# Patent

## Title
Compounds that inhibit human DNA ligases and methods of treating cancer

## Abstract

Methods for treating cancer using compounds that inhibit human DNA ligases. Methods for using compounds that inhibit human DNA ligases to provide insights into the reaction mechanisms of human DNA ligases, for example to identify the human DNA ligase involved in different DNA repair pathways. Screening methods for compounds that inhibit human DNA ligases.

## Background
CROSS REFERENCES TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 13/864,916 filed Apr. 17, 2013, now U.S. Pat. No. 9,073,896, which is a divisional of U.S. patent application Ser. No. 12/576,410 filed Oct. 9, 2009, now U.S. Pat. No. 8,445,537, which is a continuation-in-part of International Patent Application No. PCT/US2008/059931 filed Apr. 10, 2008, which claims benefit of U.S. Provisional Application 60/911,000, filed Apr. 10, 2007, all applications of which are herein incorporated by reference in their entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with the support of the U.S. government under Grant Numbers GM057479, GM047251, ES012512, CA092584 and CA102428 from National Institutes of Health (NIH). The U.S. government has certain rights in this invention.


NAMES OF PARTIES OF A JOINT RESEARCH AGREEMENT
Not Applicable
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC
Not Applicable
BACKGROUND OF THE INVENTION
Despite many years of research, there still exists a compelling need to develop novel and more effective therapeutic strategies for human cancer. The use of many agents used in cancer treatment is limited because of their cytotoxic effects on normal tissues and cells. This is a particular concern for agents that kill cells by damaging DNA and/or inhibiting DNA replication. Moreover, it is becoming more and more evident that the simultaneous or sequential attack on different aspects of cancer cell metabolism by combinations of agents is more effective than the use of a single agent. This highlights the need to develop a wider variety of therapeutic agents that hit different molecular targets in cancer cells.
As mentioned above, therapeutic agents such as ionizing radiation and temozolomide, which damage DNA, have cytotoxic effects on normal tissues and cells as well as cancer cells. Despite the frequent use of agents that either damage DNA or inhibit DNA replication, there are relatively few available compounds that specifically target DNA repair and/or DNA replication-related proteins (1-3). Topoisomerase-I inhibitors, for example alter the capacity of a key DNA replication enzyme to proceed along an entire chromosome. Although cytotoxic, this class of compounds is currently being used to treat human cancer. There are also inhibitors of DNA damage response proteins, including the checkpoint kinase Chk1, poly(ADP) ribose polymerase, DNA dependent protein kinase, ATM kinase, MGMT and AP endonuclease (1-3) that are in preclinical or early clinical evaluation as cancer therapeutics. Applicants specifically contemplate that certain types of inhibitors of DNA repair pathways will have therapeutic utility because they will potentiate the cytotoxic effects of other treatments of cancer, for example, ionizing radiation and chemotherapeutic agents that damage DNA. This may permit specific targeting of tumors and/or the use of lower doses of DNA damaging agent, thereby reducing toxicity to normal tissues and cells. In addition, there is evidence that the DNA repair capabilities of cancer cells may be different than those of normal cells. For example, BRCA2-deficient cells established from individuals with an inherited predisposition to breast cancer are extremely sensitive to inhibitors of poly (ADP-ribose) polymerase because they are defective in homologous recombination (4,5). Thus, inhibitors of DNA repair proteins may specifically target cancer cells as compared to normal cell populations.
Under normal circumstances, the genome is propagated and maintained by the combination of a highly accurate DNA replication machinery and a network of DNA repair pathways. The increased incidence of cancer associated with DNA repair-deficient human syndromes illustrates the role of these pathways in protecting against deleterious genetic changes that contribute to cancer formation. There is growing interest in the identification of DNA repair inhibitors that will enhance the cytotoxicity of DNA-damaging agents because combinations of DNA-damaging agents and DNA repair inhibitors have the potential to concomitantly increase the killing of cancer cells and reduce damage to normal tissues and cells if either the damaging agent or the inhibitor could be selectively delivered to the cancer cells (2). Because DNA ligation is required during replication and is the last step of almost all DNA repair pathways, DNA ligase-deficient cell lines exhibit sensitivity to a wide range of DNA-damaging agents (6). Thus, DNA ligase inhibitors may have pleiotropic effects on cell proliferation and sensitivity to DNA damage.
DNA ligases catalyze the joining of interruptions in the phosphodiester backbone of double-stranded DNA, making them essential enzymes for DNA repair and replication. In addition, they are an indispensable reagent in molecular biology research for generating recombinant DNA. DNA ligases are members of the larger nucleotidyl transferase family that also includes RNA ligases and mRNA capping enzymes. In the first step of the ligation reaction, DNA ligases react with a nucleotide co-factor, either NAD+ or ATP, to form the covalent enzyme-AMP intermediate. Next the AMP moiety is transferred to the 5′ phosphate termini in duplex DNA, forming the DNA adenylate intermediate. Finally, the non-adenylated enzyme catalyzes phosphodiester bond formation between the 3′ hydroxyl and 5′ phosphate termini.
BRIEF SUMMARY OF THE INVENTION
Because of their involvement in DNA replication and DNA repair, DNA ligase inhibitors are likely to be anti-proliferative and to potentiate the cytotoxicity of DNA damaging agents, properties that may have clinical utility in the treatment of cancer, in particular malignancies with an altered DNA damage response. DNA joining by a DNA ligase is required to link together Okazaki fragments during DNA replication and to complete almost all DNA repair pathways (6). Applicants' invention involves inhibitors of DNA ligases that: (i) inhibit cell growth and/or kill cells; (ii) potentiate the cytotoxic effects of many DNA damaging agents that introduce a wide variety of different types of DNA lesions, including agents such as ionizing radiation, topoisomerase inhibitors (irinotecan and topotecan), PARP inhibitors, 5-fluorouracil, mitomycin C, bleomycin, melphalan, cyclosphosphamide, platinum compounds, cis-platinum and BCNU (Carmustine) and temozolomide that are currently being used to treat human cancer; and/or (iii) potentiate the cytotoxic effects of pro-apoptotic agents and inhibitors of signaling pathways that promote proliferation, for example a bcr-abl kinase inhibitor (imatinib (GLEEVAC)) that may be used to treat human cancer.
The combination therapy methods of the present invention are contemplated to be administered at the same time or at separate times to a subject in need thereof.
Applicants' invention includes methods for screening of individual chemical compounds, a synthetic chemical collection and/or a natural product library to identify compounds that inhibit human DNA ligase.
The human genome contains three genes, LIG1, LIG3 and LIG4, which encode ATP-dependent DNA ligases (6). These enzymes have unique cellular functions, but they also appear to have some functional redundancy. Although these enzymes have a conserved catalytic domain and use the same reaction mechanism, they are directed to participate in different DNA transactions by specific protein-protein interactions (6). DNA ligase I (hLigI) has a major role in the joining of Okazaki fragments during DNA replication and also in DNA excision repair. Based on the cellular phenotype of human DNA ligase I-deficient cells (7-9), specific inhibitors of DNA ligase I should inhibit cell growth and/or kill cells. Since cancer cells in general tend to be more proliferative than normal cells, it is conceivable that they will be more susceptible to the effects of DNA ligase I inhibitors. In support of this, it has been shown that human cancer lines grown either in vitro or as tumors in nude mice and human tumor specimens have elevated levels of DNA ligase I (10). In addition, human DNA ligase I deficient cells are hypersensitive to DNA alkylating agents and an inhibitor of poly(ADP-ribose) polymerase (PARP), 3 aminobenzamide. (8-10). Applicants' invention involves DNA ligase I specific inhibitors that will preferentially kill rapidly proliferating cancer cells and potentiate the cytotoxic effects of DNA alkylating agents such as temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) that are used clinically, and, when used in combination with PARP inhibitors (several of which are in clinical trials), will synergistically enhance cytotoxicity.
Several distinct DNA ligase polypeptides that function in nuclear DNA repair, mitochondrial DNA metabolism and germ cell development are encoded by the LIG3 gene (6). It appears that all cells express a single mRNA, DNA ligase IIIα mRNA that encodes mitochondrial and nuclear forms of DNA ligase IIIα. In addition, there is an alternative splicing event in male germ cells that generates DNA ligase IIIβ. Nuclear DNA ligase IIIα participates in the repair of DNA single strand breaks and the short patch subpathway of base excision repair. This appears to underlie the sensitivity of DNA ligase III-deficient cells to DNA alkylating agents and ionizing radiation (11,12). Applicants' invention also involves DNA ligase III inhibitors that will potentiate the cytotoxic effects of DNA alkylating agents such as temozolomide and BCNU, and ionizing radiation, all of which are used to treat human cancer. Furthermore, it has been shown that depletion of human DNA ligase III by RNAi disrupts mitochondrial function (13,14), raising the possibility that DNA ligase III inhibitors may cause cytotoxic effects via effects on mitochondrial function and may potentiate the effects of pro-apoptotic drugs. Since many tumors cells have alterations in energy metabolism, it is possible that they may be more susceptible to agents that target mitochondria. There is emerging evidence that DNA ligase I and DNA ligase IIIα participate in different subpathways of base and nucleotide excision repair and that these subpathways may be, at least in a part, functionally redundant (15,16). Applicants' invention also involves compounds that inhibit both DNA ligase I and DNA ligase III. These compounds will potentiate the cytotoxicity of DNA damaging agents such as 5-fluorouracil, temozolomide, BCNU, platinum compounds and mitomycin C that introduce lesions that are removed by excision repair to a greater extent than compounds that are specific for either DNA ligase I or DNA ligase III.
The LIG4 gene product, hLigIV, completes the repair of DNA double strand breaks (DSBs) by nonhomologous end joining (NHEJ) and V(D)J recombination events that generate diversity in immunoglobulin and T-cell receptor loci during immune system development (6). Notably, DSBs are the major cytotoxic lesion introduced by ionizing radiation, which is the most commonly used modality in the treatment of human cancer. Furthermore, NHEJ is the major DSB repair pathway in human cells and inactivation of the LIG4 gene has a more severe effect than inactivation of genes encoding proteins that act earlier in the NHEJ pathway (17), suggesting that, once DSBs are committed to the NHEJ pathway, they cannot be recovered and repaired by pathways utilizing either DNA ligase I or DNA ligase III. Applicants' invention also involves DNA ligase IV inhibitors that will dramatically potentiate cell killing by ionizing radiation and radiomimetic drugs.
In the complex formed by hLigI on DNA with a non-ligatable nick, three hLigI domains encircle and interact with the nicked DNA duplex. (18) Two of these domains, an adenylation domain (AdD) and an OB-fold domain (OBD), are present in other DNA ligases and nucleotidyl transferases. In contrast, the DNA binding domain (DBD, residues Asp262 to Ser535) is restricted to eukaryotic ATP-dependent DNA ligases (6). Notably, the DBD is the predominant DNA binding activity within hLigI and stimulates joining in trans by a hLigI fragment containing the adenylation and OB-fold domains (18). Based on these properties, Applicants chose to focus on identifying compounds that bind to the DBD and inhibite hLig1 activity by interfering with DNA binding.
Applicants' invention also includes the following.
Methods for using the inhibitors to delineate the molecular mechanisms of the DNA ligation reaction.
Methods for identifying the DNA ligase that completes different DNA repair pathways in cell extracts.
Methods for identifying the pathways involved in the repair of different DNA lesions using the ligase inhibitors in cell extract and cell culture assays.
Methods for treating cancer using compounds that inhibit human DNA ligases by inhibiting cell growth, killing tumorous cells and/or potentiate cell killing by DNA damaging agents.
Methods for treating cancer using compounds that inhibit human DNA ligase including treating epithelial-derived cancers, such as epithelia neoplams selected from the colon cancer, lung cancer, breast cancer, GI cancer, ovarian cancer and head and neck cancer.
Methods for treating cancer using compounds that inhibit human DNA ligase including treating hematological malignancies, such as hematological malignancy selected from the chronic and acute leukemias.
Methods for treating cancer using compounds that inhibit human DNA ligase including treating hematological malignancies, such as lymphomas.
The methods for treating cancer using compounds that inhibit human DNA ligase including treating skin cancer, melanoma, gliablastoma, neuroblastoma, sarcoma, colon cancer, breast cancer, cervical cancer, prostate cancer, pancreatic cancer, ovarian cancer, esophageal cancer, stomach cancer and lung cancer.
Identification and Characterization of Human DNA Ligase Inhibitors
One research focus has been to determine the cellular functions of the DNA ligases encoded by the human LIG1, LIG3 and LIG4 genes and to define the molecular mechanisms by which these enzymes are directed to participate in specific DNA transactions. Recently the structure of the catalytic domain of human DNA ligase I complexed with nicked DNA was determined (18). This was the first structure of a eukaryotic DNA ligase and the first structure of any DNA ligase complexed with DNA.
Attempts to identify human DNA ligase inhibitors by screening of chemical and natural product libraries have met with limited success. Previous attempts to identify DNA ligase inhibitors have involved in vitro screening of a chemical and a natural product library (19,20). Several compounds that inhibit DNA ligase I have been identified, but these compounds have not been extensively characterized in terms of their specificity and mechanism of action. A problem with screening of random chemical libraries using an in vitro DNA ligation assay is that it yields two classes of non-specific inhibitors; (i) molecules that bind to the DNA substrate; (ii) nucleotide analogs that inhibit not only ATP-dependent DNA ligases but also other nucleotide-dependent cellular enzymes. To circumvent these problems, Applicants used available structural information identifying the interfaces between DNA ligase I and nicked DNA (18) to design an in silico screening method for the identification of small molecules that disrupt the physical and functional interactions between DNA ligase and nicked DNA. In developing the screening methods, Applicants identified a binding pocket between residues His337, Arg449, and Gly453 that are located in the central region of the DBD and make direct contacts with the DNA substrate. Other residues comprising the binding site include Gly448, Arg451 and Ala455. (18, 21, 34-37) Based on amino acid sequence conservation, Applicants determined that it is likely that this binding pocket is relatively well conserved in human DNA ligases III and IV.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 shows the Structures of 9 of Applicants' inhibitors of human DNA ligases.
FIGS. 2A and 2B show the Specificity of Compounds 67, 82 and 189 as human DNA ligase inhibitors. FIG. 2A shows the IC50 values determined for Compounds 67, 82 and 189 using the fluorescence-based DNA joining assay for each of DNA ligases I, III and IV and T4 DNA ligase. FIG. 2B shows the effect of Compounds 67, 82 and 189 on DNA joining by human DNA ligases I, III and IV and T4 DNA ligase as determined using the radioactive gel-based assay.
FIGS. 3A, 3B and 3C show the mechanism of action of human DNA ligase inhibitors. FIG. 3A shows Human DNA ligases I, III and IV and T4 DNA ligase incubated with [α32P] ATP in the absence or presence of Compounds 67, 82 and 189 (100 μM). After separation by SDS-PAGE, the labeled ligase-AMP complex was detected by phosphorimaging. FIG. 3B shows labeled ligase-adenylate form of human DNA ligases I, III and IV and T4 DNA ligase incubated with a linear DNA substrate containing a single non-ligatable nick in the absence or presence of Compounds 67, 82 and 189 (100 μM). After separation by denaturing gel electrophoresis, the labeled DNA-AMP complex was detected by phosphorimaging. FIG. 3C shows non-adenylated human DNA ligases I, III and IV and T4 DNA ligase incubated with labeled DNA-AMP in the absence or presence of Compounds 67, 82 and 189 (100 μM). After separation by denaturing gel electrophoresis, labeled ligated DNA was detected by phosphorimaging.
FIGS. 4A, 4B and 4C show the effect of human DNA ligase inhibitors on replication and repair by human cell extracts. FIG. 4A shows a cell extract from the human cervical cancer cell line HeLa incubated with the indicated labeled flap substrate that mimics a common intermediate in DNA replication and long path base excision repair in the absence or presence of Compounds 67, 82, 184 and 189 (25 μM). After separation by denaturing gel electrophoresis, labeled fragments corresponding to the DNA substrate, cleaved product and fully repaired product were detected by phosphorimaging (28). FIG. 4B shows a HeLa cell incubated with labeled dTTP and the indicated linear substrate with an incised AP site that mimics an intermediate in short path base excision repair in the absence or presence of Compounds 67, 82 and 189 (25 μM). Lane 6, DNA ligase I was immunodepleted from the extract prior to the assay. Lane 7, DNA ligase III was immunodepleted from the extract prior to the assay. After separation by denaturing gel electrophoresis, labeled fragments corresponding to a single nucleotide insertion and fully repaired product were detected by phosphorimaging. FIG. 4C shows a HeLa extract incubated with a linear cohesive-ended 1 kb fragment with cohesive ends repair in the absence or presence of Compounds 67, 82 and 189 (25 μM). Lane 7, DNA ligase I was immunodepleted from the extract prior to the assay. Lane 8, DNA ligase III was immunodepleted from the extract prior to the assay. Lane 8, DNA ligases III and IV were immunodepleted from the extract prior to the assay. After separation by native agarose gel electrophoresis, the linear DNA substrate, re-circularized substrate and dimers trimers etc of the 1 kb substrate were detected by staining with ethidium bromide.
FIGS. 5A and 5B show the effect of DNA ligase inhibitors on cell growth in the absence or presence of DNA damage. FIG. 5A shows different concentrations of Compounds 67 and 151 added to asynchronous subconfluent populations of HCT116 and MCF10A cells. FIG. 5B shows asynchronous subconfluent populations of HCT116 and MCF10A cells pre-treated with different concentrations of Compound 64 prior to the addition of 3 aminobenzamide (2 mM), MMS (100 μM) or cis-platinum (1 μM). After incubation for 5 days, cell growth was measured by the MTT assay (16).
FIGS. 6A, 6B, 6C and 6D show the cytotoxicity of DNA ligase inhibitors in the absence or presence of DNA damage. FIG. 6A shows different concentrations of Compound 184 were added to asynchronous subconfluent populations of HCT116, MCF7 and MCF10A cells. Colonies were counted after two weeks. FIG. 6B shows different concentrations of Compound 151 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells in the absence or presence of MMS (50 μM). Colonies were counted after two weeks. FIG. 6C shows different concentrations of Compound 189 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells. After 1 hour, cultures were irradiated with 2 gray of ionizing radiation. Colonies were counted after two weeks. FIG. 6D shows different concentrations of Compound 67 were added to asynchronous subconfluent populations of HCT116, MCF7 and MCF10A cells. After 1 hour, cultures were irradiated with 2 gray of ionizing radiation. Different concentrations of Compound 67 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells in the absence or presence of MMS (50 μM). Colonies were counted after two weeks.
FIG. 7. Michaelis-Menten analysis of ligase inhibitors. Effect of ligase inhibitors on DNA-protein complex formation by hLigI. A, hLig1 (0.05 pmol) was incubated in the absence (o) and presence of L189 (left), L67 (middle), and L82 (right) at 25 μmol/L (open triangle) and 50 μmol/L (open square) with increasing amounts of a linear nicked DNA substrate. Lineweaver-Burk double reciprocal plots of initial reaction velocity (1/V) versus substrate concentration (1/S). B, a labeled linear substrate with a single nonligatable nick (1 pmol) was incubated with no addition (lane 1), 0.25 pmol of hLigI (lanes 2 and 3), 0.5 pmol of hLigI (lanes 4 and 5), and 1 pmol of hLigI (lanes 6 and 7) in the absence (−) or presence (+) of 100 μmol/L of L189. C, a labeled linear substrate with a single nonligatable nick (1 pmol) and hLigI (3 pmol) were incubated with either no addition (lane 2) or 100 μmol/L (lane 3), 60 μmol/L (lane 4), 50 μmol/L (lane 5), 30 μmol/L (lane 6), 20 μmol/L (lane 7), or 10 μmol/L (lane 8) of L82. Lane 1, 1 pmol of DNA substrate alone. The positions of the labeled DNA substrate and DNA-protein complexes are indicated.
FIG. 8. Characterization of the cytostatic effect of L82. A, MCF10A (▪), MCF7 (♦), HCT116 (▴), and HeLa (upsidedown triangle) cells were plated in the absence or presence of L82 (left), L67 (middle), and L189 (right). After 6 days, cell viability was measured and is expressed as a percentage of the value obtained with untreated cells. B, MCF7 cells were plated out in the absence or presence of L82 (top) and L67 (bottom) at the indicated concentrations. After 2 weeks, colonies were stained with crystal violet. C, after serum starvation for 4 days, MCF 7 cells were returned to serum-containing media either without (▪) or with 50 μmol/L of L82 (●). The cell cycle distribution at various time intervals was determined by fluorescence-activated cell sorting. D, asynchronous populations of MCF cells were either untreated (top) or treated with L82 at 10 μmol/L (middle) and 50 μmol/L (bottom). After 3 days, tubulin and DNA were visualized by fluorescence microscopy (bars, 0.5 mm).
FIG. 9. L67 and L189 are cytotoxic and potentiate the cytotoxic effects of DNA-damaging agents: altered levels of DNA ligase in cancer cells. A, effect of L67 (left) and L189 (right) on the survival of MCF7 (Δ), HCT116 (□), HeLa (⋄), and MCF10A (upsidedown triangle) cells. B, normal breast epithelial MCF10A cells (open symbols) and breast cancer MCF7 cells (filled symbols) in the absence (circles) or presence of 3 μmol/L of L67 (squares) were exposed to increasing concentrations of MMS (left). Normal breast epithelial MCF10A cells (open symbols) and colon cancer HCT116 cells (filled symbols) in the absence (circles) or presence of 20 μmol/L of L189 (squares) were exposed to increasing doses of ionizing radiation (right). C, hLigI (I), hLigIIIα (III), and hLigIV (IV) were detected in extracts (400 μg) of the indicated cell lines by immunoblotting. To control for extract loading β-actin was also detected by immunoblotting.
FIG. 10. The DNA substrate (thin tube) is encircled by three domains of human DNA Ligase I, i.e. the DNA binding domain (DBD) containing residues Asp262-Ser535 (connected large tubes-bottom), the adenylation domain (AdD) Pro536-Asp748 (wide ribbon-upper right), and the OB-fold domain (OBD) Tyr749-Ser901 (narrow ribbon-upper left). The AMP cofactor (in CPK representation) is located in AdD and the putative binding site on DBD is represented by red spheres and the three residues defining the binding pocket, His337, Arg449 and Gly453, are shown in CPK representation.
FIG. 11. Distributions of (a) the molecular weight and (b) the van der Waals attractive energy Va of the 50,000 compounds selected, via different normalizations by N the number of heavy atoms of the compound, from the preliminary screening. The normalization Va/N2/3, as shown by the blue dotted line is used to select compounds.
FIG. 12. (A) Alignment of the crystal structure and four conformations selected from the 20 ns MD simulation. (B) Orientations of three of the residues lining the binding site.
FIG. 13, Distributions of (a) the molecular weight and (b) the total interaction energy, Etot, of the 1000 compounds selected via different normalization schemes from the secondary docking.
FIG. 14. Distributions of physical and molecular properties of the 233 selected compounds.
FIG. 15. Assessment of inhibitor and DNA interaction by EtBr replacement assay. There is no binding of 82, 67 and 189 with DNA (A). In contrast, EtBr is replaced to DNA binding by 192 in a concentration (0, 50, 100 μM) dependant manner (B).
FIG. 16. The distribution of the N2/5 normalized total energy scores for the top 1000 compounds (black circles), the selected 233 compounds (red squares) and the 10 compounds that specifically inhibit hLigI activity by >50% (spikes). The scores for the 3 characterized compounds are shown as longer spikes; the wider spikes are associated with two compounds having similar scores.



DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
Chemicals.
Compounds identified by CADD screening were purchased from ChemBridge, ChemDiv, MayBridge, MDD, Nanosyn, Specs, Timtec, and Tripos. 189 was from Specs, and 82 and 67 were from Chemdiv. Tenmillimolar stocks were prepared in DMSO and stored at −20° C. The molecular masses of three compounds 67, 82 and 189 were confirmed by mass spectrometry in the University of Maryland School of Pharmacy facility.
Proteins.
Recombinant human DNA ligase I was purified as described previously (22, 29). T4 DNA ligase was purchased from NEB.
DNA Joining Assays.
Candidate ligase inhibitors identified by CADD were assayed for their ability to inhibit hLigI and T4 DNA ligase using a high-throughput, fluorescence energy transfer-based DNA joining assay (22). Duplicate reactions (30 μL) containing 10 pmol of nicked DNA substrate and either 0.25 pmol of hLigI or 10 units of T4 DNA ligase were incubated in the presence or absence of 100 μmol/L of the putative inhibitor. DNA binding by the candidate DNA ligase inhibitors was measured by displacement of ethidium bromide from DNA as previously described (30).
A radioactive gel-based DNA ligation assay was performed as previously described (22). A 25-mer (5′-CGC CAG GGT TTT CCC AGT CAC GAC C-3′), and a 5′-[32P] end-labeled 18-mer (5′-GTA AAA CGA CGG CCA GTG-3′) were annealed to a complementary 44-mer oligonucleotide, generating a linear duplex with a central nick. DNA joining reactions (30 μL) containing 0.5 pmol of labeled DNA substrate, and hLigI (0.02 pmol), hLigIIIβ (0.02 pmol), hLigIV/XRCC4 (0.1 pmol), or T4 DNA ligase (0.02 pmol) in ligation buffer were incubated in the absence or presence of ligase inhibitors at 25° C. for 30 min.
Assays for steps 2 and 3 of the ligation reaction.
To analyze step 2 of the ligation reaction, labeled ligase-AMP intermediates (10 pmol (22)) were incubated overnight at 25° C. with an unlabeled nonligatable version (dideoxy residue at the 3′-terminus of the nick) of the DNA oligonucleotide substrate (10 pmol), either in the presence or absence of the ligase inhibitors (100 μmol/L).
To analyze step 3 of the ligation reaction, an adenylated labeled version of the 18-mer was prepared as described (24). The DNA substrate containing a preadenylated nick (0.5 pmol) and hLigI (0.05 pmol), hLigIIIβ (0.05 pmol), hLigIV/XRCC4 (0.1 pmol), or T4 DNA ligase (0.05 pmol) were incubated in ligation buffer without ATP, either in the presence or absence of the ligase inhibitors (100 μmol/L). Reactions were stopped by the addition of an equal volume of gel loading dye (95% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol). After heating at 95° C. for 5 min, DNA was separated by denaturing polyacrylamide gel electrophoresis. Labeled oligonucleotides were detected and quantitated in the dried gel by phosphorImager analysis (Molecular Dynamics).
Kinetic Analysis of Ligase Inhibitors.
To measure the initial rates of ligation, hLigI (0.05 pmol) was incubated with 0.5 to 100 pmol of the fluorescent, nicked DNA substrate and various concentrations of the ligase inhibitors. Ki values were obtained from Lineweaver-Burk double reciprocal plots and curve-fitting using PRISM v3.03 (GraphPad).
Electrophoretic Mobility Shift Assay.
A labeled linear duplex with a nonligatable nick was incubated with hLig1 in ligation buffer (30 μL total volume) with or without ligase inhibitors for 120 min at 25° C. After the addition of an equal volume of native gel buffer [160 mmol/L Tris-HCl (pH 6.8), 20% glycerol, 1.4 mol/L 2-mercaptoethanol, and 0.05% bromophenol blue], samples were separated by electrophoresis through a 12% native polyacrylamide gel and detected in the dried gel by phosphorImager analysis.
Cell Extract Assay of DNA Replication and Repair.
Extracts were prepared from human cervical cancer HeLa cells as described previously (27, 31). For base excision repair (BER) assays, the extraction buffer contained 100 mmol/L of KCl, whereas for nonhomologous end-joining (NHEJ) assays, extraction buffer contained 400 mmol/L of KCl. Where indicated, DNA ligases were immunodepleted from the extracts as described (32) using protein A or G Sepharose beads (GE Healthcare) and anti-Lig1, anti-LigIII (GeneTex), or anti-LigIV (ABCAM) antibodies. Depletion was confirmed by immunoblotting.
A labeled 5′-flap substrate (0.1 pmol; (31)) was incubated with 20 μg of extract in the absence or presence of ligase inhibitors (100 μmol/L) at 25° C. for 5 min in ligation buffer (final volume, 50 μL). For short patch BER, a linear duplex containing a single uracil residue was preincised by treatment with uracil DNA glycosylase and APE1 (both from NEB) to generate a strand break with 3′ hydroxyl and 5′ deoxyribose phosphate termini. Reactions (50 μL) containing 0.3 pmol of the incised DNA substrate, 10 μCi of [α32P]dTTP, and 20 μg of extract either in the absence or presence of ligase inhibitors (100 μmol/L) were incubated at 25° C. for 2 min in ligation buffer. After separation by denaturing polyacrylamide gel electrophoresis, labeled oligonucleotides were detected in the dried gel by phosphorImager analysis (Molecular Dynamics).
To assay NHEJ (24), a 1 kb end-labeled BamHI fragment (0.1 pmol; (33)). and 20 μg of extract were incubated in ligation buffer (final volume, 20 μL), for 120 min at 25° C. either in the presence or absence of ligase inhibitors (100 μmol/L). DNA fragments were resolved by separation through a 0.8% agarose gel. Labeled DNA fragments were detected in the dried gel by phosphorImager analysis (Molecular Dynamics).
Cell culture assays. Normal human breast epithelial MCF10A cells were grown in DMEM/F-12 media (Gibco) with 20 μg/ml of EGF, 0.5 μg/ml of Hydrocortisone, 0.1 μg/ml of Cholera Toxin, 10 μg/ml of Insulin, 5% horse serum and 1% Pen/Strep. Human colon cancer HCT116 cells were grown in McCoy media (Gibco) with 10% FBS and 1% Pen/Strep. Human cervical cancer HeLa and breast cancer MCF7 cells were grown in low glucose medium (Gibco) containing 10% FBS and 1% Pen/Strep.
For survival assays, cells were plated in 6 well plates and grown either in the absence or presence of ligase inhibitors. Where indicated, either MMS (0-50 μM) was added to the medium or the cells were irradiated in a Cs-137 irradiator (0-2.5 Gy). After two weeks, colonies were stained with crystal violet (Sigma) and then counted.
FACS Analysis.
Cells (1×106) were serum starved for 4 days prior stimulation with serum containing medium either with or without ligase inhibitors. At various time intervals, cells were trypsinized, washed with PBS and then fixed in 95% ethanol overnight at −20° C. After RNAse treatment and propidium iodide (Sigma) staining, the cell cycle distribution was determined by FACS in the Flow Cytometry Shared Service of the Marlene and Stewart Greenebaum Cancer Center.
Cell Proliferation Assays.
The ability of the compounds identified by CADD to inhibit proliferation of normal mammary epithelial MCF10A cells and colon carcinoma HCT116 cells was determined using a Biomek FX Laboratory Automation Workstation (Beckman Coulter, Inc., Fullerton, Calif.). On day 0, 20 μl of complete medium containing the appropriate number of cells (150-300) was plated per well of a 384-well tissue plate (Fisher Scientific, Hampton, N.H.) and incubated overnight at 37° C. with 5% CO2 and 90% humidity. Next day, day 1, compounds were prepared by serial dilution with complete medium to yield the concentration 100 μM, and 20 μl was added to each well containing 20 μl of medium and cells yielding the final concentration 50 μM in 40 μl volume. Plates were incubated for additional 3 days (days-2-5) until control cells (0.5% DMSO) reached  ̃70-80% confluency. On day 6, 40 μl of lysis/detection solution containing 1.2% Igepal CA-630 (Sigma) and a 1:1000 dilution of SYBR Green I nucleic acid stain (Molecular Probes, Eugene, Oreg.) was added to each well. Following an overnight incubation at 37° C., total fluorescence was measured using a Fluorostar Galaxy plate reader with a 485 nm excitation filter and 520 nm emission filter set (BMG Labtech, Inc., Durham, N.C.). Data was exported to a custom program that determined growth inhibition by dividing each individual fluorescence value by the average of fluorescence values obtained with cells treated with DMSO alone. Compounds that showed at least 40% growth inhibition compared with the DMSO-only controls inhibition of one or both of the cell lines were scored as “hits”.
The activity of hits from the initial screen was further validated using the MTT assay. Briefly, MCF10A and HCT116 cells were seeded in 96 well plates at 300 and 1200 cells per well, respectively, and allowed to adhere overnight. The following day, serial dilutions of compounds in media were added to the cells in a final volume 200 μl. After incubation for 5 days, MTT reagent (3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyltatrazolium) was added and incubation was continued for 4 h. Formazan crystals generated by reduction of the MTT reagent in mitochondria were solubilized with isopropanol prior to the measurement of absorbance at 560 nm wavelength in a plate reader.
Immunocytochemistry.
The effect of ligase inhibitors on the subcellular distribution of tubulin was examined by fluorescence microscopy. Cells grown on the chamber slides were washed with PBS, fixed and then permeabilized on ice for 10 min in 2% formaldehyde and 0.1% Triton X-100. After washing with PBS, cells were incubated for 15 min with 1% BSA (Sigma-Aldrich) and then incubated with tubulin antibody (Invitrogen) for 2 h. Cells were washed with PBS prior to incubation with the AF488 conjugated secondary antibody (Invitrogen) for 1 h. After washing with PBS, cover slips were mounted on the slides with mounting media containing DAPI (Invitrogen). Cells were observed under a fluorescence microscope (Nikon Eclipse 80i) using 60× oil immersion lenses. Images were processed using NIS-Elements BR2.30 software.
Methods
CADD Screening.
The in silico identification of compounds with a high probability of binding to and inhibiting DNA ligase involved the following steps:
Identification of a putative ligand binding site on the interface between the DBD and bound DNA (FIG. 10);
molecular dynamics (MD) simulations for the generation of multiple protein conformations to address the flexibility of the binding site in the screening process;
preliminary screening of compound set,
secondary docking of compounds from the preliminary screen against the crystal structure and the MD generated structures, and
final selection of compounds for experimental assay.
Protein Structure Preparation.
The crystal structure of hLig1, obtained from the protein database bank (38) (PDB, http://www.rcsb.org/) (PDB identifier 1×9n)6 was truncated, keeping only the DBD. Hydrogen atoms were then added followed by local energy minimization with the program CHARMM (39). The minimization involved 100 conjugate gradient (CONJ) steps with the positions of atoms identified in the crystallographic structure fixed at their experimental values. Calculations were performed using the CHARMM all-atom protein force field including the CMAP modification (40, 41) with default cutoffs for the non-bond interactions. The resulting DBD structure was used in the preliminary docking (see below).
To obtain multiple conformations of the protein for secondary docking, an MD simulation was performed for 5 ns on the DBD using stochastic boundary conditions (42). First, the structure was minimized for 200 Steepest Descent (43) (SD) steps in vacuum. The binding region was then solvated by overlaying the protein with a 35 Å water sphere centered on the geometric center of the three residues, His337, Arg449, and Gly453, defining the binding pocket (see below). Water molecules within 2.8 Å to any protein non-hydrogen atom were removed. A test equilibration MD simulation showed a tendency for the water to move towards the protein and away from the surface of the sphere associated with the deletion of water overlapping the protein. Therefore, the water ball was assigned a boundary potential of 30 Å for all remaining calculations. All atoms were divided into three radial shells, i.e. the central region, an edge region from 26 to 30 Å, and an outer region beyond 30 Å, which was comprised of only protein atoms. Atoms in the outer region were constrained to their energy-minimized positions, atoms in the edge region were harmonically restrained with a force constant of 5 kcal/mol/Å and the central region was not subject to any type of restraints. The density of the water sphere was maintained using a quartic potential via the Miscellaneous Mean Field Potential (MMFP) module (44, 45) in CHARMM. Parameters defining the potential were force 0.25, droff 28.5, and pl 2.25, which yields a local well of −0.31 kcal/mol at the edge of the sphere. Non-bond interaction lists were heuristically updated out to 14 Å with the electrostatic and Lennard Jones (LJ) interactions truncated at the range of 10 to 12 Å using force switching (46). Following a 500 step SD minimization the protein was subjected to a 5 ns MD simulation at 300 K using the velocity Verlet (VVER) (47) integrator, an integration time step of 2 fs, and SHAKE to constrain all covalent bonds involving hydrogen atoms (48). Coordinates were saved every 5 ps, yielding a total of 1000 conformations from which additional structures were selected for the secondary docking. Selection of conformations for docking was performed via clustering based on pairwise root-mean square differences of the position of residues defining the binding site, i.e. the residues Glu300-Arg305 on the loop between helixes 3 and 4 according to the helix order in 1×9n.pdb, Ser334-His337 at the end of helix 5, Pro341-Asp351 on the loop following the short helix 6, and residues Gly448-Glu456 on the loop between helixes 12 and 13. Clustering was performed with NMRCLUST (49) with representative structures from the four biggest clusters chosen and used in the secondary docking.
Identification of Putative Binding Site.
A putative DNA binding site within the DBD was identified using the sphere sets used in the program DOCK (36) in combination with residues implicated in DNA binding by x-ray crystallography.
Three residues focused on, His337, Arg449, and Gly453, that are located in the central region of the DBD and make direct contacts with the DNA substrate are shown in FIG. 10. Other residues comprising the binding site include Gly448, Arg451 and Ala455.
Generation of the sphere set used the minimized crystallographic structure, with the hydrogen atoms deleted, to compute the Connolly solvent accessible surface (50, 51) via the subroutine DMS which is implemented in the program MIDAS (52) (recently updated to Chimera (53). The solvent accessible surface was computed via DMS using the surface points of the probe sphere as required when hydrogen atoms are not present, with the density of points set to 0.5 as suggested for proteins. Second, spheres ranging from radii 1.2 Å to 4 Å, complementary to the protein surface were generated by the subroutine SPHGEN implemented in the package DOCK (36). Each sphere contacts two protein surface points and lies on the normal of one of the two points. This procedure generates a very large number of spheres, which are filtered by selecting only the largest sphere associated with each surface atom. Next, spheres within 8 Å of all three reference residues, His337, Arg449, and Gly453, were selected and selected spheres on the periphery of the putative binding pocket manually deleted. This yielded the final sphere set shown in FIG. 10 which was used to direct the in silico database screening.
In Silico Compound Databases.
An in-house database of more than 4.3 million low-molecular weight compounds has been developed in the University of Maryland Computer-Aided Drug Design Center. This database is comprised of three types of files, i.e. 2D SD format files originally from the commercial vendors, 3D MOL2 format files for docking, and binary MDB format files for use in the program MOE (Chemical Computing Group Inc. Montreal, Canada). Compound preparation included removal of the smaller components in entries containing salts (e.g. counter ions), adding hydrogen atoms, assignment of the protonation state, geometry optimization using the MMFF94 (54, 55) force field level with either the SYBYL (Tripos Associates, St. Louis, Mo.) or MOE (Chemical Computing Group, Canada), and assignment of atomic partial charges based on CM2 charge model computed at the semi-empirical quantum chemical AM1 level using AMSOL (56, 57). Preliminarily screening used approximately 1,500,000 compounds from vendors chosen based on their reliability with respect to availability of compounds. Vendors include Chembridge (371,000), Chemdiv (750,000), Maybridge (60,000), MDD (33,000), Nanosyn (47,000), Specs (232,000), Timtec (165,000) and Tripos (80,000), where the values in parentheses represent the approximate number of compounds associated with each company. Recently, the compounds in the collections from these companies have been shown to typically have drug like characteristics (58).
Docking and Final Compound Selection.
Docking computations were performed using DOCK4.0 (34) with parameters previously used in our laboratory (37, 59, 60). Kollman partial atomic charges for the DBD were assigned using the program SYBYL. Database searching in DOCK is performed via a fragment-based build up procedure (61). In this approach one or more anchor fragments (e.g. rigid units, such as rings, with 5 or more atoms) are overlaid on the spheres in 200 orientations. The remainder of the ligand is then built layer by layer, with a rotation about each added bond in 10° increments to identify the most favorable orientation based on the total ligand-protein interaction energy. Thus, the docking procedure accounts for ligand flexibility while the protein is treated as rigid. From the preliminary docking using only a single conformation of the protein, the top 50,000 compounds were selected based on the normalized van der Waals attractive (Va) energy, as described below. These compounds are then subjected to a second round of docking where the crystallographic plus four additional conformations of the protein from the MD simulation (Table 1) were targeted to account for protein flexibility. The ligands were separately docked into each protein conformation, with the most favorable score from all five conformations assigned to rank that ligand. The score used in the second docking run is the total interaction energy including electrostatic and van der Waals interactions. In addition, the ligand was subjected to additional optimization by increasing the maximum anchor fragment orientations from 200 to 500, performing minimization of the anchor at each cycle of ligand buildup and minimizing the 5 inner layers upon addition of each layer of the ligand.







TABLE 1







The RMSD values in Å between each pair of the five conformations


used for database screening, including the crystal structure (1 × 9n)


and the four MD generated conformations (C2-C5). Only residues


related to the binding region are used in the calculation.













1 × 9n
C2
C3
C4
C5


















1 × 9n
0







C2
2.18
0



C3
2.29
1.82
0



C4
2.23
1.65
1.42
0



C5
2.45
2.15
1.74
1.43
0










Compound clustering was performed using the Tanimoto similarity index (62, 63) based on BIT_MACCS fingerprints (64) which is implemented in the MOE software package. The BIT_MACCS fingerprints are used to compute the pairwise Tanimoto similarity matrix which contains the similarity metric between the molecular fingerprints of compounds i and j. The matrix element S(i,j), i.e. the Tanimoto coefficient (Tc) is defined as Tc=c(i,j)/u(i,j), where c(i,j) is the number of common features in the fingerprints of molecule i and j, and u(i,j) is the number of all features in the union of the fingerprints of molecule i and j (65). Two compounds are regarded as similar if S(i,j) is equal to or greater than a predefined similarity threshold. Then, from matrix S, another binary matrix O is created where each matrix element O(i,j) has the value 1 if S(i,j) is equal to or greater than the predefined similarity threshold, or 0 otherwise. Two molecules i and j are then grouped into a cluster if the overlap between the two row vectors O(i) and O(j) is greater than or equal to a predefined overlap threshold. In the present work a similarity threshold of 70% and an overlap threshold of 40% were used.
Results
In Silico Database Screening.
A putative DNA binding pocket within the DBD of hLigI was chosen as the target for a multi-tiered in silico database screening procedure, based on regions of the DBD in direct contact with the DNA in the X-ray structure of hLigI complexed with nicked DNA (FIG. 10). In the first step of the screen to identify compounds with a high probability of binding to the DBD of hLig1, ligand posing of 1.5 million compounds was based on the total interaction energy between the ligands and the protein, with ligand ranking performed using the normalized van der Waals attractive (Va) energy. Use of the Va energy parameter selects for compounds with significant steric overlap with the binding pocket and avoids compounds with highly favorable electrostatic interactions that do not fit well into the pocket. In addition, normalization procedures correct for the tendency of compound selection based on interaction energies to bias towards high molecular weight (MW) compounds (66).
Distributions of MW using different normalization procedures and the distributions of normalized scores are shown in FIGS. 11A and 11B, respectively. Based on N2/3 normalization, a total of 50,000 compounds with a molecular weight distribution centered around 300 Daltons were selected for further analysis.
Secondary screening of the 50,000 compounds applied additional energy minimization during docking and partially addressed protein flexibility (67, 68) via the inclusion of four additional, structurally diverse conformations obtained from an MD simulation. Overall, the five conformations of the DBD are similar (FIG. 12A), indicating that significant structural changes in the protein did not occur during the MD simulation. However, a detailed comparison of the orientation of the residues lining the binding region shows that there is significant diversity across the five conformations. In FIG. 12B, the orientation of three residues, His337, Arg449, and Gly453, located in the central site of the binding region are shown. Table 1 gives the root mean square deviations (RMSD) of the residues in binding region, including residues Glu300-Arg305, Ser334-His337, Pro341-Asp351, and Gly448-Glu456. Differences in the RMSD values between the crystal structure and the four conformations from MD simulation range from 1.4 to 2.5 Å, indicating significant conformational variation in the binding pocket.
Compounds were ranked based on the most favorable normalized total interaction energy of each ligand against the five protein conformations. At this stage the total interaction energy includes electrostatic interactions as well as steric considerations in the selection process. The MW and energy distributions for different powers of N normalization of the 1000 compounds with the most favorable normalized total interaction energies are shown in FIGS. 13A and 13B. These compounds were selected based on E/(N**(2/5)) normalization, yielding a MW distribution consistent with compounds known to be pharmacologically active or used in lead compound optimization studies. (69, 70)
Final selection of compounds for in vitro biochemical assays were chosen to maximize chemical diversity and based on their drug or lead-like compound properties (69, 70). Diversity was maximized by clustering the compounds based on chemical fingerprints using the Tanimoto similarity index. This yielded approximately 200 clusters of chemically similar compounds, with 1 or 2 compounds from each cluster selected based on drug- or lead-like compound properties as defined by Lipinski's rule of 5. (71) These rules include molecular weight (MW<500), adequate solubility expressed by the octanol/water partition coefficient (−5<log P(o/w)<5), number of hydrogen bond acceptors (H-bond acceptors<10), number of hydrogen bond donors (H-bond donors<5), number of rotatable single bonds (Rotatable bonds<10), and number of rings (Ring<5). The final compounds typically also satisfy the slightly stricter rules of Oprea (69). However, for clusters in which the criteria were not met, compounds were still selected for experimental assay. From this process, 233 compounds were selected for experimental testing (Table 2). Distributions of the physical and molecular properties of the 233 compounds are presented in FIG. 14, showing them to indeed fulfill Lipinski's Rule of 5.
The method for screening compounds comprises testing the compound for the ability to inhibit human DNA ligases I, III and IV with a high throughput fluorescence-based ligation assay. In this assay, phosphodiester bond formation covalently links together two oligonucleotides, one of which is a fluorescent donor AF488 that is 11 nucleotides from the 3′ terminus and the other contains a fluorescence acceptor and quencher, BHQ1 that is 15 nucleotides from the 5′ terminus. When these oligonucleotides are present in separate duplexes and in the same nicked or intact duplex, there is no significant quenching of fluorescence because the donor and acceptor are too far apart. As single-strands neither the AF488 oligonucleotide nor the BHQ1 oligonucleotides have significant secondary structure. However, when these oligonucleotides are ligated together, the resultant single strand forms a stable hairpin structure, in which the AF488 and BHQ1 groups are very close together, resulting in efficient intra-molecular quenching of fluorescence. Thus, in the DNA joining assay, the DNA substrate is denatured after incubation with the DNA ligase and then renatured in the presence of a 20-fold excess of an oligonucleotide that is identical to the ligated strand containing the fluorescent door and acceptor except that it lacks these modifications. Using this assay, DNA joining, which results in reduced fluorescence at 518 nm, can be measured rapidly, quantitatively and with high-throughput using a fluorescence plate reader.
Experimental Assays.
192 out of the 233 compounds were screened for activity in high throughput in vitro DNA ligase assays and cell culture assays (Table 3). The screens, described in detail below, were carried out in parallel.
(i) In Vitro DNA Ligase Assay.
Applicants developed a novel high throughput fluorescence-based ligation assay by modifying the sequence of the oligos to optimize hairpin formation by the single strand ligated product of the high throughput fluorescence-based ligation assay of Chen et al (22). The ability of the 192 compounds to inhibit human DNA ligases was tested. Compound stock solutions, which were 10 mM in 0.5% DMSO, were diluted in 0.5% DMSO. Human DNA ligases I and III were assayed with a nicked DNA substrate whereas as the substrate for human DNA ligase IV was two oligonucleotide duplexes with short complementary single strand ends.
In the fluorescence-based high throughput ligation assay, 15 of the 192 compounds inhibited hLigI by >50%.
In addition, to identify compounds that may be non-specific inhibitors of DNA joining, the 192 compounds were also assayed for their ability to inhibit bacteriophage T4 DNA ligase, an enzyme that utilizes the same reaction mechanism as hLigI, has similar adenylation and OB-fold domains but lacks a DBD domain. (6) Although this is also an ATP-dependent DNA ligase, it lacks the domain containing the binding pocket targeted by the in silico screen. Compounds that inhibited T4 DNA ligase were eliminated as non-specific inhibitors that most likely bind to the DNA. The results of the screen are shown in Tables 4, 5, 5A and 6.
Of the 15 compounds that inhibited hLigI (25, 32, 64, 67, 82, 113, 123, 175, 180, 189, 190, 192, 197, 200 and 202) (Table 4), 6 were active against T4 DNA ligase by >50%. Thus, the in silico screen yielded 9 compounds that specifically inhibit hLigI, a hit rate of about 5%.
One mechanism by which a compound may non-specifically inhibit human DNA ligase is by binding to DNA rather than the ligase thereby interfering with the enzyme-substrate interaction. In accord with this idea, 192, which inhibits both hLigI and T4 DNA ligase (Table 4), reduced DNA binding of the DNA intercalating agent ethidium bromide (FIG. 6B) whereas three other compounds, 67, 82 and 189 that inhibit hLigI but not T4 DNA ligase (Table 4)), had no effect on DNA binding by ethidium bromide (FIG. 6A) indicating that 67, 82 and 189 do not bind DNA.
Because the DNA binding domains of human DNA ligases III and IV and closely related to the DNA binding domain of human DNA ligase I that was used for the in silico screening, the 192 compounds identified by the in silico screening were also assayed for activity against human DNA ligases III and IV.
The in vitro screen identified compounds that are specific for each of one the human DNA ligases (DNA ligase I, DNA ligase III and DNA ligase IV) and identified compounds that inhibit two or more of the enzymes. The in vitro screen yielded three compounds that are specific for DNA ligase I (Compounds 1, 43, 82, 151, 184, 190), one compound that is specific for DNA ligase III (Compound 209), three compounds that are specific for DNA ligase IV (Compounds 93, 122, 215), four compounds that specifically inhibit DNA ligases I and III (Compound 25, 67, 200, 213), one compound that specifically inhibits both DNA ligases I and IV (Compound 113) and three compounds that inhibit all three human DNA ligases (Compound 64, 189, 197). See Table 5A for the In Vitro and In Vivo properties of the above grouped human DNA ligase inhibitors.
(ii) In Vivo Cell Culture Assays.
The 192 human DNA ligase inhibitors were screened in parallel for effects on proliferation and the ability to potentiate the effects of several DNA damaging agents using two cell human lines, a colon cancer line HCT116 and a normal breast epithelial cell line MCF10A using a Biomek FX Laboratory Automation Workstation as described (23). The DNA damaging agents used, methyl methanesulfonate (MMS), camptothecin, cis-platinum, 3 aminobenzamide and ionizing radiation, are representatives of classes of agents used to treat cancer. Briefly, HCT116 and MCF10A cells were plated in 96 well plates such that the cultures were about 80% confluent after 5 days incubation. Compounds were added to the cultures at either 15 μM or 50 μM 1 day after plating. After incubation for 3 days, cell lysed in a solution containing 1.2% Igepal CA-630 and SYBR green that stains DNA (1:1000, Molecular Probes, Eugene, Oreg.). Subsequently, fluorescence was measured per well. The studies with DNA damaging agents were performed as follows. Cells were pre-incubated with the compounds at 75 μM for 1 hour prior to the addition of camptothecin (2 nM), cis platinum (1 μM), 3 AB (2 mM) or MMS (100 μM) that resulted in dilution of the compound to a final concentration of 50 μM. For ionizing radiation, cells were pre-incubated with the compounds at 50 μM for 1 hour prior to exposure to 2 gray of ionizing radiation. Cultures were incubated for 3 days and then treated as above. The results of the in vivo cell culture assays are summarized in Table 6.
Among the 192 compounds, seven inhibit proliferation of one or both cell lines with an IC50<20 μM (Compounds 16, 67, 78, 151, 165, 180, 195). At 50 μM 16 compounds inhibited proliferation of one or both cell lines by more than 40%. There was considerable overlap with the compounds identified as in vitro inhibitors of human DNA ligases. Notably, the nine of the in vitro inhibitors (compounds 43, 64, 67, 82, 151, 184, 189, 190, and 213) also inhibited cell proliferation.
In addition, three compounds act as radiosensitizers (Compounds 64, 151, 105), three compounds enhance the cytostatic effect of MMS (Compounds 67, 78, 151) and two compounds act synergistically with PARP inhibitors (Compound 64, 67).
Based on the combined results of the in vitro and in vivo screens, compounds 1, 25, 43, 64, 67, 78, 82, 93, 105, 113, 122, 151, 180, 184, 189, 197, 209, 213 and 215 were chosen for further analysis.
The specificity and inhibitory effects of a subset of these compounds were quantitated using the fluorescence-based ligation assay (22) and in gel-based assays (24).
The effect of Compounds 67, 82 and 189 on DNA joining by human DNA ligases I, III and IV and T4 DNA ligase was determined. The IC50 values determined for Compounds 67, 82 and 189 using the fluorescence-based DNA joining assay (22) are shown for each of the DNA ligases. See FIG. 2A. The effect of Compounds 67, 82 and 189 on DNA joining by human DNA ligases I, III and IV and T4 DNA ligase was determined using the radioactive gel-based assay (24). See FIG. 2B. Representative gels of DNA ligation assays. The results of three independent experiments are shown graphically. For clarity, the data for T4 DNA ligase, which was not significantly inhibited, has been omitted (hLig1, □, hLigIIIβ, ∘; hLigIV/XRCC4, upside down triangle).
In addition, the mechanism of inhibition was determined. DNA ligases utilize a three step reaction to catalyze phosphodiester bond formation. In the first step, human DNA ligases interact with ATP to form a covalent enzyme-adenylate complex. When this intermediate reacts with a DNA nick, the AMP group is transferred to the 5′ phosphate terminus of the nick, forming a DNA-adenylate intermediate. Finally, non-adenylated DNA ligase catalyzes phosphodiester bond formation in a reaction that is dependent upon the hydroxyl group at the 3′ terminus of the nick and results in release of the AMP group (6).
Human DNA ligases I, III and IV and T4 DNA ligase were incubated with [α32P] ATP in the absence or presence of compounds 67, 82 and 189 (100 μM). After separation by SDS-PAGE, the labeled ligase-AMP complex was detected by phosphorimaging.
Compounds 67, 82 and 189 did not inhibit the first step of the ligation reaction, which is DNA independent (FIG. 3A).
Since the final two steps of the ligation reaction involve interactions with DNA, we examined the effects of the ligase inhibitors on the second (FIG. 3B) (The labeled ligase-adenylate form of human DNA ligases I, III and IV and T4 DNA ligase were incubated with a linear DNA substrate containing a single non-ligatable nick in the absence or presence of compounds 67, 82 and 189 (100 μM). After separation by denaturing gel electrophoresis, the labeled DNA-AMP complex was detected by phosphorimaging (26)) and third steps (FIG. 3C) of the ligation reaction using established assays (25,26) (Non-adenylated human DNA ligases I, III and IV and T4 DNA ligase were incubated with labeled DNA-AMP in the absence or presence of compounds 67, 82 and 189 (100 μM). After separation by denaturing gel electrophoresis, labeled ligated DNA was detected by phosphorimaging (25)).
Based on these assays, we concluded that Compound 189 inhibits step 2 and Compounds 76 and 82 and inhibit step 3.
Next we used published cell extract assays (16,27,28) that measure different DNA replication and repair transactions to confirm the specificity of the DNA ligase inhibitors. FIG. 4A, the flap substrate (0.1 pmol) was incubated with cell extract (20 μg) in the absence (lane 2, Mock) or presence of 100 μmol/L, of L189 (lane 3), L67 (lane 4), or L82 (lane 5). hLigI (lane 6, I-dp) and hLigIIIa (lane 7, III-dp) were immunodepleted from the cell extracts prior to incubation with the DNA substrate. Lane 1, DNA substrate alone (Sub). The positions of the DNA substrate (24-mer), cleaved product (18-mer), and fully repaired product (43-mer). The flap substrate shown in FIG. 4A corresponds to an intermediate generated on the lagging strand during DNA replication and in the long patch subpathway of base excision repair. Previously it has been shown that DNA ligase I completes these two DNA transactions after removal of the flap by FEN-1 (16,28). A cell extract from the human cervical cancer cell line HeLa was incubated with the indicated labeled flap substrate that mimics a common intermediate in DNA replication and long patch base excision repair in the absence or presence of Compounds 67, 82, 184 and 189 (25 μM). After separation by denaturing gel electrophoresis, labeled fragments corresponding to the DNA substrate, cleaved product and fully repaired product were detected by phosphorimaging (28). Compounds 67, 82, and 189, all of which inhibit DNA ligase 1, inhibited the final ligation step of the repair reaction but had no effect on flap removal by FEN-1.
A natural AP site within a linear DNA molecule (FIG. 4B) is repaired primarily by short patch base excision repair that is completed by DNA ligase 111 (15). FIG. 4B, the linear DNA substrate with an incised AP site (0.3 pmol) was incubated with a cell extract (20 μg) and [a32P]dTTP in the absence (lane 1, Mock) or presence of 100 μmol/L of L189 (lane 2), L67 (lane 3), or L82 (lane 4). hLigI (lane 5, I-dp) and hLigIIIα (lane 6, III-dp) were immunodepleted from the cell extracts prior to incubation with the DNA substrate. The positions of the single nucleotide insertion reaction intermediate (31-mer, Incorporated) and the ligated product (73-mer, Repaired) are indicated. Thus, a HeLa cell was incubated with labeled dTTP and the indicated linear substrate with an incised AP site that mimics an intermediate in short path base excision repair in the absence or presence of Compounds 67, 82 and 189 (25 μM). Lane 6, DNA ligase I was immunodepleted from the extract prior to the assay. Lane 7, DNA ligase III was immunodepleted from the extract prior to the assay. After separation by denaturing gel electrophoresis, labeled fragments corresponding to a single nucleotide insertion and fully repaired product were detected by phosphorimaging (16). Accordingly, immunodepletion of DNA ligase III but not DNA ligase I inhibited the repair reaction (FIG. 4B). Compounds 67 and 189, each of which inhibits DNA ligase III, reduce repair of the natural AP site whereas Compound 82, which only inhibits DNA ligase I, has no effect (FIG. 4B).
Linear DNA molecules with short complementary single strand overhangs are repaired by non-homologous end joining that is completed by DNA ligase IV (27). A HeLa extract was incubated with a labeled linear cohesive-ended 1 kb fragment with cohesive ends repair in the absence or presence of Compounds 67, 82 and 189 (25 μM).). Lane 7, DNA ligase I was immunodepleted from the extract prior to the assay. Lane 8, DNA ligase III was immunodepleted from the extract prior to the assay. Lane 8, DNA ligase IV was immunodepleted from the extract prior to the assay. After separation by native agarose gel electrophoresis, the linear DNA substrate, re-circularized substrate and dimers trimers etc of the 1 kb substrate were detected by phosphorimaging (27). Accordingly, immunodepletion of DNA ligase IV but not DNA ligase I and III markedly inhibited the repair reaction (FIG. 4C) Compound 189, which inhibits DNA ligase IV, reduced joining of the linear DNA molecules whereas Compounds 67 and 82, which do not inhibit DNA ligase IV, had no effect (FIG. 4C). (Specifically, C, a 1 kb fragment with cohesive ends (0.1 pmol) was incubated with cell extract (20 μg) in the absence (lane 3, Mock) or presence of 100 μmol/L of L189 (lane 4), L67 (lane 5), or L82 (lane 6). hLig1 (lane 7, I-dp), hLigIIIa (lane 8, III-dp), and hLigIV (lane 9, IV-dp) were immunodepleted from the cell extracts prior to incubation with the DNA substrate. Lane 1, molecular mass standard (M). Lane 2, DNA substrate alone (Sub). The positions of the DNA substrate and dimmers and multimers of the substrate are indicated.)
To extend the results of the in vivo cell culture screening assay, a subset of compounds were tested in larger cultures. Different concentrations of Compounds 67 and 151 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells. Asynchronous subconfluent populations of HCT116 and MCF10A cells were pre-treated with different concentrations of compound 64 prior to the addition of 3 aminobenzamide (2 mM) MMS (100 μM) or cis-platinum (1 μM). After incubation for 5 days, cell growth was measured by the MTT assay (16).
An example of the growth inhibitory effects of Compounds 67 and 151 on the MCF10A and HCT116 cell lines is shown in FIG. 5A. Compound 64 markedly potentiates the growth inhibitory effect of the PARP inhibitor, 3 aminobenzamide on MCF10A cells (FIG. 5B).
To confirm that the DNA ligase inhibitors directly kill cells and potentiate cell killing by DNA damaging agents, we performed colony forming assays.
Different concentrations of Compound 184 were added to asynchronous subconfluent populations of HCT116, MCF7 and MCF10A cells. Colonies were counted after two weeks. (FIG. 6A).
Different concentrations of Compound 151 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells in the absence or presence of MMS (50 μM). Colonies were counted after two weeks. (FIG. 6B).
Different concentrations of Compound 189 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells. After 1 hour, cultures were irradiated with 2 gray of ionizing radiation. Colonies were counted after two weeks. (FIG. 6C).
Different concentrations of Compound 67 were added to asynchronous subconfluent populations of HCT116, MCF7 and MCF10A cells. After 1 hour, cultures were irradiated with 2 gray of ionizing radiation. Different concentrations of Compound 67 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells in the absence or presence of MMS (50 μM). Colonies were counted after two weeks. (FIG. 6D).
Compound 184 kills normal breast epithelial cells (MCF10A). It is also more effective at killing colon (HCT116) and breast cancer (MCF7) cell lines (FIG. 6A). Compound 151 markedly potentiates the killing of the colon cancer cell line HCT116 by MMS but has no significant effect on MMS cytotoxicity of the normal breast epithelial cell line MCF10A (FIG. 6B). Compound 189 markedly potentiates the killing of the colon cancer cell line HCT116 by ionizing radiation but has no significant effect on the MMS cytotoxicity of the normal breast epithelial cell line MCF10A (FIG. 6C). Compound 67 also potentiates the killing of the colon cancer cell line HCT116 by ionizing radiation but has no significant effect on the MMS cytotoxicity of the normal breast epithelial cell line MCF10A (FIG. 6D, left panel). Furthermore, Compound 67 potentiates the killing of the breast cancer cell line MCF7 by both MMS and ionizing radiation but has no significant effect on the MMS- and ionizing radiation-induced cytotoxicity the normal breast epithelial cell line MCF10A (FIG. 6D, right panel). Thus, DNA ligase inhibitors are cytotoxic, enhance the cytotoxicity of DNA damaging agents and are more cytotoxic to cancer cells than normal cells.
CADD Analysis of Active Compounds.
Structures of the 10 hLigI specific inhibitors are shown in FIG. 7. As may be seen, the structures are chemical diverse, as verified by the calculation of pairwise Tanimoto similarity indices between a number of active compounds (Table 7). The largest Tc value between two compounds is 69% and the majority of the values are less than 50%, indicating a low degree of similarity between compounds. Previous studies have indicated that a value of 85% or more is associated with compounds that will have similar biological activities (72). The inclusion of chemical diversity in compound selection has the desirable effect of identify structurally dissimilar compounds for drug optimization, thereby increasing the probability of identifying active compounds. This may be seen by analyzing the energy scores of the selected compounds. Presented in FIG. 16 are the distributions of the normalized total energy scores for the 1000 compounds from the secondary screen, for the 233 compounds selected from the top 1000 based on diversity and physical properties and of 9 active compounds (FIG. 1). Consideration of diversity and physical properties led to the selection of more compounds with less favorable interaction energies. Notably, many of the active compounds would not have been selected if the top 233 scoring compounds were selected based on interaction energies and so would not have been identified.
The importance of the inclusion of multiple conformations of the putative binding site from the MD simulation in the in silico screen may be determined by simply identifying the conformation from which 10 active compounds were selected. Of the 9 hLigI specific inhibitors, one (67) was based on the crystal conformation, none were based on MD conformation C2 (time point 2.015 ns of the simulation), two (82, 113) were based on MD conformation C3 (time point 2.335 ns of the simulation), three (25, 190, 197) were based on MD conformation C4 (time point 2.950 ns of the simulation) and three (64, 189, 200) on conformation C5 (time point 3.795 ns of the simulation). Thus, the inclusion of multiple conformations is leading to the identification of additional active compounds, emphasizing the utility of this component of the screening procedure.
The protein conformation is that from the crystallographic study with the orientation of the compounds extracted from the individual conformations following alignment of the protein conformations as shown in FIG. 12A. All the inhibitors occupy the targeted site, consistent with the docking methodology. However, they do sample different regions of the binding site. Such difference may contribute to differential selectivities of activity of the inhibitors for different ligases.
Three of the active compounds, 67, 82, and 189 have been subjected to more extensive biological characterization (73). Although all three compounds are predicted to bind in the putative binding site, they do exhibit some level of variability in the binding orientations. Interestingly, while all three compounds inhibit hLigI but not T4 DNA ligase (Table 4), their activity versus the other human DNA ligases differs significantly. While 82 inhibits only hLigI, 67 inhibits both hLigI and hLigIII and 189 inhibits all three human DNA ligases. Presumably, differences in the specificities of the inhibitors for the three human DNA ligases reflect a combination of differences in the binding modes of the structurally diverse inhibitors and differences in the molecular architecture of the targeted DNA binding pocket between the three human DNA ligases. Importantly, inhibitors with defined specificities for the different human DNA ligases will be invaluable reagents for elucidating the physiological roles of human DNA ligases.
Consistent with the inclusion of physical properties in the selection process, all the hLigI specific inhibitors fall into the drug-like range according to Lipinski's rule of five (Table 4) (71), while still spanning a range of physical properties.
From the in silico database Applicants have identified compounds of General Formulae I-XVI as also being DNA ligase inhibitor candidates.
General Formulae I and II are illustrative of compounds related to Compound 64. Compounds related to Compound 64 are shown in Table 8.
General Formulae IIA and IIIA are illustrative of compounds related to Compound 67. Compounds related to Compound 67 are shown in Table 9.
General Formulae III is illustrative of compounds related to Compound 78.
Compounds related to Compound 7$ are shown in Table 10.
General Formulae IV and V are illustrative of compounds related to Compound 113. Compounds related to Compound 113 are shown in Table 11.
General Formulae IX and X are illustrative of compounds related to Compound 151. Compounds related to Compound 151 are shown in Table 12.
General Formulae XI and XII are illustrative of compounds related to Compound 180. Compounds related to Compound 180 are shown in Table 13.
General Formulae XIII and XIV are illustrative of compounds related to Compound 189. Compounds related to Compound 189 are shown in Table 14.
General Formulae XV and XVI are illustrative of compounds related to Compound 197. Compounds related to Compound 197 are shown in Table 15.




where R2 is hydrogen,

R3 is hydrogen, halogen, Cl,





(where R4 is halogen or Cl and n is 0-5), or





R1 is





(where R6 is halogen or F and n is 0-5), or





(where R6 is halogen or F and n is 0-5)





where R1, R2 and R3 are the same as in formula (I)





R2 are each the same or different and are selected from —OH, alkoxy, halogen, —CH3, —OCH3 Cl, Br and F or two R2s form a fused ring and n=0-3, preferably 0-2

R3=H, alkyl, or —CH3 

R4=H, alkyl, methyl or ethyl; or one of R3 and one of R2 form a fused ring

R1=aryl,









(where R5 is halogen, alkyl, Br, Cl, or —CH3 and n5 is 0-5)





(where R5 and is halogen, alkyl, Br, Cl, or —CH3 and n5 is 0-5)

or R1 and R4 forma fused ring.





Where R1, R2, R3, R4, and n are the same or in general formula (IIA)

    
    
        R5 and n5 are same as R2 and n in general formula (IIA).
    
    






where

R1 is alkyl, alkylene, alkoxy, ethyl, methyl, alkyl substituted with aryl,





R2 is O−, halogen, alkyl, alkoxy, BR, Cl, —O—CH3, —NH2, —CH3, and when n is greater than 2, two R2s may form a fused ring, n is 1-5.





wherein R5, R5′ R5′′ and R5′′′ are selected from H, aryl, and alkyl; and preferably H

wherein R1, R1′ R1′′′ and R1′′′ are selected from





wherein each R7 is independently selected from halogen, Cl, Br, F, alkyl, and —CH3, and n7 is 0-5





where R8 and n8 are the sane as R7 and n7,





where R9 and n9 are the same as R7 and n7, alkyl-cycloalkyl,





where R10 and n10 the same as R7 and n7,





where

z is 1, 2, 4 or 5,

R10 is selected from halogen, Br and Cl,

R20 is selected from alkylene, cycloalkyl, alkyl-aryl,





    
    
        where R29, R30 and R31 are selected from halogen, Cl, I, alkyl
        n29, n30 and n31 are 0-5; preferably 0 or 1
        n10 is 0-5; preferably 0 or 1.
    
    






    
    
        wherein
        R2 is selected from —OH, O—,
    
    






alkoxy, halogen, Cl, and —O—CH3,

    
    
        n is 0-5, preferably 1 or 2,
        R3 is selected from H, alkyl, and —CH3,
        R1 is selected from H, alkyl, and CH3, and
        X is selected from N and CH.
    
    






    
    
        wherein
        R2 is selected from —OH, O—,
    
    






alkoxy, halogen, Cl, and —O—CH3,

    
    
        X1 is selected from N and CR3,
        X2 is selected from N and CR4,
        wherein R3 and R4 are independently selected from H and
    
    






    
    
        and at least one of X1 or X2 is
    
    










    
    
        R5 is selected from H, alkyl, methyl, halogen and Cl,
        R1 is selected from aryloxy, alkoxy,
    
    






where R6 is selected from alkyl, —CH3, H, alkoxy, —OCH2—CH3, halogen, Cl, Br





n6 is 0-6;

    
    
        n1 is 0-3
        R2 is selected from 1-1, alkyl, halogen, methyl, and Cl, and
        n2 is 0 to 5.
    
    






    
    
        where R1, R2 and n2 are same as defined in Formula (XI).
    
    






    
    
        R1 is selected from H, alkyl, methyl, —C≡N, halogen and Cl, or when n is greater than 2 two Ras may form a fused ring
        n is 0-5,
        R3 is selected from H alkyl and methyl,
        R2 is selected from —OH and —NH2,
        X is selected from N and CR4, where R4 is selected from —OH, N and H,
        R5 is selected from H, —OH, NH2 and S
    
    






    
    
        R1 is selected from H, alkyl, methyl, —C≡N, halogen and Cl, or when n is greater than 2 two R1s may form a fused ring
        n is 0-5,
        R2, R3 and R4 are independently selected from —O and —N.
    
    






    
    
        R1 and R2 are independently selected from
    
    






    
    
        R2 and R4 are independently selected from alkyl, alkyl, methyl and benzyl
        n1 and n2 are independently selected from 0, 1, 2.
    
    






    
    
        R1 and R2 are independently selected from
    
    






    
    
        R2 and R4 are independently selected from alkyl, alkyl, methyl and benzyl
        n1 and n2 are independently selected from 0, 1, 2.
    
    


The DNA ligase inhibitors of the present invention may also be used as research tools in addition to therapeutics. As research tools, the DNA ligase inhibitors may be used to identify the DNA ligase involved in different DNA transactions either in cell culture and/or in cell extract based assays. Including the use of DNA ligase inhibitors to provide novel insights into the reaction mechanisms of human DNA ligases; the use of DNA ligase inhibitors in cell extract assays to identify the human DNA ligase involved in DNA repair pathways; and the use of DNA ligase inhibitors in cell culture assays to identify the human DNA ligase involved in different DNA repair pathways in vivo. For example, delineating DNA ligases participating and determining which of multiple pathways are being used.
The DNA ligase inhibitors of the present invention may be used to facilitate in vitro and in vivo studies delineating the cellular functions of these enzymes.
Most DNA damaging agents introduce more than one type of DNA lesion. In addition, a specific DNA lesion may be removed by more than one DNA repair pathway. Since the DNA ligase inhibitors of the present invention inhibit DNA repair pathways, the use of the inhibitors in a combination of extract and cell culture assays may be used to delineate the DNA repair pathways involved in the repair of DNA lesions introduced by a DNA damaging agent and the DNA repair pathways involved in the repair of a specific DNA lesion.
The DNA ligase inhibitors of the present invention may be used as research tools to promote the understanding of genome stability and DNA repair, such as Lagging strand DNA replication; Telomere replication; Rescue of stalled replication forks; Damage response signaling cascade; DSB repair, BER; polβ-dependent long patch; PCNA-dependent long patch; Short patch; and NER: DNA double strand break repair by homology-dependent pathways and by the major and alternative non-homologous end-joining pathways.
The DNA ligase inhibitors of the present invention may also be used in drug development for clinical cancer treatment, such as anti-cell proliferation and Radiosensitizers.
As used herein, “treat” means alter, apply, effect, improve, care for or deal with medically or surgically, ameliorate, cure, stop and/or prevent an undesired biological (pathogenic) process. The skilled artisan is aware that a treatment may or may not cure.
As used herein, the effective amount or “therapeutically effective amounts” of the compound of the present invention to be used are those amounts effective to produce beneficial results in the recipient animal or patient. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated. Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value.
A therapeutically effective amount of a compound of the present invention as a treatment varies depending upon the host treated and the particular mode of administration. The term “body weight” is applicable when an animal is being treated. When isolated cells are being treated, “body weight” as used herein means “total cell body weight”. The term “total body weight” may be used to apply to both isolated cell and animal treatment. Those of skill in the art will recognize the utility of a variety of dosage range.













TABLE 2






ligase_1x9n_ docking_233_compounds
COMP_NAME
IDNUMBER
stru
MolW
logP





















1




chembridge000828
5103856
2335
174
1.92


 


2




chembridge004647
5133963
2335
238
0.724


 


3




chembridge004783
5135431
1x9n
226
−0.672


 


4




chembridge005314
5140822
1x9n
198
0.552


 


5




chembridge009555
5185417
2335
281
2.54


 


6




chembridge0119938
6635973
1x9n
337
1.05


 


7




chembridge0132951
5540113
2950
295
4.32


 


8




chembridge0136123
5649818
2950
316
−1.87


 


9




chembridge0137192
5679271
2950
326
0.653


 


10




chembridge0144275
5807168
1x9n
322
2.65


 


11




chembridge014881
5226945
2950
381
4.06


 


12




chembridge015172
5227796
2950
232
1.72


 


13




chembridge0171207
6102396
2335
433
2.3


 


14




chembridge0171753
6106327
2950
235
0.925


 


15




chembridge017857
5241412
1x9n
377
1.54


 


16




chembridge017892
5241504
2335
300
2.36


 


17




chembridge0183353
6203744
2950
218
0.384


 


18




chembridge0192792
6364469
2950
270
2.85


 


19




chembridge0222418
6629596
2335
275
−0.025


 


20




chembridge0250465
5925673
2950
230
0.83


 


21




chembridge0253645
6297813
2950
210
−0.982


 


22




chembridge0257297
6550468
2950
294
3.43


 


23




chembridge0266747
6841883
2335
301
0.704


 


24




chembridge0295137
7173063
1x9n
356
1.02


 


25




chembridge029819
5317419
2950
191
0.119


 


26




chembridge0307818
7296187
2950
271
1.29


 


27




chembridge0343539
7650920
2335
308
1.64


 


28




chembridge0347904
7676009
1x9n
344
2.26


 


29




chembridge0352654
7699479
2335
383
4.76


 


30




chembridge0355774
7724431
2950
318
1.63


 


31




chembridge036048
5355751
2335
293
0.343


 


32




chembridge0365296
7777402
2335
386
1.98


 


33




chembridge0374491
7812749
2950
237
3.38


 


34




chembridge0374541
7813065
2335
303
1.57


 


35




chembridge0377382
7830700
2950
233
2.01


 


36




chembridge0378150
7836125
2950
357
2.28


 


37




chembridge0382972
7863038
2335
222
0.311


 


38




chembridge0387746
7902307
2335
312
−0.012


 


39




chembridge063174
5608638
2950
305
1.95


 


40




chembridge080590
5742694
2335
315
1.71


 


41




chembridge085143
5790780
2335
304
1.91


 


42




chembridge091964
5869880
2335
301
2.79


 


43




chembridge099119
6051018
1x9n
335
4.36


 


44




chembridge102405
5356872
2950
312
1.5


 


45




chembridge103842
5154723
2335
329
3


 


46




chembridge104229
5210894
2335
339
1.61


 


47




chemdiv015291
3448-0483
2335
276
2.97


 


48




chemdiv016239
3464-8119
2335
309
1.56


 


49




chemdiv018608
2279-5355
2950
279
1.5


 


50




chemdiv0202765
000A-0636
2335
225
−0.669


 


51




chemdiv0223644
4300-0817
2335
231
1.6


 


52




chemdiv0254427
K831-0255
2950
300
0.535


 


53




chemdiv0260892
0054-0264
2950
210
0.972


 


54




chemdiv0262519
1725-0122
2335
129
−0.903


 


55




chemdiv0281075
4055-0046
2335
205
−0.375


 


56




chemdiv0283584
4114-0028
2950
305
2.15


 


57




chemdiv0283734
4130-0051
2335
260
0.684


 


58




chemdiv0297199
4596-0306
1x9n
175
0.682


 


59




chemdiv0297579
4676-0062
2950
252
2.22


 


60




chemdiv0310162
8011-6595
2950
204
0.399


 


61




chemdiv0311450
8012-0236
2335
340
3.58


 


62




chemdiv0329374
K831-0247
2950
287
0.226


 


63




chemdiv0330961
K906-0447
2950
369
1.03


 


64




chemdiv0333435
R052-1644
2950
290
2.03


 


65




chemdiv071566
0814-0180
2950
321
3.1


 


66




chemdiv093992
1535-0002
2335
224
0.103


 


67




chemdiv102812
1761-0019
1x9n
486
4.79


 


68




chemdiv103836
1761-1961
1x9n
370
0.978


 


69




chemdiv117392
2093-0008
2950
193
0.834


 


70




chemdiv137918
2595-1130
2335
219
0.647


 


71




chemdiv172782
8007-0302
2950
320
0.727


 


72




chemdiv189691
8010-4604
2335
356
4.22


 


73




chemdiv195033
K061-1138
2950
220
0.575


 


74




chemdiv201905
K781-0461
2335
259
1.7


 


75




chemdiv202632
R095-0016
2950
306
3.66


 


76




chemdiv4000099
000A-0435
2950
205
0.146


 


77




chemdiv4000752
0519-0362
2950
331
0.823


 


78




chemdiv4003854
2672-0239
2335
321
2.85


 


79




chemdiv4003904
2684-1487
2950
309
1.16


 


80




chemdiv4004117
2764-0217
2950
259
0.825


 


81




chemdiv4006822
3350-0006
2950
266
1.75


 


82




chemdiv4014181
4300-0746
2335
310
4.72


 


83




chemdiv4016932
4469-0504
2950
375
4.66


 


84




chemdiv4016952
4470-0573
2950
310
0.754


 


85




chemdiv4017354
4483-2627
2950
321
3.86


 


86




chemdiv4030337
5167-1580
2335
328
2.46


 


87




chemdiv4032105
5227-2224
2335
219
2.2


 


88




chemdiv4047092
5720-0252
2335
182
−0.203


 


89




chemdiv4050365
5849-2464
2335
244
0.952


 


90




chemdiv4059074
6144-0830
2335
250
0.548


 


91




chemdiv4064940
6296-0075
2335
302
2.87


 


92




chemdiv4067503
6404-0306
2950
252
1.83


 


93




chemdiv4077013
7011-1829
2950
194
0.386


 


94




chemdiv4077303
7100-1148
2335
313
2.36


 


95




chemdiv4077895
7213-0775
2335
207
−1.01


 


96




chemdiv4078007
7287-0119
2335
399
0.835


 


97




chemdiv4080636
8010-0511
2950
250
2.1


 


98




chemdiv4080731
8010-5081
2335
189
0.518


 


99




chemdiv4084070
8013-6158
2335
264
0.637


 


100




chemdiv4088658
8015-2157
2335
180
−1.17


 


101




chemdiv4092369
C066-3475
2335
234
0.82


 


102




chemdiv4113108
C200-0872
2335
302
1.7


 


103




chemdiv4114091
C200-2517
2335
277
0.282


 


104




chemdiv4115701
C206-0731
2335
239
2.52


 


105




chemdiv4115760
C206-0886
2950
302
4.09


 


106




chemdiv4136188
C301-1637
2950
178
1.51


 


107




chemdiv4155887
C455-0040
2950
258
1.39


 


108




chemdiv4178771
C612-0733
2950
312
1.48


 


109




chemdiv4200659
C804-0249
2950
343
0.498


 


110




chemdiv4212928
E546-0207
2335
287
1.24


 


111




chemdiv4226558
K783-5936
2950
208
0.537


 


112




chemdiv4247207
K906-0577
2950
300
1.38


 


113




chemdiv4249937
K978-1019
2335
475
1.42


 


114




chemdiv4250212
R052-2665
2950
163
−0.321


 


115




chemdiv4250230
R052-2693
2950
174
0.921


 


116




chemdiv4256344
C200-2775
2950
475
0.953


 


117




chemdiv4257714
C430-0780
2950
227
0.861


 


118




chemdiv4262012
C800-0149
2950
244
1.26


 


119




maybridge0404348
BTB 06428
2950
247
3.81


 


120




maybridge0405401
BTB 08297
2950
323
1.9


 


121




maybridge0407919
BTB 13897
2335
207
2.38


 


122




maybridge0407972
BTB 14052
2950
216
1.27


 


123




maybridge0409092
CD 01419
2950
228
1.51


 


124




maybridge0414926
GK 01940
2335
237
1.86


 


125




maybridge0418520
HTS 03891
2950
314
2.34


 


126




maybridge0419334
HTS 05121
2950
234
0.154


 


127




maybridge0425599
JFD 00838
2950
341
1.69


 


128




maybridge0428829
KM 02107
2950
250
0.522


 


129




maybridge0430404
KM 05117
2950
207
0.114


 


130




maybridge0434449
MWP 00580
2950
189
1.07


 


131




maybridge0435308
NRB 00719
2950
289
2.87


 


132




maybridge0439282
RF 00190
2950
215
0.428


 


133




maybridge0439473
RF 00770
2950
268
2.75


 


134




maybridge0440186
RF 03622
2335
190
1.67


 


135




maybridge0441358
RH 00871
2950
239
0.994


 


136




maybridge0442630
RJC 00691
2335
183
0.161


 


137




maybridge0443763
RJC 02884
2950
244
2.06


 


138




maybridge0445673
S 00903
2335
258
4.11


 


139




maybridge0445675
S 00982
2950
227
3.37


 


140




maybridge0449798
SCR 01207
2950
342
2.75


 


141




maybridge0454605
SPB 00315
2335
167
1.14


 


142




maybridge0455528
SPB 02143
2950
346
2.91


 


143




maybridge0455775
SPB 02523
2950
198
2.39


 


144




mdd0506944
APX000008174
2335
282
3.44


 


145




mdd0507227
APX000008527
2950
254
3.16


 


146




mdd0507257
APX000008566
2950
256
1.56


 


147




mdd0525205
APX000027192
2335
179
0.51


 


148




mdd0528932
APX000030930
2335
203
0.857


 


149




mdd0531592
APX000033591
2950
317
1.52


 


150




nanosyn000678
NS49351
2335
361
3.55


 


151




nanosyn006272
NS54884
2950
306
0.108


 


152




nanosyn012438
NS15117
1x9n
304
4.26 


 


153




nanosyn019560
NS0633
1x9n
296
2.12


 


154




nanosyn020035
NS1965
2950
202
2.14


 


155




nanosyn020419
NS2538
2950
347
0.517


 


156




nanosyn022218
NS5033
2950
224
−1.3


 


157




nanosyn024054
NS7425
2335
252
1.04


 


158




nanosyn026199
NS10673
2335
288
−0.938


 


159




nanosyn028817
NS14410
2335
305
2.9


 


160




nanosyn029397
NS15310
2335
312
2.96


 


161




nanosyn032970
NS21015
2335
246
1.61


 


162




nanosyn041421
NS32415
2950
200
−1.14


 


163




nanosyn042848
NS33842
2335
254
1.25


 


164




nanosyn043053
NS34047
2950
209
1.02


 


165




nanosyn044719
NS35713
1x9n
334
4.05


 


166




nanosyn047911
NS38906
2950
195
0.555


 


167




nanosyn052735
NS44149
2335
224
0.92


 


168




nanosyn061098
NS63350
1x9n
253
2.32


 


169




nanosyn064176
NS66483
2335
289
2.57


 


170




nanosyn064661
NS67013
2335
269
1.3


 


171




specs0087646
AG-205/12145002
2335
272
0.787


 


172




specs0093768
AP-845/40883774
2950
228
1.8


 


173




specs0094412
AE-641/40197985
2950
279
1.9


 


174




specs0094816
AI-942/25121085
2950
206
0.428


 


175




specs0094999
AM-807/25050007
2335
174
0.998


 


176




specs0096856
AE-406/41056556
2335
259
1.84


 


177




specs0097907
AE-641/30103046
2335
265
4.42


 


178




specs0104444
AF-399/15335020
2335
292
1.59


 


179




specs0104985
AF-399/15393031
1x9n
355
3.6


 


180




specs0112290
AG-205/15425118
2950
301
2.44


 


181




specs0123459
AG-690/09793058
2335
200
−1.54


 


182




specs0126485
AG-690/11384661
2950
291
−0.44


 


183




specs0129149
AG-690/12002297
2335
302
2.65


 


184




specs0133795
AG-690/15436354
2950
272
−0.45


 


185




specs0140784
AG-690/40697266
2335
345
2.84


 


186




specs0142480
AG-690/40700254
2335
324
2.14


 


187




specs0142745
AG-690/40700684
2335
354
0.846


 


188




specs0154616
AI-204/31687014
2950
216
1.29


 


189




specs4002175
AC-907/34129012
2950
246
1.84


 


190




specs4002201
AC-907/34130009
2950
270
1.54


 


191




specs4003032
AE-562/12222186
2950
344
0.148


 


192




specs4003077
AE-562/12222297
2950
274
1.56


 


193




specs4004319
AE-641/06280018
2950
311
3.11


 


194




specs4007749
AE-848/30709022
2950
306
2.11


 


195




specs4020536
AG-205/08231019
2335
316
3.81


 


196




specs4021096
AG-205/08625012
2335
273
0.787


 


197




specs4028351
AG-205/32243059
2950
310
1.63


 


198




specs4030921
AG-205/33652013
2950
292
1.63


 


199




specs4036585
AG-205/40650755
2950
362
1.61


 


200




specs4039932
AG-207/37370001
2335
214
2.8


 


201




specs4043054
AG-670/31548028
2950
287
0.234


 


202




specs4044861
AG-690/08639033
1x9n
390
4.49


 


203




specs4051894
AG-690/12844905
2950
204
1.82


 


204




specs4052603
AG-690/12885209
2950
216
1.3


 


205




specs4056295
AG-690/15433670
2950
205
0.928


 


206




specs4056597
AG-690/15438954
2950
247
0.308


 


207




specs4063372
AG-690/37215010
2950
289
1.38


 


208




specs4073894
AI-237/31666029
2335
272
0.565


 


209




specs4074491
AJ-030/12105064
2335
370
−0.561


 


210




specs4074773
AJ-087/41885602
2950
241
1.57


 


211




specs4076821
AJ-292/41686278
1x9n
366
2.78


 


212




specs4078664
AJ-333/36115017
2950
212
−0.117


 


213




specs4079101
AK-087/42718317
2950
194
2.54


 


214




specs4085187
AK-918/42409851
2950
312
3.63


 


215




specs4087862
AK-968/11789151
2335
297
1.28


 


216




specs4103227
AK-968/41169454
2335
221
2.59


 


217




specs4103395
AK-968/41170109
2335
219
0.442


 


218




specs4121778
AN-465/41521127
2950
226
1.49


 


219




specs4132753
AN-829/13872035
2950
196
−0.685


 


220




specs4133908
AN-979/15013141
2335
331
1.81


 


221




specs4136100
AO-080/42479361
2950
318
3.23


 


222




speccs4156297
AR-422/41026969
2335
315
1.36


 


223




st007595
st008467
2335
197
1.3


 


224




tripos0034247
1525-00782
2950
249
0.974


 


225




tripos0034504
1525-01725
2335
279
1.36


 


226




tripos0046768
1528-02292
2335
301
0.506


 


227




tripos0047216
1528-03826
2950
342
3.44


 


228




tripos0047548
1528-04508
2335
258
2.35


 


229




tripos0049773
1528-10011
2950
336
2.6


 


230




tripos0049952
1528-10605
2950
279
2.27


 


231




tripos0052524
1533-00601
1x9n
231
1.08


 


232




tripos0053972
1533-06492
2335
423
4.12


 


233




tripos0074665
1554-07198
1x9n
362
3.09























TABLE 8






ligase64 chemdiv0333435 75
COMP NAME
IDNUMBER
MW
a
d
logP






















1




chemdiv0333435
R052-1644
288.6
2.
0.
2.15


 


2




chembridge0127433
5241477
258.6
3.
2.
0.88


 


3




chembridge017871
5241438
333.6
2.
1.
3.11


 


4




chembridge0224352
6640684
384.3
4.
0.
2.35


 


5




chembridge0252784
6208029
363.7
2.
1.
3.65


 


6




chembridge0318761
7399643
252.2
2.
2.
1.85


 


7




chembridge0350072
7684126
345.1
1.
1.
3.81


 


8




chembridge0353163
7703755
284.2
1.
1.
3.16


 


9




chembridge0361286
7749202
295.2
3.
2.
2.81


 


10




chembridge0410035
7963899
441.3
5.
0.
2.20


 


11




chembridge057754
5556669
269.2
1.
0.
2.72


 


12




chemdiv0235647
8012-5585
418.8
4.
2.
3.62


 


13




chemdiv029346
8002-5557
216.2
1.
0.
0.99


 


14




chemdiv195033
K061-1138
218.5
1.
0.
0.70


 


15




chemdiv202374
R052-0733
254.2
2.
0.
1.52


 


16




chemdiv4039985
5555-0004
216.2
2.
2.
0.94


 


17




chemdiv4228031
K784-6223
364.3
3.
2.
3.13


 


18




chemdiv4230965
K786-1157
300.6
3.
1.
1.47


 


19




chemdiv4231246
K786-1552
408.8
3.
1.
3.72


 


20




maybridge0431189
KM = 06791
257.2
3.
1.
2.02


 


21




maybridge0431220
KM = 06833
241.2
3.
1.
1.20


 


22




maybridge0431223
KM = 06837
291.2
2.
0.
3.70


 


23




maybridge0431242
KM = 06873
363.3
3.
0.
3.61


 


24




maybridge0431243
KM = 06874
345.3
3.
0.
3.45


 


25




maybridge0431901
KM = 08121
213.1
1.
0.
1.23


 


26




specs0118592
AG-227/40703560
347.7
3.
1.
3.59


 


27




specs4043497
AG-670/36154046
359.3
2.
2.
3.64


 


28




specs4099247
AK-968/37166230
294.6
1.
0.
2.66





















TABLE 9






ligase-67-chemdiv102812-similar90
COMP_NAME
IDNUMBER
Weight
logP(o/w)




















1




chemdiv102812
1761-0019
486.12
4.794


 


2




chembridge0139405
5724812
423.22
3.619


 


3




chembridge0163789
6046146
516.15
4.785


 


4




chembridge061292
5575463
407.22
3.959


 


5




chembridge091422
5863488
414.42
5.27


 


6




chemdiv0304474
8004-4208
547.36
6.79


 


7




chemdiv030710
8002-9168
449.26
3.468


 


8




chemdiv031476
8003-1205
405.3
2.642


 


9




chemdiv031594
8003-1363
542.3
5.168


 


10




chemdiv032220
8003-2671
390.78
3.338


 


11




chemdiv034545
8004-0876
407.22
3.961


 


12




chemdiv036531
8004-5914
373.32
3.096


 


13




chemdiv038132
8005-0790
421.21
3.278


 


14




chemdiv042897
8006-1915
360.3
2.672


 


15




chemdiv042898
8006-1916
376.76
3.074


 


16




chemdiv042910
8006-1930
360.3
2.672


 


17




chemdiv043057
8006-2113
515.31
5.569


 


18




chemdiv088176
1359-0042
419.23
3.514


 


19




chemdiv102882
1761-0255
486.12
4.794


 


20




chemdiv103694
1761-1563
421.25
4.704


 


21




chemdiv159407
3284-0676
378.77
3.45






















TABLE 10






ligase 78 chemdiv4003854 83
COMP NAME
IDNUNBER
cl . . .
MW
logP





















1




chemdiv4003854
2672-0239
1.00
320.
2.97


 


2




chembridge066517
5635200
12.0
399.
3.81


 


3




chembridge066825
5637177
13.0
449.
4.93


 


4




chembridge081742
5756943
15.0
384.
4.19


 


5




chembridge084110
5785385
17.0
308.
2.65


 


6




chemdiv030495
8002-8328
19.0
385.
4.24


 


7




chemdiv049350
8010-0528
16.0
358.
3.87


 


8




chemdiv049352
8010-0530
21.0
337.
1.94


 


9




chemdiv070385
0784-0310
22.0
413.
3.09


 


10




chemdiv070399
0784-0352
10.0
370.
4.10


 


11




chemdiv080501
1094-0055
30.0
319.
3.50


 


12




chemdiv080519
1094-0096
31.0
305.
3.16


 


13




chemdiv080520
1094-0097
30.0
319. 3.46



 


14




chemdiv080523
1094-0100
35.0
320.
2.49


 


15




chemdiv080527
1094-0104
1.00
320.
2.97


 


16




chemdiv080533
1094-0112
1.00
370.
4.27


 


17




chemdiv080539
1094-0122
41.0
378.
2.11


 


18




chemdiv085129
1270-0044
46.0
335.
3.12


 


19




chemdiv085130
1270-0045
47.0
385.
4.24


 


20




chemdiv105653
1783-0201
52.0
355.
4.29


 


21




chemdiv133102
2395-0012
57.0
310.
1.73


 


22




chemdiv4003850
2672-0163
10.0
370.
4.10


 


23




chemdiv4003855
2672-0240
1.00
370.
4.19


 


24




chemiv4019950
4555-4523
16.0
308.
2.65


 


25




chemdiv4021425
4632-6961
70.0
354.
3.60


 


26




chemdiv4021435
4632-6987
72.0
433.
4.44


 


27




chemdiv4021439
4632-7005
75.0
404.
4.73


 


28




specs0122994
AG-690/09504058
117. 
414.
2.97






















TABLE 11






Ligase_active#113_chemdiv4249937
COMP_NAME
IDNUMBER
MW
logP
8585







 













1




113_chemdiv4249937
K978-1019
475
1.42
1


 


2




amb_c0025720
A2357/0099549
393
3.31
1


 


3




amb_c0045243
AmblieP-559829
321
0.481
3


 


4




amb_d022599
A2357/0099549
393
3.31
1


 


5




amb_e0017501
ASKBASE/24046
321
0.481
3


 


6




amb_e0021248
ASKBASE/28478
383
2.31
6


 


7




amb_e0021385
ASKBASE/28630
318
1.6
7


 


8




amb_e0023055
ASKBASE/30341
383
2.31
8


 


9




amb_e0023957
ASKBASE/31261
383
2.34
8


 


10




amb_e0025034
ASKBASE/32342
335
0.569
10


 


11




amb_e0025080
ASKBASE/32388
353
2.19
1


 


12




amb_e0025103
ASKBASE/32411
397
2.39
1


 


13




amb_e0025149
ASKBASE/32457
332
1.68
13


 


14




asinex59633
BAS = 0138030
638
7.73
14


 


15




chembridge0216275
6584322
383
2.34
8


 


16




chembridge0216484
6585646
383
2.31
8


 


17




chembridge0218325
6599829
332
1.68
13


 


18




chembridge0258673
6620392
353
2.19
1


 


19




chembridge0266367
6831135
350
1.93
7


 


20




chembridge0318078
7390552
338
2.4
20


 


21




chembridge0368775
7788404
443
4.64
1


 


22




chembridge0372356
7802552
423
4.14
1


 


23




chembridge0408597
7957540
421
4.19
1


 


24




chemdiv0315353
8012-8697
421
4.19
1


 


25




chemdiv0324687
K781-2046
367
2.68
1


 


26




chemdiv0324691
K781-2050
401
3.28
1


 


27




chemdiv0325214
K781-4155
415
2.95
27


 


28




chemdiv0325222
K781-4171
397
2.8
1


 


29




chemdiv0325224
K781-4177
446
3.48
29


 


30




chemdiv0325227
K781-4185
411
3.1
27


 


31




chemdiv0325238
K781-4295
350
2.24
1


 


32




chemdiv0325309
K781-4963
457
3.7
32


 


33




chemdiv0325311
K781-4969
457
4.28
1


 


34




chemdiv0325312
K781-4973
488
4.81
1


 


35




chemdiv0325313
K781-4974
389
3
32


 


36




chemdiv0325316
K781-4979
431
4.32
1


 


37




chemdiv0325327
K781-4981
508
5.35
1


 


38




chemdiv0325318
K781-4982
439
4.13
1


 


39




chemdiv0326079
K783-0131
465
5.31
39


 


40




chemdiv0328145
K784-4328
432
3.39
27


 


41




chemdiv4130154
C274-3672
565
2.95
41


 


42




chemdiv4148442
C382-0316
479
5.75
39


 


43




chemdiv4148450
C382-0342
451
4.86
1


 


44




chemdiv4225320
K781-4165
411
2.89
1


 


45




chemdiv4225426
K783-0142
437
4.42
39


 


46




chemdiv4234898
K786-6828
382
2.22
46


 


47




chemdiv4238958
K788-2181
435
4.24
1


 


48




chemdiv4239059
K788-2347
465
5.22
1


 


49




chemdiv4239292
K788-2747
449
4.54
1


 


50




chemdiv4239818
K788-3519
469
4.84
1


 


51




chemdiv4239820
K788-3521
487
5.34
1


 


52




chemdiv4248907
K938-0642
419
3.81
1


 


53




chemdiv4248970
K938-0786
435
4.24
1


 


54




comgenex029401
CGX-0399491
441
4.33
1


 


55




specs0087828
AG-205/41005779
421
4.19
1


 


56




specs4072254
AH-487/42307105
435
4.84
1


 


57




specs4072408
AH-487/42483123
457
5.11
1


 


58




specs4072415
AH-487/42483920
457
4.73
1


 


59




specs4072424
AH-487/42485536
457
4.73
1


 


60




specs4077937
AJ-292/42062402
332
1.68
13


 


61




specs4077961
AJ-292/42062682
383
2.34
8


 


62




specs4108021
AK-968/41927486
515
-0.294
62


 


63




specs4135786
AO-080/41280680
393
3.31
1


 


64




timtt031879
ST031879
383
2.31
8


 


65




timtt044045
ST044045
383
2.34
8


 


66




timtt044162
ST044162
397
2.39
1


 


67




timtt044167
ST044167
332
1.68
13


 


68




timtt045785
ST045785
421
4.19
1


 


69




tripos0051110
1532-03029
396
0.623
13


 


70




tripos0051115
1532-03050
391
2.38
13





















TABLE 12






ligase 151 nanosyn006272 s85
COMP NAME
IDNUMBER
logP
MW




















1




nanosyn006272
NS54884
0.11
306.


 


2




chembridge0136023
5648015
-0.42
263.


 


3




chembridge0139327
5722560
2.90
373.


 


4




chembridge065828
5630287
0.46
304.


 


5




chembridge070734
5663902
0.45
386.


 


6




chembridge073257
5677048
-0.20
307.


 


7




chembridge074051
5681228
0.92
321.


 


8




chemdiv0227504
4487-0452
-0.19
277.


 


9




chemdiv023628
8001-0622
0.31
304.


 


10




chemdiv0308002
8008-5425
2.16
366.


 


11




chemdiv037566
8004-9459
0.31
320.


 


12




chemdiv085124
1270-0021
3.07
458.


 


13




chemdiv103764
1761-1799
2.34
366.


 


14




chemdiv103782
1761-1819
0.11
306.


 


15




chemdiv103828
1761-1952
1.01
324.


 


16




chemdiv103830
1761-1954
0.64
304.


 


17




chemdiv103836
1761-1961
1.10
368.


 


18




chemdiv103848
1761-1976
0.71
334.


 


19




chemdiv106079
1805-1308
1.21
382.


 


20




chemdiv108507
1897-1408
0.43
303.


 


21




chemdiv110779
1981-1490
1.91
427.


 


22




chemdiv119094
2144-0787
-0.45
336.


 


23




chemdiv133064
2391-2374
2.45
395.


 


24




chemdiv159730
3284-1651
1.84
316.


 


25




chemdiv197874
K089-0087
3.19
394.


 


26




chemdiv4107645
C163-0462
0.11
291.


 


27




chemdiv4107646
C163-0463
0.41
305.


 


28




mdd022784
st016688
3.25
409.


 


29




nanosyn013594
NS18706
3.40
472.


 


30




specs0169427
AK-968/15605425
1.77
399.


 


31




specs0171739
AK-968/40707401
3.14
395.


 


32




specs4086774
AK-968/11367247
2.26
369.


 


33




specs4087434
AK-968/11566598
1.72
331.


 


34




timtt017652
ST017652
3.57
460.


 


35




timtt017715
ST017715
3.04
365.


 


36




timtt017987
ST017987
4.26
415.


 


37




timtt025827
ST025827
1.05
337.


 


38




timtt4016474
ST4016474
0.39
334.






















TABLE 13






ligase 180 specs0112290 85%
COMP NAME
IDNUMBER
MW
logP
c . . .





















 1




specs0112290
AG-205/15425118
301.
2.44
1.0


 


 2




chemdiv0310191
8011-6630
362.
4.41
25.


 


 3




chemdiv0310192
8011-6631
362.
4.45
26.


 


 4




chemdiv0310201
8011-6652
300.
2.80
85.


 


 5




chemdiv0310393
8011-7163
286.
2.46
12.


 


 6




chemdiv0310717
8011-7952
270.
2.80
9.0


 


 7




chemdiv0310830
8011-8313
348.
3.82
79.


 


 8




chemdiv0310998
8011-9000
362.
4.25
10.


 


 9




chemdiv0311207
8011-9694
348.
4.11
11.


 


10




chemdiv0311221
8011-9716
378.
4.07
12.


 


11




chemdiv0311222
8011-9717
427.
4.91
80.


 


12




chemdiv0311440
8012-0207
405.
3.40
13.


 


13




chemdiv0311639
8012-0573
378.
4.11
12.


 


14




chemdiv0312704
8012-3473
405.
3.44
13.


 


15




chemdiv0313234
8012-4572
378.
3.82
81.


 


16




chemdiv0313235
8012-4573
427.
4.91
82.


 


17




chemdiv0314847
8012-7734
362.
4.45
35.


 


18




chemdiv0314848
8012-7735
315.
2.77
36.


 


19




chemdiv0315436
8012-8805
335.
3.07
37.


 


20




chemdiv0315593
8012-9041
392.
4.41
38.


 


21




chemdiv0315842
8012-9471
382.
4.71
90.


 


22




chemdiv0315970
8012-9712
376.
4.74
39.


 


23




chemdiv166863
8004-3116
359.
4.10
3.0


 


24




chemdiv167327
8004-6065
391.
4.12
91.


 


25




chemdiv4081327
8013-0295
382.
4.70
92.


 


26




chemdiv4081378
8013-0379
382.
4.74
90.


 


27




chemdiv4081817
8013-1129
382.
4.74
90.


 


28




chemdiv4082010
8013-1437
376.
4.75
42.


 


29




chemdiv4082586
8013-2547
270.
2.67
95.


 


30




chemdiv4082587
8013-2548
362.
4.31
96.


 


31




chemdiv4082589
8013-2550
364.
4.75
43.


 


32




chemdiv4082929
8013-3105
378.
4.88
44.


 


33




chemdiv4085402
8014-2098
256.
2.47
2.0


 


34




maybridge0403444
BTB = 04948
242.
1.81
76.


 


35




maybridge0403698
BTB = 05308
361.
2.76
5.0


 


36




specs0113723
AG-205/33687036
393.
4.73
77.


 


37




specs0137866
AG-690/36533026
419.
3.92
21.


 


38




specs4047979
AG-690/11629040
270.
2.77
24.


 


39




timtt049828
ST049828
362.
4.41
60.


 


40




timtt049829
ST049829
362.
4.45
61.


 


41




timtt050161
ST050161
362.
4.45
68.





















TABLE 14






mol
COMP_NAME
IDNUMBER
MW
logP




















 1




specs4002175
AC-907/34129012
246.29
1.84


 


 2




chembridge0120016
6636907
310.38
4.05


 


 3




chembridge0245406
5808234
233.29
2.14


 


 4




chembridge0273454
6955471
275.38
3.11


 


 5




chembridge0277001
6988183
297.38
3.66


 


 6




chembridge0401734
7934912
258.3
1.8


 


 7




chembridge096171
5940801
302.18
3.36


 


 8




chemdiv0281324
4065-0116
233.29
2.14


 


 9




chemdiv178113
8088-5795
281.77
3.37


 


10




maybridge0415961
GK = 03672
302.18
3.32


 


11




maybridge0438482
RDR = 01717
219.27
2.17


 


12




specs0142965
AG-690/40701022
247.32
2.44


 


13




specs4001624
AB-323/13887140
230.23
1.05


 


14




specs4002174
AC-907/34129010
214.23
0.988


 


15




specs4002176
AC-907/34129013
230.23
0.68


 


16




specs4018988
AG-205/07664054
245.24
0.375


 


17




specs4037037
AG-205/41280558
297.38
3.66


 


18




specs4064355
AG-690/40700626
258.3
1.8






















TABLE 15






ligase_197_specs4028351_similar80
COMP_NAME
IDNUMBER
MW
logP
clus





















 1




197_specs4028351
AG-205/32243059
308
1.87
1


 


 2




amb_a0005908
5908
308
1.87
17


 


 3




amb_a0034227
34248
368
2.42
23


 


 4




amb_a0041542
6653
308
1.87
26


 


 5




amb_b0092949
844
321
2.18
2


 


 6




amb_c0086983
BAS = 1172053
368
2.42
29


 


 7




amb_c0104748
A2485/0105630
307
1.85
3


 


 8




amb_c0112456
3380
321
2.18
2


 


 9




amb_d0002779
BAS = 1172053
368
2.42
29


 


10




amb_d030490
A2485/0105630
307
1.85
3


 


11




amb_e0047907
6653
308
1.87
26


 


12




chembridge0153135
5928213
308
1.87
26


 


13




chembridge0153147
5928363
294
1.66
32


 


14




chembridge0165304
6057299
370
3.53
33


 


15




chembridge0165868
6062534
368
2.42
23


 


16




chembridge0211898
6534037
280
1.44
34


 


17




chembridge0255068
6243354
307
1.85
3


 


18




chembridge0256700
6515124
308
1.87
1


 


19




chembridge0257432
6558640
308
1.87
17


 


20




chemdiv030171
8002-7527
368
2.42
23


 


21




chemdiv033499
8003-7465
308
1.87
1


 


22




chemdiv033518
8003-7503
308
1.87
17


 


23




chemdiv037027
8004-7581
370
3.53
35


 


24




chemdiv042175
8005-9535
308
1.87
17


 


25




chemdiv4080846
8011-5630
307
1.85
3


 


26




specs4027891
AG-205/14552050
307
1.85
3


 


27




timtt045056
ST045056
307
1.85
3


 


28




timtt057325
ST057325
308
1.87
1


 


29




timtt057327
ST057327
308
1.87
17


 


30




timtt204715
ST204715
308
1.87
17


 


31




timtt207481
ST207481
368
2.42
29









    1. Ding, J., Miao, Z. H., Meng, L. H., and Geng, M. Y. (2006) Trends Pharmacol Sci 27(6), 338-344
    2. Madhusudan, S., and Hickson, I. D. (2005) Trends Mol Med 11(11), 503-511
    3. Madhusudan, S., and Middleton, M. R. (2005) Cancer Treat Rev 31(8), 603-617
    4. Bryant, H. E, Schultz, N., Thomas, H. D., Parker, K. M., Flower, D., Lopez, E., Kyle, S., Meuth, M., Curtin, N. J., and Helleday, T. (2005) Nature 434(7035), 913-917
    5. Farmer, H., McCabe, N., Lord, C. J., Tutt, A. N., Johnson, D. A., Richardson, T. B., Santarosa, M., Dillon, K. J., Hickson, I., Knights, C., Martin, N. M., Jackson, S. P., Smith, G. C., and Ashworth, A. (2005) Nature 434(7035), 917-921
    6. Tomkinson, A. E., Vijayakumar, S., Pascal, J. M., and Ellenberger, T. (2006) Chem Rev 106(2), 687-699
    7. Barnes, D. E., Tomkinson, A. E., Lehmann, A. R., Webster, A. D., and Lindahl, T. (1992) Cell 69(3), 495-503
    8. Lehmann, A. R., Willis, A. E., Broughton, B. C., James, M. R., Steingrimsdottir, H., Harcourt, S. A., Arlett, C. F., and Lindahl, T. (1988) Cancer Res 48(22), 6343-6347
    9. Teo, I. A., Broughton, B. C., Day, R. S., James, M. R., Karran, P., Mayne, L. V., and Lehmann, A. R. (1983) Carcinogenesis 4(5), 559-564
    10. Sun, D., Urrabaz, R., Nguyen, M., Marty, J., Stringer, S., Cruz, E., Medina-Gundrum, L., and Weitman, S. (2001) Clin Cancer Res 7(12), 4143-4148
    11. Caldecott, K. W., McKeown, C. K., Tucker, J. D., Ljungquist, S., and Thompson, L. H. (1994) Mol Cell Biol 14(1), 68-76
    12. Caldecott, K. W., Tucker, J. D., Stanker, L. H., and Thompson, L. H. (1995) Nucleic Acids Res 23(23), 4836-4843
    13. Lakshmipathy, U., and Campbell, C. (1999) Mol Cell Biol 19(5), 3869-3876
    14. Lakshmipathy, U., and Campbell, C. (2001) Nucleic Acids Res 29(3), 668-676
    15. Frosina, G., Fortini, P., Rossi, O., Carrozzino, F., Raspaglio, G., Cox, L. S., Lane, D. P., Abbondandolo, A., and Dogliotti, E. (1996) J Biol Chem 271(16), 9573-9578
    16. Levin, D. S., McKenna, A. E., Motycka, T. A., Matsumoto, Y., and Tomkinson, A. E. (2000) Curr Biol 10(15), 919-922
    17. Adachi, N., Ishino, T., Ishii, Y., Takeda, S., and Koyama, H. (2001) Proc Natl Acad Sci USA 98(21), 12109-12113
    18. Pascal, J. M., O'Brien, P. J., Tomkinson, A. E., and Ellenberger, T. (2004) Nature 432(7016), 473-478
    19. Sun, D., and Urrabaz, R. (2004) J Biochem Biophys Methods 59(1), 49-59
    20. Tan, G. T., Lee, S., Lee, I. S., Chen, J., Leitner, P., Besterman, J. M., Kinghorn, A. D., and Pezzuto, J. M. (1996) Biochem J 314 (Pt 3), 993-1000
    21. Hancock, C. N., Macias, A., Lee, E. K., Yu, S. Y., Mackerell, A. D., Jr., and Shapiro, P. (2005) J Med Chem 48(14), 4586-4595
    22. Chen, X., Pascal, J., Vijayakumar, S., Wilson, G. M., Ellenberger, T., and Tomkinson, A. E. (2006) Methods Enrymol 409, 39-52
    23. Gallmeier, E., Hucl, T., Brody, J. R., Dezentje, D. A., Tahir, K., Kasparkova, J., Brabec, V., Bachman, K. E., and Kern, S. E. (2007) Cancer Res 67(5), 2169-2177
    24. Chen, L., Trujillo, K., Sung, P., and Tomkinson, A. E. (2000) J Biol Chem 275(34), 26196-26205
    25. Ahel, I., Rass, U., El-Khamisy, S. F., Katyal, S., Clements, P. M., McKinnon, P. J., Caldecott, K. W., and West, S. C. (2006) Nature 443(7112), 713-716
    26. Tomkinson, A. E., Tappe, N. J., and Friedberg, E. C. (1992) Biochemistry 31(47), 11762-11771
    27. Baumann, P., and West, S. C. (1998) Proc Natl Acad Sci USA 95(24), 14066-14070
    28. Zheng, L., Dai, H., Qiu, J., Huang, Q., and Shen, B. (2007) Mol Cell Biol 27(8), 3176-3186
    29. Mackey, Z. B.; Niedergang, C.; Murcia, J. M.; Leppard, J.; Au, K.; Chen, J.; de Murcia, G.; Tomkinson, A. E., DNA ligase III is recruited to DNA strand breaks by a zinc finger motif homologous to that of poly(ADP-ribose) polymerase. Identification of two functionally distinct DNA binding regions within DNA ligase III. J. Biol. Chem. 1999, 274, (31), 21679-87.
    30. Srivastava, S. K.; Dube, D.; Tewari, N.; Dwivedi, N.; Tripathi, R. P.; Ramachandran, R., Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I. Nucleic Acids Res. 2005, 33, (22), 7090-101.
    31. Wang W, Bambara R A. Human Bloom protein stimulates flap endonuclease 1 activity by resolving DNA secondary structure. J Biol Chem 2005; 280:5391-9.
    32. Di Virgilio M, Gautier J. Repair of double-strand breaks by non-homologous end joining in the absence of Mre11. J Cell Biol 2005; 171:765-71.
    33. Chen L, Trujillo K, Ramos W, Sung P, Tomkinson AE. Promotion of Dn14-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol Cell 2001; 8:1105-15.
    34. Ewing T J, Makino S, Skillman A G, Kuntz I D. DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases. J Comput Aided Mol Des 2001; 15:411-28.
    35. Huang N, Nagarsekar A, Xia G, Hayashi J, MacKerell A D, Jr. Identification of non-phosphate-containing small molecular weight inhibitors of the tyrosine kinase p 56 Lck SH2 domain via in silico screening against the pY+3 binding site. J Med Chem 2004; 47:3502-11.
    36. Kuntz I D, Blaney J M, Oatley S J, Langridge R, Ferrin T E. A geometric approach to macromolecule-ligand interactions. J Mol Biol 1982; 161:269-88.
    37. Markowitz J, Chen I, Gitti R, et al. Identification and characterization of small molecule inhibitors of the calcium-dependent S100B-p53 tumor suppressor interaction. J Med Chem 2004; 47:5085-93.
    38. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E., The Protein Data Bank. Nucleic Acids Res. 2000, 28, (1), 235-42.
    39. Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M., CHARMM: A program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 1983, 4, (2), 187-217.
    40. MacKerell, A. D., Jr.; Bashford, D.; Bellott, M.; Dunbrack, R. L.; Evanseck, I. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stow, R.; Straub, J.; Watanabe, M.; Wiorkiewicz-Kuczera, J.; Yin, D.; Karplus, M., All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 1998, 102, (18), 3586-3616.
    41. Mackerell, A. D., Jr., Empirical force fields for biological macromolecules: overview and issues. J. Comput. Chem. 2004, 25, (13), 1584-604.
    42. Brooks, C. L., III; Karplus, M., Deformable Stochastic Boundaries in Molecular Dynamics. J. Chem. Phys. 1983, 79, (12), 6312-6325.
    43. Snyman, J. A., Practical Mathematical Optimization: An Introduction to Basic Optimization Theory and Classical and New Gradient-Based Algorithms. Springer-Verlag: New York, 2005; p 257.
    44. Beglov, D.; Roux, B., Dominant Salvation Effects from the Primary Shell of Hydration—Approximation for Molecular-Dynamics Simulations. Biopolymers 1995, 35, (2), 171-178.
    45. Beglov, D.; Roux, B., Finite Representation of an Infinite Bulk System—Solvent Boundary Potential for Computer-Simulations. J. Chem. Phys. 1994, 100, (12), 9050-9063.
    46. Steinbach, P. J.; Brooks, B. R., New Spherical-Cutoff Methods of Long-Range Forces in Macromolecular Simulations. J. Comp. Chem. 1994, 15, 667-683.
    47. Swope, W. C.; Andersen, H. C.; Berens, P. H.; Wilson, K. R., A computer simulation method for the calculation of equilibrium constants for the formation of physical clusters of molecules: Application to small water clusters. J. Chem. Phys. 1982, 76, (1), 637-649.
    48. Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C., Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-alkanes. J. Comp. Phys. 1977, 23, 327-341.
    49. Kelley, L. A.; Gardner, S. P.; Sutcliffe, M. J., An automated approach for clustering an ensemble of NMR-derived protein structures into conformationally related subfamilies. Protein Eng. 1996, 9, (11), 1063-5.
    50. Connolly, M., Analytical molecular surface calculation. J. Appl. Cryst. 1983, 16, 548-558.
    51. Connolly, M. L., Solvent-accessible surfaces of proteins and nucleic acids. Science 1983, 221, (4612), 709-13.
    52. Ferrin, T. E.; Huang, C. C.; Jarvis, L. E.; Langridge, R., The MIDAS display system. J. Mol. Graphics 1988, 6, 13-27.
    53. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E., UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, (13), 1605-12.
    54. Halgren, T. A., MMFF VI. MMFF94s option for energy minimization studies. J. Comp. Chem. 1999, 20, (7), 720-729.
    55. Halgren, T. A., MMFF VII. Characterization of MMFF94, MMFF94s, and other widely available force fields for conformational energies and for intermolecular-interaction energies and geometries. J. Comp. Chem. 1999, 20, (7), 730-748.
    56. Chambers, C. C.; Hawkins, G. D.; Cramer, C. J.; Truhlar, D. G., Model for aqueous solvation based on class IV atomic charges and first solvation shell effects. J. Phys. Chem. 1996, 100, 16385-16398.
    57. Li, J.; Zhu, T.; Cramer, C. J.; Truhlar, D. G., New class IV charges model for extracting accurate partial charges from wave functions. J. Phys. Chem. A 1998, 102, 1820-1831.
    58. Sirois, S.; Hatzakis, G.; Wei, D.; Du, Q.; Chou, K.-C., Assessment of chemical libraries for their druggability. Comp. Biol. Chem. 2005, 29, 55-67.
    59. Hancock, C. N.; Macias, A.; Lee, E. K.; Yu, S. Y.; MacKerell, A. D., Jr.; Shapiro, P., Identification of novel extracellular signal-regulated kinase docking domain inhibitors. J. Med. Chem. 2005, 48, (14), 4586-4595.
    60. Huang, N.; Nagarsekar, A.; Xia, G.; Hayashi, J.; MacKerell, A. D., Jr., Identification of non-phosphate-containing small molecular weight inhibitors of the tyrosine kinase p 56 Lck SH2 domain via in silico screening against the pY+3 binding site. J. Med. Chem. 2004, 47, (14), 3502-11.
    61. Leach, A. R.; Kuntz, I. D., Conformational analysis of flexible ligands in macromolecular receptor sites. J. Comput. Chem. 1992, 13, 730-748.
    62. Tanimoto, T., IBM Internal Report, November 1957.
    63. Godden, J. W.; Xue, L.; Bajorath, J., Combinatorial preferences affect molecular similarity/diversity calculations using binary fingerprints and Tanimoto coefficients. J. Chem. Inf. Comput. Sci. 2000, 40, (1), 163-6.
    64. Durant, J. L.; Leland, B. A.; Henry, D. R.; Nourse, J. G., Reoptimization of MDL keys for use in drug discovery. J. Chem. Inf. Comput. Sci. 2002, 42, (6), 1273-80.
    65. Willett, P.; Barnard, J. M.; Downs, G. M., Chemical similarity searching. J. Chem. Inf. Comput. Sci. 1998, 38, (6), 983-996.
    66. Pan, Y.; Huang, N.; Cho, S.; MacKerell, A. D., Jr., Consideration of Molecular Weight During Compound Selection in Virtual Target-Based Database Screening. J. Chem. Inf. Comp. Sci. 2003, 43, 267-272.
    67. Carlson, H. A., Protein flexibility and drug design: how to hit a moving target. Curr. Opin. Chem. Biol. 2002, 6, 447-452.
    68. Carlson, H. A.; McCammon, J. A., Accommodating Protein Flexibility in Computational Drug Design. Mol. Pharmacol. 2000, 57, 213-218.
    69. Oprea, T. I.; Davis, A. M.; Teague, S. J.; Leeson, P. D., Is There a Difference between Leads and Drugs? A Historical Perspective. J. Chem. Inf. Comput. Sci. 2001, 41, 1308-1315.
    70. Lipinski, C. A., Drug-like properties and the causes of poor solubility and poor permeability. J. Pharmacol. Toxicol. Methods 2000, 44, 235-249.
    71. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 2001, 46, (1-3), 3-26.
    72. Brown, R. D.; Martin, Y. C., An evaluation of structural descriptors and clustering methods for use in diversity selection. SAR QSAR Environ. Res. 1998, 8, (1-2), 23-39.
    73. Chen, X.; Zhong, S.; Zhu, X.; Dziegielewska, B.; Ellenberger, E.; Wilson, G. M.; MacKerell, A. D., Jr.; Tomkinson, A. E., Rational Design of Human DNA Ligase Inhibitors that Target Cellular DNA Replication and Repair. Cancer Res. (Submitted, December 2007).


All of the cited references are hereby specifically incorporated by reference in their entirety.
While the invention has been described with reference to certain particular embodiments thereof, the invention is not to be limited to the specific embodiments described and those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention.







TABLE 3





192 Compounds Tested


List of 192 compounds that have been assayed


































1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20


21
22
23
24
25
26
27
28
29
30
32
33
34
35
36
37
38
39
40
41


42
43
45
46
47
48
49
51
53
54
55
56
57
58
59
60
61
62
63
64


65
66
67
68
69
70
71
72
73
75
76
77
78
79
80
81
82
83
84
85


86
88
89
90
92
93
94
95
96
97
99
100
101
102
103
104
105
106
107
108


109
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129


130
132
135
136
137
138
139
140
141
142
143
150
151
153
159
161
162
165
16



170
172
173
175
176
177
178
179
180
181
182
183
184
185
186
188
189
190
191
192


193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212


213
214
215
216
217
218
219
220
221
222
223
234
















TABLE 4







Molecular properties of the 15 active compounds, their experimental 


inhibition rate targeting Ligl and the growth inhibition.























hLigI_Inh %
Growth_Inh % 
(50 μM) 


Cmpd
Rot
Ring
MW
Ha
Hd
LogP
(100 μM)
MCF10A
HCT116



















25
1
2
190.13
2
1
0.24
59.3 ± 7.5 




32
6
3
385.38
6
1
2.10
83 4 ± 3.1 




64
4
2
288.65
2
0
2.15
60.6 ± 0.54
40



67
6
2
485.11
2
2
4.91
78.3 ± 8.5 
70
70


82
4
2
308.66
3
2
4.84
59.8 ± 1.7 
70
40


113
8
2
475.38
5
2
1.42
53.4 ± 7.6 




123
4
1
228.32
4
0
1.51
66.9 ± 3.5 




 175*
1
2
173.15
2
0
1.12
54.2 * 16.5




 180*
2
3
301.21
2
1
2.44
58.3 * 6.0 
70
70


189
2
2
244.28
3
i
1.96
68.8 ± 4.9 
70
70


190
2
3
268.23
5
0
1.78
52.5 * 6.6 
70
40


 192*
2
3
274.28
2
1
1.56
68.2 * 1.5 

70


197
6
2
308.31
0
0
1.87
91.0 * 6.9 




200
2
2
212.2 
1
0
3.04
50.7 * 10.9




 202*
4
4
390.42
1
0
5.08
95.0 * 6.0 
50






*= compounds are those that also inhibit T4 DNA ligase by >50%


Rot is the number of rotatable bonds,


Ring is the number of rings,


MW is the molecular weight;


Ha is the number of H-bond acceptors,


Hd is the number of H-bond donors,


LogP is the octanol/water partition coefficient.


DNA joining and cell culture assays were performed as described.


Inhibition of joining and proliferation is expressed as a percentage of values obtained with DMSO alone.














TABLE 5







% activity Inhibition
% growth inhibition
















hLigI
hLigIII
hLigIV
LigT4
MCF10A
HCT116
MCF10A
HCT116
















Drug #
100 uM
gel
100 uM
gel
100 uM
gel
100 uM
50 uM [2 Gy] 
5-15 uM










A# Drug that inhibit human DNA Ligases but not T4 DNA ligase or more than T4


A1# Common inhibitors for hLig1, hLigIII, and hLigIV


















64
60.6 ± 0.54
50
48.5 ± 0.1 
+++
51.9 ± 1.2 
66
18.4 ± 8.4 
40
70




189
68.8 ± 4.9 
75
52.6 ± 4.8 
++
>50
80
3.2 ± 1.9
70
70




197
91.0 ± 6.9 
98
96.5 ± 3.5 
+++
>60
100
24.4 ± 5.8 











A2# Common inhibitors for hLig1 and hLigIII


















67
78.3 ± 8.5 
73
88.2 ± 11.6
++
Ø

  0 ± 10.5
70
70
✓



200
50.7 ± 10.9
53
59.5 ± 5.2 
++
Ø

0
70
70

✓


82
59.8 ± 1.7 
76
29.24 ± 7.30 
+
Ø

13.9 ± 11.4
70
40




25
59.3 ± 7.5 
+++
72.8 ± 4.8 
++
Ø

12.9 ± 6.3 






213
36.4 ± 8.6 
+
25.2 ± 0.46
±
Ø


70
70




207
20.7 ± 19.0
+
25.8 ± 10.7
±
Ø

10.0 ± 2.0 











A3# Common inhibitors for hLig1 and hLigIV


















113
53.4 ± 7.6 
+++
Ø
Ø
>50
83
5.4 ± 4.5











A4# Inhibitors unique to hLig1


















184
37.7 ± 12.4
ND
Ø
Ø
Ø

0.42 ± 1.4 
70
40

✓


190
52.5 ± 6.6 
+++
Ø
Ø
Ø

 5.4 ± 0.74
40
40




1
40.3 ± 8.6 
+++
Ø
Ø
Ø

  0 ± 3.8






43
29.2 ± 3.5 
+
Ø
Ø
Ø

0
70
70




151
25.9 ± 1.2 
+
Ø
Ø
Ø


70
70
✓
✓


35
38.7
+
Ø
ND
Ø

3.7 ± 2.3






37
25.2 ± 14.1
+
Ø
ND
Ø

3.7 ± 0  






47
24.2 ± 4.3 
+
Ø
ND
Ø

2.2 ± 0.7






51
24.3 ± 13.5
+
Ø
ND
Ø

  0 ± 2.6






53
36.4 ± 4.7 
+
Ø
ND
Ø

3.7 ± 4.8






54
32.2 ± 10.5
+
Ø
ND
Ø

 6.9 ± 0.45






55
7.5 ± 9.3
+
Ø
ND
Ø

0.58 ± 3.1 






90
20.7 ± 4.8 
+
Ø
ND
Ø

1.6 ± 3.1






103
23.1 ± 1.8 
+
Ø
ND
Ø

6.2 ± 4.3






193
28.2 ± 34.4
+
Ø
ND
Ø

0.44 ± 2.1 











A5# inhibitors unique to hLigIII


















209
Ø
ND
70.5 ± 2.6 
++
Ø








46
Ø
ND
21.57 ± 8.56 
+
Ø













A6# inhibitors unique to hLigIV


















215
Ø
ND
Ø
ND
60.7 ± 1.3 
53
Ø






93
Ø
ND
Ø
ND
37.6 ± 6.1 
Ø
Ø






122
Ø
ND
Ø
ND
16.1 ± 19.1
50
Ø




















TABLE 5A







In Vitro and In Vivo properties of identified human DNA ligase inhibitors.










In vitro
In vivo













% inhib

Cell
growth inhb


















hLigI
hLigIII
hLigIV
T4

Extract
MCF10A
HCT116
cell survival












Cmpd
100 (μM)
Mechanism
100 (μM)
% Inhb at 50 (μM)
EC50 (μM)










Inhibit I, III, IV
















64
61 ± 1
49 ± 1
52 ± 1
18 ± 8 
1st step

40

IR sensitive, EC50~50


189
69 ± 5
53 ± 5
41 ± 6
3 ± 2
2nd, 3rd step
BER/NHEJ
70
70
IR sensitive, EC50~50


197
91 ± 7
96 ± 4
57 ± 3
24 ± 6 
3rd step



no activity







Inhibit I, III
















67
78 ± 8
 88 ± 12
0
 0 ± 11
3rd step
BER
70
70
IR, MMS sensitive, EC50~10


200
 51 ± 11
60 ± 5
0
0




metabolite active


25
59 ± 8
73 ± 5
0
13 ± 6 







213
36 ± 9
25 ± 1
0
0


70
70








Inhibit I, IV












113
53 ± 8
0
83 ± 5
5 ± 5








I specific
















82
60 ± 2
29 ± 7
0
14 ± 11
3rd step
BER
70
40
cytostatic, G1, EC50~50


151
26 ± 1
0
0
0

BER
70
70
MMS sensitive, EC50~10


184
 38 ± 12
0
0
1 ± 1


70
40
EC50~10


190
53 ± 7
0
0
5 ± 1


40
40



1
40 ± 9
0
0
0 ± 4







43
29 ± 4
0
0
0


70
70








III specific













209
0
71 ± 3
0
0

EC50 > 100







IV specific













215
0
0
61 ± 1
0

EC50 > 100


122
0
0
50 ± 5
0

EC50 > 100


93
0
0
37 ± 6
0
















TABLE 6







Compounds that decreased cell growth at low concentrations:











% of inhibition @



IC50 (μM)
100 μM (Helen's data)












Compound
MCF10A
HCT116
hLigI
hLigIII
hLigIV





16t
6
NI
NI
NI
NI


67
4
8
78
88
NI


78
7.5
10
ND
>20
<20


151
6
22
25
NI
NI


165
3
NI
NI
NI
37.2


180
3
12
58
>40
NI


195*
3
NI
26
24
NI


200
ID (5)
ID (10)
51
60
NI










Radiosensitizers:











% of inhibition @



IC50 ratios
100 μM (Helen's data)












Compound
MCF10A
HCT116
hLigI
hLigIII
hLigIV





64
1.2
NI
61
49
52


151
1
2
26
NI
NI


105t
1
1.3
NI
NI
NI










For 159 no IC50 was achieved, but only slight sensitization to


IR was observed. 159 did not inhibit human ligases in vitro.





Compounds that sensitized cells to MMS:











% of inhibition @



Ratios of IC50
100 μM (Helen's data)












Compound
MCF10A
HCT116
hLigI
hLigIII
hLigIV





16t
1
NI
NI
NI
NI


67
1.2
1.4 
78
88
NI


78
1.5
1 
ND
>20
<20


151
1.8
1.7 
25
NI
NI


159t
ND
2**
NI
NI
NI


165
1
NI
NI
NI
37.2


180
1
ND
58
>40
NI


195*
1.2
NI
26
24
NI


198t
NI
2**
NI
NI
NI


200
ID (1)
ID (1)
51
60
NI


214*
NI
ID (2)
ND
42
40










IC50 ratio: IC50 value derived from growth curve of cells treated 


with compound alone compared to the IC50 derived from growth


curve of cells treated in combination with MMS (100 μM). Growth


curves were normalized to either untreated control or control 


treated with MMS alone. The ratio of IC50 of compounds


that do not sensitize cells to MMS is equal to 1.





Compounds that sensitize cells to the PARP


inhibitor 3-amino benzamide (3-AB):










IC50 ratios of non- and 
% of inhibition @



3-AB treated cells
100 μM (Helen's data)












Compound
MCF10A
HCT116
hLigI
hLigIII
hLigIV





64
2 
1.6**
61
49
52


67
1.7 
1   
78
88
NI


78
1 
ND
ND
>20
<20


198t
2**
1   
NI
NI
NI


200
ID (1.2)
ID (1)
51
60
NI


214*
ID (2)
ID (1.3)
ND
42
40










IC50 ratio: ratio of IC50 value derived from growth curve of cells


treated with compound alone and IC50 derived from growth


curve of cells treated with compounds in combination with


3-AB (2 mM). Growth curves were normalized to either DMSO


control or to DMSO and 3-AB. The ratio of IC50 of compounds


that do not sensitize cells to 3-AB is equal to 1.





Compounds that inhibit cell growth at high concentrations (~50 μM)











% of inhibition @



IC50
100 μM (Helen's data)












Compound
MCF10A
HCT116
hLigI
hLigIII
hLigIV





64
50
NI
61
49
52


105t
NI
50
NI
NI
NI


159t
NI
50
NI
NI
NI


177
ND
45
48
46
NI


198t
NI
NI
NI
NI
NI


208
40
ND
21
24
NI


212t
ND
NI
NI
NI
NI


214*
ID
ID
ND
42
40










Compounds tested in the MTT assay that at 50 μM did not or only slightly


inhibit cell proliferation of either MCF10A or HCT116:









% of inhibition @


Cell line
100 μM (Helen's data)











MCF10A
HCT116
hLigI
hLigIII
hLigIV





—
16t
NI
NI
NI


—
64
61
49
52


79t
ND
NI
NI
NI


124t
124
NI
NI
NI


130t
130
NI
NI
NI


—
165
NI
NI
37


ND
184 (30%)
38
NI
NI


193t
ND
NI
NI
NI


—
195
26
24
NI


197
197
91
97
57


198**t
198**t
NI
NI
NI


199t (30%)
ND
NI
NI
NI


ND
212t
NI
NI
NI


*214**
—
NI
42
40


221t
ND
>20
NI
NI










Compounds that inhibit growth of both cell lines: MCF10A and HCT116











% of inhibition @



IC50 (μM)
100 μM (Helen's data)












Compound
MCF10A
HCT116
hLigI
hLigIII
hLigIV





67
4
8
78
88
NI


78
7.5
10
ND
>20
<20


151
6
22
25
NI
NI


180
3
12
58
>40
NI


200
ID (5)
ID (10)
51
60
NI










Compounds active on MCF10A cell line only:










IC50 (μM)
% of inhibition @ 100 μM (Helen's data)











Compound
MCF10A
hLigI
hLigIII
hLigIV





16t
6
NI
NI
NI


64
50
61
49
52


165
3
NI
NI
37


195
3
26
24
NI


208
40
21
24
NI










Compounds active on HCT116 cells more than on MCF10A:











% of inhibition @ 



IC50 (μM)
100 μM (Helen's data)











Compound
HCT116
hLigI
hLigIII
hLigIV





105t
~50
NI
NI
NI


159t
 50
NI
NI
NI


198t
**
NI
NI
NI


214*
ID 
NI
42
40



(10-50)










Compounds active in vitro that decreased cell 


growth of either MCF10A or HCT116 cell line:











% of inhibition @



IC50
100 μM (Helen's data)












Compound
MCF10A
HCT116
hLigI
hLigIII
hLigIV





64
50
NI
61
49
52


67
4
8
78
88
NI


78
7.5
10
ND
>20
<20


151
6
22
25
NI
NI


177
ND
45
48
46
NI


165
3
NI
NI
NI
37.2


180
3
3
58
>40
NI


195*
3
NI
26
24
NI


200
ID
ID
51
60
NI



(10-150)
(10-150)





208
40
ND
21
24
NI


214*
NI
ID 
ND
42
40




(10-50)













Sensitizers to MMS and 3-AB











MMS
3-AB
% of inhibition @ 



IC50 ratios
IC50 ratios
100 μM (Helen's data)














Compound
MCF10A
HCT116
MCF10A
HCT116
hLigI
hLigIII
hLigIV





64
—
2**
2 
1.6
61
49
52


67tt
1.2
1.4 
1.7 
1
78
88
NI


78tt
1.5
1 
1 
ND
ND
>20
<20


151tt
1.8
1.7 
ND
ND
25
NI
NI


159t
Ni
2* 
ND
ND
NI
NIt
NI


195*
1.2
NI (1)
ND
ND
26
24
NI


198t
NI (1)t
2**
2**
1
NI
NIt
NI


214*
NI (1)
ID (2)
2 
1
ND
42
40





Total # of compounds tested on either cell line: 24 out of 192


t indicates compounds that are inhibit cell growth but did not show any activity to inhibit ligases under cell-free conditions.


tt indicates are the most active agents as tested on MCF10A and HCT116 cell lines


*compounds that are insoluble, form crystals in media


**compounds that alone at 50 μM did not inhibit cell growth by 50%, however sensitized cells to MMS or 3-AB


#198** active at high concentrations (50 μM) sensitized MCF10A cells to 3-AB, and HCT116 cells to MMS;


#214** sensitized MCF10A cells to 3-AB, and HCT116 to MMS.


214 is highly insoluble and undergoes self degradation in DMSO (MS data, BD).


ND- no data;


ID- inconsistent data;


NI- no inhibition













TABLE 7





Similarity between 10 active compounds (bold 


numbers) based on the Tanimoto Index (Tc %)


























25
64
67
82
113
123
189
190
197
200


25
100











64
39
100










67
38
53
100









82
42
53
69
100








113
22
34
45
30
100







123
15
32 
16
16
22
100






189
40
40
37
43
24
30
100





190
43
28
32
39
18
19
52
100




197
40
35
24
21
18
20
18
22
100



200
40
19
25
27
13
4
25
35
35
100

## Claims

1. A method of ameliorating neuroblastoma exhibiting an altered DNA damage response in a patient in need comprising administering to said patient an effective amount of an anti-cancer compound according to the chemical structure as set forth below:



in combination with a second cancer treatment comprising a DNA damaging agent.

2. The method according to claim 1 wherein said DNA damaging agent is ionizing radiation, a PARP inhibitor or a DNA alkylating agent.

3. The method according to claim 2 wherein said DNA damaging agent is a PARP inhibitor.

4. The method according to claim 3 wherein said PARP inhibitor is 3-aminobenzamide.

5. The method of claim 1, wherein said DNA damaging agent is administered at the same time as administering said compound.

6. The method of claim 1, wherein said DNA damaging agent is administered prior to administering said at least one compound.

7. The method of claim 1, wherein said DNA damaging agent is administered after administering said at least one compound.