
92
232.886
80.710
173.536
1.00
39.13
C


ATOM
364
CG
LEU
A
92
231.838
79.857
172.839
1.00
52.58
C


ATOM
365
CD1
LEU
A
92
230.636
80.726
172.548
1.00
48.33
C


ATOM
366
CD2
LEU
A
92
232.404
79.256
171.557
1.00
58.60
C


ATOM
367
N
LYS
A
93
230.469
80.053
175.856
1.00
66.30
N


ATOM
368
CA
LYS
A
93
229.650
79.022
176.486
1.00
62.11
C


ATOM
369
C
LYS
A
93
228.624
78.440
175.508
1.00
61.26
C


ATOM
370
O
LYS
A
93
227.945
79.148
174.774
1.00
77.67
O


ATOM
371
CB
LYS
A
93
228.936
79.645
177.689
1.00
79.47
C


ATOM
372
CG
LYS
A
93
228.640
78.617
178.785
1.00
69.18
C


ATOM
373
CD
LYS
A
93
227.896
79.231
179.974
1.00
74.19
C


ATOM
374
CE
LYS
A
93
227.871
78.303
181.194
1.00
107.78
C


ATOM
375
NZ
LYS
A
93
228.986
78.626
182.083
1.00
92.73
N


ATOM
376
N
MET
A
94
228.551
77.095
175.489
1.00
53.94
N


ATOM
377
CA
MET
A
94
227.639
76.436
174.560
1.00
63.34
C


ATOM
378
C
MET
A
94
226.517
75.693
175.290
1.00
67.05
C


ATOM
379
O
MET
A
94
226.740
74.805
176.101
1.00
77.93
O


ATOM
380
CB
MET
A
94
228.447
75.451
173.713
1.00
54.57
C


ATOM
381
CG
MET
A
94
229.751
76.056
173.188
1.00
66.95
C


ATOM
382
SD
MET
A
94
230.855
74.810
172.511
1.00
128.32
S


ATOM
383
CE
MET
A
94
231.935
75.912
171.587
1.00
110.84
C


ATOM
384
N
LEU
A
95
225.273
76.116
174.999
1.00
76.30
N


ATOM
385
CA
LEU
A
95
224.126
75.468
175.626
1.00
47.37
C


ATOM
386
C
LEU
A
95
223.284
74.703
174.601
1.00
61.07
C


ATOM
387
O
LEU
A
95
223.143
75.102
173.452
1.00
66.44
O


ATOM
388
CB
LEU
A
95
223.276
76.549
176.295
1.00
56.39
C


ATOM
389
CG
LEU
A
95
224.057
77.342
177.346
1.00
78.66
C


ATOM
390
CD1
LEU
A
95
223.138
78.105
178.302
1.00
71.39
C


ATOM
391
CD2
LEU
A
95
224.941
76.453
178.221
1.00
68.93
C


ATOM
392
N
GLN
A
96
222.574
73.621
174.890
1.00
60.77
N


ATOM
393
CA
GLN
A
96
221.662
73.075
173.906
1.00
63.29
C


ATOM
394
C
GLN
A
96
220.493
74.049
173.876
1.00
63.17
C


ATOM
395
O
GLN
A
96
219.998
74.481
174.915
1.00
63.30
O


ATOM
396
CB
GLN
A
96
221.193
71.700
174.326
1.00
70.27
C


ATOM
397
N
ASP
A
97
220.056
74.415
172.686
1.00
59.87
N


ATOM
398
CA
ASP
A
97
218.950
75.343
172.589
1.00
58.98
C


ATOM
399
C
ASP
A
97
217.669
74.640
173.022
1.00
73.48
C


ATOM
400
O
ASP
A
97
217.277
73.626
172.448
1.00
75.22
O


ATOM
401
CB
ASP
A
97
218.831
75.843
171.157
1.00
68.14
C


ATOM
402
CG
ASP
A
97
217.933
77.038
171.045
1.00
73.65
C


ATOM
403
OD1
ASP
A
97
217.516
77.567
172.096
1.00
68.14
O


ATOM
404
OD2
ASP
A
97
217.647
77.451
169.906
1.00
80.65
O


ATOM
405
N
CYS
A
98
217.023
75.189
174.041
1.00
67.24
N


ATOM
406
CA
CYS
A
98
215.801
74.626
174.586
1.00
52.14
C


ATOM
407
C
CYS
A
98
215.258
75.697
175.510
1.00
75.99
C


ATOM
408
O
CYS
A
98
216.018
76.483
176.068
1.00
86.44
O


ATOM
409
CB
CYS
A
98
216.122
73.415
175.421
1.00
52.47
C


ATOM
410
SG
CYS
A
98
216.922
73.906
176.969
1.00
68.25
S


ATOM
411
N
PRO
A
99
213.939
75.722
175.717
1.00
81.29
N


ATOM
412
CA
PRO
A
99
213.283
76.705
176.578
1.00
85.87
C


ATOM
413
C
PRO
A
99
213.996
77.083
177.868
1.00
82.76
C


ATOM
414
O
PRO
A
99
214.153
78.268
178.149
1.00
67.85
O


ATOM
415
CB
PRO
A
99
211.926
76.073
176.821
1.00
94.07
C


ATOM
416
CG
PRO
A
99
211.642
75.452
175.492
1.00
91.19
C


ATOM
417
CD
PRO
A
99
212.959
74.756
175.201
1.00
93.97
C


ATOM
418
N
LYS
A
100
214.435
76.098
178.648
1.00
67.78
N


ATOM
419
CA
LYS
A
100
215.100
76.408
179.912
1.00
66.60
C


ATOM
420
C
LYS
A
100
216.341
77.236
179.702
1.00
80.42
C


ATOM
421
O
LYS
A
100
216.622
78.158
180.472
1.00
69.23
O


ATOM
422
CB
LYS
A
100
215.473
75.088
180.595
1.00
84.50
C


ATOM
423
N
ALA
A
101
217.085
76.885
178.656
1.00
76.33
N


ATOM
424
CA
ALA
A
101
218.320
77.570
178.319
1.00
66.38
C


ATOM
425
C
ALA
A
101
218.055
79.028
177.964
1.00
67.87
C


ATOM
426
O
ALA
A
101
218.639
79.922
178.563
1.00
70.18
O


ATOM
427
CB
ALA
A
101
219.022
76.849
177.175
1.00
55.84
C


ATOM
428
N
ARG
A
102
217.163
79.277
177.014
1.00
63.42
N


ATOM
429
CA
ARG
A
102
216.852
80.646
176.622
1.00
64.85
C


ATOM
430
C
ARG
A
102
216.409
81.491
177.812
1.00
68.04
C


ATOM
431
O
ARG
A
102
216.523
82.715
177.785
1.00
87.14
O


ATOM
432
CB
ARG
A
102
215.764
80.665
175.541
1.00
58.29
C


ATOM
433
CG
ARG
A
102
216.118
79.877
174.290
1.00
87.87
C


ATOM
434
CD
ARG
A
102
215.938
80.688
173.003
1.00
113.57
C


ATOM
435
NE
ARG
A
102
216.532
80.000
171.853
1.00
149.75
N


ATOM
436
CZ
ARG
A
102
216.645
80.517
170.626
1.00
161.97
C


ATOM
437
NH1
ARG
A
102
216.200
81.742
170.361
1.00
169.18
N


ATOM
438
NH2
ARG
A
102
217.209
79.808
169.658
1.00
123.53
N


ATOM
439
N
ARG
A
103
215.900
80.848
178.856
1.00
68.81
N


ATOM
440
CA
ARG
A
103
215.456
81.579
180.040
1.00
80.79
C


ATOM
441
C
ARG
A
103
216.664
82.065
180.812
1.00
74.21
C


ATOM
442
O
ARG
A
103
216.757
83.229
181.192
1.00
79.04
O


ATOM
443
CB
ARG
A
103
214.620
80.684
180.943
1.00
100.01
C


ATOM
444
CG
ARG
A
103
214.334
81.302
182.299
1.00
140.54
C


ATOM
445
CD
ARG
A
103
213.451
80.396
183.152
1.00
186.85
C


ATOM
446
NE
ARG
A
103
214.159
79.246
183.721
1.00
183.90
N


ATOM
447
CZ
ARG
A
103
213.851
77.974
183.469
1.00
163.96
C


ATOM
448
NH1
ARG
A
103
212.848
77.669
182.641
1.00
83.48
N


ATOM
449
NH2
ARG
A
103
214.523
77.008
184.082
1.00
172.74
N


ATOM
450
N
GLU
A
104
217.584
81.143
181.047
1.00
62.81
N


ATOM
451
CA
GLU
A
104
218.821
81.431
181.750
1.00
74.19
C


ATOM
452
C
GLU
A
104
219.481
82.643
181.094
1.00
76.22
C


ATOM
453
O
GLU
A
104
219.642
83.699
181.711
1.00
64.24
O


ATOM
454
CB
GLU
A
104
219.722
80.210
181.633
1.00
84.57
C


ATOM
455
CG
GLU
A
104
221.043
80.286
182.342
1.00
113.52
C


ATOM
456
CD
GLU
A
104
221.832
79.002
182.167
1.00
109.63
C


ATOM
457
OE1
GLU
A
104
222.923
78.879
182.757
1.00
143.02
O


ATOM
458
OE2
GLU
A
104
221.358
78.112
181.430
1.00
133.75
O


ATOM
459
N
VAL
A
105
219.845
82.462
179.828
1.00
72.46
N


ATOM
460
CA
VAL
A
105
220.488
83.485
179.010
1.00
67.75
C


ATOM
461
C
VAL
A
105
219.830
84.846
179.164
1.00
71.54
C


ATOM
462
O
VAL
A
105
220.506
85.851
179.366
1.00
75.44
O


ATOM
463
CB
VAL
A
105
220.450
83.096
177.524
1.00
70.64
C


ATOM
464
CG1
VAL
A
105
221.057
84.183
176.695
1.00
60.07
C


ATOM
465
CG2
VAL
A
105
221.196
81.796
177.312
1.00
75.62
C


ATOM
466
N
GLU
A
106
218.511
84.879
179.055
1.00
56.08
N


ATOM
467
CA
GLU
A
106
217.795
86.126
179.198
1.00
58.27
C


ATOM
468
C
GLU
A
106
218.087
86.715
180.569
1.00
62.94
C


ATOM
469
O
GLU
A
106
218.474
87.873
180.682
1.00
69.82
O


ATOM
470
CB
GLU
A
106
216.301
85.893
179.081
1.00
60.31
C


ATOM
471
CG
GLU
A
106
215.588
86.837
178.156
1.00
75.77
C


ATOM
472
CD
GLU
A
106
215.465
86.257
176.774
1.00
90.69
C


ATOM
473
OE1
GLU
A
106
216.490
86.211
176.072
1.00
127.18
O


ATOM
474
OE2
GLU
A
106
214.352
85.823
176.393
1.00
144.79
O


ATOM
475
N
LEU
A
107
217.896
85.921
181.617
1.00
59.54
N


ATOM
476
CA
LEU
A
107
218.132
86.409
182.964
1.00
60.25
C


ATOM
477
C
LEU
A
107
219.503
87.050
183.098
1.00
79.14
C


ATOM
478
O
LEU
A
107
219.649
88.148
183.623
1.00
55.44
O


ATOM
479
CB
LEU
A
107
218.013
85.269
183.967
1.00
82.80
C


ATOM
480
CG
LEU
A
107
216.618
84.741
184.297
1.00
74.03
C


ATOM
481
CD1
LEU
A
107
216.740
83.679
185.368
1.00
68.79
C


ATOM
482
CD2
LEU
A
107
215.731
85.866
184.783
1.00
64.45
C


ATOM
483
N
HIS
A
108
220.514
86.351
182.614
1.00
68.47
N


ATOM
484
CA
HIS
A
108
221.875
86.840
182.691
1.00
60.51
C


ATOM
485
C
HIS
A
108
222.058
88.075
181.819
1.00
51.27
C


ATOM
486
O
HIS
A
108
222.721
89.035
182.208
1.00
70.90
O


ATOM
487
CB
HIS
A
108
222.817
85.732
182.252
1.00
74.21
C


ATOM
488
CG
HIS
A
108
224.261
86.055
182.447
1.00
58.20
C


ATOM
489
ND1
HIS
A
108
225.260
85.149
182.176
1.00
56.15
N


ATOM
490
CD2
HIS
A
108
224.875
87.176
182.891
1.00
56.76
C


ATOM
491
CE1
HIS
A
108
226.429
85.699
182.446
1.00
52.10
C


ATOM
492
NE2
HIS
A
108
226.224
86.928
182.881
1.00
52.02
N


ATOM
493
N
TRP
A
109
221.480
88.046
180.628
1.00
57.29
N


ATOM
494
CA
TRP
A
109
221.633
89.219
179.779
1.00
60.60
C


ATOM
495
C
TRP
A
109
220.952
90.440
180.401
1.00
49.86
C


ATOM
496
O
TRP
A
109
221.365
91.583
180.231
1.00
77.39
O


ATOM
497
CB
TRP
A
109
221.000
88.905
178.423
1.00
58.21
C


ATOM
498
CG
TRP
A
109
220.711
90.158
177.691
1.00
62.11
C


ATOM
499
CD1
TRP
A
109
219.493
90.874
177.686
1.00
77.34
C


ATOM
500
CD2
TRP
A
109
221.631
90.902
176.855
1.00
77.76
C


ATOM
501
NE1
TRP
A
109
219.557
92.006
176.931
1.00
78.01
N


ATOM
502
CE2
TRP
A
109
220.933
92.041
176.375
1.00
87.90
C


ATOM
503
CE3
TRP
A
109
222.954
90.700
176.473
1.00
105.10
C


ATOM
504
CZ2
TRP
A
109
221.572
92.943
175.540
1.00
127.04
C


ATOM
505
CZ3
TRP
A
109
223.594
91.606
175.640
1.00
107.99
C


ATOM
506
CH2
TRP
A
109
222.895
92.731
175.167
1.00
111.87
C


ATOM
507
N
ARG
A
110
219.845
90.163
181.113
1.00
63.80
N


ATOM
508
CA
ARG
A
110
219.102
91.243
181.753
1.00
60.39
C


ATOM
509
C
ARG
A
110
219.807
91.738
183.019
1.00
56.74
C


ATOM
510
O
ARG
A
110
219.416
92.713
183.648
1.00
72.39
O


ATOM
511
CB
ARG
A
110
217.714
90.704
182.108
1.00
55.11
C


ATOM
512
CG
ARG
A
110
216.630
91.781
182.038
1.00
70.18
C


ATOM
513
CD
ARG
A
110
215.241
91.177
181.803
1.00
129.88
C


ATOM
514
NE
ARG
A
110
214.340
91.500
182.914
1.00
98.93
N


ATOM
515
CZ
ARG
A
110
214.072
90.515
183.794
1.00
97.18
C


ATOM
516
NH1
ARG
A
110
213.537
90.802
184.969
1.00
106.39
N


ATOM
517
NH2
ARG
A
110
214.325
89.245
183.464
1.00
84.21
N


ATOM
518
N
ALA
A
111
220.864
90.997
183.409
1.00
61.92
N


ATOM
519
CA
ALA
A
111
221.578
91.358
184.628
1.00
62.44
C


ATOM
520
C
ALA
A
111
223.023
91.776
184.343
1.00
78.96
C


ATOM
521
O
ALA
A
111
223.808
92.067
185.237
1.00
57.41
O


ATOM
522
CB
ALA
A
111
221.559
90.151
185.567
1.00
53.16
C


ATOM
523
N
SER
A
112
223.377
91.758
183.046
1.00
76.17
N


ATOM
524
CA
SER
A
112
224.737
92.128
182.670
1.00
71.16
C


ATOM
525
C
SER
A
112
224.979
93.632
182.825
1.00
64.82
C


ATOM
526
O
SER
A
112
226.104
94.113
182.806
1.00
63.34
O


ATOM
527
CB
SER
A
112
224.963
91.711
181.216
1.00
45.39
C


ATOM
528
OG
SER
A
112
224.992
90.282
181.135
1.00
81.43
O


ATOM
529
N
GLN
A
113
223.918
94.403
182.989
1.00
73.53
N


ATOM
530
CA
GLN
A
113
224.071
95.835
183.163
1.00
77.61
C


ATOM
531
C
GLN
A
113
224.644
96.132
184.545
1.00
70.17
C


ATOM
532
O
GLN
A
113
224.884
97.280
184.884
1.00
88.91
O


ATOM
533
CB
GLN
A
113
222.716
96.537
183.006
1.00
79.72
C


ATOM
534
CG
GLN
A
113
221.947
96.134
181.754
1.00
117.37
C


ATOM
535
CD
GLN
A
113
222.616
96.582
180.448
1.00
132.38
C


ATOM
536
OE1
GLN
A
113
222.305
96.060
179.363
1.00
85.14
O


ATOM
537
NE2
GLN
A
113
223.522
97.558
180.542
1.00
73.13
N


ATOM
538
N
CYS
A
114
224.854
95.098
185.348
1.00
77.28
N


ATOM
539
CA
CYS
A
114
225.395
95.280
186.692
1.00
65.33
C


ATOM
540
C
CYS
A
114
226.896
95.029
186.750
1.00
66.81
C


ATOM
541
O
CYS
A
114
227.365
93.910
186.553
1.00
70.94
O


ATOM
542
CB
CYS
A
114
224.699
94.346
187.679
1.00
68.99
C


ATOM
543
SG
CYS
A
114
225.606
94.158
189.225
1.00
70.19
S


ATOM
544
N
PRO
A
115
227.665
96.073
187.058
1.00
57.36
N


ATOM
545
CA
PRO
A
115
229.123
96.082
187.171
1.00
46.75
C


ATOM
546
C
PRO
A
115
229.770
94.790
187.650
1.00
60.90
C


ATOM
547
O
PRO
A
115
230.735
94.315
187.046
1.00
79.32
O


ATOM
548
CB
PRO
A
115
229.382
97.226
188.140
1.00
46.54
C


ATOM
549
CG
PRO
A
115
228.325
98.199
187.764
1.00
65.08
C


ATOM
550
CD
PRO
A
115
227.093
97.329
187.569
1.00
53.52
C


ATOM
551
N
HIS
A
116
229.242
94.217
188.726
1.00
50.47
N


ATOM
552
CA
HIS
A
116
229.827
93.015
189.294
1.00
62.43
C


ATOM
553
C
HIS
A
116
229.336
91.675
188.787
1.00
62.85
C


ATOM
554
O
HIS
A
116
229.654
90.634
189.366
1.00
86.03
O


ATOM
555
CB
HIS
A
116
229.709
93.085
190.804
1.00
66.18
C


ATOM
556
CG
HIS
A
116
230.547
94.163
191.405
1.00
74.19
C


ATOM
557
ND1
HIS
A
116
231.923
94.104
191.422
1.00
58.68
N


ATOM
558
CD2
HIS
A
116
230.214
95.360
191.945
1.00
50.93
C


ATOM
559
CE1
HIS
A
116
232.404
95.220
191.945
1.00
106.08
C


ATOM
560
NE2
HIS
A
116
231.386
95.999
192.269
1.00
97.40
N


ATOM
561
N
ILE
A
117
228.574
91.693
187.703
1.00
58.07
N


ATOM
562
CA
ILE
A
117
228.075
90.472
187.097
1.00
51.42
C


ATOM
563
C
ILE
A
117
228.790
90.375
185.769
1.00
57.68
C


ATOM
564
O
ILE
A
117
228.912
91.381
185.056
1.00
72.95
O


ATOM
565
CB
ILE
A
117
226.586
90.572
186.848
1.00
60.05
C


ATOM
566
CG1
ILE
A
117
225.867
90.676
188.193
1.00
65.42
C


ATOM
567
CG2
ILE
A
117
226.111
89.371
186.050
1.00
56.83
C


ATOM
568
CD1
ILE
A
117
224.420
91.064
188.100
1.00
76.80
C


ATOM
569
N
VAL
A
118
229.275
89.188
185.427
1.00
60.67
N


ATOM
570
CA
VAL
A
118
229.979
89.028
184.160
1.00
56.50
C


ATOM
571
C
VAL
A
118
229.008
89.416
183.044
1.00
62.86
C


ATOM
572
O
VAL
A
118
227.844
89.008
183.071
1.00
61.80
O


ATOM
573
CB
VAL
A
118
230.469
87.583
183.975
1.00
59.65
C


ATOM
574
CG1
VAL
A
118
229.288
86.642
183.812
1.00
58.62
C


ATOM
575
CG2
VAL
A
118
231.389
87.503
182.776
1.00
72.68
C


ATOM
576
N
ARG
A
119
229.483
90.204
182.075
1.00
59.87
N


ATOM
577
CA
ARG
A
119
228.635
90.694
180.988
1.00
63.38
C


ATOM
578
C
ARG
A
119
228.551
89.821
179.763
1.00
57.05
C


ATOM
579
O
ARG
A
119
229.546
89.276
179.314
1.00
63.34
O


ATOM
580
CB
ARG
A
119
229.083
92.079
180.566
1.00
37.88
C


ATOM
581
N
ILE
A
120
227.348
89.698
179.217
1.00
52.84
N


ATOM
582
CA
ILE
A
120
227.135
88.928
178.002
1.00
55.21
C


ATOM
583
C
ILE
A
120
227.227
89.914
176.847
1.00
56.02
C


ATOM
584
O
ILE
A
120
226.375
90.801
176.719
1.00
61.19
O


ATOM
585
CB
ILE
A
120
225.742
88.298
177.985
1.00
63.39
C


ATOM
586
CG1
ILE
A
120
225.707
87.100
178.931
1.00
59.03
C


ATOM
587
CG2
ILE
A
120
225.368
87.898
176.570
1.00
45.00
C


ATOM
588
CD1
ILE
A
120
224.390
86.384
178.896
1.00
71.76
C


ATOM
589
N
VAL
A
121
228.259
89.766
176.013
1.00
69.54
N


ATOM
590
CA
VAL
A
121
228.440
90.647
174.863
1.00
53.54
C


ATOM
591
C
VAL
A
121
227.432
90.354
173.743
1.00
53.36
C


ATOM
592
O
VAL
A
121
226.823
91.254
173.173
1.00
67.91
O


ATOM
593
CB
VAL
A
121
229.875
90.482
174.354
1.00
52.53
C


ATOM
594
CG1
VAL
A
121
230.153
91.471
173.224
1.00
91.60
C


ATOM
595
CG2
VAL
A
121
230.854
90.737
175.486
1.00
71.15
C


ATOM
596
N
ASP
A
122
227.299
89.060
173.389
1.00
45.30
N


ATOM
597
CA
ASP
A
122
226.149
88.676
172.567
1.00
76.20
C


ATOM
598
C
ASP
A
122
225.984
87.152
172.459
1.00
52.20
C


ATOM
599
O
ASP
A
122
226.852
86.378
172.842
1.00
62.83
O


ATOM
600
CB
ASP
A
122
226.337
89.280
171.173
1.00
81.39
C


ATOM
601
CG
ASP
A
122
227.687
88.850
170.615
1.00
72.92
C


ATOM
602
OD1
ASP
A
122
228.224
87.860
171.115
1.00
121.42
O


ATOM
603
OD2
ASP
A
122
228.183
89.500
169.698
1.00
55.20
O


ATOM
604
N
VAL
A
123
224.777
86.769
171.999
1.00
50.00
N


ATOM
605
CA
VAL
A
123
224.398
85.361
171.982
1.00
55.40
C


ATOM
606
C
VAL
A
123
223.988
84.917
170.574
1.00
50.66
C


ATOM
607
O
VAL
A
123
223.350
85.650
169.830
1.00
50.76
O


ATOM
608
CB
VAL
A
123
223.215
85.196
172.943
1.00
45.71
C


ATOM
609
CG1
VAL
A
123
222.819
83.726
173.058
1.00
66.35
C


ATOM
610
CG2
VAL
A
123
223.581
85.730
174.316
1.00
57.34
C


ATOM
611
N
TYR
A
124
224.384
83.682
170.271
1.00
54.76
N


ATOM
612
CA
TYR
A
124
224.108
83.092
168.964
1.00
59.15
C


ATOM
613
C
TYR
A
124
223.286
81.813
169.047
1.00
61.11
C


ATOM
614
O
TYR
A
124
223.356
81.063
170.021
1.00
77.18
O


ATOM
615
CB
TYR
A
124
225.412
82.768
168.216
1.00
54.42
C


ATOM
616
CG
TYR
A
124
226.292
83.973
167.929
1.00
64.97
C


ATOM
617
CD1
TYR
A
124
227.031
84.574
168.941
1.00
36.17
C


ATOM
618
CD2
TYR
A
124
226.352
84.532
166.649
1.00
68.56
C


ATOM
619
CE1
TYR
A
124
227.793
85.681
168.700
1.00
56.00
C


ATOM
620
CE2
TYR
A
124
227.117
85.653
166.393
1.00
49.47
C


ATOM
621
CZ
TYR
A
124
227.835
86.220
167.428
1.00
51.06
C


ATOM
622
OH
TYR
A
124
228.608
87.330
167.202
1.00
71.62
O


ATOM
623
N
GLU
A
125
222.497
81.576
168.010
1.00
65.81
N


ATOM
624
CA
GLU
A
125
221.697
80.374
167.910
1.00
49.40
C


ATOM
625
C
GLU
A
125
222.164
79.747
166.601
1.00
50.33
C


ATOM
626
O
GLU
A
125
221.610
80.007
165.535
1.00
63.99
O


ATOM
627
CB
GLU
A
125
220.210
80.728
167.851
1.00
56.44
C


ATOM
628
CG
GLU
A
125
219.285
79.525
167.952
1.00
64.02
C


ATOM
629
CD
GLU
A
125
218.600
79.176
166.637
1.00
103.79
C


ATOM
630
OE1
GLU
A
125
217.665
79.900
166.223
1.00
113.21
O


ATOM
631
OE2
GLU
A
125
219.002
78.172
166.009
1.00
118.03
O


ATOM
632
N
ASN
A
126
223.217
78.948
166.675
1.00
57.09
N


ATOM
633
CA
ASN
A
126
223.752
78.304
165.480
1.00
63.39
C


ATOM
634
C
ASN
A
126
223.370
76.842
165.453
1.00
68.35
C


ATOM
635
O
ASN
A
126
222.770
76.334
166.393
1.00
78.79
O


ATOM
636
CB
ASN
A
126
225.271
78.413
165.451
1.00
53.33
C


ATOM
637
CG
ASN
A
126
225.738
79.824
165.237
1.00
50.86
C


ATOM
638
OD1
ASN
A
126
226.934
80.105
165.254
1.00
53.47
O


ATOM
639
ND2
ASN
A
126
224.786
80.732
165.026
1.00
57.45
N


ATOM
640
N
LEU
A
127
223.729
76.167
164.370
1.00
72.36
N


ATOM
641
CA
LEU
A
127
223.431
74.753
164.222
1.00
56.01
C


ATOM
642
C
LEU
A
127
224.712
73.983
164.467
1.00
63.01
C


ATOM
643
O
LEU
A
127
225.637
74.025
163.663
1.00
79.72
O


ATOM
644
CB
LEU
A
127
222.926
74.467
162.817
1.00
57.56
C


ATOM
645
CG
LEU
A
127
221.810
73.443
162.741
1.00
56.21
C


ATOM
646
CD1
LEU
A
127
220.660
73.944
163.564
1.00
63.97
C


ATOM
647
CD2
LEU
A
127
221.386
73.235
161.311
1.00
50.73
C


ATOM
648
N
TYR
A
128
224.768
73.281
165.588
1.00
85.19
N


ATOM
649
CA
TYR
A
128
225.949
72.511
165.932
1.00
78.91
C


ATOM
650
C
TYR
A
128
225.657
71.015
166.036
1.00
99.93
C


ATOM
651
O
TYR
A
128
224.960
70.571
166.956
1.00
83.06
O


ATOM
652
CB
TYR
A
128
226.513
72.996
167.259
1.00
73.34
C


ATOM
653
CG
TYR
A
128
227.849
72.396
167.562
1.00
113.80
C


ATOM
654
CD1
TYR
A
128
229.000
72.939
167.024
1.00
118.71
C


ATOM
655
CD2
TYR
A
128
227.959
71.253
168.341
1.00
128.83
C


ATOM
656
CE1
TYR
A
128
230.223
72.372
167.247
1.00
148.93
C


ATOM
657
CE2
TYR
A
128
229.183
70.667
168.569
1.00
160.37
C


ATOM
658
CZ
TYR
A
128
230.316
71.239
168.018
1.00
162.93
C


ATOM
659
OH
TYR
A
128
231.559
70.688
168.244
1.00
162.00
O


ATOM
660
N
ALA
A
129
226.204
70.240
165.098
1.00
112.77
N


ATOM
661
CA
ALA
A
129
226.016
68.788
165.077
1.00
119.60
C


ATOM
662
C
ALA
A
129
224.556
68.445
164.843
1.00
117.04
C


ATOM
663
O
ALA
A
129
223.981
67.637
165.575
1.00
102.48
O


ATOM
664
CB
ALA
A
129
226.482
68.181
166.398
1.00
105.12
C


ATOM
665
N
GLY
A
130
223.957
69.056
163.823
1.00
115.24
N


ATOM
666
CA
GLY
A
130
222.550
68.810
163.553
1.00
115.49
C


ATOM
667
C
GLY
A
130
221.728
69.250
164.756
1.00
107.44
C


ATOM
668
O
GLY
A
130
220.501
69.259
164.722
1.00
95.78
O


ATOM
669
N
ARG
A
131
222.425
69.616
165.828
1.00
89.82
N


ATOM
670
CA
ARG
A
131
221.791
70.067
167.055
1.00
92.75
C


ATOM
671
C
ARG
A
131
221.754
71.593
167.105
1.00
80.22
C


ATOM
672
O
ARG
A
131
222.756
72.265
166.892
1.00
63.19
O


ATOM
673
CB
ARG
A
131
222.546
69.516
168.271
1.00
79.11
C


ATOM
674
N
LYS
A
132
220.574
72.121
167.381
1.00
68.38
N


ATOM
675
CA
LYS
A
132
220.349
73.553
167.487
1.00
64.95
C


ATOM
676
C
LYS
A
132
220.944
74.013
168.828
1.00
56.62
C


ATOM
677
O
LYS
A
132
220.449
73.648
169.889
1.00
63.01
O


ATOM
678
CB
LYS
A
132
218.839
73.782
167.429
1.00
50.70
C


ATOM
679
CG
LYS
A
132
218.340
75.188
167.516
1.00
60.30
C


ATOM
680
CD
LYS
A
132
216.821
75.123
167.476
1.00
72.13
C


ATOM
681
CE
LYS
A
132
216.171
76.486
167.371
1.00
106.61
C


ATOM
682
NZ
LYS
A
132
214.692
76.354
167.313
1.00
65.96
N


ATOM
683
N
CYS
A
133
222.011
74.805
168.787
1.00
77.15
N


ATOM
684
CA
CYS
A
133
222.648
75.263
170.022
1.00
74.16
C


ATOM
685
C
CYS
A
133
222.555
76.745
170.320
1.00
65.05
C


ATOM
686
O
CYS
A
133
222.157
77.543
169.471
1.00
81.10
O


ATOM
687
CB
CYS
A
133
224.114
74.877
170.024
1.00
92.23
C


ATOM
688
SG
CYS
A
133
224.343
73.126
169.850
1.00
106.91
S


ATOM
689
N
LEU
A
134
222.947
77.099
171.540
1.00
67.21
N


ATOM
690
CA
LEU
A
134
222.921
78.478
171.986
1.00
67.45
C


ATOM
691
C
LEU
A
134
224.326
78.865
172.431
1.00
63.74
C


ATOM
692
O
LEU
A
134
224.804
78.446
173.488
1.00
71.31
O


ATOM
693
CB
LEU
A
134
221.953
78.634
173.152
1.00
51.96
C


ATOM
694
CG
LEU
A
134
221.107
79.902
173.189
1.00
72.36
C


ATOM
695
CD1
LEU
A
134
220.129
79.885
172.025
1.00
66.47
C


ATOM
696
CD2
LEU
A
134
220.342
79.981
174.503
1.00
63.46
C


ATOM
697
N
LEU
A
135
224.992
79.663
171.608
1.00
69.76
N


ATOM
698
CA
LEU
A
135
226.338
80.103
171.910
1.00
48.46
C


ATOM
699
C
LEU
A
135
226.262
81.450
172.608
1.00
60.20
C


ATOM
700
O
LEU
A
135
225.639
82.381
172.101
1.00
49.35
O


ATOM
701
CB
LEU
A
135
227.130
80.192
170.609
1.00
69.61
C


ATOM
702
CG
LEU
A
135
227.191
78.851
169.873
1.00
59.09
C


ATOM
703
CD1
LEU
A
135
227.585
79.058
168.425
1.00
80.07
C


ATOM
704
CD2
LEU
A
135
228.173
77.943
170.579
1.00
92.22
C


ATOM
705
N
ILE
A
136
226.896
81.530
173.775
1.00
39.79
N


ATOM
706
CA
ILE
A
136
226.913
82.744
174.581
1.00
48.18
C


ATOM
707
C
ILE
A
136
228.321
83.324
174.691
1.00
54.20
C


ATOM
708
O
ILE
A
136
229.219
82.672
175.204
1.00
63.12
O


ATOM
709
CB
ILE
A
136
226.398
82.453
176.006
1.00
57.17
C


ATOM
710
CG1
ILE
A
136
224.997
81.864
175.934
1.00
54.12
C


ATOM
711
CG2
ILE
A
136
226.364
83.720
176.829
1.00
45.37
C


ATOM
712
CD1
ILE
A
136
224.893
80.475
176.545
1.00
94.13
C


ATOM
713
N
VAL
A
137
228.527
84.539
174.200
1.00
47.11
N


ATOM
714
CA
VAL
A
137
229.812
85.185
174.376
1.00
51.92
C


ATOM
715
C
VAL
A
137
229.827
86.081
175.615
1.00
62.30
C


ATOM
716
O
VAL
A
137
228.925
86.871
175.875
1.00
65.38
O


ATOM
717
CB
VAL
A
137
230.091
85.979
173.099
1.00
55.29
C


ATOM
718
CG1
VAL
A
137
231.461
86.640
173.162
1.00
49.20
C


ATOM
719
CG2
VAL
A
137
230.064
85.035
171.910
1.00
33.49
C


ATOM
720
N
MET
A
138
230.893
85.889
176.417
1.00
63.64
N


ATOM
721
CA
MET
A
138
231.026
86.598
177.685
1.00
47.48
C


ATOM
722
C
MET
A
138
232.339
87.381
177.769
1.00
63.41
C


ATOM
723
O
MET
A
138
233.371
86.975
177.246
1.00
60.31
O


ATOM
724
CB
MET
A
138
231.044
85.548
178.796
1.00
76.63
C


ATOM
725
CG
MET
A
138
229.707
84.836
178.965
1.00
72.52
C


ATOM
726
SD
MET
A
138
229.779
83.570
180.236
1.00
81.89
S


ATOM
727
CE
MET
A
138
228.109
82.937
180.029
1.00
180.26
C


ATOM
728
N
GLU
A
139
232.281
88.555
178.427
1.00
44.14
N


ATOM
729
CA
GLU
A
139
233.550
89.154
178.798
1.00
46.81
C


ATOM
730
C
GLU
A
139
234.381
88.099
179.511
1.00
61.98
C


ATOM
731
O
GLU
A
139
233.876
87.285
180.272
1.00
68.61
O


ATOM
732
CB
GLU
A
139
233.291
90.346
179.732
1.00
40.20
C


ATOM
733
CG
GLU
A
139
233.444
89.999
181.216
1.00
70.67
C


ATOM
734
CD
GLU
A
139
233.140
91.231
182.059
1.00
76.30
C


ATOM
735
OE1
GLU
A
139
231.972
91.501
182.306
1.00
89.15
O


ATOM
736
OE2
GLU
A
139
234.079
91.912
182.459
1.00
100.67
O


ATOM
737
N
CYS
A
140
235.664
88.142
179.172
1.00
69.16
N


ATOM
738
CA
CYS
A
140
236.625
87.167
179.666
1.00
69.69
C


ATOM
739
C
CYS
A
140
237.322
87.529
180.968
1.00
60.14
C


ATOM
740
O
CYS
A
140
238.227
88.349
180.990
1.00
82.48
O


ATOM
741
CB
CYS
A
140
237.676
86.903
178.587
1.00
74.24
C


ATOM
742
SG
CYS
A
140
238.996
85.811
179.117
1.00
73.10
S


ATOM
743
N
LEU
A
141
236.912
86.895
182.055
1.00
71.80
N


ATOM
744
CA
LEU
A
141
237.524
87.159
183.348
1.00
79.00
C


ATOM
745
C
LEU
A
141
238.785
86.304
183.509
1.00
76.59
C


ATOM
746
O
LEU
A
141
238.736
85.081
183.360
1.00
77.59
O


ATOM
747
CB
LEU
A
141
236.523
86.847
184.462
1.00
81.97
C


ATOM
748
CG
LEU
A
141
235.203
87.610
184.350
1.00
76.77
C


ATOM
749
CD1
LEU
A
141
234.214
87.100
185.382
1.00
96.50
C


ATOM
750
CD2
LEU
A
141
235.469
89.086
184.535
1.00
47.85
C


ATOM
751
N
ASP
A
142
239.913
86.940
183.818
1.00
90.64
N


ATOM
752
CA
ASP
A
142
241.158
86.197
183.977
1.00
92.77
C


ATOM
753
C
ASP
A
142
241.771
86.152
185.366
1.00
84.21
C


ATOM
754
O
ASP
A
142
242.387
85.153
185.725
1.00
120.09
O


ATOM
755
CB
ASP
A
142
242.202
86.698
182.984
1.00
92.82
C


ATOM
756
CG
ASP
A
142
241.951
86.182
181.586
1.00
110.94
C


ATOM
757
OD1
ASP
A
142
241.710
84.961
181.450
1.00
82.18
O


ATOM
758
OD2
ASP
A
142
242.003
86.987
180.632
1.00
115.53
O


ATOM
759
N
GLY
A
143
241.616
87.220
186.142
1.00
76.67
N


ATOM
760
CA
GLY
A
143
242.178
87.249
187.486
1.00
84.99
C


ATOM
761
C
GLY
A
143
242.128
85.940
188.269
1.00
83.56
C


ATOM
762
O
GLY
A
143
242.909
85.744
189.194
1.00
94.38
O


ATOM
763
N
GLY
A
144
241.216
85.040
187.913
1.00
81.49
N


ATOM
764
CA
GLY
A
144
241.132
83.778
188.621
1.00
72.81
C


ATOM
765
C
GLY
A
144
240.064
83.784
189.700
1.00
88.08
C


ATOM
766
O
GLY
A
144
239.527
84.839
190.045
1.00
76.11
O


ATOM
767
N
GLU
A
145
239.759
82.603
190.235
1.00
72.60
N


ATOM
768
CA
GLU
A
145
238.746
82.464
191.268
1.00
80.92
C


ATOM
769
C
GLU
A
145
239.050
83.249
192.539
1.00
85.24
C


ATOM
770
O
GLU
A
145
240.206
83.446
192.908
1.00
76.66
O


ATOM
771
CB
GLU
A
145
238.543
80.993
191.605
1.00
61.62
C


ATOM
772
CG
GLU
A
145
237.797
80.215
190.539
1.00
89.57
C


ATOM
773
CD
GLU
A
145
238.090
78.727
190.618
1.00
126.57
C


ATOM
774
OE1
GLU
A
145
237.257
77.922
190.143
1.00
97.67
O


ATOM
775
OE2
GLU
A
145
239.165
78.367
191.156
1.00
103.63
O


ATOM
776
N
LEU
A
146
237.988
83.679
193.210
1.00
97.07
N


ATOM
777
CA
LEU
A
146
238.095
84.479
194.419
1.00
86.04
C


ATOM
778
C
LEU
A
146
239.174
84.065
195.402
1.00
75.32
C


ATOM
779
O
LEU
A
146
240.049
84.852
195.732
1.00
72.90
O


ATOM
780
CB
LEU
A
146
236.743
84.530
195.138
1.00
83.71
C


ATOM
781
CG
LEU
A
146
236.768
85.402
196.395
1.00
84.94
C


ATOM
782
CD1
LEU
A
146
237.338
86.754
196.044
1.00
92.90
C


ATOM
783
CD2
LEU
A
146
235.382
85.551
196.975
1.00
116.24
C


ATOM
784
N
PHE
A
147
239.127
82.829
195.867
1.00
80.55
N


ATOM
785
CA
PHE
A
147
240.104
82.385
196.847
1.00
87.51
C


ATOM
786
C
PHE
A
147
241.526
82.244
196.332
1.00
92.39
C


ATOM
787
O
PHE
A
147
242.478
82.546
197.051
1.00
104.70
O


ATOM
788
CB
PHE
A
147
239.611
81.094
197.495
1.00
62.88
C


ATOM
789
CG
PHE
A
147
238.388
81.297
198.330
1.00
83.58
C


ATOM
790
CD1
PHE
A
147
237.514
80.260
198.589
1.00
80.71
C


ATOM
791
CD2
PHE
A
147
238.102
82.557
198.845
1.00
82.88
C


ATOM
792
CE1
PHE
A
147
236.375
80.476
199.342
1.00
93.95
C


ATOM
793
CE2
PHE
A
147
236.971
82.778
199.596
1.00
50.19
C


ATOM
794
CZ
PHE
A
147
236.105
81.740
199.844
1.00
97.28
C


ATOM
795
N
SER
A
148
241.680
81.805
195.090
1.00
81.24
N


ATOM
796
CA
SER
A
148
243.008
81.657
194.525
1.00
74.75
C


ATOM
797
C
SER
A
148
243.758
82.970
194.714
1.00
83.42
C


ATOM
798
O
SER
A
148
244.679
83.053
195.529
1.00
88.34
O


ATOM
799
CB
SER
A
148
242.902
81.301
193.051
1.00
76.50
C


ATOM
800
OG
SER
A
148
242.158
80.106
192.902
1.00
74.94
O


ATOM
801
N
ARG
A
149
243.347
83.999
193.978
1.00
81.76
N


ATOM
802
CA
ARG
A
149
243.974
85.316
194.077
1.00
102.63
C


ATOM
803
C
ARG
A
149
244.325
85.650
195.512
1.00
107.57
C


ATOM
804
O
ARG
A
149
245.399
86.169
195.796
1.00
115.11
O


ATOM
805
CB
ARG
A
149
243.038
86.396
193.537
1.00
92.17
C


ATOM
806
CG
ARG
A
149
242.950
86.427
192.033
1.00
96.27
C


ATOM
807
CD
ARG
A
149
244.278
86.851
191.433
1.00
127.89
C


ATOM
808
NE
ARG
A
149
244.141
88.079
190.656
1.00
105.03
N


ATOM
809
CZ
ARG
A
149
243.698
89.227
191.155
1.00
106.80
C


ATOM
810
NH1
ARG
A
149
243.350
89.307
192.434
1.00
91.41
N


ATOM
811
NH2
ARG
A
149
243.597
90.293
190.377
1.00
81.30
N


ATOM
812
N
ILE
A
150
243.410
85.352
196.423
1.00
117.64
N


ATOM
813
CA
ILE
A
150
243.658
85.636
197.821
1.00
111.89
C


ATOM
814
C
ILE
A
150
244.890
84.907
198.320
1.00
111.27
C


ATOM
815
O
ILE
A
150
245.841
85.543
198.769
1.00
125.08
O


ATOM
816
CB
ILE
A
150
242.447
85.262
198.691
1.00
102.67
C


ATOM
817
CG1
ILE
A
150
241.501
86.458
198.783
1.00
81.93
C


ATOM
818
CG2
ILE
A
150
242.900
84.866
200.081
1.00
93.62
C


ATOM
819
CD1
ILE
A
150
241.279
87.159
197.457
1.00
131.92
C


ATOM
820
N
GLN
A
151
244.898
83.583
198.221
1.00
93.05
N


ATOM
821
CA
GLN
A
151
246.045
82.836
198.708
1.00
112.82
C


ATOM
822
C
GLN
A
151
247.325
83.240
197.992
1.00
130.96
C


ATOM
823
O
GLN
A
151
248.373
83.378
198.624
1.00
146.77
O


ATOM
824
CB
GLN
A
151
245.825
81.329
198.565
1.00
96.24
C


ATOM
825
CG
GLN
A
151
246.123
80.751
197.203
1.00
107.91
C


ATOM
826
CD
GLN
A
151
246.134
79.231
197.224
1.00
135.63
C


ATOM
827
OE1
GLN
A
151
246.326
78.584
196.193
1.00
152.64
O


ATOM
828
NE2
GLN
A
151
245.929
78.654
198.406
1.00
63.64
N


ATOM
829
N
ASP
A
152
247.241
83.452
196.682
1.00
131.86
N


ATOM
830
CA
ASP
A
152
248.416
83.833
195.898
1.00
121.88
C


ATOM
831
C
ASP
A
152
248.697
85.331
195.974
1.00
119.03
C


ATOM
832
O
ASP
A
152
249.617
85.765
196.678
1.00
108.04
O


ATOM
833
CB
ASP
A
152
248.226
83.420
194.436
1.00
120.82
C


ATOM
834
CG
ASP
A
152
247.643
82.015
194.296
1.00
145.66
C


ATOM
835
OD1
ASP
A
152
248.152
81.088
194.964
1.00
161.64
O


ATOM
836
OD2
ASP
A
152
246.679
81.834
193.515
1.00
172.99
O


TER
836

ASP
A
152


ATOM
837
N
THR
A
159
244.002
91.956
205.011
1.00
65.30
N


ATOM
838
CA
THR
A
159
243.414
92.956
205.898
1.00
99.51
C


ATOM
839
C
THR
A
159
241.893
92.871
205.964
1.00
87.22
C


ATOM
840
O
THR
A
159
241.221
92.755
204.944
1.00
90.56
O


ATOM
841
CB
THR
A
159
243.790
94.404
205.473
1.00
95.19
C


ATOM
842
OG1
THR
A
159
243.610
94.560
204.061
1.00
103.02
O


ATOM
843
CG2
THR
A
159
245.229
94.718
205.831
1.00
121.85
C


ATOM
844
N
GLU
A
160
241.360
92.931
207.178
1.00
90.46
N


ATOM
845
CA
GLU
A
160
239.921
92.876
207.395
1.00
95.08
C


ATOM
846
C
GLU
A
160
239.243
93.816
206.400
1.00
97.10
C


ATOM
847
O
GLU
A
160
238.191
93.498
205.844
1.00
77.77
O


ATOM
848
CB
GLU
A
160
239.606
93.302
208.836
1.00
84.05
C


ATOM
849
CG
GLU
A
160
238.135
93.255
209.218
1.00
102.30
C


ATOM
850
CD
GLU
A
160
237.888
93.636
210.675
1.00
107.68
C


ATOM
851
OE1
GLU
A
160
238.512
93.022
211.566
1.00
102.89
O


ATOM
852
OE2
GLU
A
160
237.063
94.542
210.931
1.00
116.03
O


ATOM
853
N
ARG
A
161
239.872
94.966
206.169
1.00
91.94
N


ATOM
854
CA
ARG
A
161
239.359
95.979
205.252
1.00
98.89
C


ATOM
855
C
ARG
A
161
239.188
95.480
203.826
1.00
94.53
C


ATOM
856
O
ARG
A
161
238.251
95.874
203.129
1.00
84.43
O


ATOM
857
CB
ARG
A
161
240.276
97.204
205.245
1.00
94.61
C


ATOM
858
CG
ARG
A
161
239.886
98.251
204.206
1.00
116.99
C


ATOM
859
CD
ARG
A
161
240.724
99.506
204.347
1.00
131.31
C


ATOM
860
NE
ARG
A
161
240.601
100.069
205.688
1.00
142.10
N


ATOM
861
CZ
ARG
A
161
241.205
101.180
206.092
1.00
132.03
C


ATOM
862
NH1
ARG
A
161
241.982
101.855
205.257
1.00
142.54
N


ATOM
863
NH2
ARG
A
161
241.026
101.620
207.330
1.00
138.86
N


ATOM
864
N
GLU
A
162
240.107
94.628
203.389
1.00
86.92
N


ATOM
865
CA
GLU
A
162
240.040
94.067
202.050
1.00
96.77
C


ATOM
866
C
GLU
A
162
238.899
93.063
201.991
1.00
93.54
C


ATOM
867
O
GLU
A
162
238.101
93.078
201.052
1.00
98.52
O


ATOM
868
CB
GLU
A
162
241.360
93.385
201.699
1.00
101.33
C


ATOM
869
CG
GLU
A
162
242.463
94.360
201.317
1.00
125.60
C


ATOM
870
CD
GLU
A
162
243.848
93.755
201.448
1.00
135.51
C


ATOM
871
OE1
GLU
A
162
244.823
94.405
201.011
1.00
144.57
O


ATOM
872
OE2
GLU
A
162
243.959
92.636
201.999
1.00
130.61
O


ATOM
873
N
ALA
A
163
238.822
92.199
203.002
1.00
93.78
N


ATOM
874
CA
ALA
A
163
237.772
91.187
203.071
1.00
86.54
C


ATOM
875
C
ALA
A
163
236.426
91.889
202.976
1.00
87.93
C


ATOM
876
O
ALA
A
163
235.509
91.418
202.297
1.00
94.67
O


ATOM
877
CB
ALA
A
163
237.871
90.419
204.371
1.00
72.07
C


ATOM
878
N
SER
A
164
236.319
93.021
203.662
1.00
75.33
N


ATOM
879
CA
SER
A
164
235.098
93.809
203.651
1.00
81.45
C


ATOM
880
C
SER
A
164
234.765
94.222
202.223
1.00
92.32
C


ATOM
881
O
SER
A
164
233.708
93.892
201.679
1.00
89.51
O


ATOM
882
CB
SER
A
164
235.281
95.057
204.511
1.00
61.52
C


ATOM
883
OG
SER
A
164
234.233
95.977
204.289
1.00
85.01
O


ATOM
884
N
GLU
A
165
235.696
94.949
201.622
1.00
106.88
N


ATOM
885
CA
GLU
A
165
235.537
95.436
200.259
1.00
107.60
C


ATOM
886
C
GLU
A
165
235.138
94.312
199.306
1.00
98.22
C


ATOM
887
O
GLU
A
165
234.309
94.509
198.421
1.00
80.74
O


ATOM
888
CB
GLU
A
165
236.834
96.102
199.807
1.00
117.73
C


ATOM
889
CG
GLU
A
165
237.296
97.198
200.763
1.00
121.43
C


ATOM
890
CD
GLU
A
165
238.640
97.776
200.381
1.00
129.44
C


ATOM
891
OE1
GLU
A
165
239.620
96.998
200.285
1.00
132.54
O


ATOM
892
OE2
GLU
A
165
238.711
99.007
200.172
1.00
114.59
O


ATOM
893
N
ILE
A
166
235.722
93.134
199.483
1.00
87.57
N


ATOM
894
CA
ILE
A
166
235.354
92.020
198.628
1.00
78.81
C


ATOM
895
C
ILE
A
166
233.887
91.705
198.905
1.00
85.13
C


ATOM
896
O
ILE
A
166
233.068
91.644
197.975
1.00
86.94
O


ATOM
897
CB
ILE
A
166
236.207
90.755
198.905
1.00
76.17
C


ATOM
898
CG1
ILE
A
166
237.628
90.972
198.376
1.00
76.65
C


ATOM
899
CG2
ILE
A
166
235.567
89.534
198.255
1.00
77.07
C


ATOM
900
CD1
ILE
A
166
238.532
89.759
198.515
1.00
56.87
C


ATOM
901
N
MET
A
167
233.546
91.520
200.179
1.00
80.82
N


ATOM
902
CA
MET
A
167
232.166
91.195
200.521
1.00
77.70
C


ATOM
903
C
MET
A
167
231.228
92.224
199.941
1.00
73.89
C


ATOM
904
O
MET
A
167
230.152
91.895
199.452
1.00
72.21
O


ATOM
905
CB
MET
A
167
231.978
91.110
202.035
1.00
67.42
C


ATOM
906
CG
MET
A
167
232.544
89.845
202.620
1.00
65.04
C


ATOM
907
SD
MET
A
167
232.087
88.398
201.648
1.00
75.11
S


ATOM
908
CE
MET
A
167
230.294
88.399
201.807
1.00
53.41
C


ATOM
909
N
LYS
A
168
231.659
93.472
199.962
1.00
54.97
N


ATOM
910
CA
LYS
A
168
230.830
94.534
199.449
1.00
60.76
C


ATOM
911
C
LYS
A
168
230.456
94.386
197.990
1.00
70.78
C


ATOM
912
O
LYS
A
168
229.307
94.607
197.629
1.00
66.11
O


ATOM
913
CB
LYS
A
168
231.502
95.876
199.659
1.00
65.32
C


ATOM
914
CG
LYS
A
168
230.616
97.027
199.293
1.00
49.49
C


ATOM
915
CD
LYS
A
168
231.208
98.313
199.780
1.00
103.77
C


ATOM
916
CE
LYS
A
168
230.191
99.429
199.714
1.00
87.12
C


ATOM
917
NZ
LYS
A
168
230.644
100.674
200.407
1.00
118.45
N


ATOM
918
N
SER
A
169
231.403
94.023
197.136
1.00
82.60
N


ATOM
919
CA
SER
A
169
231.061
93.885
195.723
1.00
89.11
C


ATOM
920
C
SER
A
169
230.176
92.665
195.484
1.00
81.07
C


ATOM
921
O
SER
A
169
229.262
92.726
194.657
1.00
75.01
O


ATOM
922
CB
SER
A
169
232.327
93.796
194.863
1.00
73.74
C


ATOM
923
OG
SER
A
169
233.058
92.620
195.165
1.00
77.68
O


ATOM
924
N
ILE
A
170
230.448
91.560
196.191
1.00
75.29
N


ATOM
925
CA
ILE
A
170
229.630
90.349
196.034
1.00
74.32
C


ATOM
926
C
ILE
A
170
228.232
90.743
196.437
1.00
63.33
C


ATOM
927
O
ILE
A
170
227.255
90.334
195.818
1.00
57.44
O


ATOM
928
CB
ILE
A
170
230.053
89.177
196.951
1.00
68.61
C


ATOM
929
CG1
ILE
A
170
231.437
88.663
196.571
1.00
59.95
C


ATOM
930
CG2
ILE
A
170
229.091
88.015
196.782
1.00
46.17
C


ATOM
931
CD1
ILE
A
170
231.768
87.318
197.192
1.00
76.19
C


ATOM
932
N
GLY
A
171
228.154
91.560
197.484
1.00
56.09
N


ATOM
933
CA
GLY
A
171
226.870
92.026
197.958
1.00
75.47
C


ATOM
934
C
GLY
A
171
226.110
92.700
196.835
1.00
69.45
C


ATOM
935
O
GLY
A
171
224.947
92.390
196.568
1.00
76.12
O


ATOM
936
N
GLU
A
172
226.792
93.621
196.166
1.00
71.58
N


ATOM
937
CA
GLU
A
172
226.212
94.368
195.066
1.00
73.09
C


ATOM
938
C
GLU
A
172
225.715
93.446
193.960
1.00
69.34
C


ATOM
939
O
GLU
A
172
224.645
93.653
193.396
1.00
74.21
O


ATOM
940
CB
GLU
A
172
227.243
95.332
194.499
1.00
62.80
C


ATOM
941
CG
GLU
A
172
228.028
96.080
195.547
1.00
91.94
C


ATOM
942
CD
GLU
A
172
228.717
97.298
194.973
1.00
112.10
C


ATOM
943
OE1
GLU
A
172
228.051
98.349
194.825
1.00
116.39
O


ATOM
944
OE2
GLU
A
172
229.919
97.199
194.649
1.00
121.47
O


ATOM
945
N
ALA
A
173
226.487
92.427
193.635
1.00
50.91
N


ATOM
946
CA
ALA
A
173
226.039
91.525
192.599
1.00
55.42
C


ATOM
947
C
ALA
A
173
224.666
90.969
192.995
1.00
69.00
C


ATOM
948
O
ALA
A
173
223.779
90.793
192.156
1.00
68.62
O


ATOM
949
CB
ALA
A
173
227.036
90.392
192.429
1.00
59.45
C


ATOM
950
N
ILE
A
174
224.491
90.709
194.287
1.00
59.68
N


ATOM
951
CA
ILE
A
174
223.240
90.163
194.793
1.00
55.23
C


ATOM
952
C
ILE
A
174
222.137
91.199
194.912
1.00
66.27
C


ATOM
953
O
ILE
A
174
220.991
90.943
194.542
1.00
59.24
O


ATOM
954
CB
ILE
A
174
223.439
89.528
196.157
1.00
72.24
C


ATOM
955
CG1
ILE
A
174
224.492
88.438
196.055
1.00
44.08
C


ATOM
956
CG2
ILE
A
174
222.132
88.924
196.636
1.00
52.34
C


ATOM
957
CD1
ILE
A
174
224.090
87.355
195.102
1.00
105.89
C


ATOM
958
N
GLN
A
175
222.473
92.368
195.444
1.00
46.22
N


ATOM
959
CA
GLN
A
175
221.477
93.407
195.586
1.00
60.91
C


ATOM
960
C
GLN
A
175
220.804
93.676
194.240
1.00
48.25
C


ATOM
961
O
GLN
A
175
219.581
93.742
194.155
1.00
62.61
O


ATOM
962
CB
GLN
A
175
222.121
94.674
196.144
1.00
49.40
C


ATOM
963
CG
GLN
A
175
221.309
95.931
195.912
1.00
71.12
C


ATOM
964
CD
GLN
A
175
221.661
97.042
196.892
1.00
104.15
C


ATOM
965
OE1
GLN
A
175
222.825
97.446
197.017
1.00
76.77
O


ATOM
966
NE2
GLN
A
175
220.645
97.544
197.600
1.00
111.29
N


ATOM
967
N
TYR
A
176
221.602
93.798
193.184
1.00
72.76
N


ATOM
968
CA
TYR
A
176
221.025
94.078
191.873
1.00
74.81
C


ATOM
969
C
TYR
A
176
220.105
92.946
191.416
1.00
74.26
C


ATOM
970
O
TYR
A
176
218.933
93.142
191.122
1.00
61.26
O


ATOM
971
CB
TYR
A
176
222.168
94.255
190.874
1.00
79.19
C


ATOM
972
CG
TYR
A
176
221.628
94.703
189.563
1.00
53.89
C


ATOM
973
CD1
TYR
A
176
221.692
96.048
189.209
1.00
73.14
C


ATOM
974
CD2
TYR
A
176
221.054
93.785
188.683
1.00
78.08
C


ATOM
975
CE1
TYR
A
176
221.197
96.471
187.985
1.00
58.55
C


ATOM
976
CE2
TYR
A
176
220.550
94.209
187.463
1.00
60.19
C


ATOM
977
CZ
TYR
A
176
220.616
95.546
187.115
1.00
70.05
C


ATOM
978
OH
TYR
A
176
220.124
95.975
185.898
1.00
98.35
O


ATOM
979
N
LEU
A
177
220.626
91.729
191.357
1.00
69.60
N


ATOM
980
CA
LEU
A
177
219.824
90.582
190.986
1.00
65.69
C


ATOM
981
C
LEU
A
177
218.485
90.616
191.705
1.00
62.46
C


ATOM
982
O
LEU
A
177
217.437
90.563
191.057
1.00
73.48
O


ATOM
983
CB
LEU
A
177
220.562
89.297
191.333
1.00
59.94
C


ATOM
984
CG
LEU
A
177
221.671
88.955
190.357
1.00
54.02
C


ATOM
985
CD1
LEU
A
177
222.252
87.585
190.679
1.00
59.89
C


ATOM
986
CD2
LEU
A
177
221.078
88.962
188.964
1.00
77.33
C


ATOM
987
N
HIS
A
178
218.518
90.708
193.035
1.00
61.49
N


ATOM
988
CA
HIS
A
178
217.285
90.746
193.816
1.00
57.69
C


ATOM
989
C
HIS
A
178
216.419
91.952
193.466
1.00
56.16
C


ATOM
990
O
HIS
A
178
215.204
91.831
193.368
1.00
71.55
O


ATOM
991
CB
HIS
A
178
217.590
90.741
195.313
1.00
58.67
C


ATOM
992
CG
HIS
A
178
218.154
89.446
195.805
1.00
66.35
C


ATOM
993
ND1
HIS
A
178
218.554
89.259
197.110
1.00
49.65
N


ATOM
994
CD2
HIS
A
178
218.437
88.292
195.154
1.00
50.67
C


ATOM
995
CE1
HIS
A
178
219.068
88.049
197.241
1.00
77.64
C


ATOM
996
NE2
HIS
A
178
219.010
87.442
196.069
1.00
44.53
N


ATOM
997
N
SER
A
179
217.037
93.107
193.259
1.00
62.56
N


ATOM
998
CA
SER
A
179
216.270
94.292
192.906
1.00
56.93
C


ATOM
999
C
SER
A
179
215.575
94.094
191.567
1.00
44.23
C


ATOM
1000
O
SER
A
179
214.830
94.956
191.136
1.00
68.45
O


ATOM
1001
CB
SER
A
179
217.160
95.518
192.803
1.00
41.33
C


ATOM
1002
OG
SER
A
179
217.835
95.504
191.554
1.00
86.61
O


ATOM
1003
N
ILE
A
180
215.842
92.997
190.876
1.00
60.70
N


ATOM
1004
CA
ILE
A
180
215.135
92.778
189.629
1.00
66.56
C


ATOM
1005
C
ILE
A
180
214.483
91.409
189.667
1.00
76.38
C


ATOM
1006
O
ILE
A
180
214.223
90.783
188.641
1.00
67.28
O


ATOM
1007
CB
ILE
A
180
216.041
92.928
188.368
1.00
65.72
C


ATOM
1008
CG1
ILE
A
180
217.061
91.804
188.280
1.00
98.00
C


ATOM
1009
CG2
ILE
A
180
216.766
94.248
188.413
1.00
65.05
C


ATOM
1010
CD1
ILE
A
180
217.787
91.783
186.962
1.00
42.99
C


ATOM
1011
N
ASN
A
181
214.229
90.940
190.880
1.00
62.76
N


ATOM
1012
CA
ASN
A
181
213.556
89.672
191.081
1.00
64.37
C


ATOM
1013
C
ASN
A
181
214.257
88.447
190.552
1.00
64.72
C


ATOM
1014
O
ASN
A
181
213.624
87.605
189.919
1.00
76.26
O


ATOM
1015
CB
ASN
A
181
212.156
89.726
190.464
1.00
52.97
C


ATOM
1016
CG
ASN
A
181
211.274
90.765
191.118
1.00
91.87
C


ATOM
1017
OD1
ASN
A
181
210.607
91.549
190.435
1.00
70.51
O


ATOM
1018
ND2
ASN
A
181
211.261
90.779
192.456
1.00
66.84
N


ATOM
1019
N
ILE
A
182
215.550
88.325
190.800
1.00
69.03
N


ATOM
1020
CA
ILE
A
182
216.251
87.140
190.343
1.00
58.61
C


ATOM
1021
C
ILE
A
182
217.060
86.582
191.510
1.00
64.49
C


ATOM
1022
O
ILE
A
182
217.652
87.335
192.289
1.00
75.24
O


ATOM
1023
CB
ILE
A
182
217.217
87.452
189.175
1.00
67.77
C


ATOM
1024
CG1
ILE
A
182
216.498
88.204
188.058
1.00
51.81
C


ATOM
1025
CG2
ILE
A
182
217.765
86.162
188.604
1.00
71.00
C


ATOM
1026
CD1
ILE
A
182
217.397
88.520
186.870
1.00
51.04
C


ATOM
1027
N
ALA
A
183
217.068
85.261
191.639
1.00
74.62
N


ATOM
1028
CA
ALA
A
183
217.907
84.570
192.612
1.00
86.67
C


ATOM
1029
C
ALA
A
183
218.970
83.710
191.924
1.00
88.25
C


ATOM
1030
O
ALA
A
183
218.683
82.841
191.110
1.00
87.52
O


ATOM
1031
CB
ALA
A
183
217.006
83.691
193.480
1.00
82.75
C


ATOM
1032
N
HIS
A
184
220.241
84.008
192.248
1.00
80.72
N


ATOM
1033
CA
HIS
A
184
221.331
83.245
191.654
1.00
76.94
C


ATOM
1034
C
HIS
A
184
221.282
81.775
192.077
1.00
80.75
C


ATOM
1035
O
HIS
A
184
221.411
80.858
191.277
1.00
72.26
O


ATOM
1036
CB
HIS
A
184
222.652
83.872
192.102
1.00
71.67
C


ATOM
1037
CG
HIS
A
184
223.777
83.319
191.264
1.00
61.47
C


ATOM
1038
ND1
HIS
A
184
224.599
84.095
190.515
1.00
83.49
N


ATOM
1039
CD2
HIS
A
184
224.158
81.983
191.101
1.00
84.93
C


ATOM
1040
CE1
HIS
A
184
225.456
83.245
189.918
1.00
77.60
C


ATOM
1041
NE2
HIS
A
184
225.215
81.974
190.250
1.00
94.64
N


ATOM
1042
N
ARG
A
185
221.128
81.570
193.401
1.00
76.53
N


ATOM
1043
CA
ARG
A
185
221.070
80.207
193.915
1.00
77.64
C


ATOM
1044
C
ARG
A
185
222.301
79.398
193.501
1.00
85.59
C


ATOM
1045
O
ARG
A
185
222.247
78.190
193.308
1.00
76.00
O


ATOM
1046
CB
ARG
A
185
219.805
79.546
193.368
1.00
60.05
C


ATOM
1047
CG
ARG
A
185
218.538
80.313
193.747
1.00
69.87
C


ATOM
1048
CD
ARG
A
185
217.293
79.421
193.714
1.00
88.79
C


ATOM
1049
NE
ARG
A
185
216.984
79.018
192.339
1.00
74.62
N


ATOM
1050
CZ
ARG
A
185
216.704
77.720
192.125
1.00
102.74
C


ATOM
1051
NH1
ARG
A
185
216.704
76.865
193.132
1.00
105.16
N


ATOM
1052
NH2
ARG
A
185
216.428
77.299
190.884
1.00
124.15
N


ATOM
1053
N
ASP
A
186
223.476
80.000
193.397
1.00
85.17
N


ATOM
1054
CA
ASP
A
186
224.677
79.238
193.128
1.00
65.94
C


ATOM
1055
C
ASP
A
186
225.857
80.161
193.327
1.00
63.69
C


ATOM
1056
O
ASP
A
186
226.821
80.122
192.573
1.00
76.80
O


ATOM
1057
CB
ASP
A
186
224.651
78.670
191.712
1.00
64.22
C


ATOM
1058
CG
ASP
A
186
225.560
77.461
191.555
1.00
69.83
C


ATOM
1059
OD1
ASP
A
186
225.629
76.654
192.503
1.00
114.36
O


ATOM
1060
OD2
ASP
A
186
226.197
77.304
190.492
1.00
103.36
O


ATOM
1061
N
VAL
A
187
225.757
80.991
194.364
1.00
65.08
N


ATOM
1062
CA
VAL
A
187
226.794
81.952
194.717
1.00
62.87
C


ATOM
1063
C
VAL
A
187
227.923
81.305
195.507
1.00
61.46
C


ATOM
1064
O
VAL
A
187
228.134
81.605
196.677
1.00
76.23
O


ATOM
1065
CB
VAL
A
187
226.218
83.132
195.539
1.00
56.36
C


ATOM
1066
CG1
VAL
A
187
227.311
84.156
195.831
1.00
45.04
C


ATOM
1067
CG2
VAL
A
187
225.099
83.791
194.774
1.00
49.15
C


ATOM
1068
N
LYS
A
188
228.644
80.407
194.852
1.00
71.26
N


ATOM
1069
CA
LYS
A
188
229.766
79.731
195.482
1.00
71.74
C


ATOM
1070
C
LYS
A
188
231.033
80.392
194.985
1.00
71.11
C


ATOM
1071
O
LYS
A
188
231.049
80.975
193.905
1.00
76.85
O


ATOM
1072
CB
LYS
A
188
229.770
78.246
195.123
1.00
59.58
C


ATOM
1073
CG
LYS
A
188
229.709
77.962
193.639
1.00
61.04
C


ATOM
1074
CD
LYS
A
188
228.939
76.679
193.367
1.00
96.00
C


ATOM
1075
CE
LYS
A
188
228.669
76.509
191.869
1.00
141.45
C


ATOM
1076
NZ
LYS
A
188
227.746
75.371
191.579
1.00
122.86
N


ATOM
1077
N
PRO
A
189
232.115
80.318
195.776
1.00
66.86
N


ATOM
1078
CA
PRO
A
189
233.409
80.911
195.433
1.00
82.94
C


ATOM
1079
C
PRO
A
189
233.746
80.698
193.967
1.00
81.19
C


ATOM
1080
O
PRO
A
189
233.989
81.641
193.231
1.00
92.51
O


ATOM
1081
CB
PRO
A
189
234.359
80.199
196.381
1.00
77.38
C


ATOM
1082
CG
PRO
A
189
233.511
80.063
197.620
1.00
79.38
C


ATOM
1083
CD
PRO
A
189
232.197
79.582
197.049
1.00
84.38
C


ATOM
1084
N
GLU
A
190
233.743
79.447
193.553
1.00
79.12
N


ATOM
1085
CA
GLU
A
190
234.019
79.080
192.178
1.00
82.41
C


ATOM
1086
C
GLU
A
190
233.441
80.092
191.159
1.00
81.32
C


ATOM
1087
O
GLU
A
190
234.121
80.487
190.213
1.00
102.09
O


ATOM
1088
CB
GLU
A
190
233.432
77.689
191.920
1.00
94.71
C


ATOM
1089
CG
GLU
A
190
233.784
76.609
192.978
1.00
121.01
C


ATOM
1090
CD
GLU
A
190
232.689
76.364
194.043
1.00
135.59
C


ATOM
1091
OE1
GLU
A
190
231.485
76.334
193.693
1.00
113.86
O


ATOM
1092
OE2
GLU
A
190
233.039
76.185
195.236
1.00
133.59
O


ATOM
1093
N
ASN
A
191
232.196
80.527
191.350
1.00
80.04
N


ATOM
1094
CA
ASN
A
191
231.584
81.457
190.405
1.00
59.50
C


ATOM
1095
C
ASN
A
191
231.991
82.905
190.569
1.00
72.63
C


ATOM
1096
O
ASN
A
191
231.360
83.796
189.998
1.00
60.72
O


ATOM
1097
CB
ASN
A
191
230.068
81.364
190.471
1.00
59.13
C


ATOM
1098
CG
ASN
A
191
229.566
79.975
190.189
1.00
57.02
C


ATOM
1099
OD1
ASN
A
191
228.797
79.413
190.959
1.00
107.48
O


ATOM
1100
ND2
ASN
A
191
230.005
79.406
189.077
1.00
62.85
N


ATOM
1101
N
LEU
A
192
233.042
83.149
191.342
1.00
64.74
N


ATOM
1102
CA
LEU
A
192
233.524
84.514
191.552
1.00
58.75
C


ATOM
1103
C
LEU
A
192
234.931
84.655
190.987
1.00
67.25
C


ATOM
1104
O
LEU
A
192
235.906
84.314
191.644
1.00
63.30
O


ATOM
1105
CB
LEU
A
192
233.512
84.856
193.047
1.00
64.92
C


ATOM
1106
CG
LEU
A
192
232.127
84.840
193.709
1.00
76.84
C


ATOM
1107
CD1
LEU
A
192
232.285
84.706
195.207
1.00
63.79
C


ATOM
1108
CD2
LEU
A
192
231.350
86.108
193.349
1.00
49.09
C


ATOM
1109
N
LEU
A
193
235.022
85.173
189.767
1.00
76.60
N


ATOM
1110
CA
LEU
A
193
236.299
85.334
189.077
1.00
68.12
C


ATOM
1111
C
LEU
A
193
236.765
86.779
189.008
1.00
67.18
C


ATOM
1112
O
LEU
A
193
235.957
87.700
189.001
1.00
70.12
O


ATOM
1113
CB
LEU
A
193
236.165
84.801
187.657
1.00
57.38
C


ATOM
1114
CG
LEU
A
193
235.346
83.517
187.573
1.00
67.03
C


ATOM
1115
CD1
LEU
A
193
235.242
83.061
186.137
1.00
74.99
C


ATOM
1116
CD2
LEU
A
193
235.985
82.449
188.445
1.00
80.12
C


ATOM
1117
N
TYR
A
194
238.073
86.982
188.950
1.00
66.38
N


ATOM
1118
CA
TYR
A
194
238.603
88.333
188.846
1.00
70.20
C


ATOM
1119
C
TYR
A
194
238.833
88.697
187.382
1.00
65.05
C


ATOM
1120
O
TYR
A
194
239.219
87.856
186.578
1.00
55.59
O


ATOM
1121
CB
TYR
A
194
239.903
88.449
189.636
1.00
71.53
C


ATOM
1122
CG
TYR
A
194
239.692
88.807
191.085
1.00
83.55
C


ATOM
1123
CD1
TYR
A
194
239.221
90.068
191.446
1.00
60.55
C


ATOM
1124
CD2
TYR
A
194
239.959
87.889
192.095
1.00
54.69
C


ATOM
1125
CE1
TYR
A
194
239.026
90.402
192.766
1.00
77.54
C


ATOM
1126
CE2
TYR
A
194
239.764
88.216
193.423
1.00
72.23
C


ATOM
1127
CZ
TYR
A
194
239.300
89.472
193.749
1.00
80.69
C


ATOM
1128
OH
TYR
A
194
239.118
89.812
195.068
1.00
97.12
O


ATOM
1129
N
THR
A
195
238.585
89.952
187.035
1.00
56.71
N


ATOM
1130
CA
THR
A
195
238.754
90.389
185.660
1.00
63.48
C


ATOM
1131
C
THR
A
195
240.193
90.206
185.176
1.00
82.40
C


ATOM
1132
O
THR
A
195
240.436
89.542
184.164
1.00
96.10
O


ATOM
1133
CB
THR
A
195
238.334
91.865
185.494
1.00
66.82
C


ATOM
1134
OG1
THR
A
195
239.028
92.668
186.452
1.00
70.80
O


ATOM
1135
CG2
THR
A
195
236.829
92.022
185.688
1.00
54.17
C


ATOM
1136
N
SER
A
196
241.145
90.790
185.897
1.00
84.27
N


ATOM
1137
CA
SER
A
196
242.552
90.672
185.532
1.00
77.62
C


ATOM
1138
C
SER
A
196
243.388
90.202
186.716
1.00
88.08
C


ATOM
1139
O
SER
A
196
242.865
89.980
187.805
1.00
101.07
O


ATOM
1140
CB
SER
A
196
243.089
92.019
185.033
1.00
78.67
C


ATOM
1141
OG
SER
A
196
243.063
92.995
186.055
1.00
92.46
O


ATOM
1142
N
LYS
A
197
244.688
90.038
186.490
1.00
106.23
N


ATOM
1143
CA
LYS
A
197
245.601
89.609
187.542
1.00
100.72
C


ATOM
1144
C
LYS
A
197
246.055
90.885
188.230
1.00
93.19
C


ATOM
1145
O
LYS
A
197
246.509
90.870
189.373
1.00
91.43
O


ATOM
1146
CB
LYS
A
197
246.792
88.882
186.936
1.00
90.18
C


ATOM
1147
N
ARG
A
198
245.911
91.985
187.500
1.00
76.02
N


ATOM
1148
CA
ARG
A
198
246.275
93.318
187.959
1.00
111.08
C


ATOM
1149
C
ARG
A
198
245.815
93.469
189.412
1.00
105.33
C


ATOM
1150
O
ARG
A
198
244.993
92.696
189.885
1.00
104.59
O


ATOM
1151
CB
ARG
A
198
245.582
94.345
187.048
1.00
118.16
C


ATOM
1152
CG
ARG
A
198
246.321
95.666
186.798
1.00
144.07
C


ATOM
1153
CD
ARG
A
198
245.911
96.211
185.435
1.00
152.31
C


ATOM
1154
NE
ARG
A
198
246.262
97.611
185.210
1.00
171.50
N


ATOM
1155
CZ
ARG
A
198
245.920
98.290
184.115
1.00
162.32
C


ATOM
1156
NH1
ARG
A
198
245.223
97.692
183.152
1.00
96.38
N


ATOM
1157
NH2
ARG
A
198
246.265
99.565
183.984
1.00
146.82
N


ATOM
1158
N
PRO
A
199
246.347
94.461
190.144
1.00
115.43
N


ATOM
1159
CA
PRO
A
199
245.943
94.653
191.545
1.00
123.33
C


ATOM
1160
C
PRO
A
199
244.581
95.315
191.719
1.00
123.60
C


ATOM
1161
O
PRO
A
199
243.991
95.259
192.794
1.00
135.30
O


ATOM
1162
CB
PRO
A
199
247.061
95.524
192.105
1.00
121.70
C


ATOM
1163
CG
PRO
A
199
247.387
96.397
190.921
1.00
133.24
C


ATOM
1164
CD
PRO
A
199
247.411
95.409
189.775
1.00
125.16
C


ATOM
1165
N
ASN
A
200
244.090
95.943
190.656
1.00
122.57
N


ATOM
1166
CA
ASN
A
200
242.810
96.629
190.719
1.00
108.91
C


ATOM
1167
C
ASN
A
200
241.726
95.826
190.023
1.00
95.83
C


ATOM
1168
O
ASN
A
200
240.606
96.310
189.835
1.00
73.96
O


ATOM
1169
CB
ASN
A
200
242.923
98.019
190.090
1.00
118.71
C


ATOM
1170
N
ALA
A
201
242.059
94.598
189.637
1.00
75.18
N


ATOM
1171
CA
ALA
A
201
241.093
93.725
188.979
1.00
84.90
C


ATOM
1172
C
ALA
A
201
239.789
93.860
189.751
1.00
91.91
C


ATOM
1173
O
ALA
A
201
239.800
94.214
190.929
1.00
89.51
O


ATOM
1174
CB
ALA
A
201
241.579
92.278
189.022
1.00
81.83
C


ATOM
1175
N
ILE
A
202
238.660
93.607
189.097
1.00
78.80
N


ATOM
1176
CA
ILE
A
202
237.378
93.698
189.795
1.00
91.70
C


ATOM
1177
C
ILE
A
202
236.697
92.326
189.886
1.00
95.28
C


ATOM
1178
O
ILE
A
202
236.837
91.488
188.995
1.00
88.21
O


ATOM
1179
CB
ILE
A
202
236.454
94.707
189.121
1.00
76.30
C


ATOM
1180
CG1
ILE
A
202
235.623
94.029
188.059
1.00
78.45
C


ATOM
1181
CG2
ILE
A
202
237.278
95.772
188.468
1.00
61.72
C


ATOM
1182
CD1
ILE
A
202
234.681
94.991
187.390
1.00
169.28
C


ATOM
1183
N
LEU
A
203
235.975
92.102
190.981
1.00
93.25
N


ATOM
1184
CA
LEU
A
203
235.308
90.821
191.231
1.00
66.28
C


ATOM
1185
C
LEU
A
203
233.935
90.746
190.587
1.00
67.61
C


ATOM
1186
O
LEU
A
203
233.137
91.681
190.695
1.00
68.70
O


ATOM
1187
CB
LEU
A
203
235.195
90.599
192.745
1.00
73.90
C


ATOM
1188
CG
LEU
A
203
234.947
89.189
193.273
1.00
52.16
C


ATOM
1189
CD1
LEU
A
203
236.016
88.244
192.783
1.00
61.94
C


ATOM
1190
CD2
LEU
A
203
234.956
89.228
194.779
1.00
85.31
C


ATOM
1191
N
LYS
A
204
233.665
89.628
189.919
1.00
58.34
N


ATOM
1192
CA
LYS
A
204
232.392
89.434
189.243
1.00
58.89
C


ATOM
1193
C
LYS
A
204
231.788
88.047
189.423
1.00
73.88
C


ATOM
1194
O
LYS
A
204
232.490
87.027
189.406
1.00
67.59
O


ATOM
1195
CB
LYS
A
204
232.540
89.740
187.750
1.00
68.02
C


ATOM
1196
CG
LYS
A
204
232.844
91.199
187.461
1.00
64.40
C


ATOM
1197
CD
LYS
A
204
232.603
91.552
186.008
1.00
62.63
C


ATOM
1198
CE
LYS
A
204
232.786
93.033
185.788
1.00
71.16
C


ATOM
1199
NZ
LYS
A
204
232.247
93.468
184.487
1.00
94.63
N


ATOM
1200
N
LEU
A
205
230.472
88.023
189.586
1.00
66.05
N


ATOM
1201
CA
LEU
A
205
229.750
86.781
189.782
1.00
63.28
C


ATOM
1202
C
LEU
A
205
229.296
86.241
188.452
1.00
53.73
C


ATOM
1203
O
LEU
A
205
228.884
87.001
187.583
1.00
55.72
O


ATOM
1204
CB
LEU
A
205
228.540
87.024
190.680
1.00
64.69
C


ATOM
1205
CG
LEU
A
205
227.514
85.905
190.813
1.00
47.19
C


ATOM
1206
CD1
LEU
A
205
228.150
84.657
191.394
1.00
81.96
C


ATOM
1207
CD2
LEU
A
205
226.397
86.387
191.695
1.00
64.88
C


ATOM
1208
N
THR
A
206
229.374
84.926
188.300
1.00
53.81
N


ATOM
1209
CA
THR
A
206
228.976
84.363
187.015
1.00
65.26
C


ATOM
1210
C
THR
A
206
228.239
83.033
187.185
1.00
53.82
C


ATOM
1211
O
THR
A
206
228.098
82.495
188.276
1.00
71.37
O


ATOM
1212
CB
THR
A
206
230.236
84.153
186.174
1.00
61.13
C


ATOM
1213
OG1
THR
A
206
231.023
83.115
186.762
1.00
57.86
O


ATOM
1214
CG2
THR
A
206
231.064
85.441
186.137
1.00
74.17
C


ATOM
1215
N
ASP
A
207
227.721
82.523
186.051
1.00
79.42
N


ATOM
1216
CA
ASP
A
207
227.033
81.239
186.082
1.00
59.92
C


ATOM
1217
C
ASP
A
207
225.577
81.381
186.532
1.00
58.53
C


ATOM
1218
O
ASP
A
207
225.272
81.747
187.660
1.00
80.39
O


ATOM
1219
CB
ASP
A
207
227.785
80.312
187.037
1.00
60.66
C


ATOM
1220
CG
ASP
A
207
227.400
78.869
186.743
1.00
80.61
C


ATOM
1221
OD1
ASP
A
207
226.540
78.671
185.884
1.00
83.40
O


ATOM
1222
OD2
ASP
A
207
227.955
77.969
187.369
1.00
77.29
O


ATOM
1223
N
PHE
A
208
224.661
81.117
185.582
1.00
57.58
N


ATOM
1224
CA
PHE
A
208
223.242
81.177
185.911
1.00
65.42
C


ATOM
1225
C
PHE
A
208
222.554
79.836
185.650
1.00
55.77
C


ATOM
1226
O
PHE
A
208
221.342
79.748
185.504
1.00
71.33
O


ATOM
1227
CB
PHE
A
208
222.597
82.267
185.055
1.00
41.42
C


ATOM
1228
CG
PHE
A
208
222.872
83.615
185.655
1.00
55.64
C


ATOM
1229
CD1
PHE
A
208
224.177
84.088
185.707
1.00
30.42
C


ATOM
1230
CD2
PHE
A
208
221.825
84.390
186.125
1.00
49.87
C


ATOM
1231
CE1
PHE
A
208
224.432
85.349
186.229
1.00
68.91
C


ATOM
1232
CE2
PHE
A
208
222.089
85.654
186.645
1.00
47.56
C


ATOM
1233
CZ
PHE
A
208
223.390
86.139
186.697
1.00
56.42
C


ATOM
1234
N
GLY
A
209
223.362
78.788
185.594
1.00
61.07
N


ATOM
1235
CA
GLY
A
209
222.825
77.461
185.343
1.00
72.68
C


ATOM
1236
C
GLY
A
209
221.723
77.080
186.309
1.00
73.62
C


ATOM
1237
O
GLY
A
209
220.958
76.161
186.039
1.00
77.98
O


ATOM
1238
N
PHE
A
210
221.656
77.777
187.441
1.00
83.69
N


ATOM
1239
CA
PHE
A
210
220.624
77.527
188.439
1.00
69.57
C


ATOM
1240
C
PHE
A
210
219.818
78.787
188.732
1.00
81.37
C


ATOM
1241
O
PHE
A
210
218.872
78.738
189.507
1.00
58.29
O


ATOM
1242
CB
PHE
A
210
221.225
77.037
189.753
1.00
67.31
C


ATOM
1243
CG
PHE
A
210
222.004
75.774
189.633
1.00
73.06
C


ATOM
1244
CD1
PHE
A
210
221.429
74.645
189.085
1.00
114.78
C


ATOM
1245
CD2
PHE
A
210
223.306
75.703
190.097
1.00
90.32
C


ATOM
1246
CE1
PHE
A
210
222.134
73.459
189.000
1.00
109.98
C


ATOM
1247
CE2
PHE
A
210
224.021
74.522
190.017
1.00
108.27
C


ATOM
1248
CZ
PHE
A
210
223.432
73.398
189.467
1.00
132.84
C


ATOM
1249
N
ALA
A
211
220.208
79.913
188.137
1.00
81.89
N


ATOM
1250
CA
ALA
A
211
219.490
81.170
188.341
1.00
69.02
C


ATOM
1251
C
ALA
A
211
218.028
80.898
188.075
1.00
58.89
C


ATOM
1252
O
ALA
A
211
217.700
80.102
187.206
1.00
66.98
O


ATOM
1253
CB
ALA
A
211
219.992
82.228
187.381
1.00
73.43
C


ATOM
1254
N
LYS
A
212
217.147
81.552
188.817
1.00
66.85
N


ATOM
1255
CA
LYS
A
212
215.726
81.329
188.624
1.00
74.67
C


ATOM
1256
C
LYS
A
212
214.892
82.598
188.785
1.00
69.94
C


ATOM
1257
O
LYS
A
212
215.106
83.387
189.709
1.00
68.45
O


ATOM
1258
CB
LYS
A
212
215.242
80.249
189.600
1.00
70.15
C


ATOM
1259
CG
LYS
A
212
213.744
80.022
189.558
1.00
104.51
C


ATOM
1260
CD
LYS
A
212
213.371
78.669
188.952
1.00
157.53
C


ATOM
1261
CE
LYS
A
212
211.877
78.354
189.164
1.00
119.37
C


ATOM
1262
NZ
LYS
A
212
211.408
77.209
188.336
1.00
93.54
N


ATOM
1263
N
GLU
A
213
214.141
82.794
187.868
1.00
15.00


ATOM
1264
CA
GLU
A
213
213.459
84.043
187.550
1.00
15.00


ATOM
1265
CB
GLU
A
213
213.339
84.210
186.033
1.00
15.00


ATOM
1266
CG
GLU
A
213
212.159
83.475
185.419
1.00
15.00


ATOM
1267
CD
GLU
A
213
212.043
83.702
183.925
1.00
15.00


ATOM
1268
OE1
GLU
A
213
211.180
83.058
183.289
1.00
15.00


ATOM
1269
OE2
GLU
A
213
212.815
84.522
183.385
1.00
15.00


ATOM
1270
C
GLU
A
213
212.074
84.087
188.185
1.00
15.00


ATOM
1271
O
GLU
A
213
211.548
83.061
188.604
1.00
15.36


ATOM
1272
N
THR
A
214
211.491
85.288
188.256
1.00
15.00


ATOM
1273
CA
THR
A
214
210.119
85.515
188.693
1.00
15.00


ATOM
1274
CB
THR
A
214
209.149
84.520
188.029
1.00
15.00


ATOM
1275
OG1
THR
A
214
209.187
84.691
186.606
1.00
15.00


ATOM
1276
CG2
THR
A
214
207.729
84.751
188.525
1.00
15.00


ATOM
1277
C
THR
A
214
209.999
85.380
190.208
1.00
15.00


ATOM
1278
O
THR
A
214
208.821
85.220
190.684
1.00
20.56


ATOM
1279
N
THR
A
215
211.054
85.475
190.986
1.00
15.00


ATOM
1280
CA
THR
A
215
210.986
85.364
192.438
1.00
15.00


ATOM
1281
CB
THR
A
215
212.132
84.493
192.987
1.00
15.00


ATOM
1282
OG1
THR
A
215
212.031
83.170
192.444
1.00
15.00


ATOM
1283
CG2
THR
A
215
212.062
84.420
194.505
1.00
15.00


ATOM
1284
C
THR
A
215
211.057
86.739
193.095
1.00
15.00


ATOM
1285
O
THR
A
215
212.111
87.316
193.358
1.00
17.54


ATOM
1286
N
SER
A
216
209.845
87.327
193.310
1.00
15.00


ATOM
1287
CA
SER
A
216
209.645
88.633
193.871
1.00
15.00


ATOM
1288
CB
SER
A
216
208.582
89.393
193.087
1.00
15.00


ATOM
1289
OG
SER
A
216
207.344
88.725
193.050
1.00
15.00


ATOM
1290
C
SER
A
216
209.352
88.649
195.380
1.00
15.00


ATOM
1291
O
SER
A
216
209.553
89.753
195.944
1.00
24.37


ATOM
1292
N
HIS
A
217
208.923
87.546
195.889
1.00
15.00


ATOM
1293
CA
HIS
A
217
208.587
87.117
197.213
1.00
15.00


ATOM
1294
CB
HIS
A
217
208.248
88.231
198.187
1.00
15.00


ATOM
1295
CG
HIS
A
217
208.489
87.994
199.627
1.00
15.00


ATOM
1296
CD2
HIS
A
217
209.559
88.383
200.390
1.00
15.00


ATOM
1297
ND1
HIS
A
217
207.605
87.415
200.500
1.00
15.00


ATOM
1298
CE1
HIS
A
217
208.130
87.392
201.715
1.00
15.00


ATOM
1299
NE2
HIS
A
217
209.316
87.977
201.671
1.00
15.00


ATOM
1300
C
HIS
A
217
207.430
86.096
197.115
1.00
15.00


ATOM
1301
O
HIS
A
217
206.272
86.389
196.895
1.00
28.08


ATOM
1302
N
ASN
A
218
207.933
84.916
197.149
1.00
15.00


ATOM
1303
CA
ASN
A
218
208.098
83.782
196.497
1.00
15.00


ATOM
1304
CB
ASN
A
218
207.843
83.860
194.969
1.00
15.00


ATOM
1305
CG
ASN
A
218
206.412
83.605
194.590
1.00
15.00


ATOM
1306
OD1
ASN
A
218
205.835
82.541
194.841
1.00
15.00


ATOM
1307
ND2
ASN
A
218
205.794
84.575
193.913
1.00
15.00


ATOM
1308
C
ASN
A
218
209.199
82.856
196.804
1.00
15.00


ATOM
1309
O
ASN
A
218
210.327
83.158
197.160
1.00
16.81


ATOM
1310
N
SER
A
219
208.738
81.608
196.607
1.00
15.00


ATOM
1311
CA
SER
A
219
209.612
80.496
196.960
1.00
15.00


ATOM
1312
CB
SER
A
219
208.946
79.616
198.021
1.00
15.00


ATOM
1313
OG
SER
A
219
207.780
78.995
197.509
1.00
15.00


ATOM
1314
C
SER
A
219
209.953
79.658
195.732
1.00
15.00


ATOM
1315
O
SER
A
219
209.421
79.706
194.646
1.00
13.95


ATOM
1316
N
LEU
A
220
211.012
78.785
195.962
1.00
15.00


ATOM
1317
CA
LEU
A
220
211.564
77.825
195.013
1.00
15.00


ATOM
1318
CB
LEU
A
220
212.993
78.219
194.633
1.00
15.00


ATOM
1319
CG
LEU
A
220
213.161
79.569
193.932
1.00
15.00


ATOM
1320
CD1
LEU
A
220
214.636
79.928
193.843
1.00
15.00


ATOM
1321
CD2
LEU
A
220
212.537
79.516
192.547
1.00
15.00


ATOM
1322
C
LEU
A
220
211.553
76.415
195.594
1.00
15.00


ATOM
1323
O
LEU
A
220
211.679
76.279
196.816
1.00
14.38


ATOM
1324
N
THR
A
221
211.526
75.374
194.729
1.00
15.00


ATOM
1325
CA
THR
A
221
211.397
74.011
195.231
1.00
15.00


ATOM
1326
CB
THR
A
221
210.079
73.365
194.763
1.00
15.00


ATOM
1327
OG1
THR
A
221
208.970
74.121
195.264
1.00
15.00


ATOM
1328
CG2
THR
A
221
209.981
71.933
195.268
1.00
15.00


ATOM
1329
C
THR
A
221
212.562
73.144
194.764
1.00
15.00


ATOM
1330
O
THR
A
221
212.736
72.026
195.385
1.00
15.81


TER
1330

THR
A
221


ATOM
1331
N
TPO
A
222
213.399
73.479
193.800
1.00
15.00


ATOM
1332
CA
TPO
A
222
214.459
72.638
193.257
1.00
15.00


ATOM
1333
CB
TPO
A
222
215.056
73.250
191.976
1.00
15.00


ATOM
1334
OG1
TPO
A
222
214.002
73.557
191.055
1.00
15.00


ATOM
1335
CG2
TPO
A
222
216.025
72.275
191.324
1.00
15.00


ATOM
1336
P
TPO
A
222
213.808
74.828
190.542
1.00
15.80
P


ATOM
1337
O1P
TPO
A
222
214.914
75.324
189.669
1.00
15.57
O


ATOM
1338
O2P
TPO
A
222
213.482
75.613
191.784
1.00
15.91
O


ATOM
1339
O3P
TPO
A
222
212.591
74.609
189.572
1.00
15.73
O


ATOM
1340
C
TPO
A
222
215.578
72.444
194.276
1.00
15.00


ATOM
1341
O
TPO
A
222
216.123
73.375
194.857
1.00
12.56


ATOM
1342
N
PRO
A
223
215.917
71.098
194.555
1.00
15.00


ATOM
1343
CD
PRO
A
223
215.209
69.889
194.261
1.00
15.00


ATOM
1344
CA
PRO
A
223
217.121
70.849
195.355
1.00
15.00


ATOM
1345
CB
PRO
A
223
216.915
69.424
195.868
1.00
15.00


ATOM
1346
CG
PRO
A
223
216.042
68.783
194.845
1.00
15.00


ATOM
1347
C
PRO
A
223
218.396
70.958
194.525
1.00
15.00


ATOM
1348
O
PRO
A
223
218.639
70.105
193.676
1.00
12.12


ATOM
1349
N
CYS
A
224
219.010
72.137
194.641
1.00
15.00


ATOM
1350
CA
CYS
A
224
220.180
72.438
193.826
1.00
15.00


ATOM
1351
CB
CYS
A
224
219.754
73.079
192.503
1.00
15.00


ATOM
1352
SG
CYS
A
224
219.284
71.899
191.217
1.00
15.00


ATOM
1353
C
CYS
A
224
221.138
73.367
194.565
1.00
15.00


ATOM
1354
O
CYS
A
224
220.831
73.947
195.595
1.00
9.31


ATOM
1355
N
TYR
A
225
222.314
73.544
193.951
1.00
15.00


ATOM
1356
CA
TYR
A
225
223.376
74.375
194.506
1.00
15.00


ATOM
1357
CB
TYR
A
225
223.004
74.838
195.916
1.00
15.00


ATOM
1358
CG
TYR
A
225
222.663
73.709
196.863
1.00
15.00


ATOM
1359
CD1
TYR
A
225
223.543
72.652
197.060
1.00
15.00


ATOM
1360
CE1
TYR
A
225
223.236
71.620
197.924
1.00
15.00


ATOM
1361
CD2
TYR
A
225
221.461
73.699
197.556
1.00
15.00


ATOM
1362
CE2
TYR
A
225
221.146
72.667
198.422
1.00
15.00


ATOM
1363
CZ
TYR
A
225
222.036
71.631
198.602
1.00
15.00


ATOM
1364
OH
TYR
A
225
221.725
70.605
199.464
1.00
15.00


ATOM
1365
C
TYR
A
225
224.699
73.618
194.543
1.00
15.00


ATOM
1366
O
TYR
A
225
224.872
72.695
193.743
1.00
11.69


ATOM
1367
N
THR
A
226
225.564
74.018
195.383
1.00
15.00


ATOM
1368
CA
THR
A
226
226.833
73.340
195.622
1.00
15.00


ATOM
1369
CB
THR
A
226
228.026
74.279
195.362
1.00
15.00


ATOM
1370
OG1
THR
A
226
229.137
73.880
196.175
1.00
15.00


ATOM
1371
CG2
THR
A
226
227.653
75.716
195.691
1.00
15.00


ATOM
1372
C
THR
A
226
226.916
72.827
197.056
1.00
15.00


ATOM
1373
O
THR
A
226
226.576
73.621
197.967
1.00
9.55


ATOM
1374
N
PRO
A
227
227.140
71.546
197.345
1.00
15.00


ATOM
1375
CD
PRO
A
227
228.192
71.043
196.515
1.00
15.00


ATOM
1376
CA
PRO
A
227
227.065
70.837
198.626
1.00
15.00


ATOM
1377
CB
PRO
A
227
227.911
69.587
198.389
1.00
15.00


ATOM
1378
CG
PRO
A
227
228.885
69.992
197.336
1.00
15.00


ATOM
1379
C
PRO
A
227
227.621
71.670
199.777
1.00
15.00


ATOM
1380
O
PRO
A
227
226.859
71.868
200.794
1.00
10.86


ATOM
1381
N
TYR
A
228
228.838
72.168
199.777
1.00
15.00


ATOM
1382
CA
TYR
A
228
229.509
72.872
200.863
1.00
15.00


ATOM
1383
CB
TYR
A
228
230.999
73.029
200.552
1.00
15.00


ATOM
1384
CG
TYR
A
228
231.748
71.718
200.461
1.00
15.00


ATOM
1385
CD1
TYR
A
228
232.077
71.168
199.228
1.00
15.00


ATOM
1386
CE1
TYR
A
228
232.762
69.972
199.141
1.00
15.00


ATOM
1387
CD2
TYR
A
228
232.124
71.030
201.607
1.00
15.00


ATOM
1388
CE2
TYR
A
228
232.808
69.831
201.527
1.00
15.00


ATOM
1389
CZ
TYR
A
228
233.124
69.307
200.293
1.00
15.00


ATOM
1390
OH
TYR
A
228
233.805
68.114
200.210
1.00
15.00


ATOM
1391
C
TYR
A
228
228.883
74.243
201.095
1.00
15.00


ATOM
1392
O
TYR
A
228
229.096
74.833
202.195
1.00
10.82


ATOM
1393
N
TYR
A
229
228.075
74.767
200.161
1.00
15.00


ATOM
1394
CA
TYR
A
229
227.513
76.111
200.221
1.00
15.00


ATOM
1395
CB
TYR
A
229
227.822
76.871
198.929
1.00
15.00


ATOM
1396
CG
TYR
A
229
229.281
77.238
198.768
1.00
15.00


ATOM
1397
CD1
TYR
A
229
229.951
76.991
197.577
1.00
15.00


ATOM
1398
CE1
TYR
A
229
231.283
77.325
197.425
1.00
15.00


ATOM
1399
CD2
TYR
A
229
229.987
77.829
199.807
1.00
15.00


ATOM
1400
CE2
TYR
A
229
231.320
78.165
199.661
1.00
15.00


ATOM
1401
CZ
TYR
A
229
231.962
77.911
198.470
1.00
15.00


ATOM
1402
OH
TYR
A
229
233.289
78.245
198.324
1.00
15.00


ATOM
1403
C
TYR
A
229
226.005
76.064
200.443
1.00
15.00


ATOM
1404
O
TYR
A
229
225.390
77.180
200.548
1.00
10.79


ATOM
1405
N
VAL
A
230
225.363
74.929
200.540
1.00
15.00


ATOM
1406
CA
VAL
A
230
223.918
74.808
200.689
1.00
15.00


ATOM
1407
CB
VAL
A
230
223.468
73.336
200.596
1.00
15.00


ATOM
1408
CG1
VAL
A
230
224.223
72.492
201.612
1.00
15.00


ATOM
1409
CG2
VAL
A
230
221.967
73.230
200.819
1.00
15.00


ATOM
1410
C
VAL
A
230
223.457
75.378
202.027
1.00
15.00


ATOM
1411
O
VAL
A
230
224.048
75.172
203.074
1.00
9.09


ATOM
1412
CB
ALA
A
231
220.726
77.807
202.673
1.00
15.00


ATOM
1413
C
ALA
A
231
220.871
75.607
203.851
1.00
15.00


ATOM
1414
O
ALA
A
231
220.417
74.655
203.184
1.00
9.49


ATOM
1415
N
ALA
A
231
222.349
76.103
201.918
1.00
15.00


ATOM
1416
CA
ALA
A
231
221.655
76.680
203.104
1.00
15.00


ATOM
1417
N
PRO
A
232
220.755
75.645
205.185
1.00
15.00


ATOM
1418
CD
PRO
A
232
220.885
76.873
205.909
1.00
15.00


ATOM
1419
CA
PRO
A
232
220.064
74.616
205.968
1.00
15.00


ATOM
1420
CB
PRO
A
232
220.102
75.171
207.392
1.00
15.00


ATOM
1421
CG
PRO
A
232
220.173
76.648
207.213
1.00
15.00


ATOM
1422
C
PRO
A
232
218.629
74.402
205.498
1.00
15.00


ATOM
1423
O
PRO
A
232
218.155
73.251
205.520
1.00
10.53


ATOM
1424
N
GLU
A
233
217.931
75.458
205.055
1.00
15.00


ATOM
1425
CA
GLU
A
233
216.540
75.343
204.633
1.00
15.00


ATOM
1426
CB
GLU
A
233
215.917
76.732
204.474
1.00
15.00


ATOM
1427
CG
GLU
A
233
214.503
76.844
205.020
1.00
15.00


ATOM
1428
CD
GLU
A
233
213.601
77.678
204.133
1.00
15.00


ATOM
1429
OE1
GLU
A
233
212.376
77.703
204.384
1.00
15.00


ATOM
1430
OE2
GLU
A
233
214.114
78.309
203.185
1.00
15.00


ATOM
1431
C
GLU
A
233
216.428
74.573
203.321
1.00
15.00


ATOM
1432
O
GLU
A
233
215.392
73.897
203.095
1.00
10.95


ATOM
1433
N
VAL
A
234
217.465
74.585
202.462
1.00
15.00


ATOM
1434
CA
VAL
A
234
217.484
73.810
201.227
1.00
15.00


ATOM
1435
CB
VAL
A
234
218.603
74.293
200.282
1.00
15.00


ATOM
1436
CG1
VAL
A
234
218.713
73.363
199.084
1.00
15.00


ATOM
1437
CG2
VAL
A
234
218.331
75.720
199.832
1.00
15.00


ATOM
1438
C
VAL
A
234
217.690
72.327
201.515
1.00
15.00


ATOM
1439
O
VAL
A
234
217.255
71.447
200.787
1.00
10.86


ATOM
1440
N
LEU
A
235
218.265
72.046
202.723
1.00
15.00


ATOM
1441
CA
LEU
A
235
218.512
70.679
203.169
1.00
15.00


ATOM
1442
CB
LEU
A
235
219.760
70.629
204.053
1.00
15.00


ATOM
1443
CG
LEU
A
235
221.077
71.046
203.392
1.00
15.00


ATOM
1444
CD1
LEU
A
235
222.165
71.181
204.445
1.00
15.00


ATOM
1445
CD2
LEU
A
235
221.473
70.027
202.337
1.00
15.00


ATOM
1446
C
LEU
A
235
217.313
70.129
203.934
1.00
15.00


ATOM
1447
O
LEU
A
235
217.361
69.014
204.500
1.00
10.16


ATOM
1448
N
GLY
A
236
216.252
70.887
203.946
1.00
15.00


ATOM
1449
CA
GLY
A
236
214.958
70.501
204.475
1.00
15.00


ATOM
1450
C
GLY
A
236
213.924
70.292
203.386
1.00
15.00


ATOM
1451
O
GLY
A
236
214.223
70.745
202.251
1.00
15.43


ATOM
1452
N
PRO
A
237
212.800
69.571
203.652
1.00
15.00


ATOM
1453
CD
PRO
A
237
212.368
68.973
204.879
1.00
15.00


ATOM
1454
CA
PRO
A
237
211.813
69.401
202.581
1.00
15.00


ATOM
1455
CB
PRO
A
237
210.885
68.315
203.124
1.00
15.00


ATOM
1456
CG
PRO
A
237
210.988
68.445
204.605
1.00
15.00


ATOM
1457
C
PRO
A
237
211.047
70.688
202.295
1.00
15.00


ATOM
1458
O
PRO
A
237
211.186
71.681
202.987
1.00
19.34


ATOM
1459
N
GLU
A
238
210.242
70.629
201.243
1.00
15.00


ATOM
1460
CA
GLU
A
238
209.352
71.651
200.704
1.00
15.00


ATOM
1461
CB
GLU
A
238
208.401
72.153
201.793
1.00
15.00


ATOM
1462
CG
GLU
A
238
207.415
71.107
202.287
1.00
15.00


ATOM
1463
CD
GLU
A
238
206.523
71.625
203.397
1.00
15.00


ATOM
1464
OE1
GLU
A
238
205.595
70.894
203.807
1.00
15.00


ATOM
1465
OE2
GLU
A
238
206.747
72.764
203.860
1.00
15.00


ATOM
1466
C
GLU
A
238
210.146
72.820
200.131
1.00
15.00


ATOM
1467
O
GLU
A
238
211.191
72.629
199.449
1.00
25.64


ATOM
1468
N
LYS
A
239
209.723
74.116
200.341
1.00
15.00


ATOM
1469
CA
LYS
A
239
210.244
75.246
199.582
1.00
15.00


ATOM
1470
CB
LYS
A
239
209.094
76.047
198.966
1.00
15.00


ATOM
1471
CG
LYS
A
239
208.369
75.328
197.840
1.00
15.00


ATOM
1472
CD
LYS
A
239
207.259
76.189
197.261
1.00
15.00


ATOM
1473
CE
LYS
A
239
206.534
75.469
196.134
1.00
15.00


ATOM
1474
NZ
LYS
A
239
205.442
76.301
195.556
1.00
15.00


ATOM
1475
C
LYS
A
239
211.090
76.155
200.466
1.00
15.00


ATOM
1476
O
LYS
A
239
211.196
76.171
201.667
1.00
16.81


ATOM
1477
N
TYR
A
240
211.840
77.115
199.704
1.00
15.00


ATOM
1478
CA
TYR
A
240
212.736
78.087
200.316
1.00
15.00


ATOM
1479
CB
TYR
A
240
214.163
77.536
200.363
1.00
15.00


ATOM
1480
CG
TYR
A
240
214.299
76.138
199.801
1.00
15.00


ATOM
1481
CD1
TYR
A
240
214.813
75.929
198.526
1.00
15.00


ATOM
1482
CE1
TYR
A
240
214.940
74.655
198.009
1.00
15.00


ATOM
1483
CD2
TYR
A
240
213.913
75.030
200.542
1.00
15.00


ATOM
1484
CE2
TYR
A
240
214.038
73.752
200.030
1.00
15.00


ATOM
1485
CZ
TYR
A
240
214.550
73.570
198.765
1.00
15.00


ATOM
1486
OH
TYR
A
240
214.674
72.298
198.252
1.00
15.00


ATOM
1487
C
TYR
A
240
212.717
79.406
199.552
1.00
15.00


ATOM
1488
O
TYR
A
240
212.436
79.469
198.354
1.00
13.90


ATOM
1489
N
ASP
A
241
213.113
80.515
200.250
1.00
15.00


ATOM
1490
CA
ASP
A
241
213.048
81.860
199.690
1.00
15.00


ATOM
1491
CB
ASP
A
241
212.482
82.836
200.725
1.00
15.00


ATOM
1492
CG
ASP
A
241
211.007
82.608
200.994
1.00
15.00


ATOM
1493
OD1
ASP
A
241
210.289
82.190
200.062
1.00
15.00


ATOM
1494
OD2
ASP
A
241
210.567
82.847
202.138
1.00
15.00


ATOM
1495
C
ASP
A
241
214.426
82.330
199.237
1.00
15.00


ATOM
1496
O
ASP
A
241
215.421
81.629
199.180
1.00
12.46


ATOM
1497
N
LYS
A
242
214.597
83.587
198.906
1.00
106.74
N


ATOM
1498
CA
LYS
A
242
215.786
84.284
198.467
1.00
86.50
C


ATOM
1499
C
LYS
A
242
216.900
84.299
199.494
1.00
99.35
C


ATOM
1500
O
LYS
A
242
217.948
84.893
199.254
1.00
106.37
O


ATOM
1501
CB
LYS
A
242
215.446
85.726
198.107
1.00
97.76
C


ATOM
1502
CG
LYS
A
242
214.108
86.236
198.608
1.00
71.36
C


ATOM
1503
CD
LYS
A
242
213.651
87.417
197.771
1.00
103.00
C


ATOM
1504
CE
LYS
A
242
212.555
88.212
198.477
1.00
138.19
C


ATOM
1505
NZ
LYS
A
242
212.180
89.484
197.778
1.00
123.67
N


ATOM
1506
N
SER
A
243
216.681
83.659
200.637
1.00
97.75
N


ATOM
1507
CA
SER
A
243
217.683
83.627
201.704
1.00
84.81
C


ATOM
1508
C
SER
A
243
218.986
82.926
201.333
1.00
82.48
C


ATOM
1509
O
SER
A
243
220.025
83.174
201.948
1.00
74.56
O


ATOM
1510
CB
SER
A
243
217.097
82.973
202.940
1.00
77.67
C


ATOM
1511
OG
SER
A
243
215.950
83.691
203.344
1.00
121.59
O


ATOM
1512
N
CYS
A
244
218.944
82.044
200.343
1.00
67.22
N


ATOM
1513
CA
CYS
A
244
220.098
81.217
200.052
1.00
78.41
C


ATOM
1514
C
CYS
A
244
221.259
82.130
199.674
1.00
64.58
C


ATOM
1515
O
CYS
A
244
222.377
81.930
200.139
1.00
67.24
O


ATOM
1516
CB
CYS
A
244
219.790
80.254
198.911
1.00
64.99
C


ATOM
1517
SG
CYS
A
244
219.163
81.050
197.435
1.00
91.08
S


ATOM
1518
N
ASP
A
245
220.988
83.129
198.834
1.00
49.99
N


ATOM
1519
CA
ASP
A
245
222.016
84.076
198.416
1.00
45.00
C


ATOM
1520
C
ASP
A
245
222.720
84.635
199.651
1.00
60.39
C


ATOM
1521
O
ASP
A
245
223.952
84.702
199.688
1.00
67.65
O


ATOM
1522
CB
ASP
A
245
221.410
85.236
197.620
1.00
69.55
C


ATOM
1523
CG
ASP
A
245
221.092
84.874
196.180
1.00
60.27
C


ATOM
1524
OD1
ASP
A
245
221.535
83.802
195.715
1.00
73.75
O


ATOM
1525
OD2
ASP
A
245
220.410
85.679
195.506
1.00
70.27
O


ATOM
1526
N
MET
A
246
221.937
85.033
200.659
1.00
43.54
N


ATOM
1527
CA
MET
A
246
222.494
85.577
201.898
1.00
64.86
C


ATOM
1528
C
MET
A
246
223.266
84.526
202.684
1.00
67.48
C


ATOM
1529
O
MET
A
246
224.198
84.855
203.425
1.00
67.31
O


ATOM
1530
CB
MET
A
246
221.401
86.146
202.790
1.00
52.62
C


ATOM
1531
CG
MET
A
246
220.635
87.263
202.163
1.00
60.79
C


ATOM
1532
SD
MET
A
246
221.708
88.445
201.385
1.00
66.30
S


ATOM
1533
CE
MET
A
246
222.403
89.309
202.804
1.00
55.43
C


ATOM
1534
N
TRP
A
247
222.868
83.265
202.536
1.00
54.14
N


ATOM
1535
CA
TRP
A
247
223.551
82.177
203.215
1.00
62.36
C


ATOM
1536
C
TRP
A
247
224.934
82.015
202.592
1.00
62.90
C


ATOM
1537
O
TRP
A
247
225.951
82.071
203.287
1.00
62.42
O


ATOM
1538
CB
TRP
A
247
222.748
80.888
203.074
1.00
69.04
C


ATOM
1539
CG
TRP
A
247
223.470
79.670
203.559
1.00
86.20
C


ATOM
1540
CD1
TRP
A
247
224.327
78.896
202.844
1.00
72.11
C


ATOM
1541
CD2
TRP
A
247
223.430
79.110
204.885
1.00
75.65
C


ATOM
1542
NE1
TRP
A
247
224.827
77.885
203.636
1.00
81.90
N


ATOM
1543
CE2
TRP
A
247
224.293
77.999
204.893
1.00
68.57
C


ATOM
1544
CE3
TRP
A
247
222.749
79.446
206.064
1.00
91.13
C


ATOM
1545
CZ2
TRP
A
247
224.500
77.221
206.030
1.00
68.02
C


ATOM
1546
CZ3
TRP
A
247
222.952
78.669
207.193
1.00
82.48
C


ATOM
1547
CH2
TRP
A
247
223.821
77.572
207.166
1.00
74.62
C


ATOM
1548
N
SER
A
248
224.959
81.829
201.274
1.00
77.02
N


ATOM
1549
CA
SER
A
248
226.203
81.676
200.531
1.00
73.53
C


ATOM
1550
C
SER
A
248
227.156
82.786
200.892
1.00
80.75
C


ATOM
1551
O
SER
A
248
228.369
82.583
200.943
1.00
84.06
O


ATOM
1552
CB
SER
A
248
225.930
81.731
199.043
1.00
67.92
C


ATOM
1553
OG
SER
A
248
225.112
80.642
198.668
1.00
118.25
O


ATOM
1554
N
LEU
A
249
226.593
83.967
201.130
1.00
76.04
N


ATOM
1555
CA
LEU
A
249
227.378
85.132
201.506
1.00
82.09
C


ATOM
1556
C
LEU
A
249
228.081
84.869
202.845
1.00
84.27
C


ATOM
1557
O
LEU
A
249
229.298
85.035
202.960
1.00
83.90
O


ATOM
1558
CB
LEU
A
249
226.473
86.363
201.610
1.00
85.62
C


ATOM
1559
CG
LEU
A
249
227.049
87.629
200.964
1.00
77.42
C


ATOM
1560
CD1
LEU
A
249
227.250
87.346
199.493
1.00
68.42
C


ATOM
1561
CD2
LEU
A
249
226.119
88.832
201.153
1.00
95.64
C


ATOM
1562
N
GLY
A
250
227.318
84.449
203.851
1.00
64.22
N


ATOM
1563
CA
GLY
A
250
227.915
84.165
205.147
1.00
62.72
C


ATOM
1564
C
GLY
A
250
229.103
83.231
204.998
1.00
63.71
C


ATOM
1565
O
GLY
A
250
230.220
83.544
205.418
1.00
57.98
O


ATOM
1566
N
VAL
A
251
228.855
82.076
204.387
1.00
57.35
N


ATOM
1567
CA
VAL
A
251
229.902
81.091
204.152
1.00
72.77
C


ATOM
1568
C
VAL
A
251
231.130
81.779
203.587
1.00
65.76
C


ATOM
1569
O
VAL
A
251
232.239
81.624
204.093
1.00
72.08
O


ATOM
1570
CB
VAL
A
251
229.474
80.052
203.129
1.00
72.02
C


ATOM
1571
CG1
VAL
A
251
230.627
79.119
202.852
1.00
48.81
C


ATOM
1572
CG2
VAL
A
251
228.255
79.312
203.617
1.00
45.51
C


ATOM
1573
N
ILE
A
252
230.927
82.544
202.525
1.00
65.58
N


ATOM
1574
CA
ILE
A
252
232.036
83.248
201.899
1.00
68.61
C


ATOM
1575
C
ILE
A
252
232.722
84.197
202.876
1.00
77.12
C


ATOM
1576
O
ILE
A
252
233.932
84.098
203.088
1.00
79.18
O


ATOM
1577
CB
ILE
A
252
231.564
84.008
200.634
1.00
65.58
C


ATOM
1578
CG1
ILE
A
252
231.151
82.989
199.570
1.00
42.53
C


ATOM
1579
CG2
ILE
A
252
232.679
84.891
200.102
1.00
53.46
C


ATOM
1580
CD1
ILE
A
252
230.557
83.587
198.355
1.00
59.04
C


ATOM
1581
N
MET
A
253
231.956
85.091
203.498
1.00
72.95
N


ATOM
1582
CA
MET
A
253
232.553
86.028
204.444
1.00
81.03
C


ATOM
1583
C
MET
A
253
233.367
85.286
205.497
1.00
75.68
C


ATOM
1584
O
MET
A
253
234.460
85.724
205.883
1.00
79.62
O


ATOM
1585
CB
MET
A
253
231.486
86.866
205.149
1.00
64.17
C


ATOM
1586
CG
MET
A
253
232.083
88.081
205.819
1.00
54.30
C


ATOM
1587
SD
MET
A
253
230.954
88.969
206.885
1.00
85.00
S


ATOM
1588
CE
MET
A
253
229.555
89.250
205.802
1.00
83.83
C


ATOM
1589
N
TYR
A
254
232.829
84.161
205.960
1.00
81.14
N


ATOM
1590
CA
TYR
A
254
233.513
83.370
206.969
1.00
68.03
C


ATOM
1591
C
TYR
A
254
234.879
83.008
206.419
1.00
74.50
C


ATOM
1592
O
TYR
A
254
235.901
83.519
206.879
1.00
78.65
O


ATOM
1593
CB
TYR
A
254
232.711
82.107
207.279
1.00
66.09
C


ATOM
1594
CG
TYR
A
254
233.253
81.291
208.427
1.00
68.80
C


ATOM
1595
CD1
TYR
A
254
234.285
80.388
208.230
1.00
93.85
C


ATOM
1596
CD2
TYR
A
254
232.739
81.434
209.713
1.00
66.37
C


ATOM
1597
CE1
TYR
A
254
234.797
79.640
209.281
1.00
93.85
C


ATOM
1598
CE2
TYR
A
254
233.238
80.696
210.775
1.00
60.25
C


ATOM
1599
CZ
TYR
A
254
234.270
79.797
210.553
1.00
78.16
C


ATOM
1600
OH
TYR
A
254
234.780
79.043
211.592
1.00
83.85
O


ATOM
1601
N
ILE
A
255
234.876
82.148
205.405
1.00
71.34
N


ATOM
1602
CA
ILE
A
255
236.099
81.690
204.774
1.00
61.37
C


ATOM
1603
C
ILE
A
255
237.128
82.782
204.558
1.00
76.55
C


ATOM
1604
O
ILE
A
255
238.298
82.590
204.870
1.00
92.35
O


ATOM
1605
CB
ILE
A
255
235.810
81.046
203.443
1.00
62.35
C


ATOM
1606
CG1
ILE
A
255
234.928
79.819
203.653
1.00
62.67
C


ATOM
1607
CG2
ILE
A
255
237.107
80.661
202.790
1.00
76.47
C


ATOM
1608
CD1
ILE
A
255
234.514
79.131
202.387
1.00
57.99
C


ATOM
1609
N
LEU
A
256
236.709
83.925
204.024
1.00
73.36
N


ATOM
1610
CA
LEU
A
256
237.647
85.022
203.809
1.00
76.61
C


ATOM
1611
C
LEU
A
256
238.466
85.295
205.065
1.00
80.02
C


ATOM
1612
O
LEU
A
256
239.681
85.116
205.074
1.00
95.93
O


ATOM
1613
CB
LEU
A
256
236.921
86.316
203.430
1.00
82.53
C


ATOM
1614
CG
LEU
A
256
236.503
86.623
201.991
1.00
67.94
C


ATOM
1615
CD1
LEU
A
256
236.199
88.119
201.870
1.00
83.50
C


ATOM
1616
CD2
LEU
A
256
237.615
86.248
201.038
1.00
61.15
C


ATOM
1617
N
LEU
A
257
237.780
85.718
206.124
1.00
64.99
N


ATOM
1618
CA
LEU
A
257
238.407
86.060
207.399
1.00
83.07
C


ATOM
1619
C
LEU
A
257
239.336
85.046
208.090
1.00
92.07
C


ATOM
1620
O
LEU
A
257
240.175
85.450
208.897
1.00
79.63
O


ATOM
1621
CB
LEU
A
257
237.331
86.467
208.402
1.00
65.07
C


ATOM
1622
CG
LEU
A
257
236.445
87.661
208.054
1.00
72.20
C


ATOM
1623
CD1
LEU
A
257
235.475
87.888
209.185
1.00
109.20
C


ATOM
1624
CD2
LEU
A
257
237.282
88.906
207.843
1.00
77.76
C


ATOM
1625
N
CYS
A
258
239.211
83.751
207.805
1.00
84.06
N


ATOM
1626
CA
CYS
A
258
240.068
82.782
208.486
1.00
65.38
C


ATOM
1627
C
CYS
A
258
240.811
81.822
207.572
1.00
79.35
C


ATOM
1628
O
CYS
A
258
241.865
81.305
207.936
1.00
89.35
O


ATOM
1629
CB
CYS
A
258
239.250
81.952
209.465
1.00
74.82
C


ATOM
1630
SG
CYS
A
258
238.333
80.661
208.627
1.00
88.33
S


ATOM
1631
N
GLY
A
259
240.259
81.552
206.397
1.00
77.77
N


ATOM
1632
CA
GLY
A
259
240.927
80.636
205.491
1.00
86.18
C


ATOM
1633
C
GLY
A
259
240.211
79.311
205.306
1.00
84.64
C


ATOM
1634
O
GLY
A
259
240.562
78.538
204.415
1.00
91.47
O


ATOM
1635
N
TYR
A
260
239.219
79.030
206.145
1.00
80.25
N


ATOM
1636
CA
TYR
A
260
238.454
77.788
206.024
1.00
85.40
C


ATOM
1637
C
TYR
A
260
236.969
78.064
206.208
1.00
92.78
C


ATOM
1638
O
TYR
A
260
236.587
79.064
206.813
1.00
109.20
O


ATOM
1639
CB
TYR
A
260
238.936
76.760
207.050
1.00
90.73
C


ATOM
1640
CG
TYR
A
260
239.030
77.305
208.451
1.00
78.95
C


ATOM
1641
CD1
TYR
A
260
237.942
77.261
209.312
1.00
99.14
C


ATOM
1642
CD2
TYR
A
260
240.204
77.892
208.904
1.00
82.37
C


ATOM
1643
CE1
TYR
A
260
238.023
77.785
210.585
1.00
88.16
C


ATOM
1644
CE2
TYR
A
260
240.292
78.420
210.173
1.00
97.24
C


ATOM
1645
CZ
TYR
A
260
239.201
78.364
211.007
1.00
67.63
C


ATOM
1646
OH
TYR
A
260
239.298
78.896
212.269
1.00
84.01
O


ATOM
1647
N
PRO
A
261
236.114
77.183
205.676
1.00
89.49
N


ATOM
1648
CA
PRO
A
261
234.656
77.296
205.753
1.00
103.35
C


ATOM
1649
C
PRO
A
261
234.107
77.095
207.161
1.00
96.36
C


ATOM
1650
O
PRO
A
261
234.743
76.459
207.995
1.00
100.89
O


ATOM
1651
CB
PRO
A
261
234.185
76.209
204.791
1.00
98.88
C


ATOM
1652
CG
PRO
A
261
235.208
75.152
204.996
1.00
95.80
C


ATOM
1653
CD
PRO
A
261
236.493
75.962
204.950
1.00
91.52
C


ATOM
1654
N
PRO
A
262
232.918
77.653
207.444
1.00
94.45
N


ATOM
1655
CA
PRO
A
262
232.337
77.488
208.773
1.00
85.15
C


ATOM
1656
C
PRO
A
262
232.015
76.028
209.043
1.00
68.20
C


ATOM
1657
O
PRO
A
262
232.519
75.448
209.998
1.00
106.94
O


ATOM
1658
CB
PRO
A
262
231.092
78.368
208.717
1.00
75.57
C


ATOM
1659
CG
PRO
A
262
230.721
78.337
207.278
1.00
64.63
C


ATOM
1660
CD
PRO
A
262
232.053
78.504
206.609
1.00
99.03
C


ATOM
1661
N
PHE
A
263
231.200
75.419
208.191
1.00
73.57
N


ATOM
1662
CA
PHE
A
263
230.838
74.021
208.393
1.00
79.36
C


ATOM
1663
C
PHE
A
263
231.864
73.093
207.757
1.00
106.15
C


ATOM
1664
O
PHE
A
263
231.972
73.009
206.530
1.00
105.28
O


ATOM
1665
CB
PHE
A
263
229.428
73.777
207.848
1.00
69.85
C


ATOM
1666
CG
PHE
A
263
228.420
74.759
208.374
1.00
81.28
C


ATOM
1667
CD1
PHE
A
263
228.128
75.917
207.677
1.00
113.87
C


ATOM
1668
CD2
PHE
A
263
227.845
74.573
209.613
1.00
117.67
C


ATOM
1669
CE1
PHE
A
263
227.285
76.867
208.205
1.00
85.69
C


ATOM
1670
CE2
PHE
A
263
227.004
75.520
210.145
1.00
108.39
C


ATOM
1671
CZ
PHE
A
263
226.729
76.670
209.437
1.00
117.73
C


ATOM
1672
N
TYR
A
264
232.620
72.408
208.622
1.00
134.25
N


ATOM
1673
CA
TYR
A
264
233.681
71.497
208.197
1.00
147.77
C


ATOM
1674
C
TYR
A
264
233.192
70.094
207.848
1.00
140.09
C


ATOM
1675
O
TYR
A
264
232.006
69.787
207.979
1.00
138.93
O


ATOM
1676
CB
TYR
A
264
234.775
71.405
209.284
1.00
153.86
C


ATOM
1677
CG
TYR
A
264
236.191
71.629
208.760
1.00
157.09
C


ATOM
1678
CD1
TYR
A
264
237.265
70.886
209.250
1.00
193.79
C


ATOM
1679
CD2
TYR
A
264
236.448
72.574
207.768
1.00
140.55
C


ATOM
1680
CE1
TYR
A
264
238.553
71.074
208.764
1.00
220.80
C


ATOM
1681
CE2
TYR
A
264
237.734
72.771
207.279
1.00
201.56
C


ATOM
1682
CZ
TYR
A
264
238.782
72.015
207.781
1.00
222.82
C


ATOM
1683
OH
TYR
A
264
240.069
72.185
207.307
1.00
220.52
O


ATOM
1684
N
SER
A
265
234.118
69.246
207.409
1.00
141.69
N


ATOM
1685
CA
SER
A
265
233.785
67.877
207.044
1.00
135.35
C


ATOM
1686
C
SER
A
265
234.560
66.896
207.915
1.00
131.47
C


ATOM
1687
O
SER
A
265
235.792
66.945
207.976
1.00
126.66
O


ATOM
1688
CB
SER
A
265
234.113
67.628
205.566
1.00
138.65
C


ATOM
1689
OG
SER
A
265
233.591
66.386
205.130
1.00
107.77
O


TER
1689

SER
A
265


ATOM
1690
N
GLY
A
274
229.307
63.721
209.431
1.00
106.41
N


ATOM
1691
CA
GLY
A
274
228.030
64.307
209.816
1.00
119.68
C


ATOM
1692
C
GLY
A
274
227.987
65.812
209.615
1.00
113.16
C


ATOM
1693
O
GLY
A
274
227.553
66.551
210.496
1.00
95.55
O


ATOM
1694
N
MET
A
275
228.446
66.261
208.449
1.00
110.29
N


ATOM
1695
CA
MET
A
275
228.463
67.676
208.112
1.00
95.81
C


ATOM
1696
C
MET
A
275
227.067
68.162
207.746
1.00
94.19
C


ATOM
1697
O
MET
A
275
226.633
69.206
208.220
1.00
75.71
O


ATOM
1698
CB
MET
A
275
229.412
67.938
206.943
1.00
90.51
C


ATOM
1699
CG
MET
A
275
229.404
69.380
206.472
1.00
83.11
C


ATOM
1700
SD
MET
A
275
230.324
69.611
204.942
1.00
99.84
S


ATOM
1701
CE
MET
A
275
229.028
69.352
203.750
1.00
79.14
C


ATOM
1702
N
LYS
A
276
226.362
67.411
206.904
1.00
80.06
N


ATOM
1703
CA
LYS
A
276
225.018
67.816
206.511
1.00
81.42
C


ATOM
1704
C
LYS
A
276
224.233
68.145
207.770
1.00
67.18
C


ATOM
1705
O
LYS
A
276
223.297
68.931
207.738
1.00
87.18
O


ATOM
1706
CB
LYS
A
276
224.327
66.705
205.731
1.00
70.05
C


ATOM
1707
N
THR
A
277
224.641
67.552
208.886
1.00
90.20
N


ATOM
1708
CA
THR
A
277
223.982
67.776
210.167
1.00
86.79
C


ATOM
1709
C
THR
A
277
224.402
69.103
210.790
1.00
88.87
C


ATOM
1710
O
THR
A
277
223.570
69.987
211.004
1.00
90.25
O


ATOM
1711
CB
THR
A
277
224.316
66.657
211.170
1.00
94.65
C


ATOM
1712
OG1
THR
A
277
223.953
65.387
210.614
1.00
124.83
O


ATOM
1713
CG2
THR
A
277
223.559
66.871
212.464
1.00
101.58
C


ATOM
1714
N
ARG
A
278
225.696
69.224
211.083
1.00
83.15
N


ATOM
1715
CA
ARG
A
278
226.273
70.425
211.683
1.00
85.59
C


ATOM
1716
C
ARG
A
278
225.653
71.666
211.039
1.00
80.18
C


ATOM
1717
O
ARG
A
278
225.537
72.716
211.673
1.00
83.67
O


ATOM
1718
CB
ARG
A
278
227.792
70.413
211.479
1.00
76.56
C


ATOM
1719
CG
ARG
A
278
228.346
68.998
211.313
1.00
94.91
C


ATOM
1720
CD
ARG
A
278
229.398
68.600
212.352
1.00
109.01
C


ATOM
1721
NE
ARG
A
278
230.699
69.199
212.072
1.00
121.75
N


ATOM
1722
CZ
ARG
A
278
231.384
69.003
210.944
1.00
131.02
C


ATOM
1723
NH1
ARG
A
278
230.892
68.216
209.988
1.00
101.83
N


ATOM
1724
NH2
ARG
A
278
232.556
69.613
210.763
1.00
135.84
N


ATOM
1725
N
ILE
A
279
225.250
71.516
209.778
1.00
83.10
N


ATOM
1726
CA
ILE
A
279
224.615
72.579
209.001
1.00
82.30
C


ATOM
1727
C
ILE
A
279
223.167
72.765
209.421
1.00
76.46
C


ATOM
1728
O
ILE
A
279
222.778
73.847
209.847
1.00
90.20
O


ATOM
1729
CB
ILE
A
279
224.609
72.257
207.498
1.00
84.92
C


ATOM
1730
CG1
ILE
A
279
226.006
72.436
206.913
1.00
77.12
C


ATOM
1731
CG2
ILE
A
279
223.611
73.144
206.789
1.00
57.01
C


ATOM
1732
CD1
ILE
A
279
226.093
72.021
205.455
1.00
73.72
C


ATOM
1733
N
ARG
A
280
222.371
71.709
209.270
1.00
68.55
N


ATOM
1734
CA
ARG
A
280
220.965
71.757
209.640
1.00
66.51
C


ATOM
1735
C
ARG
A
280
220.886
72.218
211.090
1.00
62.26
C


ATOM
1736
O
ARG
A
280
220.039
73.038
211.456
1.00
69.34
O


ATOM
1737
CB
ARG
A
280
220.326
70.383
209.475
1.00
49.45
C


ATOM
1738
N
MET
A
281
221.787
71.693
211.912
1.00
62.31
N


ATOM
1739
CA
MET
A
281
221.846
72.058
213.323
1.00
71.30
C


ATOM
1740
C
MET
A
281
222.348
73.489
213.438
1.00
78.13
C


ATOM
1741
O
MET
A
281
222.151
74.152
214.450
1.00
79.48
O


ATOM
1742
CB
MET
A
281
222.794
71.122
214.075
1.00
73.13
C


ATOM
1743
CG
MET
A
281
222.253
69.721
214.315
1.00
98.38
C


ATOM
1744
SD
MET
A
281
220.850
69.703
215.434
1.00
82.33
S


ATOM
1745
CE
MET
A
281
221.659
70.065
216.963
1.00
94.31
C


ATOM
1746
N
GLY
A
282
223.000
73.953
212.379
1.00
80.51
N


ATOM
1747
CA
GLY
A
282
223.536
75.298
212.354
1.00
70.03
C


ATOM
1748
C
GLY
A
282
224.577
75.481
213.435
1.00
89.35
C


ATOM
1749
O
GLY
A
282
224.566
76.482
214.146
1.00
77.94
O


ATOM
1750
N
GLN
A
283
225.479
74.514
213.576
1.00
81.75
N


ATOM
1751
CA
GLN
A
283
226.509
74.624
214.602
1.00
104.61
C


ATOM
1752
C
GLN
A
283
227.939
74.764
214.094
1.00
94.71
C


ATOM
1753
O
GLN
A
283
228.503
73.843
213.505
1.00
88.07
O


ATOM
1754
CB
GLN
A
283
226.421
73.450
215.583
1.00
113.16
C


ATOM
1755
CG
GLN
A
283
226.575
72.075
214.982
1.00
114.78
C


ATOM
1756
CD
GLN
A
283
226.574
71.002
216.057
1.00
133.39
C


ATOM
1757
OE1
GLN
A
283
227.356
71.068
217.005
1.00
133.52
O


ATOM
1758
NE2
GLN
A
283
225.691
70.012
215.922
1.00
112.55
N


ATOM
1759
N
TYR
A
284
228.516
75.935
214.347
1.00
90.04
N


ATOM
1760
CA
TYR
A
284
229.875
76.242
213.934
1.00
64.09
C


ATOM
1761
C
TYR
A
284
230.474
77.137
215.008
1.00
65.66
C


ATOM
1762
O
TYR
A
284
229.806
77.468
215.981
1.00
88.03
O


ATOM
1763
CB
TYR
A
284
229.864
77.002
212.620
1.00
82.88
C


ATOM
1764
CG
TYR
A
284
229.040
78.259
212.690
1.00
50.94
C


ATOM
1765
CD1
TYR
A
284
227.656
78.208
212.619
1.00
72.36
C


ATOM
1766
CD2
TYR
A
284
229.645
79.496
212.856
1.00
79.70
C


ATOM
1767
CE1
TYR
A
284
226.901
79.355
212.709
1.00
69.83
C


ATOM
1768
CE2
TYR
A
284
228.896
80.646
212.951
1.00
57.96
C


ATOM
1769
CZ
TYR
A
284
227.527
80.569
212.877
1.00
57.53
C


ATOM
1770
OH
TYR
A
284
226.778
81.716
212.978
1.00
94.22
O


ATOM
1771
N
GLU
A
285
231.725
77.538
214.821
1.00
80.73
N


ATOM
1772
CA
GLU
A
285
232.399
78.388
215.785
1.00
81.22
C


ATOM
1773
C
GLU
A
285
233.397
79.305
215.101
1.00
90.67
C


ATOM
1774
O
GLU
A
285
233.713
79.119
213.926
1.00
106.16
O


ATOM
1775
CB
GLU
A
285
233.113
77.527
216.807
1.00
87.46
C


ATOM
1776
N
PHE
A
286
233.875
80.303
215.839
1.00
86.81
N


ATOM
1777
CA
PHE
A
286
234.880
81.236
215.334
1.00
97.06
C


ATOM
1778
C
PHE
A
286
236.110
80.887
216.167
1.00
100.16
C


ATOM
1779
O
PHE
A
286
236.527
81.662
217.028
1.00
110.05
O


ATOM
1780
CB
PHE
A
286
234.476
82.697
215.602
1.00
78.01
C


ATOM
1781
CG
PHE
A
286
233.214
83.127
214.904
1.00
98.13
C


ATOM
1782
CD1
PHE
A
286
232.172
83.698
215.615
1.00
92.96
C


ATOM
1783
CD2
PHE
A
286
233.064
82.959
213.538
1.00
102.41
C


ATOM
1784
CE1
PHE
A
286
231.003
84.092
214.980
1.00
96.16
C


ATOM
1785
CE2
PHE
A
286
231.892
83.353
212.897
1.00
82.36
C


ATOM
1786
CZ
PHE
A
286
230.865
83.918
213.620
1.00
71.90
C


ATOM
1787
N
PRO
A
287
236.707
79.715
215.909
1.00
96.70
N


ATOM
1788
CA
PRO
A
287
237.883
79.170
216.586
1.00
103.18
C


ATOM
1789
C
PRO
A
287
239.086
80.081
216.730
1.00
112.32
C


ATOM
1790
O
PRO
A
287
239.531
80.709
215.770
1.00
114.13
O


ATOM
1791
CB
PRO
A
287
238.210
77.940
215.754
1.00
105.25
C


ATOM
1792
CG
PRO
A
287
237.828
78.359
214.411
1.00
92.44
C


ATOM
1793
CD
PRO
A
287
236.474
78.978
214.659
1.00
100.51
C


ATOM
1794
N
ASN
A
288
239.608
80.133
217.950
1.00
119.25
N


ATOM
1795
CA
ASN
A
288
240.796
80.913
218.239
1.00
131.90
C


ATOM
1796
C
ASN
A
288
241.953
79.964
217.949
1.00
138.51
C


ATOM
1797
O
ASN
A
288
241.808
78.742
218.021
1.00
143.95
O


ATOM
1798
CB
ASN
A
288
240.826
81.347
219.708
1.00
133.89
C


ATOM
1799
CG
ASN
A
288
239.631
82.211
220.092
1.00
158.92
C


ATOM
1800
OD1
ASN
A
288
239.383
83.259
219.485
1.00
172.68
O


ATOM
1801
ND2
ASN
A
288
238.890
81.779
221.114
1.00
139.14
N


ATOM
1802
N
PRO
A
289
243.119
80.510
217.602
1.00
145.89
N


ATOM
1803
CA
PRO
A
289
243.399
81.940
217.477
1.00
147.78
C


ATOM
1804
C
PRO
A
289
242.632
82.643
216.367
1.00
140.12
C


ATOM
1805
O
PRO
A
289
242.027
83.678
216.608
1.00
145.48
O


ATOM
1806
CB
PRO
A
289
244.904
81.967
217.231
1.00
160.27
C


ATOM
1807
CG
PRO
A
289
245.107
80.724
216.415
1.00
158.40
C


ATOM
1808
CD
PRO
A
289
244.286
79.712
217.185
1.00
149.68
C


ATOM
1809
N
GLU
A
290
242.665
82.064
215.166
1.00
135.89
N


ATOM
1810
CA
GLU
A
290
242.019
82.602
213.959
1.00
122.33
C


ATOM
1811
C
GLU
A
290
241.100
83.810
214.141
1.00
111.48
C


ATOM
1812
O
GLU
A
290
241.314
84.858
213.531
1.00
94.90
O


ATOM
1813
CB
GLU
A
290
241.230
81.502
213.236
1.00
121.58
C


ATOM
1814
CG
GLU
A
290
241.934
80.155
213.147
1.00
140.70
C


ATOM
1815
CD
GLU
A
290
241.650
79.287
214.360
1.00
183.86
C


ATOM
1816
OE1
GLU
A
290
241.898
79.762
215.484
1.00
193.44
O


ATOM
1817
OE2
GLU
A
290
241.178
78.137
214.193
1.00
182.20
O


ATOM
1818
N
TRP
A
291
240.080
83.655
214.980
1.00
102.18
N


ATOM
1819
CA
TRP
A
291
239.105
84.715
215.221
1.00
106.95
C


ATOM
1820
C
TRP
A
291
239.335
85.573
216.457
1.00
115.40
C


ATOM
1821
O
TRP
A
291
238.474
86.375
216.824
1.00
120.18
O


ATOM
1822
CB
TRP
A
291
237.712
84.106
215.295
1.00
110.40
C


ATOM
1823
CG
TRP
A
291
237.364
83.374
214.056
1.00
104.11
C


ATOM
1824
CD1
TRP
A
291
237.782
82.121
213.692
1.00
84.92
C


ATOM
1825
CD2
TRP
A
291
236.556
83.857
212.982
1.00
106.19
C


ATOM
1826
NE1
TRP
A
291
237.282
81.799
212.456
1.00
103.99
N


ATOM
1827
CE2
TRP
A
291
236.529
82.848
211.996
1.00
102.71
C


ATOM
1828
CE3
TRP
A
291
235.857
85.048
212.753
1.00
70.56
C


ATOM
1829
CZ2
TRP
A
291
235.827
82.992
210.807
1.00
86.03
C


ATOM
1830
CZ3
TRP
A
291
235.165
85.189
211.578
1.00
73.88
C


ATOM
1831
CH2
TRP
A
291
235.154
84.171
210.616
1.00
114.55
C


ATOM
1832
N
SER
A
292
240.491
85.404
217.091
1.00
129.00
N


ATOM
1833
CA
SER
A
292
240.851
86.158
218.287
1.00
124.04
C


ATOM
1834
C
SER
A
292
240.910
87.670
218.051
1.00
123.03
C


ATOM
1835
O
SER
A
292
240.661
88.455
218.966
1.00
113.67
O


ATOM
1836
CB
SER
A
292
242.199
85.658
218.825
1.00
125.10
C


ATOM
1837
OG
SER
A
292
243.196
85.669
217.817
1.00
135.63
O


ATOM
1838
N
GLU
A
293
241.237
88.081
216.829
1.00
123.41
N


ATOM
1839
CA
GLU
A
293
241.321
89.508
216.513
1.00
127.66
C


ATOM
1840
C
GLU
A
293
240.108
90.026
215.724
1.00
121.92
C


ATOM
1841
O
GLU
A
293
240.091
91.178
215.285
1.00
115.91
O


ATOM
1842
CB
GLU
A
293
242.600
89.792
215.737
1.00
138.28
C


ATOM
1843
N
VAL
A
294
239.094
89.181
215.555
1.00
111.91
N


ATOM
1844
CA
VAL
A
294
237.898
89.568
214.818
1.00
95.15
C


ATOM
1845
C
VAL
A
294
236.811
90.136
215.721
1.00
103.25
C


ATOM
1846
O
VAL
A
294
236.506
89.573
216.771
1.00
98.28
O


ATOM
1847
CB
VAL
A
294
237.322
88.378
214.066
1.00
92.93
C


ATOM
1848
CG1
VAL
A
294
236.182
88.840
213.168
1.00
91.77
C


ATOM
1849
CG2
VAL
A
294
238.418
87.705
213.269
1.00
94.47
C


ATOM
1850
N
SER
A
295
236.218
91.249
215.292
1.00
84.68
N


ATOM
1851
CA
SER
A
295
235.168
91.914
216.056
1.00
92.73
C


ATOM
1852
C
SER
A
295
233.919
91.072
216.244
1.00
88.74
C


ATOM
1853
O
SER
A
295
233.568
90.250
215.399
1.00
88.58
O


ATOM
1854
CB
SER
A
295
234.745
93.201
215.365
1.00
102.34
C


ATOM
1855
OG
SER
A
295
233.464
93.030
214.784
1.00
114.40
O


ATOM
1856
N
GLU
A
296
233.244
91.301
217.362
1.00
92.37
N


ATOM
1857
CA
GLU
A
296
232.008
90.600
217.658
1.00
104.61
C


ATOM
1858
C
GLU
A
296
230.970
91.166
216.694
1.00
112.98
C


ATOM
1859
O
GLU
A
296
229.956
90.523
216.406
1.00
105.91
O


ATOM
1860
CB
GLU
A
296
231.593
90.857
219.108
1.00
99.76
C


ATOM
1861
CG
GLU
A
296
230.316
90.154
219.543
1.00
121.36
C


ATOM
1862
CD
GLU
A
296
230.324
88.663
219.236
1.00
136.85
C


ATOM
1863
OE1
GLU
A
296
231.304
87.970
219.609
1.00
131.43
O


ATOM
1864
OE2
GLU
A
296
229.340
88.185
218.619
1.00
136.46
O


ATOM
1865
N
GLU
A
297
231.237
92.379
216.203
1.00
112.50
N


ATOM
1866
CA
GLU
A
297
230.357
93.045
215.247
1.00
99.85
C


ATOM
1867
C
GLU
A
297
230.420
92.241
213.960
1.00
92.47
C


ATOM
1868
O
GLU
A
297
229.396
91.854
213.400
1.00
93.48
O


ATOM
1869
CB
GLU
A
297
230.838
94.472
214.998
1.00
98.76
C


ATOM
1870
CG
GLU
A
297
230.226
95.146
213.787
1.00
102.64
C


ATOM
1871
CD
GLU
A
297
230.489
96.642
213.773
1.00
129.34
C


ATOM
1872
OE1
GLU
A
297
229.777
97.379
214.497
1.00
134.48
O


ATOM
1873
OE2
GLU
A
297
231.415
97.081
213.049
1.00
104.72
O


ATOM
1874
N
VAL
A
298
231.638
91.993
213.499
1.00
80.38
N


ATOM
1875
CA
VAL
A
298
231.843
91.200
212.305
1.00
87.49
C


ATOM
1876
C
VAL
A
298
231.282
89.809
212.579
1.00
80.88
C


ATOM
1877
O
VAL
A
298
230.690
89.176
211.702
1.00
71.80
O


ATOM
1878
CB
VAL
A
298
233.333
91.057
211.993
1.00
64.02
C


ATOM
1879
CG1
VAL
A
298
233.542
89.953
210.980
1.00
84.65
C


ATOM
1880
CG2
VAL
A
298
233.873
92.364
211.466
1.00
112.61
C


ATOM
1881
N
LYS
A
299
231.479
89.331
213.801
1.00
79.83
N


ATOM
1882
CA
LYS
A
299
230.990
88.015
214.162
1.00
75.16
C


ATOM
1883
C
LYS
A
299
229.478
87.983
214.138
1.00
80.77
C


ATOM
1884
O
LYS
A
299
228.872
87.052
213.618
1.00
62.14
O


ATOM
1885
CB
LYS
A
299
231.517
87.616
215.539
1.00
74.57
C


ATOM
1886
CG
LYS
A
299
232.979
87.229
215.509
1.00
75.11
C


ATOM
1887
CD
LYS
A
299
233.432
86.563
216.790
1.00
92.06
C


ATOM
1888
CE
LYS
A
299
234.841
86.005
216.623
1.00
125.81
C


ATOM
1889
NZ
LYS
A
299
235.368
85.378
217.867
1.00
126.47
N


ATOM
1890
N
MET
A
300
228.872
89.021
214.691
1.00
78.00
N


ATOM
1891
CA
MET
A
300
227.428
89.116
214.735
1.00
80.36
C


ATOM
1892
C
MET
A
300
226.822
89.120
213.327
1.00
69.48
C


ATOM
1893
O
MET
A
300
225.732
88.587
213.118
1.00
75.70
O


ATOM
1894
CB
MET
A
300
227.033
90.378
215.502
1.00
87.97
C


ATOM
1895
CG
MET
A
300
226.011
90.131
216.601
1.00
118.90
C


ATOM
1896
SD
MET
A
300
226.231
88.508
217.393
1.00
128.71
S


ATOM
1897
CE
MET
A
300
224.516
87.872
217.267
1.00
113.92
C


ATOM
1898
N
LEU
A
301
227.538
89.707
212.369
1.00
73.89
N


ATOM
1899
CA
LEU
A
301
227.079
89.782
210.972
1.00
63.96
C


ATOM
1900
C
LEU
A
301
227.048
88.406
210.301
1.00
70.75
C


ATOM
1901
O
LEU
A
301
226.145
88.086
209.532
1.00
60.38
O


ATOM
1902
CB
LEU
A
301
227.977
90.735
210.177
1.00
63.62
C


ATOM
1903
CG
LEU
A
301
227.617
91.019
208.720
1.00
52.63
C


ATOM
1904
CD1
LEU
A
301
226.130
91.284
208.596
1.00
65.57
C


ATOM
1905
CD2
LEU
A
301
228.414
92.212
208.225
1.00
73.24
C


ATOM
1906
N
ILE
A
302
228.046
87.588
210.592
1.00
67.57
N


ATOM
1907
CA
ILE
A
302
228.090
86.250
210.037
1.00
61.70
C


ATOM
1908
C
ILE
A
302
226.900
85.455
210.582
1.00
75.97
C


ATOM
1909
O
ILE
A
302
226.226
84.741
209.837
1.00
58.38
O


ATOM
1910
CB
ILE
A
302
229.422
85.565
210.402
1.00
70.53
C


ATOM
1911
CG1
ILE
A
302
230.562
86.271
209.659
1.00
76.98
C


ATOM
1912
CG2
ILE
A
302
229.369
84.086
210.071
1.00
65.01
C


ATOM
1913
CD1
ILE
A
302
231.930
85.670
209.869
1.00
71.99
C


ATOM
1914
N
ARG
A
303
226.630
85.611
211.878
1.00
73.53
N


ATOM
1915
CA
ARG
A
303
225.526
84.913
212.532
1.00
61.85
C


ATOM
1916
C
ARG
A
303
224.162
85.201
211.898
1.00
74.05
C


ATOM
1917
O
ARG
A
303
223.370
84.286
211.695
1.00
79.80
O


ATOM
1918
CB
ARG
A
303
225.461
85.279
214.009
1.00
61.93
C


ATOM
1919
CG
ARG
A
303
226.666
84.917
214.852
1.00
78.75
C


ATOM
1920
CD
ARG
A
303
226.402
85.392
216.284
1.00
71.93
C


ATOM
1921
NE
ARG
A
303
227.608
85.725
217.038
1.00
101.37
N


ATOM
1922
CZ
ARG
A
303
228.488
84.827
217.457
1.00
93.09
C


ATOM
1923
NH1
ARG
A
303
228.286
83.540
217.191
1.00
90.11
N


ATOM
1924
NH2
ARG
A
303
229.561
85.211
218.140
1.00
76.11
N


ATOM
1925
N
ASN
A
304
223.865
86.458
211.596
1.00
61.15
N


ATOM
1926
CA
ASN
A
304
222.575
86.775
210.982
1.00
77.76
C


ATOM
1927
C
ASN
A
304
222.517
86.277
209.535
1.00
74.95
C


ATOM
1928
O
ASN
A
304
221.449
86.208
208.929
1.00
67.98
O


ATOM
1929
CB
ASN
A
304
222.312
88.291
211.016
1.00
83.64
C


ATOM
1930
CG
ASN
A
304
222.000
88.806
212.421
1.00
112.22
C


ATOM
1931
OD1
ASN
A
304
222.399
89.919
212.802
1.00
84.27
O


ATOM
1932
ND2
ASN
A
304
221.269
88.004
213.197
1.00
69.02
N


ATOM
1933
N
LEU
A
305
223.671
85.943
208.973
1.00
63.99
N


ATOM
1934
CA
LEU
A
305
223.711
85.458
207.605
1.00
54.76
C


ATOM
1935
C
LEU
A
305
223.590
83.952
207.666
1.00
73.10
C


ATOM
1936
O
LEU
A
305
222.929
83.313
206.841
1.00
58.22
O


ATOM
1937
CB
LEU
A
305
225.032
85.860
206.948
1.00
52.05
C


ATOM
1938
CG
LEU
A
305
225.127
87.316
206.481
1.00
58.75
C


ATOM
1939
CD1
LEU
A
305
226.511
87.621
205.932
1.00
69.80
C


ATOM
1940
CD2
LEU
A
305
224.075
87.560
205.421
1.00
47.92
C


ATOM
1941
N
LEU
A
306
224.240
83.401
208.681
1.00
57.81
N


ATOM
1942
CA
LEU
A
306
224.256
81.975
208.915
1.00
59.22
C


ATOM
1943
C
LEU
A
306
223.087
81.525
209.779
1.00
70.17
C


ATOM
1944
O
LEU
A
306
223.205
80.586
210.565
1.00
86.15
O


ATOM
1945
CB
LEU
A
306
225.581
81.582
209.559
1.00
54.83
C


ATOM
1946
CG
LEU
A
306
226.752
81.718
208.599
1.00
56.28
C


ATOM
1947
CD1
LEU
A
306
228.035
81.262
209.237
1.00
46.67
C


ATOM
1948
CD2
LEU
A
306
226.445
80.883
207.385
1.00
50.52
C


ATOM
1949
N
LYS
A
307
221.960
82.212
209.648
1.00
70.52
N


ATOM
1950
CA
LYS
A
307
220.772
81.832
210.394
1.00
64.84
C


ATOM
1951
C
LYS
A
307
220.251
80.593
209.676
1.00
65.05
C


ATOM
1952
O
LYS
A
307
220.153
80.570
208.449
1.00
73.22
O


ATOM
1953
CB
LYS
A
307
219.720
82.930
210.337
1.00
60.26
C


ATOM
1954
CG
LYS
A
307
220.137
84.247
210.952
1.00
76.35
C


ATOM
1955
CD
LYS
A
307
219.620
84.411
212.371
1.00
75.87
C


ATOM
1956
CE
LYS
A
307
220.487
83.696
213.389
1.00
92.33
C


ATOM
1957
NZ
LYS
A
307
219.963
83.886
214.773
1.00
94.73
N


ATOM
1958
N
THR
A
308
219.925
79.558
210.439
1.00
87.36
N


ATOM
1959
CA
THR
A
308
219.432
78.314
209.864
1.00
86.54
C


ATOM
1960
C
THR
A
308
218.057
78.489
209.219
1.00
80.07
C


ATOM
1961
O
THR
A
308
217.722
77.809
208.248
1.00
80.48
O


ATOM
1962
CB
THR
A
308
219.382
77.198
210.945
1.00
80.31
C


ATOM
1963
OG1
THR
A
308
220.615
76.457
210.929
1.00
85.50
O


ATOM
1964
CG2
THR
A
308
218.211
76.265
210.701
1.00
108.80
C


ATOM
1965
N
GLU
A
309
217.264
79.407
209.755
1.00
86.56
N


ATOM
1966
CA
GLU
A
309
215.934
79.647
209.217
1.00
80.34
C


ATOM
1967
C
GLU
A
309
215.964
80.820
208.253
1.00
80.14
C


ATOM
1968
O
GLU
A
309
216.353
81.933
208.614
1.00
72.43
O


ATOM
1969
CB
GLU
A
309
214.937
79.945
210.345
1.00
79.03
C


ATOM
1970
CG
GLU
A
309
213.461
79.716
209.976
1.00
107.32
C


ATOM
1971
CD
GLU
A
309
213.028
78.264
210.160
1.00
128.86
C


ATOM
1972
OE1
GLU
A
309
213.354
77.691
211.227
1.00
100.32
O


ATOM
1973
OE2
GLU
A
309
212.364
77.699
209.252
1.00
91.75
O


ATOM
1974
N
PRO
A
310
215.554
80.585
207.002
1.00
69.20
N


ATOM
1975
CA
PRO
A
310
215.536
81.644
205.988
1.00
75.56
C


ATOM
1976
C
PRO
A
310
214.955
82.953
206.540
1.00
73.13
C


ATOM
1977
O
PRO
A
310
215.697
83.886
206.848
1.00
70.20
O


ATOM
1978
CB
PRO
A
310
214.675
81.041
204.888
1.00
58.28
C


ATOM
1979
CG
PRO
A
310
215.049
79.598
204.957
1.00
61.31
C


ATOM
1980
CD
PRO
A
310
215.073
79.312
206.442
1.00
64.66
C


ATOM
1981
N
THR
A
311
213.633
83.004
206.683
1.00
57.71
N


ATOM
1982
CA
THR
A
311
212.952
84.188
207.194
1.00
63.44
C


ATOM
1983
C
THR
A
311
213.671
84.899
208.338
1.00
65.40
C


ATOM
1984
O
THR
A
311
213.424
86.078
208.585
1.00
70.08
O


ATOM
1985
CB
THR
A
311
211.552
83.839
207.666
1.00
52.68
C


ATOM
1986
OG1
THR
A
311
211.650
82.936
208.763
1.00
76.00
O


ATOM
1987
CG2
THR
A
311
210.781
83.179
206.574
1.00
44.79
C


ATOM
1988
N
GLN
A
312
214.551
84.198
209.043
1.00
61.23
N


ATOM
1989
CA
GLN
A
312
215.271
84.831
210.145
1.00
58.30
C


ATOM
1990
C
GLN
A
312
216.527
85.518
209.629
1.00
63.30
C


ATOM
1991
O
GLN
A
312
216.995
86.509
210.189
1.00
65.70
O


ATOM
1992
CB
GLN
A
312
215.675
83.798
211.202
1.00
78.77
C


ATOM
1993
CG
GLN
A
312
215.203
84.120
212.625
1.00
101.21
C


ATOM
1994
CD
GLN
A
312
215.952
83.321
213.691
1.00
105.49
C


ATOM
1995
OE1
GLN
A
312
217.112
83.612
214.004
1.00
110.73
O


ATOM
1996
NE2
GLN
A
312
215.294
82.305
214.243
1.00
82.13
N


ATOM
1997
N
ARG
A
313
217.057
84.974
208.545
1.00
75.75
N


ATOM
1998
CA
ARG
A
313
218.278
85.463
207.924
1.00
58.86
C


ATOM
1999
C
ARG
A
313
218.184
86.894
207.391
1.00
51.81
C


ATOM
2000
O
ARG
A
313
217.125
87.341
206.952
1.00
85.05
O


ATOM
2001
CB
ARG
A
313
218.663
84.495
206.807
1.00
52.47
C


ATOM
2002
CG
ARG
A
313
220.131
84.401
206.516
1.00
74.86
C


ATOM
2003
CD
ARG
A
313
220.355
83.299
205.506
1.00
84.86
C


ATOM
2004
NE
ARG
A
313
219.851
82.018
205.984
1.00
53.52
N


ATOM
2005
CZ
ARG
A
313
219.467
81.032
205.186
1.00
62.95
C


ATOM
2006
NH1
ARG
A
313
219.528
81.192
203.874
1.00
85.30
N


ATOM
2007
NH2
ARG
A
313
219.039
79.888
205.697
1.00
49.87
N


ATOM
2008
N
MET
A
314
219.318
87.590
207.430
1.00
75.49
N


ATOM
2009
CA
MET
A
314
219.450
88.981
206.980
1.00
62.90
C


ATOM
2010
C
MET
A
314
219.115
89.160
205.496
1.00
68.20
C


ATOM
2011
O
MET
A
314
219.267
88.229
204.694
1.00
78.91
O


ATOM
2012
CB
MET
A
314
220.888
89.463
207.248
1.00
79.70
C


ATOM
2013
CG
MET
A
314
221.145
90.951
206.994
1.00
74.01
C


ATOM
2014
SD
MET
A
314
222.873
91.447
207.330
1.00
84.44
S


ATOM
2015
CE
MET
A
314
222.742
92.071
208.957
1.00
104.75
C


ATOM
2016
N
THR
A
315
218.660
90.357
205.135
1.00
63.73
N


ATOM
2017
CA
THR
A
315
218.319
90.652
203.750
1.00
64.98
C


ATOM
2018
C
THR
A
315
219.470
91.347
203.030
1.00
68.09
C


ATOM
2019
O
THR
A
315
220.317
91.969
203.658
1.00
63.59
O


ATOM
2020
CB
THR
A
315
217.075
91.545
203.661
1.00
62.15
C


ATOM
2021
OG1
THR
A
315
217.322
92.813
204.286
1.00
58.05
O


ATOM
2022
CG2
THR
A
315
215.929
90.868
204.338
1.00
61.97
C


ATOM
2023
N
ILE
A
316
219.498
91.236
201.707
1.00
65.76
N


ATOM
2024
CA
ILE
A
316
220.566
91.854
200.935
1.00
66.14
C


ATOM
2025
C
ILE
A
316
220.564
93.351
201.167
1.00
71.26
C


ATOM
2026
O
ILE
A
316
221.618
93.981
201.192
1.00
67.36
O


ATOM
2027
CB
ILE
A
316
220.417
91.583
199.426
1.00
60.35
C


ATOM
2028
CG1
ILE
A
316
221.714
91.937
198.710
1.00
52.24
C


ATOM
2029
CG2
ILE
A
316
219.314
92.435
198.854
1.00
56.27
C


ATOM
2030
CD1
ILE
A
316
222.942
91.410
199.405
1.00
55.83
C


ATOM
2031
N
THR
A
317
219.381
93.926
201.346
1.00
60.50
N


ATOM
2032
CA
THR
A
317
219.301
95.356
201.591
1.00
58.94
C


ATOM
2033
C
THR
A
317
219.858
95.658
202.964
1.00
61.73
C


ATOM
2034
O
THR
A
317
220.533
96.659
203.161
1.00
69.91
O


ATOM
2035
CB
THR
A
317
217.865
95.875
201.554
1.00
68.58
C


ATOM
2036
OG1
THR
A
317
217.301
95.636
200.259
1.00
67.56
O


ATOM
2037
CG2
THR
A
317
217.844
97.364
201.860
1.00
50.05
C


ATOM
2038
N
GLU
A
318
219.577
94.794
203.923
1.00
54.23
N


ATOM
2039
CA
GLU
A
318
220.082
95.027
205.266
1.00
69.88
C


ATOM
2040
C
GLU
A
318
221.586
94.796
205.348
1.00
72.99
C


ATOM
2041
O
GLU
A
318
222.262
95.329
206.221
1.00
64.78
O


ATOM
2042
CB
GLU
A
318
219.336
94.138
206.269
1.00
82.03
C


ATOM
2043
CG
GLU
A
318
217.903
94.596
206.496
1.00
64.99
C


ATOM
2044
CD
GLU
A
318
217.152
93.742
207.486
1.00
85.92
C


ATOM
2045
OE1
GLU
A
318
216.415
94.327
208.315
1.00
91.71
O


ATOM
2046
OE2
GLU
A
318
217.288
92.497
207.424
1.00
88.17
O


ATOM
2047
N
PHE
A
319
222.103
94.005
204.421
1.00
75.36
N


ATOM
2048
CA
PHE
A
319
223.519
93.700
204.380
1.00
69.31
C


ATOM
2049
C
PHE
A
319
224.279
94.883
203.800
1.00
79.85
C


ATOM
2050
O
PHE
A
319
225.281
95.324
204.359
1.00
86.43
O


ATOM
2051
CB
PHE
A
319
223.744
92.455
203.523
1.00
78.91
C


ATOM
2052
CG
PHE
A
319
225.193
92.144
203.250
1.00
82.05
C


ATOM
2053
CD1
PHE
A
319
225.978
91.538
204.210
1.00
64.69
C


ATOM
2054
CD2
PHE
A
319
225.757
92.432
202.011
1.00
68.65
C


ATOM
2055
CE1
PHE
A
319
227.287
91.219
203.940
1.00
55.30
C


ATOM
2056
CE2
PHE
A
319
227.060
92.114
201.744
1.00
75.06
C


ATOM
2057
CZ
PHE
A
319
227.826
91.506
202.711
1.00
57.56
C


ATOM
2058
N
MET
A
320
223.805
95.404
202.679
1.00
64.36
N


ATOM
2059
CA
MET
A
320
224.489
96.527
202.066
1.00
69.16
C


ATOM
2060
C
MET
A
320
224.525
97.753
202.973
1.00
78.25
C


ATOM
2061
O
MET
A
320
225.304
98.673
202.745
1.00
86.13
O


ATOM
2062
CB
MET
A
320
223.848
96.889
200.725
1.00
73.27
C


ATOM
2063
CG
MET
A
320
224.120
95.877
199.635
1.00
72.98
C


ATOM
2064
SD
MET
A
320
225.832
95.315
199.691
1.00
87.32
S


ATOM
2065
CE
MET
A
320
226.586
96.413
198.528
1.00
70.20
C


ATOM
2066
N
ASN
A
321
223.695
97.772
204.006
1.00
77.28
N


ATOM
2067
CA
ASN
A
321
223.691
98.907
204.912
1.00
79.74
C


ATOM
2068
C
ASN
A
321
224.489
98.692
206.174
1.00
76.50
C


ATOM
2069
O
ASN
A
321
224.868
99.651
206.836
1.00
95.86
O


ATOM
2070
CB
ASN
A
321
222.271
99.296
205.267
1.00
79.55
C


ATOM
2071
CG
ASN
A
321
221.783
100.428
204.427
1.00
104.33
C


ATOM
2072
OD1
ASN
A
321
222.191
101.578
204.622
1.00
115.09
O


ATOM
2073
ND2
ASN
A
321
220.926
100.121
203.456
1.00
119.81
N


ATOM
2074
N
HIS
A
322
224.744
97.439
206.521
1.00
82.50
N


ATOM
2075
CA
HIS
A
322
225.527
97.179
207.707
1.00
80.78
C


ATOM
2076
C
HIS
A
322
226.837
97.948
207.555
1.00
84.39
C


ATOM
2077
O
HIS
A
322
227.483
97.895
206.506
1.00
83.28
O


ATOM
2078
CB
HIS
A
322
225.812
95.691
207.851
1.00
75.27
C


ATOM
2079
CG
HIS
A
322
226.690
95.370
209.013
1.00
76.67
C


ATOM
2080
ND1
HIS
A
322
227.927
95.950
209.189
1.00
84.96
N


ATOM
2081
CD2
HIS
A
322
226.504
94.548
210.070
1.00
67.42
C


ATOM
2082
CE1
HIS
A
322
228.464
95.502
210.308
1.00
85.58
C


ATOM
2083
NE2
HIS
A
322
227.622
94.649
210.862
1.00
86.63
N


ATOM
2084
N
PRO
A
323
227.234
98.686
208.603
1.00
91.61
N


ATOM
2085
CA
PRO
A
323
228.447
99.501
208.680
1.00
84.82
C


ATOM
2086
C
PRO
A
323
229.697
98.867
208.087
1.00
79.23
C


ATOM
2087
O
PRO
A
323
230.318
99.436
207.193
1.00
85.59
O


ATOM
2088
CB
PRO
A
323
228.567
99.772
210.170
1.00
74.59
C


ATOM
2089
CG
PRO
A
323
227.135
99.952
210.553
1.00
86.91
C


ATOM
2090
CD
PRO
A
323
226.484
98.767
209.868
1.00
92.14
C


ATOM
2091
N
TRP
A
324
230.066
97.692
208.578
1.00
81.53
N


ATOM
2092
CA
TRP
A
324
231.247
97.002
208.070
1.00
80.47
C


ATOM
2093
C
TRP
A
324
231.260
97.039
206.541
1.00
76.75
C


ATOM
2094
O
TRP
A
324
232.288
97.293
205.917
1.00
63.50
O


ATOM
2095
CB
TRP
A
324
231.225
95.553
208.537
1.00
79.62
C


ATOM
2096
CG
TRP
A
324
232.549
94.881
208.546
1.00
68.42
C


ATOM
2097
CD1
TRP
A
324
233.579
95.118
209.408
1.00
97.23
C


ATOM
2098
CD2
TRP
A
324
232.973
93.806
207.704
1.00
91.77
C


ATOM
2099
NE1
TRP
A
324
234.614
94.254
209.162
1.00
96.32
N


ATOM
2100
CE2
TRP
A
324
234.268
93.437
208.120
1.00
90.15
C


ATOM
2101
CE3
TRP
A
324
232.382
93.118
206.639
1.00
85.73
C


ATOM
2102
CZ2
TRP
A
324
234.979
92.411
207.515
1.00
55.96
C


ATOM
2103
CZ3
TRP
A
324
233.091
92.099
206.038
1.00
78.54
C


ATOM
2104
CH2
TRP
A
324
234.375
91.755
206.475
1.00
83.38
C


ATOM
2105
N
ILE
A
325
230.099
96.785
205.952
1.00
61.98
N


ATOM
2106
CA
ILE
A
325
229.956
96.764
204.513
1.00
63.34
C


ATOM
2107
C
ILE
A
325
229.832
98.156
203.920
1.00
81.46
C


ATOM
2108
O
ILE
A
325
230.518
98.483
202.956
1.00
101.97
O


ATOM
2109
CB
ILE
A
325
228.723
95.941
204.092
1.00
64.13
C


ATOM
2110
CG1
ILE
A
325
229.156
94.621
203.470
1.00
57.81
C


ATOM
2111
CG2
ILE
A
325
227.909
96.703
203.076
1.00
79.69
C


ATOM
2112
CD1
ILE
A
325
230.005
93.796
204.357
1.00
99.33
C


ATOM
2113
N
MET
A
326
228.971
98.986
204.497
1.00
86.84
N


ATOM
2114
CA
MET
A
326
228.766
100.327
203.962
1.00
97.80
C


ATOM
2115
C
MET
A
326
229.941
101.291
204.108
1.00
88.82
C


ATOM
2116
O
MET
A
326
230.023
102.271
203.375
1.00
96.42
O


ATOM
2117
CB
MET
A
326
227.509
100.951
204.573
1.00
99.22
C


ATOM
2118
CG
MET
A
326
227.134
102.287
203.957
1.00
109.19
C


ATOM
2119
SD
MET
A
326
225.425
102.771
204.309
1.00
131.16
S


ATOM
2120
CE
MET
A
326
224.679
102.571
202.683
1.00
123.49
C


ATOM
2121
N
GLN
A
327
230.847
101.016
205.042
1.00
99.42
N


ATOM
2122
CA
GLN
A
327
232.005
101.880
205.253
1.00
100.47
C


ATOM
2123
C
GLN
A
327
233.330
101.120
205.290
1.00
109.31
C


ATOM
2124
O
GLN
A
327
234.060
101.184
206.279
1.00
116.79
O


ATOM
2125
CB
GLN
A
327
231.856
102.668
206.557
1.00
90.31
C


ATOM
2126
CG
GLN
A
327
230.780
103.743
206.538
1.00
124.03
C


ATOM
2127
CD
GLN
A
327
230.929
104.713
205.370
1.00
165.27
C


ATOM
2128
OE1
GLN
A
327
232.047
105.051
204.958
1.00
160.87
O


ATOM
2129
NE2
GLN
A
327
229.795
105.178
204.841
1.00
148.79
N


ATOM
2130
N
SER
A
328
233.644
100.406
204.215
1.00
109.39
N


ATOM
2131
CA
SER
A
328
234.888
99.643
204.157
1.00
117.56
C


ATOM
2132
C
SER
A
328
236.084
100.539
204.457
1.00
126.63
C


ATOM
2133
O
SER
A
328
236.892
100.239
205.332
1.00
122.39
O


ATOM
2134
CB
SER
A
328
235.055
99.008
202.777
1.00
106.48
C


ATOM
2135
OG
SER
A
328
233.976
98.139
202.490
1.00
101.32
O


ATOM
2136
N
THR
A
329
236.189
101.642
203.725
1.00
147.22
N


ATOM
2137
CA
THR
A
329
237.283
102.582
203.919
1.00
153.16
C


ATOM
2138
C
THR
A
329
237.668
102.713
205.393
1.00
151.36
C


ATOM
2139
O
THR
A
329
238.849
102.680
205.736
1.00
165.48
O


ATOM
2140
CB
THR
A
329
236.918
103.986
203.365
1.00
159.02
C


ATOM
2141
OG1
THR
A
329
237.929
104.930
203.748
1.00
164.35
O


ATOM
2142
CG2
THR
A
329
235.553
104.443
203.893
1.00
171.23
C


ATOM
2143
N
LYS
A
330
236.670
102.840
206.262
1.00
138.67
N


ATOM
2144
CA
LYS
A
330
236.916
102.994
207.691
1.00
128.38
C


ATOM
2145
C
LYS
A
330
236.937
101.670
208.457
1.00
134.38
C


ATOM
2146
O
LYS
A
330
236.425
101.586
209.580
1.00
138.61
O


ATOM
2147
CB
LYS
A
330
235.867
103.931
208.298
1.00
134.91
C


ATOM
2148
N
VAL
A
331
237.526
100.637
207.860
1.00
122.49
N


ATOM
2149
CA
VAL
A
331
237.608
99.340
208.531
1.00
120.28
C


ATOM
2150
C
VAL
A
331
239.066
98.890
208.689
1.00
111.19
C


ATOM
2151
O
VAL
A
331
239.850
98.899
207.739
1.00
85.41
O


ATOM
2152
CB
VAL
A
331
236.770
98.239
207.785
1.00
120.15
C


ATOM
2153
CG1
VAL
A
331
237.342
97.971
206.422
1.00
125.69
C


ATOM
2154
CG2
VAL
A
331
236.740
96.946
208.595
1.00
124.20
C


ATOM
2155
N
PRO
A
332
239.433
98.496
209.916
1.00
102.76
N


ATOM
2156
CA
PRO
A
332
240.737
98.021
210.385
1.00
105.52
C


ATOM
2157
C
PRO
A
332
241.528
97.215
209.363
1.00
112.97
C


ATOM
2158
O
PRO
A
332
241.016
96.262
208.779
1.00
111.93
O


ATOM
2159
CB
PRO
A
332
240.369
97.193
211.609
1.00
105.70
C


ATOM
2160
CG
PRO
A
332
239.247
97.970
212.184
1.00
122.25
C


ATOM
2161
CD
PRO
A
332
238.418
98.332
210.971
1.00
100.61
C


ATOM
2162
N
GLN
A
333
242.782
97.604
209.154
1.00
109.39
N


ATOM
2163
CA
GLN
A
333
243.645
96.908
208.206
1.00
106.56
C


ATOM
2164
C
GLN
A
333
244.265
95.714
208.913
1.00
99.10
C


ATOM
2165
O
GLN
A
333
245.347
95.251
208.554
1.00
97.72
O


ATOM
2166
CB
GLN
A
333
244.744
97.842
207.696
1.00
117.29
C


ATOM
2167
CG
GLN
A
333
244.224
99.042
206.922
1.00
121.73
C


ATOM
2168
CD
GLN
A
333
244.771
99.095
205.510
1.00
144.41
C


ATOM
2169
OE1
GLN
A
333
244.442
99.996
204.735
1.00
153.63
O


ATOM
2170
NE2
GLN
A
333
245.613
98.124
205.165
1.00
138.94
N


ATOM
2171
N
THR
A
334
243.565
95.229
209.931
1.00
88.60
N


ATOM
2172
CA
THR
A
334
244.023
94.081
210.693
1.00
112.85
C


ATOM
2173
C
THR
A
334
244.310
92.878
209.797
1.00
123.95
C


ATOM
2174
O
THR
A
334
243.438
92.392
209.086
1.00
128.29
O


ATOM
2175
CB
THR
A
334
242.989
93.668
211.738
1.00
105.19
C


ATOM
2176
OG1
THR
A
334
243.011
92.243
211.887
1.00
102.93
O


ATOM
2177
CG2
THR
A
334
241.607
94.120
211.315
1.00
106.97
C


ATOM
2178
N
PRO
A
335
245.549
92.372
209.832
1.00
124.58
N


ATOM
2179
CA
PRO
A
335
245.911
91.224
209.005
1.00
124.62
C


ATOM
2180
C
PRO
A
335
245.058
89.999
209.317
1.00
119.67
C


ATOM
2181
O
PRO
A
335
244.513
89.878
210.416
1.00
108.06
O


ATOM
2182
CB
PRO
A
335
247.383
91.002
209.358
1.00
134.66
C


ATOM
2183
CG
PRO
A
335
247.857
92.390
209.673
1.00
120.03
C


ATOM
2184
CD
PRO
A
335
246.730
92.881
210.547
1.00
120.76
C


ATOM
2185
N
LEU
A
336
244.926
89.110
208.333
1.00
110.96
N


ATOM
2186
CA
LEU
A
336
244.175
87.867
208.499
1.00
98.45
C


ATOM
2187
C
LEU
A
336
245.087
86.726
208.066
1.00
87.77
C


ATOM
2188
O
LEU
A
336
245.851
86.877
207.117
1.00
92.64
O


ATOM
2189
CB
LEU
A
336
242.909
87.852
207.632
1.00
95.92
C


ATOM
2190
CG
LEU
A
336
241.694
88.725
207.963
1.00
81.15
C


ATOM
2191
CD1
LEU
A
336
241.641
88.972
209.461
1.00
94.01
C


ATOM
2192
CD2
LEU
A
336
241.773
90.034
207.215
1.00
82.79
C


ATOM
2193
N
HIS
A
337
244.999
85.595
208.762
1.00
86.05
N


ATOM
2194
CA
HIS
A
337
245.813
84.410
208.469
1.00
100.05
C


ATOM
2195
C
HIS
A
337
245.401
83.709
207.170
1.00
103.25
C


ATOM
2196
O
HIS
A
337
246.075
82.782
206.712
1.00
110.52
O


ATOM
2197
CB
HIS
A
337
245.687
83.390
209.610
1.00
118.04
C


ATOM
2198
CG
HIS
A
337
246.205
83.874
210.930
1.00
151.49
C


ATOM
2199
ND1
HIS
A
337
246.241
85.210
211.271
1.00
169.76
N


ATOM
2200
CD2
HIS
A
337
246.675
83.198
212.010
1.00
160.51
C


ATOM
2201
CE1
HIS
A
337
246.713
85.337
212.502
1.00
129.99
C


ATOM
2202
NE2
HIS
A
337
246.984
84.132
212.971
1.00
150.26
N


ATOM
2203
N
THR
A
338
244.294
84.167
206.589
1.00
111.23
N


ATOM
2204
CA
THR
A
338
243.713
83.590
205.375
1.00
105.16
C


ATOM
2205
C
THR
A
338
244.649
82.822
204.440
1.00
106.58
C


ATOM
2206
O
THR
A
338
244.708
81.589
204.504
1.00
102.89
O


ATOM
2207
CB
THR
A
338
242.957
84.658
204.557
1.00
100.36
C


ATOM
2208
OG1
THR
A
338
242.218
85.494
205.451
1.00
103.82
O


ATOM
2209
CG2
THR
A
338
241.961
83.993
203.605
1.00
97.07
C


ATOM
2210
N
SER
A
339
245.370
83.532
203.573
1.00
100.67
N


ATOM
2211
CA
SER
A
339
246.276
82.877
202.622
1.00
106.75
C


ATOM
2212
C
SER
A
339
246.957
81.641
203.202
1.00
117.17
C


ATOM
2213
O
SER
A
339
246.862
80.555
202.634
1.00
126.71
O


ATOM
2214
CB
SER
A
339
247.341
83.855
202.137
1.00
100.87
C


ATOM
2215
OG
SER
A
339
246.771
84.880
201.348
1.00
116.32
O


ATOM
2216
N
ARG
A
340
247.643
81.811
204.329
1.00
108.64
N


ATOM
2217
CA
ARG
A
340
248.325
80.697
204.971
1.00
101.87
C


ATOM
2218
C
ARG
A
340
247.391
79.508
205.121
1.00
94.03
C


ATOM
2219
O
ARG
A
340
247.554
78.480
204.454
1.00
80.82
O


ATOM
2220
CB
ARG
A
340
248.848
81.112
206.347
1.00
117.73
C


ATOM
2221
CG
ARG
A
340
250.041
82.055
206.292
1.00
162.36
C


ATOM
2222
CD
ARG
A
340
249.770
83.353
207.068
1.00
208.75
C


ATOM
2223
NE
ARG
A
340
249.607
83.107
208.504
1.00
216.90
N


ATOM
2224
CZ
ARG
A
340
249.378
84.053
209.416
1.00
203.42
C


ATOM
2225
NH1
ARG
A
340
249.279
85.330
209.055
1.00
186.56
N


ATOM
2226
NH2
ARG
A
340
249.255
83.719
210.696
1.00
191.24
N


ATOM
2227
N
VAL
A
341
246.412
79.653
206.004
1.00
84.97
N


ATOM
2228
CA
VAL
A
341
245.455
78.585
206.239
1.00
88.40
C


ATOM
2229
C
VAL
A
341
244.913
78.128
204.891
1.00
93.56
C


ATOM
2230
O
VAL
A
341
244.917
76.939
204.566
1.00
86.14
O


ATOM
2231
CB
VAL
A
341
244.269
79.077
207.078
1.00
86.60
C


ATOM
2232
CG1
VAL
A
341
243.692
77.928
207.866
1.00
93.99
C


ATOM
2233
CG2
VAL
A
341
244.700
80.214
207.975
1.00
87.95
C


ATOM
2234
N
LEU
A
342
244.463
79.101
204.107
1.00
98.12
N


ATOM
2235
CA
LEU
A
342
243.887
78.847
202.798
1.00
97.33
C


ATOM
2236
C
LEU
A
342
244.771
77.929
201.976
1.00
103.21
C


ATOM
2237
O
LEU
A
342
244.280
77.134
201.181
1.00
108.03
O


ATOM
2238
CB
LEU
A
342
243.666
80.171
202.065
1.00
101.09
C


ATOM
2239
CG
LEU
A
342
242.513
80.212
201.065
1.00
102.01
C


ATOM
2240
CD1
LEU
A
342
241.211
79.865
201.761
1.00
89.86
C


ATOM
2241
CD2
LEU
A
342
242.428
81.595
200.463
1.00
76.62
C


ATOM
2242
N
LYS
A
343
246.078
78.031
202.177
1.00
118.91
N


ATOM
2243
CA
LYS
A
343
246.980
77.154
201.447
1.00
128.99
C


ATOM
2244
C
LYS
A
343
247.211
75.800
202.144
1.00
129.81
C


ATOM
2245
O
LYS
A
343
247.730
74.859
201.559
1.00
124.36
O


ATOM
2246
CB
LYS
A
343
248.308
77.894
201.320
1.00
131.78
C


ATOM
2247
CG
LYS
A
343
249.233
77.265
200.274
1.00
133.47
C


ATOM
2248
CD
LYS
A
343
250.528
78.061
200.106
1.00
161.36
C


ATOM
2249
CE
LYS
A
343
251.591
77.288
199.318
1.00
189.16
C


ATOM
2250
NZ
LYS
A
343
252.882
77.968
199.425
1.00
166.57
N


ATOM
2251
N
GLU
A
344
246.842
75.714
203.442
1.00
128.07
N


ATOM
2252
CA
GLU
A
344
247.254
74.549
204.240
1.00
135.55
C


ATOM
2253
C
GLU
A
344
246.429
73.272
203.985
1.00
137.12
C


ATOM
2254
O
GLU
A
344
246.915
72.156
204.102
1.00
147.11
O


ATOM
2255
CB
GLU
A
344
247.196
74.935
205.720
1.00
144.51
C


ATOM
2256
CG
GLU
A
344
248.480
74.589
206.485
1.00
182.52
C


ATOM
2257
CD
GLU
A
344
249.414
75.782
206.484
1.00
205.75
C


ATOM
2258
OE1
GLU
A
344
249.252
76.650
207.336
1.00
195.59
O


ATOM
2259
OE2
GLU
A
344
250.270
75.861
205.607
1.00
217.38
O


ATOM
2260
N
ASP
A
345
245.127
73.456
203.702
1.00
134.97
N


ATOM
2261
CA
ASP
A
345
244.310
72.298
203.321
1.00
153.39
C


ATOM
2262
C
ASP
A
345
243.429
72.611
202.107
1.00
164.28
C


ATOM
2263
O
ASP
A
345
243.779
73.413
201.250
1.00
177.09
O


ATOM
2264
CB
ASP
A
345
243.435
71.892
204.514
1.00
147.36
C


ATOM
2265
CG
ASP
A
345
242.851
70.495
204.291
1.00
174.56
C


ATOM
2266
OD1
ASP
A
345
243.455
69.731
203.533
1.00
191.84
O


ATOM
2267
OD2
ASP
A
345
241.820
70.182
204.883
1.00
169.86
O


TER
2267

ASP
A
345


ATOM
1
N
LEU
C
4
242.399
73.184
199.539
1.00
15.00


ATOM
2
CA
LEU
C
4
240.997
73.382
199.193
1.00
15.00


ATOM
3
CB
LEU
C
4
240.688
74.874
199.065
1.00
15.00


ATOM
4
CG
LEU
C
4
239.462
75.245
198.226
1.00
15.00


ATOM
5
CD1
LEU
C
4
238.302
75.613
199.137
1.00
15.00


ATOM
6
CD2
LEU
C
4
239.801
76.397
197.295
1.00
15.00


ATOM
7
C
LEU
C
4
240.650
72.668
197.890
1.00
15.00


ATOM
8
O
LEU
C
4
241.075
72.982
196.773
1.00
22.73


ATOM
9
N
GLN
C
5
239.765
71.671
197.981
1.00
15.00


ATOM
10
CA
GLN
C
5
239.268
70.862
196.875
1.00
15.00


ATOM
11
CB
GLN
C
5
238.690
69.547
197.399
1.00
15.00


ATOM
12
CG
GLN
C
5
238.232
69.602
198.847
1.00
15.00


ATOM
13
CD
GLN
C
5
238.424
68.283
199.571
1.00
15.00


ATOM
14
OE1
GLN
C
5
237.494
67.758
200.184
1.00
15.00


ATOM
15
NE2
GLN
C
5
239.634
67.743
199.504
1.00
15.00


ATOM
16
C
GLN
C
5
238.205
71.615
196.082
1.00
15.00


ATOM
17
O
GLN
C
5
237.316
72.263
196.691
1.00
15.04


ATOM
18
N
ARG
C
6
238.236
71.564
194.739
1.00
18.35
N


ATOM
19
CA
ARG
C
6
237.243
72.209
193.873
1.00
18.05
C


ATOM
20
C
ARG
C
6
235.863
71.634
194.126
1.00
17.37
C


ATOM
21
O
ARG
C
6
235.711
70.424
194.303
1.00
16.29
O


ATOM
22
CB
ARG
C
6
237.578
72.015
192.398
1.00
20.66
C


ATOM
23
CG
ARG
C
6
238.842
72.678
191.953
1.00
23.47
C


ATOM
24
CD
ARG
C
6
238.840
72.882
190.445
1.00
23.04
C


ATOM
25
NE
ARG
C
6
237.880
73.892
189.981
1.00
22.74
N


ATOM
26
CZ
ARG
C
6
237.975
75.204
190.210
1.00
20.22
C


ATOM
27
NH1
ARG
C
6
238.987
75.699
190.913
1.00
19.12
N


ATOM
28
NH2
ARG
C
6
237.069
76.031
189.710
1.00
20.28
N


ATOM
29
N
GLN
C
7
234.864
72.520
194.186
1.00
15.00


ATOM
30
CA
GLN
C
7
233.488
72.105
194.427
1.00
15.00


ATOM
31
CB
GLN
C
7
232.904
72.869
195.617
1.00
15.00


ATOM
32
CG
GLN
C
7
233.852
72.996
196.798
1.00
15.00


ATOM
33
CD
GLN
C
7
233.258
73.799
197.940
1.00
15.00


ATOM
34
OE1
GLN
C
7
232.067
74.116
197.937
1.00
15.00


ATOM
35
NE2
GLN
C
7
234.086
74.132
198.923
1.00
15.00


ATOM
36
C
GLN
C
7
232.623
72.336
193.191
1.00
15.00


ATOM
37
O
GLN
C
7
232.724
73.384
192.538
1.00
20.09


ATOM
38
N
LEU
C
8
231.805
71.324
192.833
1.00
15.00


ATOM
39
CA
LEU
C
8
230.922
71.415
191.677
1.00
15.00


ATOM
40
CB
LEU
C
8
231.160
70.233
190.734
1.00
15.00


ATOM
41
CG
LEU
C
8
231.208
70.558
189.238
1.00
15.00


ATOM
42
CD1
LEU
C
8
232.592
70.252
188.686
1.00
15.00


ATOM
43
CD2
LEU
C
8
230.147
69.759
188.500
1.00
15.00


ATOM
44
C
LEU
C
8
229.459
71.446
192.108
1.00
15.00


ATOM
45
O
LEU
C
8
229.098
70.977
193.190
1.00
13.73


ATOM
46
N
SER
C
9
228.611
72.005
191.242
1.00
13.08
N


ATOM
47
CA
SER
C
9
227.183
72.104
191.526
1.00
12.87
C


ATOM
48
C
SER
C
9
226.402
70.843
191.151
1.00
13.16
C


ATOM
49
O
SER
C
9
226.930
69.943
190.490
1.00
14.95
O


ATOM
50
CB
SER
C
9
226.569
73.323
190.823
1.00
14.61
C


ATOM
51
OG
SER
C
9
226.326
73.072
189.444
1.00
14.77
O


ATOM
52
N
ILE
C
10
225.174
70.780
191.613
1.00
15.00


ATOM
53
CA
ILE
C
10
224.249
69.694
191.311
1.00
15.00


ATOM
54
CB
ILE
C
10
223.837
68.939
192.591
1.00
15.00


ATOM
55
CG2
ILE
C
10
225.025
68.817
193.532
1.00
15.00


ATOM
56
CG1
ILE
C
10
222.681
69.664
193.280
1.00
15.00


ATOM
57
CD1
ILE
C
10
222.397
69.168
194.682
1.00
15.00


ATOM
58
C
ILE
C
10
222.992
70.220
190.626
1.00
15.00


ATOM
59
O
ILE
C
10
222.755
71.452
190.654
1.00
12.25


ATOM
60
N
ALA
C
11
222.224
69.342
190.029
1.00
14.49
N


ATOM
61
CA
ALA
C
11
220.983
69.676
189.342
1.00
16.79
C


ATOM
62
C
ALA
C
11
220.122
68.421
189.365
1.00
19.44
C


ATOM
63
O
ALA
C
11
220.552
67.360
188.901
1.00
19.58
O


ATOM
64
CB
ALA
C
11
221.252
70.125
187.902
1.00
15.87
C


TER
64

ALA
C
11


END
















TABLE 2







Pairs of contacting atoms in the MAPKAP kinase-2/peptide complex











Atom in activated





MAPKAP kinase-2
Atom in peptide
Distance







ILE74 CD1
SER9 OG
2.90275



GLU145 OE1
ARG6 NH2
1.94903



LYS188 NZ
SER9 CB
2.48016



GLU190 CD
GLN7 O
3.33866



PHE210 CE2
SER9 OG
2.78278



PHE210 CZ
ILE10 O
2.37786



CYS224 SG
ALA11 CA
3.36808



TYR225 O
SER9 CA
3.25655



TYR225 O
ILE10 N
2.88016



THR 226 OG1
GLN 7 OE1
3.42713



PRO 227 CD
LEU 8 O
3.44686



TYR 228 CB
GLN 7 OE1
3.02662



TYR 229 CE1
GLN 7 OE1
3.34283



ASP 345 O
LEU 4 N
2.21006










RNA interference (RNAi) and recombinant DNA. siRNA duplexes consisting of twenty-one base pairs with a two-base deoxynucleotide overhang were purchased from Dharmacon Research. Cells were transfected with siRNAs using oligofectamine (Invitrogen) according to the manufacturer's instructions. Cells were typically harvested for further experiments after forty-eight hours. U2OS cells stably expressing shRNA constructs were generated by lentiviral gene transfer. The RNAi hairpins were cloned into the multiple cloning site of the lentiviral transfer vector pLentiLox-3.7puro or -3.7GFP. Amphotropic VSV-G pseudotyped lentivirus was used for all infections in a BL2+ facility. All transfer and packaging constructs were a kind gift from C.P. Dillon, (MIT). Targeted cells were selected in 8 μg/ml puromycin for four days. Sequences used for RNAi were: luciferase (shRNA), 5′pTGA CCA GGC ATT CAC AGA AAT TCA AGA GAT TTC TGT GAA TGC CTG GTC TTT TTT C-3′ (SEQ ID NO: 24); hMAPKAP kinase-2 (shRNA), 5′-pTTG ACC ATC ACC GAG TTT ATT TCA AGA GAA TA AAC TCG GTG ATG GTC ATT TTT TC-3′ (SEQ ID NO: 25); mMAPKAP kinase-2 (shRNA), 5′-pTCG ATG CGT GTT GAC TAT GAT TCA AGA GAT CAT AGT CAA CAC GCA TCG TTT TTT C-3′ (SEQ ID NO: 26); GFP (siRNA) sense 5′-UCC CGG CUA UGU GCA GGA GdTdT-3′ (SEQ ID NO: 27) and antisense strand 5′-CUCCUG CAC AUA GCC GGG AdTdT-3′ (SEQ ID NO: 28); mMAPKAP kinase-2 (siRNA), sense 5′-CGA UGC GUG UUG ACU AUG AdTdT-3′ (SEQ ID NO: 29) and antisense strand 5′-UCA UAG UCA ACA CGC AUC GdTdT-3′ (SEQ ID NO: 30); hMAPKAP kinase-2 (siRNA), sense 5′-UGA CCA UCA CCG AGU UWA UdTdT-3′ (SEQ ID NO: 31) and anti-sense strand 5′-AUA AAC UCG GUG AUG GUC AdTdT-3′ (SEQ ID NO: 32); Chk1 (siRNA), 5′-UGG CAA CAG UAU UUC GGU AdTdT-3′ (SEQ ID NO: 33) and antisense strand 5′-UAC CGA AAU ACU GUU GCC AdTdT-3′ (SEQ ID NO: 34).
For overexpression studies, FLAG-6×His-tagged human Chk1 cDNA was PCR amplified and subcloned into the Mlu-1 and Not-1 sites of pHURRA downstream from the CMV promoter. pHURRA was a kind gift from Dr. H. Pavenstadt (U. of Munster).
Therapy
Therapy according to the invention may be performed alone or in conjunction with another therapy and may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment generally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed. The duration of the therapy depends on the age and condition of the patient, the stage of the patient's disease or disorder, and how the patient responds to the treatment. Additionally, a person having a greater risk of developing a disease or disorder that may be treated by the methods of the invention (e.g., a person who is genetically predisposed) may receive prophylactic treatment to inhibit or delay symptoms of the disease. Drug administration may be performed at different intervals (e.g., daily, weekly, or monthly). Therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to build healthy new cells and regain its strength. Therapy may be used to extend the patient's lifespan.
For cancer treatment, depending on the type of cancer and its stage of development, the therapy can be used to slow the spreading of the cancer, to slow the cancer's growth, to kill or arrest cancer cells that may have spread to other parts of the body from the original tumor, to relieve symptoms caused by the cancer, or to prevent cancer in the first place.
Administration of Therapeutic Compounds
By selectively disrupting or preventing a compound from binding to its natural partner(s) through its binding site, one may inhibit the biological activity or the biological function of a MAPKAP kinase-2 polypeptide. The methods of the invention feature the use of compounds that inhibit an activity of a MAPKAP kinase-2 polypeptide, whether by reducing expression of the polypeptide (e.g., RNAi or antisense therapy), or by binding directly to a MAPKAP kinase-2 polypeptide and inhibiting its substrate-binding activity. In some instances, MAPKAP kinase-2 inhibitory compounds are administered to patients having one or more p53-deficient cells, e.g., tumor cells. Exemplary inhibitory compounds will be described further below.
Diseases or disorders characterized by inappropriate cell cycle regulation include cellular proliferative disorders, such as neoplasias. Examples of neoplasias include, without limitation, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, acute erythroleukemia, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, colon cancer, colon carcinoma, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing's tumor, glioma, heavy chain disease, hemangioblastoma, hepatoma, Hodgkin's disease, large cell carcinoma, leiomyosarcoma, liposarcoma, lung cancer, lung carcinoma, lymphangioendotheliosarcoma, lymphangiosarcoma, macroglobulinemia, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, neuroblastoma, non-Hodgkin's disease, oligodendroglioma, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rhabdomyosarcoma, renal cell carcinoma, retinoblastoma, schwannoma, sebaceous gland carcinoma, seminoma, small cell lung carcinoma, squamous cell carcinoma, sweat gland carcinoma, synovioma, testicular cancer, uterine cancer, Waldenstrom's fibrosarcoma, and Wilm's tumor. Any of these diseases or disorders can include, or be associated with, one or more p53-deficient cells, e.g., tumor cells.
A MAPKAP kinase-2-binding peptide, small molecule, or other compound may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease that is caused by excessive cell proliferation. Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Combination Therapy
As described above, if desired, treatment with compounds that inhibit MAPKAP kinase-2 polypeptides may be combined with therapies for the treatment of proliferative disease, such as radiotherapy, surgery, or chemotherapy. Chemotherapeutic agents that may be administered with compounds (e.g., UCN-01) that interact with a MAPKAP kinase-2 polypeptide include, but are not limited to, alemtuzumab, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, bicalutamide, busulfan, capecitabine, carboplatin, carmustine, celecoxib, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine phosphate, etodolac, etoposide, exemestane, floxuridine, fludarabine, 5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab, goserelin, hexamethylmelamine, hydroxyurea, hypericin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leuporelin, lomustine, mechlorethamine, melphalen, mercaptopurine, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, paclitaxel, pentostatin, procarbazine, raltitrexed, rituximab, rofecoxib, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, toremofine, trastuzumab, vinblastine, vincristine, vindesine, and vinorelbine. One or more chemotherapeutic agents may be administered in combination with one or more compounds that inhibit MAPKAP kinase-2 polypeptides. In some instances, combination therapy is administered to patients having one or more p53-deficient cells, e.g., tumor cells.
In the combination therapies of the invention, the therapy components are administered simultaneously, or within twenty-eight days of each other, in amounts sufficient to inhibit the growth of said neoplasm.
Depending on the type of cancer and its stage of development, the combination therapy can be used to treat cancer, to slow the spreading of the cancer, to slow the cancer's growth, to kill or arrest cancer cells that may have spread to other parts of the body from the original tumor, to relieve symptoms caused by the cancer, or to prevent cancer in the first place. Combination therapy can also help people live more comfortably by eliminating cancer cells that cause pain or discomfort.
The administration of a combination of the present invention allows for the administration of lower doses of each compound, providing similar efficacy and lower toxicity compared to administration of either compound alone. Alternatively, such combinations result in improved efficacy in treating neoplasms with similar or reduced toxicity.
RNA Interference Therapy
The invention features the novel and therapeutically important discovery that the use of RNA interference (RNAi) to reduce MAPKAP kinase-2 expression renders cells more susceptible to chemotherapeutic agents. Accordingly, using the methods of the invention, nucleobase oligomers may be employed in double-stranded RNAs for RNAi-mediated knockdown of MAPKAP kinase-2 expression. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002). In RNAi, gene silencing is typically triggered post-transcriptionally by the presence of double-stranded RNA (dsRNA) in a cell. This dsRNA is processed intracellularly into shorter pieces called small interfering RNAs (siRNAs). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.
In one embodiment of the invention, a double-stranded RNA (dsRNA) molecule is made. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 995515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference. Small hairpin RNAs consist of a stem-loop structure with optional 3′ UU-overhangs. While there may be variation, stems can range from twenty-one to thirty-one base pairs (desirably twenty-five to twenty-nine base pairs), and the loops can range from four to thirty base pairs (desirably four to twenty-three base pairs). For expression of shRNAs within cells, plasmid vectors containing, e.g., the polymerase III H1-RNA or U6 promoter, a cloning site for the stem-looped RNA insert, and a 4-5-thymidine transcription termination signal can be employed. The Polymerase III promoters generally have well-defined initiation and stop sites and their transcripts lack poly(A) tails. The termination signal for these promoters is defined by the polythymidine tract, and the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3′ UU overhang in the expressed shRNA, which is similar to the 3′ overhangs of synthetic siRNAs. Additional methods for expressing the shRNA in mammalian cells are described in the references cited above.
Computer programs that employ rational design of oligos are useful in predicting regions of the MAPKAP kinase-2 sequence that may be targeted by RNAi. For example, see Reynolds et al., Nat. Biotechnol., 22:326-330, 2004, for a description of the Dharmacon siDESIGN tool. Table 3 lists several exemplary nucleotide sequences within MAPKAP kinase-2 that may be targeted for purposes of RNA interference. siRNA or shRNA oligos may be made corresponding to the sequences shown and including an overhang, e.g., a 3′ dTdT overhang and/or a loop.







TABLE 3







MAPKAP kinase-2 RNAi target sequences










Sequence (5′ to 3′)
SEQ ID NO:















GACCAGGCATTCACAGAAA
35




 



TTGACCACTCCTTGTTATA
36



 



GACCACTCCTTGTTATACA
37



 



TGACCATCACCGAGTTTAT
38



 



TCACCGAGTTTATGAACCA
39



 



TCAAGAAGAACGCCATCAT
40



 



AAGCATCCGAAATCATGAA
41



 



AGTATCTGCATTCAATCAA
42



 



CTTTGACCACTCCTTGTTA
43



 



TTTGACCACTCCTTGTTAT
44



 



TACGGATCGTGGATGTGTA
45



 



GGACGGTGGAGAACTCTTT
46



 



CTTGTTATACACCGTACTA
47



 



GACGGTGGAGAACTCTTTA
48



 



GGAGAACTCTTTAGCCGAA
49










Antisense Therapy
As an alternative to RNAi-based approaches, therapeutic strategies utilizing MAPKAP kinase-2 antisense nucleic acids may be employed in the methods of the invention. The technique is based on the principle that sequence-specific suppression of gene expression can be achieved by intracellular hybridization between mRNA and a complementary antisense species. The formation of a hybrid RNA duplex may then interfere with the processing/transport/translation and/or stability of the target MAPKAP kinase-2 mRNA. Antisense strategies may use a variety of approaches, including the use of antisense oligonucleotides and injection of antisense RNA. An exemplary approach features transfection of antisense RNA expression vectors into targeted cells. Antisense effects can be induced by control (sense) sequences; however, the extent of phenotypic changes are highly variable. Phenotypic effects induced by antisense effects are based on changes in criteria such as protein levels, protein activity measurement, and target mRNA levels.
Computer programs such as OLIGO (previously distributed by National Biosciences Inc.) may be used to select candidate nucleobase oligomers for antisense therapy based on the following criteria:

    
    
        1) no more than 75% GC content, and no more than 75% AT content;
        2) preferably no nucleobase oligomers with four or more consecutive G residues (due to reported toxic effects, although one was chosen as a toxicity control);
        3) no nucleobase oligomers with the ability to form stable dimers or hairpin structures; and
        4) sequences around the translation start site are a preferred region.

In addition, accessible regions of the target mRNA may be predicted with the help of the RNA secondary structure folding program MFOLD (M. Zuker, D. H. Mathews & D. H. Turner, Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide. In: RNA Biochemistry and Biotechnology, J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers, (1999). Sub-optimal folds with a free energy value within 5% of the predicted most stable fold of the mRNA may be predicted using a window of 200 bases within which a residue can find a complimentary base to form a base pair bond. Open regions that do not form a base pair may be summed together with each suboptimal fold, and areas that consistently are predicted as open may be considered more accessible to the binding to nucleobase oligomers. Additional nucleobase oligomer that only partially fulfill some of the above selection criteria may also be chosen as possible candidates if they recognize a predicted open region of the target mRNA.

    
    


Therapeutically Useful Compounds and Pharmaceutical Compositions
Any compound or pharmaceutical composition that inhibits an activity of MAPKAP kinase-2 may be useful in the methods of the invention. The model of the activated MAPKAP kinase-2/peptide complex described above (Table 1) indicates that peptides, or peptide-like compounds, e.g., peptidomimetics, may be useful for inhibiting MAPKAP kinase-2. Such compounds achieve this effect by mimicking the natural peptide substrate of MAPKAP kinase-2 and decreasing the extent or rate with which a MAPKAP kinase-2 polypeptide is able to bind to its natural substrates in vivo. Accordingly, methods of synthesis or modification of peptides and peptide-like compounds is described below.
Peptide Synthesis and Conjugation
A compound of the invention that includes a peptide is prepared as detailed above. Alternatively, peptides can be prepared using standard FMOC chemistry on 2-chlorotrityl chloride resin (Int. J. Pept. Prot. Res. 38, 1991, 555-61). Cleavage from the resin is performed using 20% acetic acid in dichloromehane (DCM), which leaves the side chain still blocked. Free terminal carboxylate peptide is then coupled to 4′(aminomethy)-fluorescein (Molecular Probes, A-1351; Eugene, Oreg.) using excess diisopropylcarbodiimide (DIC) in dimethylformamide (DMF) at room temperature. The fluorescent N—C blocked peptide is purified by silica gel chromatography (10% methanol in DCM). The N terminal FMOC group is then removed using piperidine (20%) in DMF, and the N-free peptide, purified by silica gel chromatography (20% methanol in DCM, 0.5% HOAc). Finally, any t-butyl side chain protective groups are removed using 95% trifluoroacetic acid containing 2.5% water and 2.5% triisopropyl silane. The peptide obtained in such a manner should give a single peak by HPLC and is sufficiently pure for carrying on with the assay described below.
Peptide Modifications and Unnatural Amino Acids
It is understood that modifications can be made to the amino acid residues of the peptide-containing compounds of the invention, to enhance or prolong the therapeutic efficacy and/or bioavailability of the compound. Accordingly, α-amino acids having the following general formula (I):




where R defines the specific amino acid residue, may undergo various modifications. Exemplary modifications of α-amino acids, include, but are not limited to, the following formula (II):





R1, R2, R3, R4, and R5, are independently hydrogen, hydroxy, nitro, halo, C1-5 branched or linear alkyl, C1-5 alkaryl, heteroaryl, and aryl; wherein the alkyl, alkaryl, heteroaryl, and aryl may be unsubstituted or substituted by one or more substituents selected from the group consisting of C1-5 alkyl, hydroxy, halo, nitro, C1-5 alkoxy, C1-5 alkylthio, trihalomethyl, C1-5 acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, C1-5 alkoxycarbonyl, oxo, arylalkyl (wherein the alkyl group has from 1 to 5 carbon atoms) and heteroarylalkyl (wherein the alkyl group has from 1 to 5 carbon atoms); alternatively, R1 and R2 are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl; or R2 and R3 are joined to form a C3-8 cyclic ring, optionally substituted by hydroxyl and optionally including oxygen, sulfinur, C1-5 aminoalkyl, or C1-5 alkyl.

A compound of the invention that includes a peptide may include, but it is not limited to, an unnatural N-terminal amino acid of the formula (III):




where A1 is an amino acid or peptide chain linked via an α-amino group; R1 and R3 are independently hydrogen, C1-5 branched or linear C1-5 alkyl, C1-5 alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of C1-5 alkyl, 1 to 3 of halogen, 1 to 2 of —OR5, N(R5)(R6), SR5, N—C(NR5)NR6R7, methylenedioxy, —S(O)mR5, 1 to 2 of —CF3, —OCF3, nitro, —N(R5)C(O)(R6), —C(O)OR5, —C(O)N(R5)(R6), -1H-tetrazol-5-yl, —SO2N(R5)(R6), —N(R5)SO2 aryl, or —N(R5)SO2R6; R5, R6 and R7 are independently selected from hydrogen, C1-5 linear or branched alkyl, C1-5 alkaryl, aryl, heteroaryl, and C3-7 cycloalkyl, and where two C1-5 alkyl groups are present on one atom, they optionally are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl; R2 is hydrogen, F, C1-5 linear or branched alkyl, C1-5 alkaryl; or R2 and R1 are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur, or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl, or R2 and R3 are joined to form a C3-8 cyclic ring, optionally substituted by hydroxyl and optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl; R2 is hydrogen, F, C1-5 linear or branched alkyl, C1-5 alkaryl; and R4 is hydrogen, C1-5 branched or linear C1-5 alkyl, C1-5 alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of C1-5 alkyl, 1 to 3 of halogen, 1 to 2 of —OR5, N(R5)(R6), N—C(NR5)NR6R7, methylenedioxy, —S(O)mR5 (where m is 0-2), 1 to 2 of —CF3, —OCF3, nitro, —N(R5)C(O)(R6), —N(R5)C(O)(OR6), —C(O)OR5, —C(O)N(R5)(R6), -1H-tetrazol-5-yl, —SO2N(R5)(R6), —N(R5)SO2 aryl, or —N(R5)SO2R6, R5, R6 and R7 are independently selected from hydrogen, C1-5 linear or branched alkyl, C1-5 alkaryl, aryl, heteroaryl, and C3-7 cycloalkyl, and where two C1-5 alkyl groups are present on one atom, they optionally are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl.

A compound of the invention may also include an unnatural internal amino acid of the formula:




where A2 is an amino acid or peptide chain linked via an α-carboxy group; A1 is an amino acid or peptide chain linked via an α-amino group; R1 and R3 are independently hydrogen, C1-5 branched or linear C1-5 alkyl, C1-5 alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of C1-5 alkyl, 1 to 3 of halogen, 1 to 2 of —OR5, N(R5)(R6), SR5, N—C(NR5)NR6R7, methylenedioxy, —S(O)mR5 (m is 1-2), 1 to 2 of —CF3, —OCF3, nitro, —N(R5)C(O)(R6), —C(O)OR5, —C(O)N(R5)(R6), -1H-tetrazol-5-yl, —SO2N(R5)(R6), —N(R5)SO2 aryl, or —N(R5)SO2R6; R1, R6 and R7 are independently selected from hydrogen, C1-5 linear or branched alkyl, C1-5 alkaryl, aryl, heteroaryl, and C3-7 cycloalkyl, and where two C1-5 alkyl groups are present on one atom, they optionally are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl; and R2 is hydrogen, F, C1-5 linear or branched alkyl, C1-5 alkaryl; or R2 and R1 are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl, or R2 and R3 are joined to form a C3-8 cyclic ring, optionally substituted by hydroxyl and optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl.

The invention also includes modifications of the peptide-containing compounds of the invention, wherein an unnatural internal amino acid of the formula:




is present, where A2 is an amino acid or peptide chain linked via an α-carboxy group; A1 is an amino acid or peptide chain linked via an α-amino group; R1 and R3 are independently hydrogen, C1-5 branched or linear C1-5 alkyl, and C1-5 alkaryl; R2 is hydrogen, F, C1-5 linear or branched alkyl, C1-5 alkaryl; or R2 and R1 are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl; X is O or S; and R5 and R6 are independently selected from hydrogen, C1-5 linear or branched alkyl, C1-5 alkaryl, aryl, heteroaryl, and C3-7 cycloalkyl, and where two C1-5 alkyl groups are present on one atom, they optionally are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl; or R5 and R6 are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl.

A compound of the invention may also include a C-terminal unnatural internal amino acid of the formula:




where A2 is an amino acid or peptide chain linked via an α-carboxy group; R1 and R3 are independently hydrogen, C1-5 branched or linear C1-5 alkyl, C1-5 alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of C1-5 alkyl, 1 to 3 of halogen, 1 to 2 of —OR5, N(R5)(R6), SR5, N—C(NR5)NR6R7, methylenedioxy, S(O)mR5, 1 to 2 of —CF3, —OCF3, nitro, —N(R5)C(O)(R6), —C(O)OR5, —C(O)N(R5)(R6), -1H-tetrazol-5-yl, —SO2N(R5)(R6), —N(R5)SO2 aryl, or —N(R5)SO2R6; R5, R6 and R7 are independently selected from hydrogen, C1-5 linear or branched alkyl, C1-5 alkaryl, aryl, heteroaryl, and C3-7 cycloalkyl, and where two C1-5 alkyl groups are present on one atom, they optionally are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl; R2 is hydrogen, F, C1-5 linear or branched alkyl, C1-5 alkaryl; or R2 and R1 are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl; or R2 and R3 are joined to form a C3-8 cyclic ring, optionally substituted by hydroxyl and optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl; R2 is hydrogen, F, C1-5 linear or branched alkyl, C1-5 alkaryl; and Q is OH, OR5, or NR5R6, where R5, R6 are independently selected from hydrogen, C1-5 linear or branched alkyl, C1-5 alkaryl, aryl, heteroaryl, and C3-7 cycloalkyl, and where two C1-5 alkyl groups are present on one atom, they optionally are joined to form a C3-8 cyclic ring, optionally including oxygen, sulfur or NR7, where R7 is hydrogen, or C1-5 alkyl, optionally substituted by hydroxyl. Methods well known in the art for modifying peptides are found, for example, in “Remington: The Science and Practice of Pharmacy,” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia).

Peptidomimetics
Peptide derivatives (e.g., peptidomimetics) include cyclic peptides, peptides obtained by substitution of a natural amino acid residue by the corresponding D-stereoisomer, or by an unnatural amino acid residue, chemical derivatives of the peptides, dual peptides, multimers of the peptides, and peptides fused to other proteins or carriers. A cyclic derivative of a peptide of the invention is one having two or more additional amino acid residues suitable for cyclization. These residues are often added at the carboxyl terminus and at the amino terminus. A peptide derivative may have one or more amino acid residues replaced by the corresponding D-amino acid residue. In one example, a peptide or peptide derivative of the invention is all-L, all-D, or a mixed D,L-peptide. In another example, an amino acid residue is replaced by an unnatural amino acid residue. Examples of unnatural or derivatized unnatural amino acids include Na-methyl amino acids, Cα-methyl amino acids, and β-methyl amino acids.
A chemical derivative of a peptide of the invention includes, but is not limited to, a derivative containing additional chemical moieties not normally a part of the peptide. Examples of such derivatives include: (a) N-acyl derivatives of the amino terminal or of another free amino group, where the acyl group may be either an alkanoyl group, e.g., acetyl, hexanoyl, octanoyl, an aroyl group, e.g., benzoyl, or a blocking group such as Fmoc (fluorenylmethyl-O—CO—), carbobenzoxy(benzyl-O—CO—), monomethoxysuccinyl, naphthyl-NH—CO—, acetylamino-caproyl, adamantyl-NH—CO—; (b) esters of the carboxyl terminal or of another free carboxyl or hydroxy groups; (c) amides of the carboxyl terminal or of another free carboxyl groups produced by reaction with ammonia or with a suitable amine; (d) glycosylated derivatives; (e) phosphorylated derivatives; (f) derivatives conjugated to lipophilic moieties, e.g., caproyl, lauryl, stearoyl; and (g) derivatives conjugated to an antibody or other biological ligand. Also included among the chemical derivatives are those derivatives obtained by modification of the peptide bond —CO—NH—, for example, by: (a) reduction to —CH2—NH—; (b) alkylation to —CO—N(alkyl)—; and (c) inversion to —NH—CO—. Peptidomimetics may also comprise phosphonate or sulfonate moieties.
A dual peptide of the invention consists of two of the same, or two different, peptides of the invention covalently linked to one another, either directly or through a spacer.
Multimers of the invention consist of polymer molecules formed from a number of the same or different peptides or derivatives thereof.
In one example, a peptide derivative is more resistant to proteolytic degradation than the corresponding non-derivatized peptide. For example, a peptide derivative having D-amino acid substitution(s) in place of one or more L-amino acid residue(s) resists proteolytic cleavage.
In another example, the peptide derivative has increased permeability across a cell membrane as compared to the corresponding non-derivatized peptide. For example, a peptide derivative may have a lipophilic moiety coupled at the amino terminus and/or carboxyl terminus and/or an internal site. Such derivatives are highly preferred when targeting intracellular protein-protein interactions, provided they retain the desired functional activity.
In another example, a peptide derivative binds with increased affinity to a ligand (e.g., a MAPKAP kinase-2 polypeptide).
The peptides or peptide derivatives of the invention are obtained by any method of peptide synthesis known to those skilled in the art, including synthetic and recombinant techniques. For example, the peptides or peptide derivatives can be obtained by solid phase peptide synthesis which, in brief, consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids. The protecting groups may be any such groups known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed. The coupling of amino acids to appropriate resins has been described by Rivier et al. (U.S. Pat. No. 4,244,946). Such solid phase syntheses have been described, for example, by Merrifield, J. Am. Chem. Soc. 85:2149, 1964; Vale et al., Science 213:1394-1397, 1984; Marki et al., J. Am. Chem. Soc. 10:3178, 1981, and in U.S. Pat. Nos. 4,305,872 and 4,316,891. Desirably, an automated peptide synthesizer is employed.
Purification of the synthesized peptides or peptide derivatives is carried out by standard methods, including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, hydrophobicity, or by any other standard technique for the purification of proteins. In one embodiment, thin layer chromatography is employed. In another embodiment, reverse phase HPLC (high performance liquid chromatography) is employed.
Finally, structure-function relationships determined from the peptides, peptide derivatives, and other small molecules of the invention may also be used to prepare analogous molecular structures having similar properties. Thus, the invention is contemplated to include molecules in addition to those expressly disclosed that share the structure, hydrophobicity, charge characteristics and side chain properties of the specific embodiments exemplified herein.
In one example, such derivatives or analogs that have the desired binding activity can be used for binding to a molecule or other target of interest, such as any MAPKAP kinase-2 polypeptide. Derivatives or analogs that retain, or alternatively lack or inhibit, a desired property-of-interest (e.g., inhibit MAPKAP kinase-2 binding to a natural ligand), can be used to inhibit the biological activity of a MAPKAP kinase-2 polypeptide.
In particular, peptide derivatives are made by altering amino acid sequences by substitutions, additions, or deletions that provide for functionally equivalent molecules, or for functionally enhanced or diminished molecules, as desired. Due to the degeneracy of the genetic code, other nucleic acid sequences that encode substantially the same amino acid sequence may be used for the production of recombinant peptides. These include, but are not limited to, nucleotide sequences comprising all or portions of a peptide of the invention that is altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
The derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations that result in their production can occur at the gene or protein level. For example, a cloned nucleic acid sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
MAPKAP Kinase-2 Inhibitors
Based on the present discovery that RNAi knockdown of MAPKAP kinase-2 expression sensitizes cells to chemotherapeutic agents, any compound that inhibits MAPKAP kinase-2, whether specifically or nonspecifically, may be of utility in antineoplastic therapy. Suitable MAPKAP kinase-2 inhibitors that may be used in the methods and compositions of the invention include UCN-01, 2-(3-aminopropyl)-8-(methylthio)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylic acid dihydrochloride, 2-(3-aminopropyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylic acid hydrochloride, 2-(3-{[2-(4-bromophenyl)ethyl]amino}propyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylic acid hydrochloride, 2-(2-aminoethyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylic acid hydrochloride, 8-(allylthio)-2-(3-aminopropyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylic acid dihydrochloride, 2-(3-aminopropyl)-8-(benzylthio)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylic acid dihydrochloride, 2-{3-[(2-thien-2-ylethyl)amino]propyl}-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylic acid, 2-{3-[(2-thien-3-ylethyl)amino]propyl}-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylic acid hydrochloride, ethyl 2-(3-{[2-(4-bromophenyl)ethyl]amino}propyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylate, 2-(3-aminopropyl)-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid dihydrochloride, 2-(3-aminopropyl)-8-(1,3-benzodioxol-5-yl)-4,5-dihydro-2H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid hydrochloride, 2-(3-aminopropyl)-8-phenyl-4,5-dihydro-2H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid hydrochloride, 2-quinolin-3-yl-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one, 2-pyridin-3-yl-5,6,8,9,10,11-hexahydro-7H-(1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one, 8-quinolin-3-yl-2-[3-(tritylamino)propyl]-4,5-dihydro-2H-pyrazolo[4,3-h]quinazoline-3-carboxylic acid hydrochloride, 2-(1,3-benzodioxol-5-yl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one, 2-(4-methoxyphenyl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one, 2-pyridin-4-yl-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one hydrochloride, 2-(3-aminopropyl)-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-(3-nitrophenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-(4-hydroxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid hydrobromide, 2-(3-aminopropyl)-8-(3-hydroxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid hydrobromide, 2-(3-aminopropyl)-8-(2-naphthyl)-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-(3,5-difluorophenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-(1,3-benzodioxol-5-yl)-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-(3-cyanophenyl)-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylic acid trifluoroacetate, 9-(hydroxymethyl)-2-quinolin-3-yl-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-(3-aminopropyl)-8-(4-methoxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-[3-(methyl-sulfonyl)phenyl]-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-[3-(trifluoromethyl)phenyl]-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(1H-imidazol-1-yl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-q]isoquinolin-7(8H)-one trifluoroacetate, 2-(3-aminopropyl)-8-(3-methoxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-[4-(trifluoromethyl)phenyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-anilino-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 2-(3-aminopropyl)-8-(3,4-difluorophenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(4′-carboxy-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(4-hydroxyphenyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-propyl-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 2-[3-({2-[3′-(trifluoromethyl)-1,1′-biphenyl-4-yl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-(4-tert-butylphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-({2-[4-(3-furyl)phenyl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(3′-chloro-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(4-methoxyphenyl)-5,6,8,9,10,1′-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-[3-({2-[4′-(trifluoromethyl)-1,1′-biphenyl-4-yl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-hydroxyphenyl)-5,6,9,10-tetrahydropyrazino[1′,2′-:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-(3-aminopropyl)-N-hydroxy-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f-]isoquinoline-3-carboxamide hydrochloride, 2-[(E)-2-(4-hydroxyphenyl)ethenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-quinolin-3-yl-5,6,8,9,10,1′-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-(4-hydroxyphenyl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-(3-{[2-(2′-chloro-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(4′-tert-butyl-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(3,4-dichlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[3-(3-chlorophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[(E)-2-phenylethenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-(3-{[2-(4-pyridin-4-ylphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[3-(4-bromophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[3-(4-tert-butylphenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(3′-isopropyl-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(2-thien-2-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[4-(dimethylamino)phenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-(3-{[2-(1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-methoxyphenyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]-isoquinolin-7(8H)-one, 2-(3-{[2-(4-bromophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(2,4-dichlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(benzylsulfonyl)amino]propyl}-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 9-(aminomethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-(3-nitrophenyl)-5,6,8,9,10,1′-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-(3-aminopropyl)-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxamide hydrochloride, 2-(3-{[3-(4-chlorophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-(dimethylamino)phenyl]-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-(3-{[(4-chlorobenzyl)sulfonyl]amino}propyl)-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 2-(3-{[2-(4-pyridin-3-ylphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(4-chlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(5-chlorothien-2-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[3-(4-cyanophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[3-(5-methyl-2-furyl)butyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-({2-[4-(1-benzothien-3-yl)phenyl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-ammoniopropyl)-3-carboxy-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinolin-7-ium dichloride, 2-(4-methoxyphenyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-(3-{[3-(4-acetylphenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-({3-[4-(methylsulfonyl)phenyl]propyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-(2-methoxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-({2-[3′-(aminomethyl)-1,1′-biphenyl-4-yl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(2-aminoethyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid dihydrochloride, 2-(3-{[2-(4-nitrophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-({2-[2′-(trifluoromethyl)-1,1′-biphenyl-4-yl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 9-(hydroxymethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-(3-{[2-(4-methylphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 9-{[(2-thien-2-ylethyl)amino]methyl}-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-(3-{[2-(4-ethoxyphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(1,3-benzodioxol-5-yl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-(3-{[2-(4-methoxyphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 3-[3-(1H-tetraazol-5-yl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinolin-2-yl]propan-1-amine hydrochloride, 2-(3-aminopropyl)-8-chloro-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[(1R,2S)-2-phenylcyclopropyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(3,3-diphenylpropyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(3-bromo-4-methoxyphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(4-phenylbutyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-(2,3-dihydro-1H-inden-2-ylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(2-naphthyl)-5,6,8,9,-10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-{3-[(3-phenylpropyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(4-fluorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(2-thien-3-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid dihydrochloride, 5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-[3-(glycoloylamino)propyl]-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid hydrochloride, 8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 2-(3-{[2-(4-ethylphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(2-chlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(2-ethylbutyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxamide, 2-{3-[(2-pyridin-4-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(4-chlorophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(3-chlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-(glycoloylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(3-{[2-(3,4-dimethoxyphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(1-benzofuran-2-yl)-5,6,8,9,10,1,1-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-(3-{[4-(2-aminoethyl)phenyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(1-naphthyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 8-(3-aminopropyl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one dihydrochloride, 2-anilino-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-(3-aminopropyl)-8-[2-(trifluoromethyl)phenyl]-4,5-dihydro-2H-pyrazolo[3,4-f] isoquinoline-3-carboxylic acid trifluoroacetate, 9-(azidomethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 9-({[2-(4-chlorophenyl)ethyl]amino}methyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-phenyl-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-[3-(pentylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 10-(2-aminoethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-[3-(allylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(4-aminobutyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid dihydrochloride, 2-(3-{[2-(4-aminophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[(1E)-3,3-dimethylbut-1-enyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-(8H)-one-trifluoroacetate, 10-(nitromethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 3-carboxy-2-[3-(methylammonio)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinolin-7-ium dichloride, 2-[3-({[(4-butoxyphenyl)amino]carbonyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 2-{3-[(2-pyridin-3-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(2-pyridin-2-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(cyclopropylmethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(2-thien-2-ylpropyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-methoxy-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-[3-(dimethylamino)phenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-q]isoquinolin-7(8H)-one, 2-(3-{[2-(1H-pyrrol-1-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-(benzyloxy)propyl]-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 2-{3-[(4-butoxybenzyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(1,3-benzodioxol-5-yl)-5-,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-[(E)-2-(2-fluorophenyl)ethenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-q]isoquinolin-7(8H)-one, 2-[(1E)-hex-1-enyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-anilino-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1′,5]pyrazolo[3,4-f]isoquinolin-7-one, 5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-chloro-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-[(E)-2-(4-methoxyphenyl)ethenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-(methylthio)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-{3-[(2-furylmethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-azepan-1-yl-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-(3,6-dihydropyridin-1 (2H)-yl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 9-methyl-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-(piperidin-3-ylmethyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid hydrochloride, 9-(chloromethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 2-[(4-methoxybenzyl)amino]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-(3-([2-(1H-imidazol-4-yl)-ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(benzylamino)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-(methylthio)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-{3-[(2-chlorobenzyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-[3-(benzylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate; 2-[3-({[(4-methoxyphenyl)amino]carbonyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 2-{3-[(2-phenylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(thien-2-ylmethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-benzyl-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one, 5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one, 2-{3-[(4-chlorobenzyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-{3-[(2-phenylpropyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 7-oxo-5,6,7,8,9,10-hexahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinoline-9-carboxamide trifluoroacetate, 2-(3-hydroxypropyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid, 2-(1,3-dihydro-2H-isoindol-2-yl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-{3-[(4-aminophenyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate, 2-(4-hydroxypiperidin-1-yl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one trifluoroacetate, 2-(3-aminopropyl)-7-hydroxy-8-(3-nitrophenyl)-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid trifluoroacetate, 2-(2-aminoethyl)-7-hydroxy-8-(3-nitrophenyl)-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid trifluoroacetate, 3-hydroxy-2-(3-nitrophenyl)-5,6,8,9,10,11-hexahydro-7H-benzo[g][1,4]diazepino[1,2-b]indazol-7-one trifluoroacetate, 2-(3-aminopropyl)-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid dihydrochloride, 2-(2-aminoethyl)-8-bromo-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-[2-(4-chlorophenyl)ethyl]-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid trifluoroacetate, 3-hydroxy-2-(3-nitrophenyl)-5,6,9,10-tetrahydrobenzo[g]pyrazino[1,2-b]indazol-7(8H)-one hydrobromide, 2-(3-aminopropyl)-8-bromo-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid hydrobromide, 2-(3-aminopropyl)-7-hydroxy-8-(4-nitrophenyl)-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-8-(3-cyanophenyl)-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid trifluoroacetate, 2-(3-aminopropyl)-7-hydroxy-8-[3-(trifluoromethyl)phenyl]-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid trifluoroacetate, and 2-(3-aminopropyl)-7-hydroxy-8-(3,3,3-trifluoropropyl)-4,5-dihydro-2H-benzo[g]indazole-3-carboxylic acid trifluoroacetate, 7-hydroxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 2,3,8,10,11,12-hexahydro-1H,7H-9,12-methanoazepino[3,4-b]pyrano[3,2-e]indole-8-carboxylic acid, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 7-(methylthio)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 7-(benzyloxy)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 7-(methylthio)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 2,2,2-trifluoroethyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, 2,3-dihydroxypropyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, pyridin-4-ylmethyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, 2-fluoroethyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, allyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, benzyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, 2-(methylthio)ethyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, 2-methoxyethyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylatem, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 7-hydroxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 2,3,8,10,11,12-hexahydro-1H,7H-9,12-methanoazepino[3,4-b]pyrano[3,2-e]indole-8-carboxylic acid, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 7-(methylthio)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 7-(benzyloxy)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 7-(methylthio)-3,4, 5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 6-(2-oxo-2-phenylethoxy)-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 6-methoxy-2-methyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 2,2,2-trifluoroethyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, 6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 7-hydroxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 6-hydroxy-2-methyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid, 6-methoxy-2-methyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 2,3-dihydroxypropyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate, 4-ethyl-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 6-methoxy-4-methyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 8,9,10,11-tetrahydro-7H-pyrido[3′,4′:4,5]pyrrolo[2,3-f]isoquinolin-7-one trifluoroacetate, 3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one trifluoroacetate, 3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one hydrochloride, 7-methoxy-3,4,5,10-tetrahydroazepino[3,4-b]indol-1 (2H)-one, 6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 6-methoxy-2,9-dihydro-1H-beta-carbolin-1-one, 6-hydroxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 8,9,10,11-tetrahydro-7H-pyrido[3′,4′:4,5]pyrrolo-[2,3-f]isoquinolin-7-one, 3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 6-methoxy-3-{3-[(2-phenylethyl)amino]propyl}-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, (1E)-6-methoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime, (1Z)-6-methoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime, 6-methoxy-3-{3-[(3-phenylpropyl)amino]propyl}-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, methyl 1-oxo-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-6-carboxylate, 3-(hydroxymethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 3-(3-aminopropyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 3-(2-aminoethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, ethyl 1-(hydroxyimino)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxylate, 2-methoxy-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one oxime, 3-(hydroxymethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 3-(3-aminopropyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 3-(2-aminoethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, ethyl 1-(hydroxyimino)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxylate, 2-methoxy-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one oxime, 3-[3-(benzylamino)propyl]-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 6-methoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime, 6-iodo-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime, 6-methoxy-2-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime, 3-(3-hydroxypropyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, ethyl 1-oxo-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-6-carboxylate, 6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-thione, methyl 4-oxo-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylate, and 2,3,4,9-tetrahydro-1H-carbazol-1-one oxime. Others are described in U.S. patent application Publication Nos. 2004-0127492, 2005-0101623, 2005-0137220, and 2005-0143371.
Prodrugs and Other Modified Compounds
Interaction of a molecule, e.g., a drug, with a MAPKAP kinase-2 polypeptide can be used to promote enhanced sensitivity of cells to chemotherapy or radiation treatment. The treatment, stabilization, or prevention of a disease or disorder associated with MAPKAP kinase-2 can be mediated by administering a compound, peptide, or nucleic acid molecule. In some cases, however, a compound that is effective in vitro in disrupting the interaction of a MAPKAP kinase-2 polypeptide and a natural substrate is not an effective therapeutic agent in vivo. For example, this could be due to low bioavailability of the compound. One way to circumvent this difficulty is to administer a modified drug, or prodrug, with improved bioavailability that converts naturally to the original compound following administration. Such prodrugs may undergo transformation before exhibiting their full pharmacological effects. Prodrugs contain one or more specialized protective groups that are specifically designed to alter or to eliminate undesirable properties in the parent molecule. In one embodiment, a prodrug masks one or more charged or hydrophobic groups of a parent molecule. Once administered, a prodrug is metabolized in vivo into an active compound.
Prodrugs may be useful for improving one or more of the following characteristics of a drug: solubility, absorption, distribution, metabolization, excretion, site specificity, stability, patient acceptability, reduced toxicity, or problems of formulation. For example, an active compound may have poor oral bioavailability, but by attaching an appropriately-chosen covalent linkage that may be metabolized in the body, oral bioavailability may improve sufficiently to enable the prodrug to be administered orally without adversely affecting the parent compound's activity within the body.
A prodrug may be carrier-linked, meaning that it contains a group such as an ester that can be removed enzymatically. Optimally, the additional chemical group has little or no pharmacologic activity, and the bond connecting this group to the parent compound is labile to allow for efficient in vivo activation. Such a carrier group may be linked directly to the parent compound (bipartate), or it may be bonded via a linker region (tripartate). Common examples of chemical groups attached to parent compounds to form prodrugs include esters, methyl esters, sulfates, sulfonates, phosphates, alcohols, amides, imines, phenyl carbamates, and carbonyls.
As one example, methylprednisolone is a poorly water-soluble corticosteroid drug. In order to be useful for aqueous injection or ophthalmic administration, this drug must be converted into a prodrug of enhanced solubility. Methylprednisolone sodium succinate ester is much more soluble than the parent compound, and it is rapidly and extensively hydrolysed in vivo by cholinesterases to free methylprednisolone.
Caged compounds may also be used as prodrugs. A caged compound may have, e.g., one or more photolyzable chemical groups attached that renders the compound biologically inactive. In this example, flash photolysis releases the caging group (and activates the compound) in a spatially or temporally controlled manner. Caged compounds may be made or designed by any method known to those of skill in the art.
For further description of the design and use of prodrugs, see Testa and Mayer, Hydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry and Enzymology, published by Vch. Verlagsgesellschaft Mbh. (2003). Other modified compounds are also possible in the methods of the invention. For example, a modified compound need not be metabolized to form a parent molecule. Rather, in some embodiments, a compound may contain a non-removable moiety that, e.g., increases bioavailability without substantially diminishing the activity of the parent molecule. Such a moiety could, for example, be covalently-linked to the parent molecule and could be capable of translocating across a biological membrane such as a cell membrane, in order to enhance cellular uptake. Exemplary moieties include peptides, e.g., penetratin or TAT. An exemplary penetratin-containing compound according to the invention is, e.g., a peptide comprising the sixteen amino acid sequence from the homeodomain of the Antennapedia protein (Derossi et al., J. Biol. Chem. 269:10444-10450, 1994), particularly a peptide having the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 50), or including a peptide sequence disclosed by Lin et al. (J. Biol. Chem. 270:14255-14258, 1995). Others are described in U.S. patent application Publication No. 2004-0209797 and U.S. Pat. Nos. 5,804,604, 5,747,641, 5,674,980, 5,670,617, and 5,652,122. In addition, a compound of the invention could be attached, for example, to a solid support.
Screening Assays
Fluorescence polarization assays can be used in displacement assays to identify small molecule peptidomimetics or other compounds useful in the methods of the invention. The following is an exemplary method for use of fluorescence polarization, and should not be viewed as limiting in any way. For screening, all reagents are diluted at the appropriate concentration and the working solution, kept on ice. The working stock concentration for GST and GST fusion proteins are  ̃4 ng/μL, Fluorescein-labeled peptides can be used at a concentration of 1.56 fmol/μL, while cold peptides at 25 pmol/μL. Samples are incubated at a total volume of 200 μL per well in black flat bottom plates, Biocoat, #359135 low binding (BD BioSciences; Bedford, Mass.). Assays are started with the successive addition using a Labsystem Multi-prop 96/384 device (Labsystem; Franklin, Mass.) of 50 μL test compounds, diluted in 10% DMSO (average concentration of 28 μM), 50 μL of 50 mM MES-pH 6.5, 50 μL of Fluorescein-peptide, 50 μL of GST-MAPKAP kinase-2 polypeptide, or 50 μL of unlabeled polypeptide can be used as a negative control. Once added, all the plates are placed at 4° C. Following overnight incubation at 4° C., the fluorescence polarization is measured using a Polarion plate reader (Tecan, Research Triangle Park, N.C.). A xenon flash lamp equipped with an excitation filter of 485 nm and an emission filter of 535 nm. The number of flashes is set at 30. Raw data can then be converted into a percentage of total interaction(s). All further analysis can be performed using SPOTFIRE data analysis software (SPOTFIRE, Somerville, Mass.)
Upon selection of active compounds, auto-fluorescence of the hits is measured as well as the fluorescein quenching effect, where a measurement of 2,000 or more units indicates auto-fluorescence, while a measurement of 50 units indicates a quenching effect. Confirmed hits can then be analyzed in dose-response curves (IC50) for reconfirmation. Best hits in dose-response curves can then be assessed by isothermal titration calorimetry using a GST-MAPKAP kinase-2 polypeptide fusion.
Assays with a candidate compound may be performed in the presence of a compound known to bind MAPKAP kinase-2, and the difference in binding the presence and absence of the compound known to bind may be a useful measure of the candidate compound's ability to bind to MAPKAP kinase-2. This assay may be done in any format known to those of skill in the art, e.g., as a displacement assay.
Alternate Binding and Displacement Assays
Fluorescence polarization assays are but one means to measure compound-protein interactions in a screening strategy. Alternate methods for measuring compound-protein interactions are known to the skilled artisan. Such methods include, but are not limited to mass spectrometry (Nelson and Krone, J. Mol. Recognit., 12:77-93, 1999), surface plasmon resonance (Spiga et al., FEBS Lett., 511:33-35, 2002; Rich and Mizka, J. Mol. Recognit., 14:223-8, 2001; Abrantes et al., Anal. Chem., 73:2828-35, 2001), fluorescence resonance energy transfer (FRET) (Bader et al., J. Biomol. Screen, 6:255-64, 2001; Song et al., Anal. Biochem. 291:133-41, 2001; Brockhoff et al., Cytometry, 44:338-48, 2001), bioluminescence resonance energy transfer (BRET) (Angers et al., Proc. Natl. Acad. Sci. USA, 97:3684-9, 2000; Xu et al., Proc. Natl. Acad. Sci. USA, 96:151-6, 1999), fluorescence quenching (Engelborghs, Spectrochim. Acta A. Mol. Biomol. Spectrosc., 57:2255-70, 70; Geoghegan et al., Bioconjug. Chem. 11:71-7, 2000), fluorescence activated cell scanning/sorting (Barth et al., J. Mol. Biol., 301:751-7, 2000), ELISA, and radioimmunoassay (RIA).
Pharmaceutical Compositions
The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional dissolving, lyophilizing, mixing, granulating or confectioning processes. Methods well known in the art for making compositions and formulations are found, for example, in “Remington: The Science and Practice of Pharmacy,” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia).
Solutions of the active ingredient, and also suspensions, and especially isotonic aqueous solutions or suspensions, are preferably used, it being possible, for example in the case of lyophilized compositions that comprise the active ingredient alone or together with a carrier, for example mannitol, for such solutions or suspensions to be produced prior to use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known per se, for example by means of conventional dissolving or lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing substances, such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, poly vinylpyrrolidone or gelatin.
Suspensions in oil comprise as the oil component the vegetable, synthetic or semi-synthetic oils customary for injection purposes. There may be mentioned as such especially liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms, for example lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, behenic acid or corresponding unsaturated acids, for example oleic acid, elaidic acid, erucic acid, brasidic acid or linoleic acid, if desired with the addition of anti oxidants, for example, vitamins E, β-carotene, or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of those fatty acid esters has a maximum of 6 carbon atoms and is a mono- or poly-hydroxy, for example a mono-, di- or tri-hydroxy, alcohol, for example methanol, ethanol, propanol, butanol or pentanol or the isomers thereof, but especially glycol and glycerol. The following examples of fatty acid esters are therefore to be mentioned: ethyl oleate, isopropyl myristate, isopropyl palmitate, “Labrafil M 2375” (poly oxyethylene glycerol trioleate, Gattefoss, Paris), “Miglyol 812” (triglyceride of saturated fatty acids with a chain length of C8 to C12, Huls AG, Germany), but especially vegetable oils, such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and more especially groundnut oil.
The injection compositions are prepared in customary manner under sterile conditions; the same applies also to introducing the compositions into ampoules or vials and sealing the containers.
Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, drage cores or capsules. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.
Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and binders, such as starch pastes using for example corn, wheat, rice or potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinyl-pyrrolidone, and/or, if desired, disintegrates, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate. Excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol. Drage cores are provided with suitable, optionally enteric, coatings, there being used, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as ethylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Capsules are dry-filled capsules made of gelatin and soft sealed capsules made of gelatin and a plasticiser, such as glycerol or sorbitol. The dry-filled capsules may comprise the active ingredient in the form of granules, for example with fillers, such as lactose, binders, such as starches, and/or glidants, such as talc or magnesium stearate, and if desired with stabilisers. In soft capsules the active ingredient is preferably dissolved or suspended in suitable oily excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols, it being possible also for stabilisers and/or antibacterial agents to be added. Dyes or pigments may be added to the tablets or drage coatings or the capsule casings, for example for identification purposes or to indicate different doses of active ingredient.
The pharmaceutical compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, drages, tablets or capsules.
The formulations can be administered to human patients in a therapeutically effective amount (e.g., an amount that decreases, suppresses, attenuates, diminishes, arrests, or stabilizes the development or progression of a disease, disorder, or infection in a eukaryotic host organism). The preferred dosage of therapeutic agent to be administered is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration.
For any of the methods of application described above, a compound that interacts with a MAPKAP kinase-2 polypeptide may be applied to the site of the needed therapeutic event (for example, by injection, e.g., direct injection into one or more tumors), or to tissue in the vicinity of the predicted therapeutic event or to a blood vessel supplying the cells predicted to require enhanced therapy.
The dosages of compounds that interact with a MAPKAP kinase-2 polypeptide depend on a number of factors, including the size and health of the individual patient, but, generally, between 0.1 mg and 1000 mg inclusive are administered per day to an adult in any pharmaceutically acceptable formulation. In addition, treatment by any of the approaches described herein may be combined with more traditional therapies.
EXAMPLES
Example 1
The p38MAPK-MK2 Pathway is Activated by Drugs that Directly Damage DNA
To investigate whether the p38 MAPK/MK2 pathway was involved in the DNA damage response of cells following exposure to clinically useful chemotherapeutic agents, we treated human U2OS osteosarcoma cells with the DNA crosslinking agent cis-Platinum (cisplatin), the topoisomerase I inhibitor camptothecin, or the topoisomerase II inhibitor doxorubicin (FIG. 12). p38 MAPK activation was assessed using an antibody specific for the Thr-180/Tyr-182 doubly phosphorylated active form of the kinase. Activation of MK2 was monitored by its altered mobility on SDS-PAGE, and by immunoblotting using a phospho-specific antibody for pThr-344, a site in the auto-inhibitory domain whose phosphorylation by p38MAPK results in a dramatic elevation of MK2 activity. Prior to exposure of cells to DNA damaging compounds (FIG. 12A-12C, zero hour lanes), or in cells treated with DMSO (vehicle) alone (FIG. 12D), MK2 ran as a single band that did not cross-react with the anti-pThr-344 antibody. Within one hour after exposure of the cells to cisplatin and doxorubicin, or within four hour following treatment with camptothecin, MK2 displayed a significant reduction in its electrophoretic mobility. The upshifted MK2 band appeared with the same kinetics as both the MK2 pThr-344- and the p38MAPK pThr-180/pTyr-182 immunoreactive bands. Activation of MK2 was entirely dependent on p38MAPK, since addition of the p38MAPK selective inhibitor SB203580 to the growth media 30 minutes prior to application of the DNA damaging agents completely abolished MK2 activation, while preserving activation of p38MAPK (FIG. 12E). Similar results for MK2 activation in response to cisplatin, camptothecin and doxorubicin were also observed in HeLa cervical carcinoma cells, U87MG human glioblastoma cells, and primary MEFs (FIG. 29A and data not shown). The time course of MK2 activation upon treatment with each of these drugs (FIG. 12A-12D) matched the rate of appearance of γ-H2AX nuclear foci (FIG. 12F). These data indicate that treatment of cells with these genotoxic agents results in MK2 activation, likely as a direct result of chemotherapy-induced DNA damage.
Example 2
ATM and ATR are Required for p38MAPK/MK2 Activation Following Genotoxin-Induced DNA Damage but not in Response to UV Irradiation
We analyzed the p38MAPK/MK2activation profile in ATM-deficient and ATR-defective fibroblasts (FIGS. 13A and 13B, and FIG. 12G). We also studied the effect of pharmacological inhibition of these kinases by addition of caffeine (FIG. 13C). Activation of the p38MAPK/MK2 complex in response to cisplatin, camptothecin or UV exposure occurred normally in ATM deficient fibroblasts, while doxorubicin treatment failed to activate either p38MAPK or MK2 in these cells. ATR-defective fibroblasts, on the other hand, failed to activate p38MAPK or MK2 following either cisplatin, doxorubicin, or camptothecin exposure. However, UV-induced p38MAPK/MK2 activation in these cells was unaffected. Similarly, treatment of U2OS cells with 20 mM caffeine, a concentration sufficient to inhibit ATM, ATR and DNA-PK, for thirty minutes prior to exposure to cisplatin and doxorubicin completely abrogated the p38MAPK/MK2 response, while the activation of these kinases by UV occurred normally under these conditions. Taken together, these data indicate that cisplatin, camptothecin, and doxorubicin require ATR for p38MAPK/MK2 activation, that doxorubicin also requires ATM activity, and that UV irradiation is capable of activating the p38MAPK/MK2 in a manner that is independent of ATM, ATR, or DNA-PK function.
Example 3
Loss of p53 Renders Cells Dependent on MK2 Signaling for Survival after Chemically-Induced DNA Damage
The p53 tumor suppressor protein plays an important role in the cellular response to DNA damage by transcriptionally upregulating the Cdk inhibitor p21 to induce a G1 and G2 arrest. Cancer cells frequently show disruptions in the p53 pathway, eliminating this component of the DNA damage response, and leaving the cells entirely dependent on remaining checkpoint signaling pathways. To examine whether the ATR/ATMp38MAPK-MK2 pathway was required for cell survival after genotoxin-induced DNA damage in p53 wild-type and p53−/− MEFs, we used RNAi to deplete MK2, and examined the response of these otherwise genetically identical cells to cisplatin and doxorubicin using a colony survival assay (FIG. 29). Cells were infected with lentiviruses delivering shRNAs against luciferase (control) or MK2 and analyzed 6 days later (FIG. 29C). Luciferase and MK2 knockdown MEFs were then exposed to increasing doses of cisplatin or doxorubicin. As seen in FIGS. 29A and 29B, there was no difference in the number of surviving colonies in the p53 wt/wt MEFs, regardless of the presence or absence of MK2, at any dose of cisplatin or doxorubicin examined. In contrast, downregulation of MK2 in p53−/− cells dramatically reduced the number of surviving colonies. These results demonstrate that depletion of MK2 specifically sensitizes p53-deficient cells to the anti-proliferative effects of chemotherapy-induced DNA damage. We used Western blot analysis to profile activation of the MK2 pathway and the p53 network following cisplatin and doxorubicin treatment in these four cell lines (FIG. 29C). The presence or absence of MK2 had no effect on the strong induction of p53 and p21 following exposure to cisplatin or doxorubicin in the p53 WT/WT cells. Only minimal amounts of the p53 inducer protein p19ARF were detected. Neither p53 or p21 induction was detectable in p53−/− MEFs in the presence or absence of MK2. However, the tumor suppressor p19ARF was strongly induced in these cells even in the absence of DNA damaging chemotherapy, likely reflecting a feedback response due to the inability of these cells to induce p53. Thus, we concluded that MK2 is not required for the normal p53/p21 induction or stabilization in response to DNA damage in wild-type primary cells, and that 2 is unable to induce p21 expression after DNA damage in the absence of functional p53.
Example 4
Down-Regulation of MK2 Leads to Mitotic Catastrophe after DNA Damage in p53−/− Cells
We speculated that the reduced colony formation observed in MK2-depleted p53−/− MEFs after DNA damaging chemotherapy might be due to mitotic catastrophe resulting from defective cell cycle checkpoints. A hallmark of mitotic catastrophe is entry of cells into mitosis despite the presence of damaged DNA, resulting in activation of the apoptotic cell death pathway. To investigate this, luciferase shRNA control and MK2 depleted p53 wild-type or null MEFs were treated with low doses of doxorubicin or cisplatin for thirty hours and immunostained with antibodies against histone H3 pSer−10 as a marker for mitotic entry, histone γ-H2AX as marker for persistent DNA damage, and cleaved caspase-3 as a marker for apoptosis (FIG. 30). Luciferase shRNA-treated p53 wt/wt and p53−/− cells showed robust γ-H2AX foci after exposure to DNA damaging chemotherapy. No phospho-histone H3 or cleaved caspase-3 staining was observed in MK2-containing cells, consistent with an intact DNA damage response regardless of the presence (FIG. 30A) or absence (FIG. 30C) of p53. Similarly, an intact DNA damage checkpoint response was also observed in MK2 depleted cells that contained wild-type p53 (FIG. 30B). In sharp contrast, however, in the MK2-deficient p53−/− cells, a substantial fraction of the γ-H2AX positive cells also stained positively for both phospho-histone H3 and cleaved caspase 3 (FIG. 3D). Interestingly, no caspase 3 staining was observed in γ-H2AX-positive cells that did not also contain phospho-histone H3 (FIG. 30D, arrowhead). Thus, in the absence of M2, p53 null primary cells treated with cisplatin and doxorubicin lose one or more critical cell cycle checkpoints and undergo mitotic catastrophe.
Example 5
Down-Regulation of MK2 Causes Regression of Established p53−/− Tumors In Vivo after Low Dose Treatment with DNA Damaging Agents
We investigated whether the chemo-sensitizing effect of MK2 depletion in p53 null cells in culture could also be observed when pre-existing p53 deficient tumors were treated with DNA damaging drugs in vivo. In these experiments HRas-V12 transformed p53−/− MEFs were stably transfected with control shRNA or MK2 shRNA expressed from a murine U6 promoter, using a lentiviral delivering system (FIG. 18A). The lentiviral transfer vector also encoded GFP under the control of a CMV promoter, allowing for fluorescent detection of tumors in situ. Tumors were induced by injection of 106 cells into the flanks of nude mice. Twelve days later  ̃1 cm diameter tumors had formed at all injection sites, and treatment with cisplatin, doxorubicin, or vehicle was begun (FIG. 18B). In the absence of treatment with DNA damaging drugs, the tumors arising from the MK2 depleted cells in the right flanks of these animals grew slightly larger than those of the luciferase shRNA control cells in the left flanks (FIG. 18B-18D). Following treatment with cisplatin or doxorubicin, the control tumors showed either minimal reduction in size, or slow continued growth (FIG. 18B, D, red symbols). In contrast, the MK2 depleted tumors showed a dramatic reduction in weight and diameter (FIG. 18B-18D, blue symbols). Tumors depleted of MK2 shrank from 1.3 cm to 0.4 cm over fourteen days when treated with cisplatin, and from 1.4 to 0.5 cm when treated with doxorubicin. Thus, the sensitizing effect of MK2 depletion on DNA damage induced cell death in p53-deficient primary cells observed in cell culture was also maintained in vivo. These results strongly suggest that MK2 may be a useful target for the design of new cancer treatment agents.
Example 6
MK2 is Required for the G2/M Checkpoint Following Doxorubicin Treatment in p53-Deficient Cells
To investigate the molecular mechanisms involved in MK2 dependent responses to DNA lesions, we examined cell cycle profiles of control and MK2 depleted p53−/− MEFs. Asynchronous MK2- or control knock-down p53−/− MEFs were mock treated or exposed to doxorubicin for thirty hour, and cell cycle distribution monitored by FACS. In one set of experiments the spindle poison nocodazole was added to the media three hours after addition of doxorubicin, to cause any cells progressing through the cell cycle to arrest in mitosis. DNA content was monitored by PI staining; phospho-histone-H3 staining was used as an indicator of mitotic entry. As shown in the left panels of FIG. 31A, treatment of control knock down p53−/− cells with doxorubicin led to the accumulation of cells with 4N DNA content, and a lack of phospho-histone H3 staining in either the absence or presence of nocodazole, indicative of an intact G2/M checkpoint. These cells expressing control shRNAs behaved identically to an untransfected control p53−/− cell population. In marked contrast, MK2 depleted p53−/− cells treated with doxorubicin displayed a cell cycle profile similar to that of untreated cells (FIG. 31A, right upper and middle panels), with only a small increase in the 4N peak compared to the doxorubicin-treated luciferase shRNA controls, a slightly increased S-phase population, and the appearance of a sub-G1 population indicative of apoptosis. Addition of nocodazole following doxorubicin treatment to the MK2 depleted cells caused them to accumulate in a 4N DNA containing peak, with 28.5% of the cells staining positively for phospho-histone H3 (FIG. 31A, right lower panels), a value similar to that of untreated p53−/− cells blocked in mitosis with nocodazole. Intriguingly, MK2 depletion did not alter total Chk1 levels or reduce Chk1 activation following DNA damage (FIG. 31B). These findings demonstrate that loss of MK2 prevents p53-deficient cells from establishing a functional G2/M checkpoint following doxorubicin-induced DNA damage, despite the presence of activated Chk1. Identical results were obtained using a second unrelated shRNA against MK2. Importantly, the checkpoint defect could be fully rescued in the MK2 depleted cells by expressing an shRNA-resistant form of MK2 at comparable levels to the endogenous protein.
Two Cdc25 family members, Cdc25B and C, play important roles in initiating and maintaining mitotic entry in normal cells, and are prominent targets of the G2/M checkpoint. Cdc25B is believed to function by activating Cdk1/Cyclin B at the centrosome in late G2 as an initiator of early mitotic events, while Cdc25C functions to further amplify Cdk1/CyclinB activity within a nuclear auto-amplification loop once mitosis has begun. In response to γ- or UV-irradiation-induced DNA damage, checkpoint kinases phosphorylate Cdc25B and C on Ser−323 and 216, respectively, to induce their binding to 14-3-3 proteins, sequestering them in the cytoplasm away from their cyclin/Cdk substrates. Cdc25B plays a particularly crucial role in initiating and maintaining normal cell cycle G2/M checkpoint responses, since reactivation of Cdc25B is critical for DNA-damaged cells to re-enter the cell cycle. We therefore investigated whether MK2 signaling was required for association of Cdc25B with 14-3-3 in response to DNA damage by chemotherapeutic drugs. As shown in FIG. 31C, both doxorubicin and camptothecin treatment, resulted in the generation of stable 14-3-3-binding sites on Cdc25B in the luciferase shRNA control cells. No 14-3-3 binding of Cdc25B, however, was detected in lysates from the MK2 depleted cells (FIG. 31C, lower panel). This result is in good agreement with the cell cycle studies in panel A, which showed loss of the G2/M checkpoint in MK2 depleted cells after treatment with the topoisomerase inhibitor doxorubicin. These data indicate that loss of the G2/M checkpoint after DNA lesions in MAPKAP Kinase-2-depleted p53-defective cells likely arises, at least in part, from loss of Cdc25B binding to 14-3-3 proteins.
Example 7
MK2 is Required for S-Phase Checkpoint Arrest Following Cisplatin Treatment in p53-Deficient Cells
Treatment with the DNA intra-strand cross-linker cisplatin caused p53−/− cells to predominantly accumulate in S phase of the cell cycle. RNA interference was used to investigate the role of MK2 in this process. p53−/− control knock-down cells showed an identical accumulation in S phase after cisplatin exposure (FIG. 32A, left middle panels) as that seen in untransfected p53−/− cells. Addition of nocodazole to the luciferase knock-down cells three hour following cisplatin treatment did not reveal the appearance of any mitotic cells over the ensuing twenty seven hours, as monitored by phospho-histone H3 staining (FIG. 32A, lower left panels), indicating a functionally intact S-phase checkpoint. Depletion of MK2 prior to cisplatin exposure resulted in a dramatically different result. As seen in the right panels of FIG. 32A, MK2-depleted p53−/− cells showed a cell cycle profile after cisplatin treatment that was similar to that of untreated cells, other than a slight increase in the total number of cells in S-phase and the appearance of a sub-G1 population consistent with apoptosis. Strikingly, when nocodazole was added three hours following cisplatin addition, the MK2 depleted p53-r cells accumulated in a 4N DNA containing peak with  ̃25% of the cells staining positive for phospho-histone H3. The same cell cycle defects after cisplatin exposure were observed using a second unrelated shRNA sequence against M2, and the MK2 shRNA phenotype was completely reversed by expression of an RNAi-resistant form of MK2 at physiological levels. Similar to what was observed following doxorubicin treatment (FIG. 31B), MK2 depletion did not impair activation of Chk1 after cisplatin exposure (FIG. 32B). These data imply that MK2 is essential for the cisplatin induced S-phase arrest in p53-deficient cells, and that loss of MK2 enables these cells to override the cisplatin-induced S-phase checkpoint, despite the presence of activated Chk1, and proceed into mitosis. In contrast to the 14-3-3-mediated sequestration of Cdc25B and C involved in the G2/M checkpoint response, the G1 and S phase checkpoints are largely controlled by the phosphorylation-dependent degradation of another Cdc25 isoform, Cdc25A. We therefore investigated whether MK2 was required for the degradation of Cdc25A following cisplatin-induced DNA damage (FIG. 32B). Cdc25A levels were undetectable in the control luciferase knock-down cells after treatment with cisplatin. In contrast, in the MK2 depleted cells, substantial amounts of Cdc25A remain present in the lysates after cisplatin exposure, indicating that in the absence of MAPKAP Kinase-2, p53−/− MEFs cells are defective in targeting Cdc25A for degradation in response to cisplatin induced DNA damage. This impaired ability of MK2 depleted cells to degrade Cdc25A likely explains their failure to establish a sustained G1/S checkpoint following cisplatin exposure. Interestingly, Cdc25A may be a direct target of MK2 in vivo, since both MK2 and Chk1 phosphorylate Cdc25A equivalently in vitro, Chk1 phosphorylation of Cdc25A in vivo has been shown to facilitate its ubiquitin-mediated proteolysis in a complex and incompletely understood manner, and MK2 and Chk1 phosphorylate the identical optimal sequence motifs when analyzed by oriented peptide library screening.
Example 8
MK2 and Chk1 are Activated Independently by DNA Damage, and are Both Potently Inhibited by UCN-01
Activation of MK2 by cisplatin, camptothecin, and doxorubicin is strikingly similar to the activation profile reported for Chk1. Similarly, the impaired S-phase and G2/M checkpoints seen after these DNA damaging stimuli in MK2 knock-down cells bears some resemblance to what has been previously reported for Chk1-deficient p53-defective cells. These phenotypic similarities prompted us to further investigate whether the activation of Chk1 and MK2 was interdependent. As shown in FIGS. 31B and 32B, activation of Chk1 in response to doxorubicin and cisplatin was unimpaired in MK2 depleted cells. We therefore investigated the converse—whether the activation of MK2 after DNA damage was dependent on Chk1. U2OS cells depleted of Chk1 using siRNA were exposed to cisplatin and doxorubicin, and analyzed for activation of MK2. As shown in FIG. 19, phosphorylation/activation of MK2 occurred normally after treatment with these DNA damaging agents, regardless of the presence or absence of Chk1. Thus, activation of MK2 and Chk1 after drug-induced DNA damage appears to occur independently of each other, and both kinases appear to participate in parallel DNA damage checkpoint signaling pathways that are necessary for cell survival in the absence of a strong p53 response. The staurosporine derivative 7-hydroxystaurosporin/UCN-01 has been shown to increase the cytotoxicity of chemotherapy and radiation and is currently in clinical trials. It has been demonstrated that a major target of UCN-01 is the checkpoint kinase Chk1, leading to speculation that that the increased chemo- and radiation sensitivity of cells treated with UCN-01 is a direct result of Chk1-mediated checkpoint abrogation. UCN-01 inhibits Chk1 with an IC50 that is  ̃1000 fold lower than that for Chk2, and hence has been used experimentally as a Chk1 specific inhibitor. Strong circumstantial evidence, however, suggests that UCN-01 must be inhibiting other kinases involved in cell cycle control at similar concentrations as those used for Chk1 inhibition studies. For example, Chk1-depleted cells maintain phosphorylation of Ser−216, a well characterized Chk1 target site on Cdc25C, both during asynchronous growth and following γ-irradiation. Phosphorylation at this site is lost, however, when cells are treated with low doses of UCN-01 ( ̃300 nM), indicating that UCN-01 inhibitable kinase(s) other than Chk1 participate in Cdc25C Ser−216 phosphorylation. Based on our finding that MK2 depletion results in a dramatically increased chemosensitivity of malignant cells, we asked whether MK2 might be a UCN-01 target, similar to Chk1. In vitro kinase assays were performed under identical reaction conditions with Chk1 and MK2 using the same optimal peptide substrate for both kinases with the core consensus sequence L-Q-R-Q-L-S-I, in the presence of various concentrations of UCN-01. As shown in FIG. 21A, UCN-01 potently inhibited both kinases, with an IC50 value of  ̃35 nM for Chk1 and  ̃95 nM for MAPKAP Kinase-2. The IC50 value we measured for Chk1 is in good agreement with previously published data. Importantly, the IC50 value we measured for MK2 is significantly below the concentrations of UCN-01 that are used in “Chk1-specific” checkpoint abrogation assays, suggesting that under the conditions used in those studies, both Chk1 and MK2 were being inhibited. To examine the structural basis for UCN-01 inhibition of MK2, the structure of the MK2:UCN-01 complex was modeled using coordinates from the published MK2:staurosporine structure, and compared the results with the co-crystal structure of Chk1:UCN-01 (FIG. 21B). As seen in panels 2, 3 and 5, the 7-hydroxy moiety of UCN-01 can be easily accommodated into the MK2:staurosporine structure, where its closest neighboring residues would be Val-118 (2.8 Å to Cγ2), Leu-141 (3.2 Å to Cγ1), and Thr-206 (3.6 Å to Cγ2). Lack of steric hindrance, and the overall similarity of the modeled MK2:UCN-01 structure to the Chk1:UCN-01 structure (panels 1, 4), provides a structural rationale for the tight binding observed biochemically. To verify that MK2 is a direct target of UCN-01 within cells, we measured the phosphorylation of the MK2 substrate hsp-27 after heat shock, a stimulus that activates the p38MAPK/MK2 pathway. Control or MK2 shRNA expressing U2OS cells were incubated at 42° C. or 37° C. for two hours in the presence or absence of 250 nM UCN-01, and phosphorylation of hsp-27 monitored by immunoblotting. FIG. 21C shows that hsp27 is phosphorylated on Ser−82 when the control luciferase shRNA cells were placed at 42° C. (lane 1). This phosphorylation was completely abrogated by treatment with UCN-01 (lane 2). No phosphorylation was observed in MK2 knock-down cells placed at 42° C. regardless of the presence or absence of UCN-01 (lanes 3, 4), and no signal was observed in both the control and MK2 knock-down cells that were maintained at 37° C., with or without UCN-01 treatment (lane 5-8). Heat shock was equally effective in promoting the phosphorylation of hsp-27 on Ser−82, and UCN-01 was equally effective in blocking Ser−82 phosphorylation in cells that were depleted of Chk1 (FIG. 21D, lanes 1-4). Thus, UCN-01 inhibits MK2 in vivo, and this effect is independent of Chk1 function. This data demonstrates that UCN-01 is a direct inhibitor of MK2 within cells, and indicates that the clinical efficacy of UCN-01 in cancer treatment, particularly in p53-defective tumors, likely arises from the simultaneous inhibition of both the Chk1 and MK2 signaling pathways
Example 9
A Model for Re-Wiring of Cell Cycle Checkpoint Pathways in p53-Proficient and Deficient Cells
Checkpoint function in p53-proficient cells is mediated primarily through a robust, sustained p53 response downstream of ATM, together with Chk1 (FIG. 33A). Although not shown explicitly in the diagram, Chk1 has been shown to also directly phosphorylate p53. Under these conditions the presence of MK2 is not required for cell survival after exposure to DNA damaging agents. In p53-deficient cancer cells, checkpoint signaling following exposure to DNA damaging agents is mediated through the combined action of both the Chk1 and the p38 MAPK/MK2 pathways (FIG. 33B). In this situation the p38MAPK/MK2 branch of checkpoint signaling becomes essential for cell survival after DNA damage. Both pathways are simultaneously inhibited by the indolocarbazole drug UCN-01.
Other Embodiments
All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference. In addition, U.S. patent application Publication Nos. US 2005-0196808 and US 2006-0052951 are hereby incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.
Other embodiments are in the claims.

## Claims

1. A method for treating a cellular proliferative disorder in a patient having a p53-deficient cell, said method comprising administering to the patient a peptide comprising the amino acid sequence LQRQLSI (SEQ ID NO: 16), wherein the peptide comprises no more than 50 amino acids, and wherein the peptide is capable of inhibiting an activity of a MAPKAP kinase-2 polypeptide.

2. The method of claim 1, wherein said patient has a tumor, and wherein said peptide is administered to said tumor.

3. The method of claim 2, wherein said peptide is administered to said tumor by direct injection.

4. The method of claim 2, wherein said administration results in a reduction in size of said tumor, in comparison to a control patient to whom said peptide is not administered.

5. The method of claim 1, wherein said administering induces increased sensitivity of said p53-deficient cell to a chemotherapeutic agent, in comparison to a p53-deficient control cell in a control patient to whom said peptide is not administered.

6. The method of claim 5, wherein said chemotherapeutic agent is selected from the group consisting of alemtuzumab, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, bicalutamide, busulfan, capecitabine, carboplatin, carmustine, celecoxib, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine phosphate, etodolac, etoposide, exemestane, floxuridine, fludarabine, 5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab, goserelin, hexamethylmelamine, hydroxyurea, hypericin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leuporelin, lomustine, mechlorethamine, melphalen, mercaptopurine, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, paclitaxel, pentostatin, procarbazine, raltitrexed, rituximab, rofecoxib, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, toremofine, trastuzumab, vinblastine, vincristine, vindesine, and vinorelbine.

7. The method of claim 5, wherein said administering alters a DNA damage-responsive cell cycle checkpoint of said p53-deficient cell in comparison to said control cell.

8. The method of claim 7, wherein said DNA damage-responsive cell cycle checkpoint is G1/S phase arrest.

9. The method of claim 8, wherein Cdc25a degradation in a p53-deficient cell is impaired in comparison to a p53-deficient control cell in a control patient.

10. The method of claim 7, wherein said DNA damage-responsive cell cycle checkpoint is G2/M phase arrest.

11. The method of claim 10, wherein the interaction between Cdc25b and a 14-3-3 protein is reduced in comparison to a p53-deficient control cell in a control patient.

12. The method of claim 7, wherein said cell cycle checkpoint alteration induces cell death.

13. The method of claim 12, wherein said cell death is by apoptosis.

14. The method of claim 13, wherein said apoptosis is at least partially dependent on caspase-3 activation.