# Patent

## Title
Compositions and methods for inhibiting G2 cell cycle arrest and sensitizing cells to DNA damaging agents

## Abstract

The invention provides compositions and methods for inhibiting Chk1 and/or Chk2 kinases. Also provided are compositions and methods for inhibiting G2 cell arrest checkpoint, particularly in mammalian, e.g., human, cells. The compositions and methods of the invention are also used to treat disorders of cell growth, such as cancer. In particular, the invention provides methods for selectively sensitizing G1 checkpoint impaired cancer cells to DNA damaging agents and treatments. Also provided are methods for screening for compounds able to interact with, e.g., inhibit, enzymes involved in the G2 cell cycle arrest checkpoint, such as Chk1 and/or Chk2/Cds1 kinase.

## Background
This application is divisional of U.S. patent application Ser. No. 09/667,365, filed Sep. 21, 2000, now U.S. Pat. No. 6,881,575 which claims priority to Japanese Patent Application Nos. 11-269398, filed Sep. 22, 1999, and 11-340322, filed Nov. 30, 1999.


TECHNICAL FIELD
This invention generally pertains to the fields of medicine and cancer therapeutics. In particular, this invention provides novel genes and polypeptides and methods for making and using them. Specifically, the compositions and methods of the invention are used to treat disorders of cell growth, such as cancer. In particular, the invention provides methods for selectively sensitizing G1 checkpoint impaired cancer cells to DNA damaging agents and treatments. Also provided are methods for screening for compounds able to interact with, e.g., inhibit, enzymes involved in the G2 cell cycle arrest checkpoint, such as Chk1 and/or Chk2/Cds1 kinase.
BACKGROUND
It is a continuing challenge to develop anti-cancer agents that are capable of inhibiting the growth of, or killing, cancer cells, without affecting normal cells. Researchers have focused on genetic mutations in cancer cells to find clues to discover such new anti-cancer drugs.
Many cancer cells have mutations in genes involved in the G1 cell cycle arrest checkpoint. Such genes include impaired tumor suppressor genes, e.g., p53, Rb, p16INK4, and p19ARF. Alternatively, such mutations can cause expression of oncogenes, e.g., MDM-2 and cyclin D. In addition to these, excessive growth factor signaling can be caused by the over expression of growth factors. Together with these gain-of-function mutations, growth factor receptors or downstream signal-transducing molecules can cause cell transformation by overriding the G1 checkpoint. In contrast, few cancers have disrupted G2 cell cycle arrest checkpoints. Thus, the G2 checkpoint is usually retained in cancer cells with the impaired G1 checkpoint.
If the G2 checkpoint could be selectively disrupted, cancer cells with an impaired G1 checkpoint would become more sensitive to DNA-damaging treatment, as compared to normal cells (with intact G1), since progression through G1 and G2 without repairing such damage induces apoptosis.
The mechanism that promotes the cell cycle G2 arrest after DNA damage is conserved among species from yeast to human. In the presence of damaged DNA, Cdc2/Cyclin B kinase is kept inactive because of inhibitory phosphorylation of threonine-14 and tyrosine-15 residues on Cdc2 kinase. At the onset of mitosis, the dual phosphatase Cdc25 kinase removes these inhibitory phosphates and thereby activates Cdc2/Cyclin B kinase.
In fission yeast, the protein kinase Chk1 is required for the cell cycle arrest in response to damaged DNA. Chk1 kinase acts downstream of several rad gene products and is modified by the phosphorylation upon DNA damage. The kinases Rad53 of budding yeast and Cds1 of fission yeast are known to conduct signals from unreplicated DNA. It appears that there is some redundancy between Chk1 and Cds1 because elimination of both Chk1 and Cds1 was culminated in disruption of the G2 arrest induced by damaged DNA. Interestingly, both Chk1 and Cds1 phosphorylate Cdc25 kinase and promote Rad24 binding to Cdc25, which sequesters Cdc25 to cytosol and prevents Cdc2/Cyclin B activation. Therefore Cdc25 appears to be a common target of theses kinases and presumably an indispensable factor in the G2 checkpoint.
In humans, both hChk1, a human homologue of fission yeast Chk1, and Chk2/HuCds1, a human homologue of the budding yeast Rad53 and fission yeast Cds1, phosphorylate Cdc25C at serine-216, a critical regulatory site, in response to DNA damage. This phosphorylation creates a binding site for small acidic proteins 14-3-3s, human homologues of Rad24 and Rad25 of fission yeast (Lopez-Girona (1999) Nature 397:172-175). The regulatory role of this phosphorylation was clearly indicated by the fact that substitution of serine-216 to alanine on Cdc25C disrupted cell cycle G2 arrest in human cells (Peng (1997) Science 277:1501-1505).
SUMMARY
This invention provides nucleic acids and polypeptides which can be used to treat cell proliferative disorders, such as those associated with benign and malignant tumor cells. While the invention is not limited to any particular mechanisms, the polypeptides of the invention can function by inhibiting the G2 cell cycle arrest checkpoint. Thus, the invention also provides compositions and methods for selectively sensitizing a cell with an impaired G1 cell cycle arrest checkpoint, e.g., a cancer cell, to a DNA damaging agent
The invention provides an isolated or recombinant polypeptide comprising the amino acid sequence: X1 X2 X3 X4 X5 X6 X7 X8X9 X10 X11, wherein X1 is L, F, W, M, R, I, V, Y, K, or absent, X2 is Y, F, A, W, S or T, X3 is any amino acid, X4 is any amino acid, X5 is any amino acid, X6 is S, A, N, H or P, X7 is any amino acid, X8 is any amino acid, X9 is any amino acid or absent, X10 is N, G, L, S, M, P, N, A or absent, and X11 is L or absent, wherein the polypeptide when administered to or expressed in a cell disrupts the G2 cell cycle arrest checkpoint.
In alternative embodiments, for the isolated or recombinant polypeptide of the invention: X1 is L, F, W, M, R or absent or X1 is L, F or W; X2 is Y, F, A; X3 is R, T, S, H, D, G, A, L, K, A, N, Q or P, or, X3 is R, T, S, H, D, G, A or L, or, X3 is R, T, S or H; X4 is S, T, G, A, L, R, I, M, V, P, or, X4 is S, T, G, A, L, R, or, X4 is S; X5 is P, A, G, S or T, or, X5 is P; X6 is S, N, H, P, A, G or T, or, X6 is S, N or H, or, X6 is S; X7 is M, F, Y, D, E, N, Q, H, G, I, L, V, A, P, N or W, or, X7 is M, F, Y, D, E, N, Q or H, or, X7 is M, F, Y, Q or H; X8 is P, F, Y, W, L, G, M, D, E, N, Q, H, I, V, A or P, or, X8 is P, F, Y or W, or, X8 is Y; X9 is E, G, L, S, M, P, N, D, A, T, P or absent; X10 is absent; X11 is absent.
In one embodiment, the invention provides a polypeptide wherein X2 is Y, X5 is P, and X10 is N. In one embodiment, the invention provides a polypeptide wherein X3 is R, X8 is P, and X11 is L. In one embodiment, the invention provides a polypeptide wherein X4 is S, X5 is P, X6 is S, X9 is E, X10 is N and X11 is L.
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises Y G G P G G G G N (SEQ ID NO: 1895); R Y S L P P E L S N M (SEQ ID NO: 1); L A R S A S M P E A L (SEQ ID NO: 1896); L Y R S P S M P E N L (SEQ ID NO: 2); L Y R S P A M P E N L (SEQ ID NO: 1897); W Y R S P S F Y E N L (SEQ ID NO: 904); W Y R S P S Y Y E N L (SEQ ID NO: 908); or, W Y R S P S Y Y (SEQ ID NO: 1898).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L Y R S P S Y P E N L (SEQ ID NO: 10), L Y R S P S Y F E N L (SEQ ID NO: 11), L Y R S P S Y Y E N L (SEQ ID NO: 12), or L Y R S P S Y W E N L (SEQ ID NO: 13).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L Y R S P S N P E N L (SEQ ID NO: 22), L Y R S P S N F E N L (SEQ ID NO: 23), L Y R S P S N Y E N L (SEQ ID NO: 24), or L Y R S P S N W E N L (SEQ ID NO: 25).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L Y R S P S H P E N L (SEQ ID NO: 30), L Y R S P S H F E N L (SEQ ID NO: 31), L Y R S P S H Y E N L (SEQ ID NO: 32), L Y R S P S H W E N L (SEQ ID NO: 33), L Y S S P S M P E N L (SEQ ID NO: 34), L Y S S P S M F E N L (SEQ ID NO: 35), L Y S S P S M Y E N L (SEQ ID NO: 36), L Y S S P S M W E N L (SEQ ID NO: 37), L Y S S PS F P E N L (SEQ ID NO: 38), L Y S S P S F P E N L (SEQ ID NO: 38), L Y S S P S F F E N L (SEQ ID NO: 39), L Y S S P S F Y E N L (SEQ ID NO: 40), L Y S S P S F W E N L (SEQ ID NO: 41), L Y S S P S Y P E N L (SEQ ID NO: 42), L Y S S P S Y F E N L (SEQ ID NO: 43), L Y S S P S Y Y E N L (SEQ ID NO: 44), or L Y S S P S Y W E N L (SEQ ID NO: 45).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L Y S S P S Q P E N L (SEQ ID NO: 58), L Y S S P S Q W E N L (SEQ ID NO: 61), L Y S S P S H P E N L (SEQ ID NO: 62), L Y S S P S H F E N L (SEQ ID NO: 63), L Y S S P S H Y E N L (SEQ ID NO: 64), L Y S S P S H W E N L (SEQ ID NO: 65), L Y T S P S M P E N L (SEQ ID NO: 66), L Y T S P S M F E N L (SEQ ID NO: 67), L Y T S P S M Y E N L (SEQ ID NO: 68), L Y T S P S M W E N L (SEQ ID NO: 69), L Y T S P S F P E N L (SEQ ID NO: 70), L Y T S P S F F E N L (SEQ ID NO: 71), L Y T S P S F Y E N L (SEQ ID NO: 72), L Y T S P S F W E N L (SEQ ID NO: 73), L Y T S P S Y P E N L (SEQ ID NO: 74), L Y T S P S Y F E N L (SEQ ID NO: 75), L Y T S P S Y Y E N L (SEQ ID NO: 76), or L Y T S P S Y W E N L (SEQ ID NO: 77).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L Y T S P S N P E N L (SEQ ID NO: 86), L Y T S P S N F E N L (SEQ ID NO: 87), L Y T S P S N Y E N L (SEQ ID NO: 88)or L Y T S P S N W E N L (SEQ ID NO: 89).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L Y T S P S H P E N L (SEQ ID NO: 94), L Y T S P S H F E N L (SEQ ID NO: 95), L Y T S P S H Y E N L (SEQ ID NO: 96)or L Y T S P S H W E N L (SEQ ID NO: 97).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L Y H S P S Y P E N L (SEQ ID NO: 106), L Y H S P S Y F E N L (SEQ ID NO: 107), L Y H S P S Y Y E N L (SEQ ID NO: 108) or L Y H S P S Y W E N L (SEQ ID NO: 109).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L F T S P S Y P E N L (SEQ ID NO: 298), L F T S P S Y F EN L (SEQ ID NO: 299), L F T S P S Y Y E N L (SEQ ID NO: 300)or L F T S P S Y W E N L (SEQ ID NO: 301).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises F Y S S P S H P E N L (SEQ ID NO: 510), F Y S S P S H F E N L (SEQ ID NO: 511), F Y S S P S H Y E N L (SEQ ID NO: 512), F Y S S P S H W E N L (SEQ ID NO: 513), F Y T S P S M P E N L (SEQ ID NO: 514), F Y T S P S M F E N L (SEQ ID NO: 515), F Y T S P S M Y E N L (SEQ ID NO: 516), F Y T S P S M W E N L (SEQ ID NO: 517), F Y T S P S F P E N L (SEQ ID NO: 518), F Y T S P S F F E N L (SEQ ID NO: 519), F Y T S P S F Y E N L (SEQ ID NO: 520), F Y T S P S F W E N L (SEQ ID NO: 521), F Y T S P S Y P E N L (SEQ ID NO: 522), F Y T S P S Y F E N L (SEQ ID NO: 523), F Y T S P S Y Y E N L (SEQ ID NO: 524) or F Y T S P S Y W E N L (SEQ ID NO: 525).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises W Y R S P S M P E N L (SEQ ID NO: 898), W Y R S P S M F E N L (SEQ ID NO: 899), W Y R S P S M Y E N L (SEQ ID NO: 900), W Y R S P S M W E N L (SEQ ID NO: 901), W Y R S P S F P E N L (SEQ ID NO: 902), W Y R S P S F F E N L (SEQ ID NO: 903), W Y R S P S F Y E N L (SEQ ID NO: 904), W Y R S P S F W E N L (SEQ ID NO: 905), W Y R S P S Y P E N L (SEQ ID NO: 906), W Y R S P S Y F E N L (SEQ ID NO: 907), W Y R S P S Y Y E N L (SEQ ID NO: 908) or W Y R S P S Y W E N L (SEQ ID NO: 909).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises W Y T S P S M P E N L (SEQ ID NO: 962), W Y T S P S M F E N L (SEQ ID NO: 963), W Y T S P S M Y E N L (SEQ ID NO: 964), W Y T S P S M W E N L (SEQ ID NO: 965), W Y T S P S F P E N L (SEQ ID NO: 966), W Y T S P S F F E N L (SEQ ID NO: 967), W Y T S P S F Y E N L (SEQ ID NO: 968), W Y T S P S F W E N L (SEQ ID NO: 969), W Y T S P S Y P E N L (SEQ ID NO: 970), W Y T S P S Y F E N L (SEQ ID NO: 971), W Y T S P S Y Y E N L (SEQ ID NO: 972) or W Y T S P S Y W E N L (SEQ ID NO: 973).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises W Y T S P S H P E N L (SEQ ID NO: 990), W Y T S P S H F E N L (SEQ ID NO: 991), W Y T S P S H Y E N L (SEQ ID NO: 992) or W Y T S P S H W E N L (SEQ ID NO: 993).
In alternative embodiments, the invention provides an isolated or recombinant polypeptide wherein the amino acid sequence comprises L K R S P S M P E N L (SEQ ID NO: 1826), L Y I S P S M P E N L (SEQ ID NO: 1844) or L Y R S P S M V E N L (SEQ ID NO: 1894).
In one embodiment, the invention provides an isolated or recombinant polypeptide wherein the polypeptide when administered to or expressed in a cell disrupts the G2 cell cycle arrest checkpoint, wherein the cell is a mammalian cell. The cell can be a human cell, a yeast cell, an insect cell, a bacterial cell, a plant cell, and the like.
In one embodiment, the invention provides an isolated or recombinant polypeptide further comprising a cell membrane permeant. The cell membrane permeant can comprise a polypeptide, such as a TAT protein transduction domain, e.g., comprising a sequence Y G R K K R R Q R R R (SEQ ID NO: 1899). Alternatively, the cell membrane permeant can comprise a lipid, such as a liposome.
The invention provides a chimeric polypeptide comprising a first domain comprising a polypeptide of the invention and a second domain comprising a cell membrane permeant, wherein the polypeptide when administered to or expressed in a cell disrupts the G2 cell cycle arrest checkpoint. The chimeric polypeptide can be a recombinant fusion protein.
The invention provides an isolated or recombinant nucleic acid encoding a polypeptide or a chimeric polypeptide of the invention, wherein the polypeptide, when administered to or expressed in a cell, disrupts the G2 cell cycle arrest checkpoint.
The invention provides an expression vector comprising a nucleic acid encoding a polypeptide or a chimeric polypeptide of the invention, wherein the polypeptide, when administered to or expressed in a cell, disrupts the G2 cell cycle arrest checkpoint.
The invention provides a cell comprising a nucleic acid or an expression vector of the invention. The cell can be a bacterial, a yeast, an insect, a plant, or a mammalian cell.
The invention provides a pharmaceutical composition comprising a polypeptide of the invention, a nucleic acid of the invention, an expression vector of the invention, or a cell of the invention; and, a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition can comprise a liposome.
The invention provides a method for inhibiting a the activity of a Chk1 kinase or a Chk2 kinase comprising contacting the kinase with a polypeptide of the invention or a pharmaceutical composition of the invention, in an amount sufficient to inhibit the activity of the Chk1 or Chk2 kinase.
The invention provides a method for disrupting a cell G2 cell cycle arrest checkpoint comprising contacting the cell with a polypeptide of the invention or a pharmaceutical composition of the invention in an amount sufficient to disrupt the G2 cell cycle arrest checkpoint. In alternative embodiments the cell is a mammalian cell, a human cell or a cancer cell.
The invention provides a method for sensitizing a cell to a DNA damaging agent comprising contacting the cell with a polypeptide of the invention or a pharmaceutical composition of the invention in an amount sufficient to disrupt the G2 cell cycle arrest checkpoint, thereby sensitizing the cell to the DNA damaging agent. In alternative embodiments the cell is a mammalian cell, a human cell or a cancer cell. The cancer cell can have an impaired G1 cell cycle arrest checkpoint.
The invention provides a method for selectively sensitizing a cell with an impaired G1 cell cycle arrest checkpoint to a DNA damaging agent comprising contacting the cell with a polypeptide of the invention or a pharmaceutical composition of the invention, in an amount sufficient to disrupt the G2 cell cycle arrest checkpoint, thereby sensitizing the cell to the DNA damaging agent. In alternative embodiments the cell is a mammalian cell, a human cell or a cancer cell.
The invention provides a method for inducing apoptosis in a cell in an individual comprising a administering a polypeptide of the invention or a pharmaceutical composition of the invention, in an amount sufficient to disrupt the G2 cell cycle arrest checkpoint in the cancer cell, thereby sensitizing the cancer cell to a DNA damaging agent, and administering a DNA damaging agent. In alternative embodiments the cell is a mammalian cell, a human cell or a cancer cell. The cancer cell can have an impaired G1 cell cycle arrest checkpoint. The DNA damaging agent can be 5-fluorouracil (5-FU), rebeccamycin, adriamycin, bleomycin, cisplatin, hyperthermia, UV irradiation or gamma-irradiation.
The invention provides a method for screening for compounds capable of modulating the activity of a Chk1 kinase or a Chk2 kinase comprising the following steps: (a) providing a test compound; (b) providing a Chk1 kinase or a Chk2 kinase; (c) providing a polypeptide of the invention, wherein the polypeptide binds to the Chk1 kinase or the Chk2 kinase; and, (d) contacting the test compound with the kinase and the polypeptide and measuring the ability of the test compound to prevent binding of the polypeptide to the kinase.
The invention provides a method for screening for compounds capable of modulating the activity of a Chk1 kinase or a Chk2 kinase comprising the following steps: (a) providing a test compound; (b) providing a Chk1 kinase or a Chk2 kinase; (c), providing a polypeptide of the invention, wherein the polypeptide is phosphorylated by the Chk1 kinase or the Chk2 kinase; and, (d) contacting the test compound with the kinase and the polypeptide and measuring the ability of the test compound to inhibit or abrogate phosphorylation of the polypeptide by the kinase. The method can further comprising providing a full length human Cdc25C. In one embodiment, of the method, the polypeptide of step (c) comprises-amino acid residue serine 216 of human Cdc25C, such as comprising from about amino acid residue 200 to about amino acid residue 250 of human Cdc25C. In one embodiment of the method, the polypeptide of step (c) further comprises glutathione-S-transferase.
In one embodiment of the methods of the invention, including the screening methods, the polypeptide of the invention is immobilized.
The invention provides a method for screening for compounds capable of specifically inhibiting the G2 cell cycle checkpoint comprising the following steps: (a) providing a test compound and a polypeptide of the invention; (b) providing a G1 checkpoint impaired cell; (c) contacting the cell of step (b) with the test compound or the polypeptide of step (a) plus a DNA damaging treatment, such as 5-fluorouracil (5-FU), rebeccamycin, adriamycin, bleomycin, cisplatin, hyperthermia, UV irradiation or gamma-irradiation, or, or an M phase checkpoint activator; and, (d) measuring the amount of DNA in the cells after the contacting of step (c) to determine if the test compound has inhibited the G2 cell cycle checkpoint, wherein the polypeptide of step (a) acts as a G2-checkpoint-inhibiting positive control. In alternative embodiments the cell is a mammalian cell, a human cell or a cancer cell. In one embodiment, the amount of DNA is measured using propidium iodide by, e.g., a FACS analysis, or equivalent. In one embodiment, the amount of DNA is measured after about 10 to about 72 hours after the contacting of step (c).
In one embodiment, the method comprises contacting the cell of step (b) with an M phase checkpoint activator alone (as a substitute for a DNA damaging agent) and the test compound or the polypeptide of step (a), wherein a test compound that has not inhibited or abrogated the arrest at the M phase checkpoint of the cell cycle after contacting the cell with an M phase activator is a specific inhibitor of the G2 cell cycle checkpoint (because it did not affect M phase checkpoint or it was not a non-specific phenomenon). In one embodiment, the M phase checkpoint activator is colchicine or nocodazole.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications, GenBank sequences and ATCC deposits, cited herein are hereby expressly incorporated by reference for all purposes.



DESCRIPTION OF DRAWINGS
FIG. 1 shows chimeric peptides used in and results of experiments demonstrating that TAT-S216A and TAT-S216 peptides inhibit hChk1 and Chk2/HuCds1 kinase activity in vitro, as described in Example 1, below. FIG. 1A shows a schematic diagram of the fusion/chimeric peptides TAT-control (SEQ ID NO: 1934), TAT-S216A (SEQ ID NO: 1933) and TAT-S216 (SEQ ID NO: 1932). FIG. 1B shows SDS-PAGE autoradiograms demonstrating the results of in vitro Cdc25C phosphorylation assays using TAT-S216A and TAT-S216 peptides to inhibit purified hChk1 activity; amino acid residues 200 to 256 of Cdc25C (SEQ ID NO:1) were used as a substrate at a concentration of 1 μM. FIG. 1C shows SDS-PAGE autoradiograms demonstrating the results of in vitro Cdc25C phosphorylation assays using TAT-S216A peptide to inhibit purified hChk1 and Chk2/HuCds1 activity; amino acid residues 211 to 220 of Cdc25C (SEQ ID NO:1) were used as a substrate at a concentration of 10 μM.
FIG. 2 the results of experiments demonstrating that TAT-S216A and TAT-S216 peptides can abrogate DNA damage-induced G2 arrest in Jurkat cells. FIG. 2A shows the results of a FACS analysis of Jurkat cells treated with bleomycin (10 μg/ml) and TAT-S216A and TAT-S216 peptides (10 μM each). FIG. 2B shows the results of an SDS-PAGE of cell lysates from a histone H1 kinase analysis; lysates were prepared from cells treated with the indicated reagent for six hours. FIG. 2C shows the results a FACS analysis of colchicines—(5 μg/ml) and peptide—(10 μM each) treated cells; Jurkat cells were treated for 20 hours.
FIG. 3 shows the results of experiments demonstrating that TAT-S216A and TAT-S216 peptides can specifically sensitize cancer cells to bleomycin, but not colchicine. FIG. 3A shows the results of trypan blue dye exclusion analysis of Jurkat cells treated with bleomycin with or without the TAT-S216A and TAT-S216 peptides. FIG. 3B shows the results of trypan blue dye exclusion (survival) analysis of Jurkat cells treated with colchicine with or without the TAT-S216A and TAT-S216 peptides. FIG. 3C shows the results of trypan blue dye exclusion (survival) analysis of PHA blasts treated with bleomycin with or without the TAT-S216A and TAT-S216 peptides. FIG. 3D shows the results of FACS analysis PHA blasts treated with bleomycin with or without the TAT-S216A and TAT-S216 peptides (vertical axis is DNA content indicated by propidium iodide staining).
FIG. 4 shows the results of experiments demonstrating that TAT-S216A and TAT-S216 peptides can sensitize cancer cells to bleomycin. FIG. 4A shows the results of X-TT analysis of PANC1 cells treated with bleomycin with or without the TAT-S216A and TAT-S216 peptides. FIG. 4B shows the results of X-TT analysis of MIA PaCa2 cells treated with bleomycin with or without the TAT-S216A and TAT-S216 peptides.
FIG. 5 shows a schematic 3-dimensional structure of human Chk2 interacting with exemplary G2-abrogating peptides of the invention, as described in Example 2, below.
FIG. 6 shows the results of FACS analysis of the amount of DNA in cells to determine the number of cells in one of the four cell cycle phases after incubating these cells with bleomycin and exemplary peptides of the invention, as described in Example 3, below.
FIG. 7 shows the results of FACS analysis of the amount of DNA in cells to determine the number of cells in one of the four cell cycle phases after incubating these cells with colchicine and exemplary peptides of the invention, as described in Example 3, below.
FIG. 8 shows the sequences of peptides (SEQ ID NOS 1935-1948) used in experiments described in Example 4, below.
FIG. 9 shows a summary of results of experiments as described in Example 4, below.
FIG. 10 shows the results of experiments demonstrating that a peptide of the invention (as a S216-containing fusion protein) administered to an animal in vivo effectively sensitized cancer cells to a DNA damaging agent.
FIG. 11 shows the results of experiments demonstrating that a peptide of the invention (as a R-II-containing fusion protein) administered to an animal in vivo effectively sensitized cancer cells to a DNA damaging agent.



Like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION
The genes and polypeptides of the invention provide a novel means to treat cell proliferative disorders, including, e.g., to stop the growth of, or kill, cancer cells. While the invention is not limited by any particular mechanism of action, administration of the polypeptides of the invention will delay or abrogate G2 cell cycle arrest checkpoint in cells. The genes and polypeptides of the invention can also be used to inhibit Chk1 and/or Chk2/Cds1 kinase activity. Inhibition of Chk1 and/or Chk2/Cds1 kinase may be the mechanism by which the G2 checkpoint is inhibited. The invention also provides methods for selectively sensitizing G1 checkpoint impaired cancer cells to DNA damaging agents and treatments. Also provided are methods for screening for compounds able to interact with, e.g., inhibit, enzymes involved in the G2 cell cycle arrest checkpoint, such as Chk1 and/or Chk2/Cds1 kinases. Thus, the invention provides methods to screen for compounds that inhibit or abrogate cell cycle G2 checkpoint.
The invention for the first time describes amino acid peptide motifs in the human Cdc25C (hCdc25C) polypeptide (SEQ ID NO:1) that are the substrate motifs for human Chk1 (hChk1) (SEQ ID NO:3) and human Chk2/ human Cds1 (Chk2/HuCds1) (SEQ ID NO:4) kinase activity. The kinase-inhibitory polypeptides and nucleic acids of the invention are modeled on these hCdc25C peptide motifs. Wild-type hCdc25C is phosphorylated by hChk1 (SEQ ID NO:3) and Chk2/HuCds1 (SEQ ID NO:4).
Phosphorylation of Cdc25C is necessary for the cell's arrest at G2 checkpoint. Thus, the polypeptides and peptides of the invention, by inhibiting the phosphorylation of Cdc25C (by enzymes which probably include Chk1 and Chk2/HuCds1), can inhibit or abrogate the cell's G2 checkpoint capability. The lack of an effective G2 checkpoint after DNA damage becomes fatal to the cell (see, e.g., Maity (1994) Radiother. Oncol. 31:1-13). If a cell progresses through G2 without sufficient repair of DNA damage it becomes apoptotic. Thus, the compositions of the invention can be used to sensitize cells, such as tumor cells, to DNA damaging agents. In fact, as discussed below, the compositions of the invention can sensitize cancer cells to the apoptotic effects of DNA-damaging agents with little or no cytotoxic effect on normal cells.
Example 1, below, describes the synthesis and use of two exemplary polypeptides of the invention. Two peptides corresponding to amino acids 211 to 221 of human Cdc25C (SEQ ID NO:1) fused with a part of HIV-1-TAT (SEQ ID NO:5). These peptides were demonstrated to inhibit hChk1 kinase (SEQ ID NO:3) and Chk2/HuCds1 kinase (SEQ ID NO:4) activity in vitro and to specifically abrogate the G2 checkpoint in vivo. These peptides sensitized p53-defective cancer cell lines to the apoptotic effects of DNA-damaging agents without obvious cytotoxic effect on normal cells. These results clearly demonstrate that the polypeptides comprising the motifs of the invention can be used to specifically inhibit or abrogate the cell cycle G2 checkpoint. These results demonstrate that the compositions of the invention can be used to screen for compositions that inhibit Chk1 or Chk2 kinase activity. These results also demonstrate that the compositions of the invention can be used for cancer therapy. While the invention is not limited by any particular mechanism of action, the polypeptides and peptides of the invention can be used to target and inhibit hChk1 (SEQ ID NO:3) and Chk2/HuCds1 (SEQ ID NO:4) kinases.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
The term “cell membrane permeant” as used herein means any composition which, when associated with a peptide or polypeptide of the invention, or a nucleic acid of the invention, causes, or assists in, the internalization of the composition into a cell. The association can be covalent (e.g., a linking reagent, or, as a fusion protein) or non-covalent (e.g., as with liposomes). For example, in one embodiment, a cell membrane permeant domain is linked to a peptide or polypeptide of the invention as a fusion protein domain, e.g., a TAT protein transduction domain (see, e.g., Vives (1997) J. Biol. Chem. 272:16010-16017). Other cell membrane permeant domains include, e.g., the PreS2- and S-domain of the hepatitis-B virus surface antigens, see, e.g., Oess (2000) Gene Ther. 7:750-758.
The term “human Cdc25C” or “hCdc25C” as used herein means, depending on the context, the human Cdc25C polypeptide (SEQ ID NO:1) or the human Cdc25C polypeptide (SEQ ID NO:1) message (cDNA) (SEQ ID NO:2) or gene (see, e.g., Peng (1997) Science 277:1501-1505). The term also includes all functional variations of hCdc25C, including, e.g., allelic variations, functional mutations, variations with additions, deletions, substitutions that retain functional activity. A Cdc25C polypeptide that has functional activity has the same activity as wild type Cdc25C, i.e., when appropriately phosphorylated, it can act in concert with other cell cycle control polypeptides to arrest cell growth at G2 under the proper conditions, e.g., under conditions in which sufficient DNA damage has incurred to induce apoptosis if the cell passes through the G2 checkpoint.
The terms “DNA damaging treatment” or “DNA damaging agent” include any treatments or agents that will cause DNA damage to a cell, including a drug, a radiation, an environmental shock, and the like, including, e.g., hyperthermia, UV radiation or gamma-radiation, in addition to the known DNA damaging drugs, e.g., 5-fluorouracil (5-FU), rebeccamycin, adriamycin, bleomycin, cisplatin and the like.
The term “disrupt the cell cycle G2 checkpoint” or “inhibit the cell cycle G2 checkpoint” means the ability of a peptide or polypeptide of the invention to inhibit (including abrogate) a Chk1 kinase and/or Chk2 kinase activity, e.g., a mammalian kinase, such as a human Chk1 (hChk1) kinase (SEQ ID NO:3) (see, e.g., Yin (2000) Mol. Pharmacol. 57:453-459) or a human Chk2/human Cds1 kinase (Chk2/HuCds1) (SEQ ID NO:4) (see, e.g., Hirao (2000) Science 287:1824-1827), or, to disrupt (including abrogate) the ability of a cell to arrest growth at the G2 checkpoint under appropriate conditions, e.g., where conditions in the cell otherwise would cause G2 cell cycle arrest, such as the accumulation of DNA damage by, e.g., some anti-tumor agents.
The ability of a peptide or polypeptide of the invention to modulate or inhibit a Chk1 kinase and/or a Chk2 kinase activity can be easily tested in vitro or in vivo as, for example, in the assays, or variations thereof, described in Example 1, below. A peptide or polypeptide is considered an effective inhibitor if, e.g., it binds the kinase to inhibit or abrogate kinase activity. Alternatively, a peptide or polypeptide is also considered an effective inhibitor of kinase activity if it acts as a phosphorylation substrate and prevents phosphorylation of natural substrate, e.g., wild type Cdc25C, thereby disrupt the ability of a cell to arrest growth at the G2 checkpoint under appropriate conditions.
The ability of exemplary peptides or polypeptides of the invention to disrupt the ability of a cell to arrest growth at the G2 checkpoint, i.e., to act in concert with other cell cycle control polypeptides to arrest cell growth at G2 under the proper conditions, e.g., under conditions in which sufficient DNA damage has incurred to induce apoptosis if the cell passes through the G2 checkpoint can be easily tested in vivo, e.g., cell culture, is demonstrated in Example 1, below
The term “expression cassette” as used herein refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence) in a host compatible with such sequences. Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers. “Operably linked” as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. Thus, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. A “vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and includes both the expression and nonexpression plasmids. Where a recombinant microorganism or cell culture is described as hosting an “expression vector” this includes both extrachromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
The term “chemically linked” refers to any chemical bonding of two moieties, e.g., as in one embodiment of the invention, a polypeptide comprising at least two peptide motifs of the invention. Such chemical linking includes the peptide bonding of a recombinantly or in vivo generated fusion protein.
The term “chimeric protein” or “fusion protein” refers to a composition comprising at least one polypeptide or peptide domain or motif which is associated with a second polypeptide or peptide domain or motif. For example, in one embodiment, the invention provides an isolated or recombinant nucleic acid molecule encoding a fusion protein comprising at least two domains, wherein the first domain comprises one kinase-inhibiting or G2-checkpoint inhibiting motif and the second domain comprising a second motif with the same or similar activity (for example, on motif may have a high binding affinity for the kinase, whilst the second motif has high kinase inhibitory activity). Additional domains can comprise a polypeptide, peptide, polysaccharide, or the like. The “fusion” can be an association generated by a peptide bond, a chemical linking, a charge interaction (e.g., electrostatic attractions, such as salt bridges, H-bonding, etc.) or the like. If the polypeptides are recombinant, the “fusion protein” can be translated from a common message. Alternatively, the compositions of the domains can be linked by any chemical or electrostatic means. The chimeric molecules of the invention can also include additional sequences, e.g., linkers, epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals, and the like. Alternatively, a peptide can be linked to a carrier simply to facilitate manipulation or identification/ location of the peptide.
The term “G2 checkpoint inhibitory activity” as used herein means any amount of inhibition of the G2 checkpoint.
The term “isolated” as used herein, when referring to a molecule or composition, such as, e.g., a nucleic acid or polypeptide of the invention, means that the molecule or composition is separated from at least one other compound, such as a protein, other nucleic acids (e.g., RNAs), or other contaminants with which it is associated in vivo or in its naturally occurring state. Thus, a nucleic acid or polypeptide is considered isolated when it has been isolated from any other component with which it is naturally associated, e.g., cell membrane, as in a cell extract. An isolated composition can, however, also be substantially pure. An isolated composition can be in a homogeneous state and can be in a dry or an aqueous solution. Purity and homogeneity can be determined, for example, using analytical chemistry techniques such as polyacrylamide gel electrophoresis (SDS-PAGE) or high performance liquid chromatography (HPLC). Thus, the isolated compositions of this invention do not contain materials normally associated with their in situ environment. Even where a protein has been isolated to a homogenous or dominant band, there can be trace contaminants which co-purify with the desired protein.
The terms “polypeptide,” “protein,” and “peptide” include compositions of the invention that also include “analogs,” or “conservative variants” and “mimetics” or “peptidomimetics” with structures and activity that substantially correspond to the polypeptide from which the variant was derived, including, e.g., variations of the peptides and polypeptides of the invention which can either inhibit a mammalian Chk1 and/or Chk2 kinase, or, inhibit a mammalian G2 checkpoint.
The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use, e.g., as an anti-cancer agent, in a subject. The pharmaceutical compositions of this invention are formulations that comprise a pharmacologically effective amount of a composition comprising, e.g., a peptide, polypeptide, nucleic acid, vector, or cell of the invention, and a pharmaceutically acceptable carrier.
The term “promoter” is an array of nucleic acid control sequences which direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter which is active under most environmental and developmental conditions. An “inducible” promoter is a promoter which is under environmental or developmental regulation. A “tissue specific” promoter is active in certain tissue types of an organism, but not in other tissue types from the same organism. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
The term “recombinant” refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide. For example, recombinant peptides or polypeptides or nucleic acids can be used to practice the methods of the invention. “Recombinant means” also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of polypeptide coding sequences in the vectors used to practice this invention.
Nucleic Acids and Expression Vectors
This invention provides novel nucleic acids, including expression vectors, for use in the treatment of uncontrolled cell growth, such as cancer, and means to make and express those nucleic acids. As the genes and vectors of the invention can be made and expressed in vitro or in vivo, the invention provides for a variety of means of making and expressing these genes and vectors. One of skill will recognize that desired levels of expression of the polypeptides of the invention can be obtained by modulating the expression or activity of the genes and nucleic acids (e.g., promoters) within the vectors of the invention. Any of the known methods described for increasing or decreasing expression or activity, including tissue-specific expression, can be used for this invention. The invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature.
General Techniques
The nucleic acid sequences of the invention and other nucleic acids used to practice this invention, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to bacterial cells, e.g., mammalian, yeast, insect or plant cell expression systems.
Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
Techniques for the manipulation of nucleic acids, such as, e.g., generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g. fluid or gel precipitin reactions, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography. Amplification methods include, e.g., polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicase amplification (see, e.g., Smith (1997) J. Clin. Microbiol. 35:1477-1491), automated Q-beta replicase amplification assay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-564.
Once amplified, the libraries can be cloned, if desired, into any of a variety of vectors using routine molecular biological methods; methods for cloning in vitro amplified nucleic acids are described, e.g., U.S. Pat. No. 5,426,039. To facilitate cloning of amplified sequences, restriction enzyme sites can be “built into” the PCR primer pair.
The invention provides libraries of expression vectors encoding polypeptides and peptides of the invention. These nucleic acids may be introduced into a genome or into the cytoplasm or a nucleus of a cell and expressed by a variety of conventional techniques, well described in the scientific and patent literature. See, e.g., Roberts (1987) Nature 328:731; Schneider (1995) Protein Expr. Purif. 6435:10; Sambrook, Tijssen or Ausubel. The vectors can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods. For example, the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses which are stably or transiently expressed in cells (e.g., episomal expression systems). Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences. For example, selection markers can code for episomal maintenance and replication such that integration into the host genome is not required.
In one embodiment, the nucleic acids of the invention are administered in vivo for in situ expression of the peptides or polypeptides of the invention. The nucleic acids can be administered as “naked DNA” (see, e.g., U.S. Pat. No. 5,580,859) or in the form of an expression vector, e.g., a recombinant virus. The nucleic acids can be administered by any route, including peri- or intra-tumorally, as described below. Vectors administered in vivo can be derived from viral genomes, including recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxviridae, adenoviridiae, or picornnaviridiae. Chimeric vectors may also be employed which exploit advantageous merits of each of the parent vector properties (See e.g., Feng (1997) Nature Biotechnology 15:866-870). Such viral genomes may be modified by recombinant DNA techniques to include the nucleic acids of the invention; and may be further engineered to be replication deficient, conditionally replicating or replication competent. In alternative embodiments, vectors are derived from the adenoviral (e.g., replication incompetent vectors derived from the human adenovirus genome, see, e.g., U.S. Pat. Nos. 6,096,718; 6,110,458; 6,113,913; 5,631,236); adeno-associated viral and retroviral genomes. Retroviral vectors can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof; see, e.g., U.S. Pat. Nos. 6,117,681; 6,107,478; 5,658,775; 5,449,614; Buchscher (1992) J. Virol. 66:2731-2739; Johann (1992) J. Virol. 66:1635-1640). Adeno-associated virus (AAV)-based vectors can be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures; see, e.g., U.S. Pat. Nos. 6,110,456; 5,474,935; Okada (1996) Gene Ther. 3:957-964.
The peptides and polypeptides of the invention are derived from, or, based on, the structure of the kinase Cdc25C. The cDNA nucleic acid sequence for hCdc25C is







(SEQ ID NO: 1900) 








1
caggaagact ctgagtccga cgttggccta cccagtcgga



aggcagagct gcaatctagt


 


61
taactacctc ctttccccta gatttccttt cattctgctc



aagtcttcgc ctgtgtccga


 


121
tccctatcta ctttctctcc tcttgtagca agcctcagac



tccaggcttg agctaggttt


 


181
tgtttttctc ctggtgagaa ttcgaagacc atgtctacgg



aactcttctc atccacaaga


 


241
gaggaaggaa gctctggctc aggacccagt tttaggtcta



atcaaaggaa aatgttaaac


 


301
ctgctcctgg agagagacac ttcctttacc gtctgtccag



atgtccctag aactccagtg


 


361
ggcaaatttc ttggtgattc tgcaaaccta agcattttgt



ctggaggaac cccaaaatgt


 


421
tgcctcgatc tttcgaatct tagcagtggg gagataactg



ccactcagct taccacttct


 


481
gcagaccttg atgaaactgg tcacctggat tcttcaggac



ttcaggaagt gcatttagct


 


541
gggatgaatc atgaccagca cctaatgaaa tgtagcccag



cacagcttct ttgtagcact


 


601
ccgaatggtt tggaccgtgg ccatagaaag agagatgcaa



tgtgtagttc atctgcaaat


 


661
aaagaaaatg acaatggaaa cttggtggac agtgaaatga



aatatttggg cagtcccatt


 


721
actactgttc caaaattgga taaaaatcca aacctaggag



aagaccaggc agaagagatt


 


781
tcagatgaat taatggagtt ttccctgaaa gatcaagaag



caaaggtgag cagaagtggc


 


841
ctatatcgct ccccgtcgat gccagagaac ttgaacaggc



caagactgaa gcaggtggaa


 


901
aaattcaagg acaacacaat accagataaa gttaaaaaaa



agtatttttc tggccaagga


 


961
aagctcagga agggcttatg tttaaagaag acagtctctc



tgtgtgacat tactatcact


 


1021
cagatgctgg aggaagattc taaccagggg cacctgattg



gtgatttttc caaggtatgt


 


1081
gcgctgccaa ccgtgtcagg gaaacaccaa gatctgaagt



atgtcaaccc agaaacagtg


 


1141
gctgccttac tgtcggggaa gttccagggt ctgattgaga



agttttatgt cattgattgt


 


1201
cgctatccat atgagtatct gggaggacac atccagggag



ccttaaactt atatagtcag


 


1261
gaagaactgt ttaacttctt tctgaagaag cccatcgtcc



ctttggacac ccagaagaga


 


1321
ataatcatcg tgttccactg tgaattctcc tcagagaggg



gcccccgaat gtgccgctgt


 


1381
ctgcgtgaag aggacaggtc tctgaaccag tatcctgcat



tgtactaccc agagctatat


 


1441
atccttaaag gcggctacag agacttcttt ccagaatata



tggaactgtg tgaaccacag


 


1501
agctactgcc ctatgcatca tcaggaccac aagactgagt



tgctgaggtg tcgaagccag


 


1561
agcaaagtgc aggaagggga gcggcagctg cgggagcaga



ttgcccttct ggtgaaggac


 


1621
atgagcccat gataacattc cagccactgg ctgctaacaa



gtcaccaaaa agacactgca


 


1681
gaaaccctga gcagaaagag gccttctgga tggccaaacc



caagattatt aaaagatgtc


 


1741
tctgcaaacc aacaggctac caacttgtat ccaggcctgg



gaatggatta ggtttcagca


 


1801
gagctgaaag ctggtggcag agtcctggag ctggctctat



aaggcagcct tgagttgcat


 


1861
agagatttgt attggttcag ggaactctgg cattcctttt



cccaactcct catgtcttct


 


1921
cacaagccag ccaactcttt ctctctgggc ttcgggctat



gcaagagcgt tgtctacctt


 


1981
ctttctttgt attttccttc tttgtttccc cctctttctt



ttttaaaaat ggaaaaataa


 


2041
acactacaga atgag






The amino acid sequence of human hCdc25C is









MSTELFSSTREEGSSGSGPSFRSNQRKMLNLLLERDTSFTVCPD
(SEQ ID NO: 1901)



 


VPRTPVGKFLGDSANLSILSGGTPKCCLDLSNLSSGEITATQLTTSADLDETGHLDSS



 


LQEVHLAGMNHDQHLMKCSPAQLLCSTPNGLDRGHRKRDAMCSSSANKENDNGNLVD



 


SEMKYLGSPITTVPKLDKNPNLGEDQAEEISDELMEFSLKDQEAKVSRSGLYRSPSMP



 


ENLNRPRLKQVEKFKDNTIPDKVKKKYFSGQGKLRKGLCLKKTVSLCDITITQMLEED



 


SNQGHLIGDFSKVCALPTVSGKHQDLKYVNPETVAALLSGKFQGLIEKFYVIDCRYPY



 


EYLGGHIQGALNLYSQEELFNFFLKKPIVPLDTQKRIIIVFHCEFSSERGPRMCRCLR



 


EEDRSLNQYPALYYPELYILKGGYRDFFPEYMELCEPQSYCPMHHQDHKTELLRCRSQ



 


SKVQEGERQLREQIALLVKDMSP







See also, e.g., GenBank Accession Nos. NP 001781 (protein) and NM 001790 (nucleic acid, cDNA) and Sadhu (1990) Proc. Natl. Acad. Sci. U.S.A. 87:5139-5143.
Peptides and Polypeptides
The peptides and polypeptides of the invention can be administered to treat cell proliferative disorders, including, e.g., to stop the growth of, or kill, cancer cells. The peptides and polypeptides of the invention can be used to inhibit (e.g., delay) or abrogate G2 cell cycle arrest checkpoint in cells. The peptides and polypeptides of the invention can also be used to inhibit Chk1 and/or Chk2/Cds1 kinase activity.
While the peptides and polypeptides of the invention can be expressed recombinantly in vivo after administration of nucleic acids, as described above, they can also be administered directly, e.g., as a pharmaceutical composition.
Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa. For example, peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
The peptides and polypeptides of the invention, as defined above, include all “mimetic” and “peptidomimetic” forms. The terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. As with polypeptides of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Thus, a mimetic composition is within the scope of the invention if, when administered to or expressed in a cell, it disrupts the G2 cell cycle arrest checkpoint. A mimetic composition can also be within the scope of the invention if it can inhibit Chk1 and/or Chk2/Cds1 kinase activity, or, bind to the active site of either of these enzymes.
Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g., —C(═O)—CH2— for —C(═O)—NH—), aminomethylene (CH2—NH), ethylene, olefin (CH═CH), ether (CH2—O), thioether (CH2—S), tetrazole (CN4—), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).
A polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below. Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
Mimetics of acidic amino acids can be generated by substitution by, e.g., non-carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R′—N—C—N—R′) such as, e.g., 1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile derivative (e.g., containing the CN-moiety in place of COOH) can be substituted for asparagine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
A component of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form.
The skilled artisan will recognize that individual synthetic residues and polypeptides incorporating these mimetics can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY. Peptides and peptide mimetics of the invention can also be synthesized using combinatorial methodologies. Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymol. 267:220-234. Modified peptides of the invention can be further produced by chemical modification methods, see, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkei (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896.
Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like. Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification. For example, an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-14). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
The invention provides methods for inhibiting a the activity of a Chk1 kinase or a Chk2 kinase. The invention also provides methods for screening for compositions that inhibit the activity of, or bind to (e.g., bind to the active site), Chk1 kinase and/or a Chk2 kinase. The amino acid sequence of human Chk1 kinase is









MAVPFVEDWDLVQTLGEGAYGEVQLAVNRVTEEAVAVKIVDMKR
(SEQ ID NO: 1902)



 


AVDCPENIKKEICINKMLNHENVVKFYGHRREGNIQYLFLEYCSGGELFDRIEPDIGM



 


PEPDAQRFFHQLMAGVVYLHGIGITHRDIKPENLLLDERDNLKISDFGLATVFRYNNR



 


ERLLNKMCGTLPYVAPELLKRREFHAEPVDVWSCGIVLTAMLAGELPWDQPSDSCQEY



 


SDWKEKKTYLNPWKKIDSAPLALLHKILVENPSARITIPDIKKDRWYNKPLKKGAKRP



 


RVTSGGVSESPSGFSKHIQSNLDFSPVNSASSEENVKYSSSQPEPRTGLSLWDTSPSY



 


IDKLVQGISFSQPTCPDHMLLNSQLLGTPGSSQNPWQRLVKRMTRFFTKLDADKSYQC



 


LKETCEKLGYQWKKSCMNQVTISTTDRRNNKLIFKVNLLEMDDKILVDFRLSKGDGLE



 


FKRHFLKIKGKLIDIVSSQKVWLPAT







See also, Sanchez (1997) Science 277:1497-1501; Genbank Accession Nos. AF 016582; AAC 51736; NP 001265, NM 001274.

The amino acid sequence of human Chk2 kinase is









MSRESDVEAQQSHGSSACSQPHGSVTQSQGSSSQSQGISSSSTS
(SEQ ID NO: 1903)



 


MPNSSQSSHSSSGTLSSLETVSTQELYSIPEDQEPEDQEPEEPTPAPWARLWALQDG



 


FANLECVNDNYWFGRDKSCEYCFDEPLLKRTDKYRTYSKKHFRIFREVGPKNSYIAYI



 


EDHSGNGTFVNTELVGKGKRRPLNNNSEIALSLSRNKVFVFFDLTVDDQSVYPKALRD



 


EYIMSKTLGSGACGEVKLAFERKTCKKVAIKIISKRKFAIGSAREADPALNVETEIEI



 


LKKLNHPCIIKIKNFFDAEDYYIVLELMEGGELFDKVVGNKRLKEATCKLYFYQMLLA



 


VQYLHENGIIHRDLKPENVLLSSQEEDCLIKITDFGHSKILGETSLMRTLCGTPTYLA



 


PEVLVSVGTAGYNRAVDCWSLGVILFICLSGYPPFSEHRTQVSLKDQITSGKYNFIPE



 


VWAEVSEKALDLVKKLLVVDPKARFTTEEALRHPWLQDEDMKRKFQDLLSEENESTAL



 


PQVLAQPSTSRKRPREGEAEGAETTKRPAVCAAVL







See also Brown (1999) Proc. Natl. Acad. Sci. USA 96:3745-3750; Chaturvedi (1999) Oncogene 18:4047-4054; Genbank Accession Nos. NP 009125; NM 007194.

Antibody Generation

The invention provides antibodies that specifically bind to the peptides and polypeptides of the invention. These antibodies can be used to identify the presence of these peptides and polypeptides. The peptides and polypeptides of the invention can be used as immunogens to generate antibodies specific for a corresponding Cdc25C phosphatase. The anti-peptide antibodies of the invention can be used to generate anti-idiotype antibodies that specifically bind to active sites of Chk1 or Chk2 kinase.
Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y. (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Huse (1989) Science 246:1275; Ward (1989) Nature 341:544; Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45. Human antibodies can be generated in mice engineered to produce only human antibodies, as described by, e.g., U.S. Pat. Nos. 5,877,397; 5,874,299; 5,789,650; and 5,939,598. B-cells from these mice can be immortalized using standard techniques (e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line) to produce a monoclonal human antibody-producing cell. See, e.g., U.S. Pat. Nos. 5,916,771; 5,985,615. For making chimeric, e.g., “humanized,” antibodies, see e.g., U.S. Pat. Nos. 5,811,522; 5,789,554; 5,861,155. Alternatively, recombinant antibodies can also be expressed by transient or stable expression vectors in mammalian, including human, cells as in Norderhaug (1997) J. Immunol. Methods 204:77-87; Boder (1997) Nat. Biotechnol. 15:553-557; see also U.S. Pat. No. 5,976,833
Screening for Candidate Compounds
The invention provides compositions and methods for screening for potential therapeutic compounds (“candidate compounds”) to inhibit or abrogate Chk1 and/or Chk2/Cds1 kinase activity and/or the G2 cell cycle arrest checkpoint. For example, the screening can involve in vitro or in vivo assays wherein Chk1 and Chk2/Cds1 kinases phosphorylate peptides and polypeptides comprising the motifs of the invention; see Example 1, below. Inhibitors of peptide phosphorylation are candidate compounds. Alternatively, assays incorporating the experiments, or variations thereof, as set forth in Example 1, below, can be designed to assay for candidate compounds which can inhibit or abrogate Chk1 and/or Chk2/Cds1 kinase activity and/or the G2 cell cycle arrest checkpoint.
In one embodiment, the peptides and polypeptides of the invention can be bound to a solid support. Solid supports can include, e.g., membranes (e.g., nitrocellulose or nylon), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dip stick (e.g., glass, PVC, polypropylene, polystyrene, latex and the like), a microfuge tube, or a glass, silica, plastic, metallic or polymer bead or other substrate such as paper. One solid support uses a metal (e.g., cobalt or nickel)-comprising column which binds with specificity to a histidine tag engineered onto a peptide.
Adhesion of peptides to a solid support can be direct (i.e. the protein contacts the solid support) or indirect (a particular compound or compounds are bound to the support and the target protein binds to this compound rather than the solid support). Peptides can be immobilized either covalently (e.g., utilizing single reactive thiol groups of cysteine residues (see, e.g., Colliuod (1993) Bioconjugate Chem. 4:528-536) or non-covalently but specifically (e.g., via immobilized antibodies (see, e.g., Schuhmann (1991) Adv. Mater. 3:388-391; Lu (1995) Anal. Chem. 67:83-87; the biotin/strepavidin system (see, e.g., Iwane (1997) Biophys. Biochem. Res. Comm. 230:76-80); metal chelating, e.g., Langmuir-Blodgett films (see, e.g., Ng (1995) Langmuir 11:4048-55); metal-chelating self-assembled monolayers (see, e.g., Sigal (1996) Anal. Chem. 68:490-497) for binding of polyhistidine fusions.
Indirect binding can be achieved using a variety of linkers which are commercially available. The reactive ends can be any of a variety of functionalities including, but not limited to: amino reacting ends such as N-hydroxysuccinimide (NHS) active esters, imidoesters, aldehydes, epoxides, sulfonyl halides, isocyanate, isothiocyanate, and nitroaryl halides; and thiol reacting ends such as pyridyl disulfides, maleimides, thiophthalimides, and active halogens. The heterobifunctional crosslinking reagents have two different reactive ends, e.g., an amino-reactive end and a thiol-reactive end, while homobifunctional reagents have two similar reactive ends, e.g., bismaleimidohexane (BMH) which permits the cross-linking of sulfhydryl-containing compounds. The spacer can be of varying length and be aliphatic or aromatic. Examples of commercially available homobifunctional cross-linking reagents include, but are not limited to, the imidoesters such as dimethyl adipimidate dihydrochloride (DMA); dimethyl pimelimidate dihydrochloride (DMP); and dimethyl suberimidate dihydrochloride (DMS). Heterobifunctional reagents include commercially available active halogen-NHS active esters coupling agents such as N-succinimidyl bromoacetate and N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) and the sulfosuccinimidyl derivatives such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) (Pierce). Another group of coupling agents is the heterobifunctional and thiol cleavable agents such as N-succinimidyl 3-(2-pyridyidithio)propionate (SPDP) (Pierce Chemicals, Rockford, Ill.).
Antibodies can be used for binding polypeptides and peptides of the invention to a solid support. This can be done directly by binding peptide-specific antibodies to the column or it can be done by creating fusion protein chimeras comprising motif-containing peptides linked to, e.g., a known epitope (e.g., a tag (e.g., FLAG, myc) or an appropriate immunoglobulin constant domain sequence (an “immunoadhesin,” see, e.g., Capon (1989) Nature 377:525-531 (1989).
There are a variety of assay formats that can be used to screen for “candidate compounds” to inhibit or abrogate Chk1 and/or Chk2/Cds1 kinase activity and/or the G2 cell cycle arrest checkpoint. For example, as discussed above, compounds that inhibit the phosphorylation of the motif-comprising peptides of the invention can be candidate compounds. Alternatively, compounds that specifically bind to the motifs of the invention can be candidate compounds. For a general description of different formats for binding assays, see, e.g., BASIC AND CLINICAL IMMUNOLOGY, 7th Ed. (D. Stiles and A. Terr, ed.)(1991); ENZYME IMMUNOASSAY, E. T. Maggio, ed., CRC Press, Boca Raton, Fla. (1980); and “Practice and Theory of Enzyme Immunoassays” in P. Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, Elsevier Science Publishers, B.V. Amsterdam (1985).
Combinatorial Chemical Libraries
Combinatorial chemical libraries are one means to assist in the generation of new chemical compound leads, i.e., compounds that inhibit Chk1 and/or Chk2/Cds1 kinase and/or inhibit or abrogate the G2 cell cycle arrest checkpoint. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (see, e.g., Gallop et al. (1994) 37(9): 1233-1250). Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art, see, e.g., U.S. Pat. Nos. 6,004,617; 5,985,356. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88). Other chemistries for generating chemical diversity libraries include, but are not limited to: peptoids (see, e.g., WO 91/19735), encoded peptides (see, e.g., WO 93/20242), random bio-oligomers (see, e.g., WO 92/00091), benzodiazepines (see, e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (see, e.g., Hobbs (1993) Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (see, e.g., Hagihara (1992) J. Amer. Chem. Soc. 114: 6568), non-peptidal peptidomimetics with a Beta-D-Glucose scaffolding (see, e.g., Hirschmann (1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic syntheses of small compound libraries (see, e.g., Chen (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (see, e.g., Cho (1993) Science 261:1303), and/or peptidyl phosphonates (see, e.g., Campbell (1994) J. Org. Chem. 59: 658). See also Gordon (1994) J. Med. Chem. 37:1385; for nucleic acid libraries, peptide nucleic acid libraries, see, e.g., U.S. Pat. No. 5,539,083; for antibody libraries, see, e.g., Vaughn (1996) Nature Biotechnology 14:309-314; for carbohydrate libraries, see, e.g., Liang et al. (1996) Science 274: 1520-1522, U.S. Pat. No. 5,593,853; for small organic molecule libraries, see, e.g., for isoprenoids U.S. Pat. No. 5,569,588; for thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; for pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; for morpholino compounds, U.S. Pat. No. 5,506,337; for benzodiazepines U.S. Pat. No. 5,288,514.
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., U.S. Pat. Nos. 6,045,755; 5,792,431; 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). A number of robotic systems have also been developed for solution phase chemistries. These systems include automated workstations, e.g., like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
Formulation and Administration of Pharmaceutical Compositions
In one embodiment, the peptides and polypeptides of the invention are combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts to, e.g., stabilize, or increase or decrease the absorption or clearance rates of the pharmaceutical compositions of the invention. Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the peptides or polypeptides, or excipients or other stabilizers and/or buffers. Detergents can also used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. Pharmaceutically acceptable carriers and formulations for peptides and polypeptide are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. (“Remington's”).
Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g., phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the peptide or polypeptide of the invention and on its particular physio-chemical characteristics.
In one embodiment, a solution of peptide or polypeptide of the invention is dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier if the composition is water-soluble. Examples of aqueous solutions that can be used in formulations for enteral, parenteral or transmucosal drug delivery include, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions and the like. The formulations can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. Additives can also include additional active ingredients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The concentration of peptide in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
Solid formulations can be used for enteral (oral) administration. They can be formulated as, e.g., pills, tablets, powders or capsules. For solid compositions, conventional nontoxic solid carriers can be used which include, e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10% to 95% of active ingredient (e.g., peptide). A non-solid formulation can also be used for enteral administration. The carrier can be selected from various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients include e.g., starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol.
Peptides and polypeptides of the invention, when administered orally, can be protected from digestion. This can be accomplished either by complexing the peptide or polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the peptide or complex in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are well known in the art, see, e.g., Fix (1996) Pharm Res. 13:1760-1764; Samanen (1996) J. Pharm. Pharmacol. 48:119-135; U.S. Pat. No. 5,391,377, describing lipid compositions for oral delivery of therapeutic agents (liposomal delivery is discussed in further detail, infra).
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories. See, e.g., Sayani (1996) “Systemic delivery of peptides and proteins across absorptive mucosae” Crit. Rev. Ther. Drug Carrier Syst. 13:85-184. For topical, transdermal administration, the agents are formulated into ointments, creams, salves, powders and gels. Transdermal delivery systems can also include, e.g., patches.
The peptides and polypeptide complexes can also be administered in sustained delivery or sustained release mechanisms, which can deliver the formulation internally. For example, biodegradeable microspheres or capsules or other biodegradeable polymer configurations capable of sustained delivery of a peptide can be included in the formulations of the invention (see, e.g., Putney (1998) Nat. Biotechnol. 16:153-157).
For inhalation, the peptide or polypeptide can be delivered using any system known in the art, including dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like. See, e.g., Patton (1998) Biotechniques 16:141-143; product and inhalation delivery systems for polypeptide macromolecules by, e.g., Dura Pharmaceuticals (San Diego, Calif.), Aradigm (Hayward, Calif.), Aerogen (Santa Clara, Calif.), Inhale Therapeutic Systems (San Carlos, Calif.), and the like. For example, the pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another embodiment, the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include, e.g., air jet nebulizers.
In preparing pharmaceuticals of the present invention, a variety of formulation modifications can be used and manipulated to alter pharmacokinetics and biodistribution. A number of methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art. Examples of such methods include protection of the complexes in vesicles composed of substances such as proteins, lipids (for example, liposomes, see below), carbohydrates, or synthetic polymers (discussed above). For a general discussion of pharmacokinetics, see, e.g., Remington's, Chapters 37-39.
The peptide and polypeptide complexes used in the methods of the invention can be delivered alone or as pharmaceutical compositions by any means known in the art, e.g., systemically, regionally, or locally (e.g.,.directly into, or directed to, a tumor); by intraarterial, intrathecal (IT), intravenous (IV), parenteral, intra-pleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa). Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in detail in the scientific and patent literature, see e.g., Remington's. For a “regional effect,” e.g., to focus on a specific organ, one mode of administration includes intra-arterial or intrathecal (IT) injections, e.g., to focus on a specific organ, e.g., brain and CNS (see e.g., Gurun (1997) Anesth Analg. 85:317-323). For example, intra-carotid artery injection if preferred where it is desired to deliver a peptide or polypeptide complex of the invention directly to the brain. Parenteral administration is a preferred route of delivery if a high systemic dosage is needed. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in detail, in e.g., Remington's,. See also, Bai (1997) J. Neuroimmunol. 80:65-75; Warren (1997) J. Neurol. Sci. 152:31-38; Tonegawa (1997) J. Exp. Med. 186:507-515.
In one embodiment, the pharmaceutical formulations comprising peptides or polypeptides of the invention are incorporated in lipid monolayers or bilayers, e.g., liposomes, see, e.g., U.S. Pat. Nos. 6,110,490; 6,096,716; 5,283,185; 5,279,833. The invention also provides formulations in which water soluble peptides or complexes have been attached to the surface of the monolayer or bilayer. For example, peptides can be attached to hydrazide-PEG-(distearoylphosphatidyl) ethanolamine-containing liposomes (see, e.g., Zalipsky (1995) Bioconjug. Chem. 6:705-708). Liposomes or any form of lipid membrane, such as planar lipid membranes or the cell membrane of an intact cell, e.g., a red blood cell, can be used. Liposomal formulations can be by any means, including administration intravenously, transdermally (see, e.g., Vutla (1996) J. Pharm. Sci. 85:5-8), transmucosally, or orally. The invention also provides pharmaceutical preparations in which the peptides and/or complexes of the invention are incorporated within micelles and/or liposomes (see, e.g., Suntres (1994) J. Pharm. Pharmacol. 46:23-28; Woodle (1992) Pharm. Res. 9:260-265). Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art, see, e.g., Remington's; Akimaru (1995) Cytokines Mol. Ther. 1:197-210; Alving (1995) Immunol. Rev. 145:5-31; Szoka (1980) Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.
Treatment Regimens: Pharmacokinetics
The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisorial in nature and are adjusted depending on the particular therapeutic context, patient tolerance, etc. The amount of peptide or polypeptide adequate to accomplish this is defined as a “therapeutically effective dose.” The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like. See, e.g., the latest Remington's; Egleton (1997) “Bioavailability and transport of peptides and peptide drugs into the brain” Peptides 18:1431-1439; Langer (1990) Science 249:1527-1533.
In therapeutic applications, compositions are administered to a patient suffering from a cancer in an amount sufficient to at least partially arrest the disease and/or its complications. For example, in one embodiment, a soluble peptide pharmaceutical composition dosage for intravenous (IV) administration would be about 0.01 mg/hr to about 1.0 mg/hr administered over several hours (typically 1, 3, or 6 hours), which can be repeated for weeks with intermittent cycles. Considerably higher dosages (e.g., ranging up to about 10 mg/ml) can be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ, e.g., the cerebrospinal fluid (CSF).
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Administration of Peptides of the Invention to Selectively Sensitize Cancer Cells to DNA Damaging Agents
The invention provides compositions and methods for sensitizing cells, particularly cells with an impaired G1 cell cycle arrest checkpoint, such as cancer cells, to DNA damaging agents. The following example describes studies which demonstrate that the compositions and methods of the invention are effective for selectively killing cancer cells (versus normal cells, which have an unimpaired G1 checkpoint). Specifically, these experiments describes the synthesis and use of two exemplary polypeptides of the invention. Two peptides corresponding to amino acids 211 to 221 of human Cdc25C (SEQ ID NO:1) fused with a part of HIV-1-TAT (SEQ ID NO:5). These peptides were demonstrated to inhibit hChk1 kinase (SEQ ID NO:3) and Chk2/HuCds1 (SEQ ID NO:4) kinase activity in vitro and to specifically abrogate the G2 checkpoint in vivo.
Chemicals and reagents. Bleomycin and colchicine were purchased from Wako Pure Chemical Co. (Osaka, Japan). Hydroxyurea was purchased from Sigma Chemical Co. (St. Louis, Mo.). These chemicals were dissolved in distilled H2O to 10, 5 and 50 mg/ml, respectively, and stored at 4° C. Antibodies against 14-3-3β were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.) and anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Life Sciences (Arlington Heights, Ill.). Antibodies against HA and c-myc, and protein G-Sepharose were purchased from Santa Cruz Biotechnology and Amersham Pharmacia Biotech (Uppsala, Sweden), respectively.
Cell culture and plasmids. A human T-cell leukemia-derived cell line, Jurkat, was cultured in RPMI 1640 (Sigma) supplemented with 10% fetal calf serum. (IBL: Immuno-Biological Laboratories, Gunma, Japan) at 37° C./5% CO2. Human pancreatic epitheloid carcinoma-derived cell lines, MIA PaCa2 and PANC1, were cultured in Eagle's MEM (IWAKI, Chiba, Japan) and Dulbecco's modified Eagle's medium with 4 mM 1-glucose (Sigma) and 1.0 mM sodium pyruvate (Life Technologies, Inc., Grand Island, N.Y.), respectively, and supplemented with 10% fetal calf serum at 37° C./5% CO2. Normal human peripheral blood lymphocytes were collected by Ficoll-Paque (Amersham Pharmacia Biotech) density gradient. Two million cells/ml were cultured in RPMI 1640 supplemented with 10% fetal calf serum at 37° C./5% CO2 in the presence of 5 μg/ml PHA (Life Technologies, Inc.) for a week. Baculovirus lysates that include HA-tagged hChk1 (SEQ ID NO:3) or c-myc-tagged Chk2/HuCds1 (SEQ ID NO:4) and plasmid for GST-Cdc25C (amino, acid 200-256) were made as described in Matsuoka (1998) Science 282:1893-1897, and provided by Dr. Makoto Nakanishi (Department of Biochemistry, Nagoya City University.
Peptides. TAT-S216 peptide was synthesized so that it contained an NH2-terminal 11 amino acid TAT protein transduction domain (YGRKKRRQRRR (SEQ ID NO: 1899); see, e.g., Nagahara (1998) Nature Med. 4:1449-1453) followed by a corresponding amino acid 211 to 221 derived from the human Cdc25C amino acid sequence (SEQ ID NO: 2) (S216; LYRSPSMPENL). Serine-216 residue was changed to alanine in TAT-S216A (S216A; LYRSPAMPENL) (SEQ ID NO: 1897). The Cdc25C portion was partially deleted and substituted with glycine in TAT_Control (GGRSPAMPE) (SEQ ID NO: 1905). All peptides were synthesized by Sawady Technology Co. (Tokyo, Japan).
Purification of recombinant GST-Cdc25C proteins. Escherichia coli DH5α cells were transformed by GST-Cdc25C (200-256) plasmid. The cells were incubated with 0.1 mM isopropyl β-D-thiogalactoside for 2 hr, harvested, and lysed with a buffer containing 50 mM Tris HCl (pH8.0), 100 mM NaCl, 0.5% NP-40, 5 μg/ml aprotinin, 5 μg/ml pepstatin A and 5 μg/ml leupeptin. The lysate was sonicated, centrifuged for clarification and incubated with glutathione-Sepharose 4BTM beads for 1 hr at 4° C. and washed five times.
Kinase assay. HA-tagged hChk1 (SEQ ID NO:3) and c-myc-tagged Chk2/HuCds1 (SEQ ID NO:4) expressed in insect cells using recombinant baculovirus (see, e.g., Kaneko (1999) Oncogene 18:3673-3681) were purified by immunoprecipitation using anti-HA or anti-c-myc antibodies and protein G-Sepharose. Immune complex kinase reaction was done in PBS with 1 mM DTT, 1 mM MgCl2 and 100 μCi of [γ-32P] ATP (Amersham; 6000 Ci/mmol) plus purified 1 μM GST-Cdc25C or 10 μM Cdc25C peptide (amino acid 211 to 221 of Cdc25C (SEQ ID NO: 2); LYRSPSMPENL, Sawady Technology Co.) substrates at 30° C. for 15 min in the presence of 10 μM TAT-S216, TAT-S216A or TAT-Control. After the reaction, samples were separated in 12% or 15% SDS-PAGE and autoradiographed to detect GST-Cdc25C or peptide phosphorylation.
Cell-cycle analysis. The cell cycle status of the cells treated with peptides and/or bleomycin or colchicine was analyzed by FACS, as described by Kawabe (1997) Nature 385:454-458. In brief, two million Jurkat cells were re-suspended and incubated in 300 μl Krishan's solution (0.1% Sodium citrate, 50 μg/ml PI, 20 μg/ml RNase A and 0.5% NP-40; see supra) for 1 hr at 4° C. and analyzed by FACScanTM (Beckton Dickinson, Mountain View, Calif.) with the program CELLQuestTM (Beckton Dickinson).
Histone H1 kinase assay. Ten million Jurkat cells were treated with hydroxyurea (100 μg/ml), bleomycin (10 μg/ml), or colchicine (5 μg/ml) with or without addition of TAT-S216A, TAT-S216 or TAT-Control (10 μM) for 6 hr. The cells were washed in cold PBS and lysed at 4° C. in 1 ml of buffer A (50 mM Tris pH 8, 2 mM DTT, 5 mM EDTA, 100 mM NaCl, 0.5% NP40, 20 mM Na3V04, 50 mM NaF, 4 μM Okadaic acid, 5 μg/ml aprotinin, 5 μg/ml pepstatin A and 5 μg/ml leupeptin.). Twenty microliter of p13suc1 agarose beads (Upstate Biotechnology., Saranac, N.Y.) were added to the cleared lysates, incubated for 4 hr at 4° C., and washed five times with buffer A without 5 mM EDTA, 20 mM Na3V04, 50 mM NaF, 4 μM Okadaic acid. Histone HI kinase activity on the beads were analyzed by using Cdc2 kinase assay kit (Upstate Biotechnology) with [γ-32P] ATP, followed by 12% SDS-PAGE electrophoresis, and autoradiographed to detect the phosphorylated Histone H1.
Cell cytotoxicity assay. MIA PaCa2 and PANC1 cells (3×103/well) were plated in 96-well microtiter plates. After an overnight adherence, cells were treated with bleomycin (10 μg/ml) with or without the indicated TAT-peptides at various time points up to 96 hr. Cytotoxicity and cell survival were determined by the 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) (XTT) assay (Cell Proliferation Kit IITM: Boehringer Mannheim, Germany), which was done according to company's protocol and Scudiero (1988) Cancer Res. 48.4827-4833.
TAT-S216 and TAT-S216A Peptides Inhibit hChk1 and Chk2/HuCds1 Kinase Activities
To inhibit hChk1 (SEQ ID NO:3) and Chk2/HuCds1 (SEQ ID NO:4) kinase activities and to abrogate DNA damage-induced-G2 arrest, synthetic peptides comprising amino acid residues 211 to 221 of Cdc25C (SEQ ID NO:1) and a variation of the TAT protein transduction domain (YGRKKRRQRRR (SEQ ID NO: 1899) (TAT-S216) were generated.
The results are shown in FIG. 1: TAT-S216A and TAT-S216 peptides inhibit hChk1 and Chk2/HuCds1 kinase activities in vitro. FIG. 1A. sequences of the peptides. FIG. 1B, in vitro phosphorylation analysis using GST-Cdc25C and purified hChk1. GST-Cdc25C (amino acid 200-256) that was produced in E. coli (DH5α) was used as substrate (1 μM). Immune complex kinase reaction was done in the presence of TAT-S216A (10 μM) or TAT-S216 (10 μM). FIG. 1C, in vitro phosphorylation analysis of hChk1 and Chk2/HuCds1 using synthesized Cdc25C peptide corresponding amino acid 211-221 of Cdc25C (LYRSPSMPENL (SEQ ID NO: 2)) as a substrate (10 μM).
A TAT-S216A peptide (S216A; LYRSPAMPENL, (SEQ ID NO: 1897)), in which serine residue 216 was substituted by alanine was devised to stabilize the transient status of its interaction with hChk1 (SEQ ID NO:3) and Chk2/HuCds1 (SEQ ID NO:4) (FIG. 1A). This TAT peptide was included to efficiently transduce these peptides into cells (see, e.g., Nagahara (1998) supra). This sequence is known to facilitate the uptake of heterologous proteins across the cell membrane. As a control peptide, part of the Cdc25C portion of this peptide was deleted (TAT-Control).
As shown in FIG. 1B, hChk1 (SEQ ID NO:3) was capable of phosphorylating a Cdc25C protein (residues 200-256) (SEQ ID NO:1) fused to GST. Serine-216 on Cdc25C (SEQ ID NO:1) is the major phosphorylation site of this fusion protein in vivo (see, e.g., Furnari (1997) Science 277:1495-1497; Sanchez (1997) Science 277:1497-1501; Peng (1997) Science 277:1501-1505).
In FIG. 1B, both TAT-S216 and TAT-S216A inhibited the phosphorylation of Cdc25C by baculovirus-produced hChk1 (SEQ ID NO:3). TAT-S216 but not TAT-S216A was efficiently phosphorylated by hChk1, suggesting that serine-216 on TAT-S216 was phosphorylated by hChk1 and TAT-S216 would competitively inhibit substrate phosphorylation at excess molar ratio if present in great enough quantity. TAT-Control peptide did not inhibit hChk1 kinase activity.
As shown in FIG. 1C, TAT-S216A significantly inhibited phosphorylation of Cdc25C peptide (residues 200-256) (SEQ ID NO:1) mediated by hChk1 (SEQ ID NO:3) and Chk2/HuCds1 (SEQ ID NO:4) even at a low stoichiometry (at four times more molar excess of TAT-S216A peptide against substrate Cdc25C peptide).
Abrogation of DNA Damage-induced G2 Checkpoint by TAT-S216 and TAT-S216A Peptides
The cell cycle status of the cells treated with TAT-S216A or TAT-S216 upon the DNA damage-induced G2 arrest was analyzed by FACS analysis. Histone H1 kinase activities of theses cells were simultaneously monitored. Jurkat cells arrested exclusively at G2 by bleomycin (10 μg/ml) treatment, because it does not have functional p53. Results are shown in FIG. 2: abrogation of DNA damage-induced G2 arrest by TAT-S216A and TAT-S216 peptides. FIG. 2A, FACS analysis of Jurkat cells treated with bleomycin and peptides. Cells were treated with bleomycin (10 μg/ml) with or without peptides (10 μM) for 20 hr. B, histone H1 kinase analysis. Cell lysates were prepared from the cells treated with the indicated reagent for 6 hr. Concentrations used were: hydroxyurea (HU), 100 μg/ml; bleomycin (Bleo), 10 μg/ml; colchicine, 5 μg/ml; TAT-S216A and TAT-S216, 10 μM. C, FACS analysis of colchicine -and peptide-treated cells. Jurkat cells were treated with colchicine (5 μg/ml) with or without peptide (10 μM) for 20 hr.
As shown in FIG. 2A, G2 arrest was completely abrogated by the addition of TAT-S216A or TAT-S216 in response to bleomycin. G2 arrest was abrogated at any time point between 12 and 48 hr by the treatment with TAT-S216A or TAT-S216. Jurkat cells treated with bleomycin together with TAT-Control arrested at G2 similarly to the cells treated with bleomycin alone.
We also observed that either TAT-S216A or TAT-S216 also abrogated G2 arrest induced by gamma-irradiation and cisplatin (gamma-irradiation, 5 Gy; cisplatin, 1 μg/ml for 1 hr treatment). To further analyze the effect of these peptides on G2/M transition, histone H1 kinase activity was monitored. Consistent with the above findings, although histone H1 kinase activity was decreased by the treatment with bleomycin or hydroxyurea, it was unchanged or rather increased by the treatment with bleomycin in the presence of TAT-S216A or TAT-S216 (FIG. 2B). In the presence of TAT-Control peptide, the bleomycin treatment did not affect with H1 kinase activity.
As shown in FIG. 2C, The M-phase arrest of Jurkat cells induced by colchicine was not affected by the addition of TAT-S216 or TAT-S216A. These results demonstrate that TAT-S216A and TAT-S216 specifically abrogated the DNA damage-activated cell cycle G2 checkpoint by inhibiting hChk1 (SEQ ID NO:3) and/or Chk2/HuCds1 (SEQ ID NO:4) kinase activities.
Sensitization of Jurkat Cells to the Bleomycin-induced Cell Death by TAT-S216A and TAT-S216 Peptides
The effect of TAT-S216A and TAT-S216 on the cell death induced by bleomycin was examined. The results are shown in FIG. 3; Trypan blue dye exclusion analysis of Jurkat cells treated with bleomycin (A) or colchicine (B) with or without indicated peptides. Bars, SD Vertical axis, % viability of the cells; Bleo 5, bleomycin 5 μg/ml; Bleo 10, bleomycin 10 μg/ml; colchicine, 5 μg/ml; TAT-S216 or TAT-S216A, 10 μM of indicated peptide. Note that TAT-S216A and TAT-S216 peptides did not increase the cytotoxicity of bleomycin to normal cells. C, survival analysis of PHA blasts treated with bleomycin and peptides. Vertical axis, % viability of the cells determined by trypan blue dye exclusion analysis; horizontal axis, time in hours. Bleo 5, bleomycin 5 μg/ml; Bleo 10, bleomycin 10 μg/ml; TAT-S216 or TAT-S216A, 10 μM of indicated peptide. D, FACS analysis of the cells treated with bleomycin and peptides. PHA-blasts were treated with bleomycin with or without peptides for 20 hr. Vertical axis, cell number; horizontal axis, DNA content indicated by propidium iodide staining.
As shown in FIG. 3A, the addition of TAT-S216A and TAT-S216 efficiently sensitized Jurkat cells to the bleomycin-induced cell death. Whereas bleomycin treatment at 5 or μ10 g/ml killed Jurkat cells by only 27-30%, the addition of 10 μM TAT-216A or TAT-S216 killed Jurkat cells by nearly 80%. In contrast, these peptide by themselves did not show any significant cytotoxicity. In addition, a control peptide TAT-Control did not affect the viability of bleomycin-treated Jurkat cells. Moreover, as expected from the result in FIG. 2C, either TAT-S216A or TAT-S216 did not affect the cytotoxicity by colchicine (FIG. 3B). This observation indicates that the cell death induced by these peptides in the presence of bleomycin was not attributable to a nonspecific cytotoxic effect.
TAT-S216 and TAT-S216A Peptides did not Affect the Viability of Normal Cells
In order to confirm the specificity of the effect of these peptides on cancer cells in which the G1 checkpoint is abrogated; the effect of these peptides on normal human cells was investigated. Mitogen-activated normal human T lymphocytes (PHA blasts) were prepared by stimulating peripheral blood mononuclear cells obtained from a healthy donor with PHA for 1 week. These cells were treated with bleomycin (5 and 10 μg/ml) in the presence or absence of either TAT-S216A or TAT-S216.
As shown in FIG. 3C, these peptides did not augment the cytotoxic effect of bleomycin, although these cells replicated as fast as Jurkat cells. As shown in FIG. 3D, PHA blasts treated with bleomycin (5 μg/ml) arrested at G1 and S phase but not G2, presumably because of the activity of wild-type p53. When these cells were treated with TAT-S216 or TAT-S216A in addition to bleomycin, no further alteration of cell cycle pattern was observed.
Sensitization of Pancreatic Cancer Cells to the Bleomycin-induced Cell Death by TAT-S216A and TAT-S216 Peptides
The effect of these peptides on two other p53-defective pancreatic cancer cell lines, MIA-PaCa2 and PANC1 cells, was examined. FIG. 4 shows the results of survival analysis of PANC1 (A) and MIA PaCa2 (B) cells treated with bleomycin and peptides. PANC1 and MIA PaCa2 cells were treated with bleomycin with or without the indicated peptide. The cell viability was determined by the 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate assay at the indicated times after addition of bleomycin and peptide. Bleo 60, bleomycin 60 μg/ml; TAT-S216 or TAT-S216A, 10 μM of indicated peptide. Bars, SD.
Although these pancreatic cancer cells are known to be resistant to various anti-cancer reagents, these cells could also be sensitized to the bleomycin-induced cell death by TAT-S216A and TAT-S216 (FIG. 4). Similarly, these peptides could sensitize these cells to the cell death induced by other DNA-damaging agents including cisplatin and gamma-irradiation.
In summary, these experiments demonstrated for the first time that short peptides that inhibit both hChk1 and Chk2/HuCds1 kinase activities can specifically abrogate the DNA damage-induced G2 cell growth arrest checkpoint. These data also demonstrated that the specific abrogation of the G2 checkpoint sensitized cancer cells to bleomycin, a DNA-damaging agent, without obvious effect on normal cell cycle and its viability. These observations indicate that these kinases involved in G2 cell cycle checkpoint are ideal targets for the specific abrogation of G2 checkpoint and that the peptides and polypeptides of the invention and their derivatives can be used in novel cancer therapy.
Example 2
Optimization of Sequences for G2 Abrogating Peptides of the Invention
The following example describes studies which identified exemplary G2 checkpoint-abrogating peptides of the invention. This was accomplished by using a computer analysis of the structure of human Chk2 kinase (SEQ ID NO:4) and the peptides of the invention.
The 3-dimensional structure of human Chk2 was predicted by comparing the primary and 3-D structure of another serine threonine kinase, PKA (PDB protein data base, Research Collaboratory for Structural Bioinformatics (RCSB), The National Science Foundation, Arlington, Va.) (1CDK), using a computer program, MODELERTM (IMMD, Tokyo; Japan). The alignment of the peptides of the invention and hChk2 were predicted by comparing an alignment of hChk1 and various Cdc25C peptides as described by Chen (2000) “The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation,” Cell 100:681-92. By comparing the predicted structure of hChk2 with the peptides of the invention, it was predicted that there are four pockets on hChk2 that are important for the interaction with peptides, as shown in FIG. 5, P1, P2, P3 and P4. The structure of these pockets was used to design and confirm the sequences of exemplary peptides of the invention
The ability of these peptides to abrogate the activity of Chk2 kinase, thereby imbuing the ability to abrogate the G2 cell cycle checkpoint, was demonstrated by their ability to act as a phosphorylation substrate for human Chk2 kinase. Exemplary peptides were directly synthesized (immobilized) on a membrane and contacted with human Chk2 kinase. Specifically, oligo-peptides with all sequences predicted by the 3-dimensional model were directly synthesized on a membrane by using an auto-spot-peptide-synthesizer, Model ASP-22 2 (ABiMED, Germany). The amount of peptide was about 0.1 micro-mol/cm2.
The membrane was incubated with 2% Gly-Gly in PBS for 2 hours (hr) at room temperature (RT). Then, they were washed three times with 0.1% Tween-P BSTM. The “kination,” or “phosphorylation,” reaction was performed with a recombinant fusion protein Gst-Chk2 at a concentration of about 5 μg in 4 ml reaction buffer, 1 mM MgCl2, 2% Gly-Gly and γ-33P-ATP in PBS at RT for 1 hr. After the reaction, the membrane was washed 5 times with RIPA (1% SDS, 1% NP-40, 100 mM NaCl) and analyzed with a Bass 2500TM image analyzer (Fuji, Japan). The signal was graded to “−,” a “+,” a “++,” or a “+++.” Table 1 shows the peptide sequences that gave signals stronger than “++.” The peptides RYSLPPELSNM (SEQ ID NO: 1) and LYRSPSAMPENL (SEQ ID NO: 1906) gave “+” signals by this analysis.
All of the following peptides were phosphorylated by human Chk2 kinase; in position “X” (corresponding to position X8), wherein X=P, F, Y, or W, the signal was strongest (a “+++”) when X=the amino acid tyrosine (Y):










(SEQ ID NO: 1907) 37-40
LYRSPSHXENL


 



(SEQ ID NO: 1908) 52-53
LYSSPSYXENL


 



(SEQ ID NO: 1909) 92-95
LYTSPSYXENL


 



(SEQ ID NO: 1910) 117-121
LYTSPSHXENL


 



(SEQ ID NO: 1911) 132-135
LYHSPSYXENL


 



(SEQ ID NO: 1912) 1127-1130
WYRSPSFXENL


 



(SEQ ID NO: 1913) 1237-1240
WYTSPSHXENL


 



(SEQ ID NO: 1914) 372-375
LFTSPSYXENL


 



(SEQ ID NO: 1915) 637-640
FYSSPSHXENL


 



(SEQ ID NO: 1916) 642-645
FYTSPSMXENL


 



(SEQ ID NO: 1917) 648-651
FYTSPSFXENL


 



(SEQ ID NO: 1918) 652-655
FYTSPSYXENL


 



(SEQ ID NO: 1919) 1202-1205
WYTSPSMXENL


 



(SEQ ID NO: 1920) 1207-1210
WYTSPSFXENL


 



(SEQ ID NO: 1921) 1212-1215
WYTSPSYXENL






The best phosphorylation substrates were the peptides L Y R S P S Y Y E N L (SEQ ID NO: 12) and W Y T S P S Y F E N L (SEQ ID NO: 971).
The following Table 1 is a complete list of tested peptides and results of the in vitro phosphorylation by human Chk2 kinase assay. Results are presented to the right of the peptide, below: a “+++” indicates the peptide was relatively highly phosphorylated; a “++” indicates the peptide was relatively less phosphorylated, a “+” indicates the peptide was detectably significantly phosphorylated over negative control, and no indication indicates that a peptide was not significantly phosphorylated over negative control (note: the number immediately to the right of the peptide is the MW of the peptide).











TABLE 1







1
RYSLPPELSNM
1308.6
+
(SEQ ID NO: 1)


2
LYRSPSMPENL
1308.6
+
(SEQ ID NO: 2)


3
LYRSPSMFENL
1358.6
−
(SEQ ID NO: 3)


4
LYRSPSMYENL
1374.6
−
(SEQ ID NO: 4)


5
LYRSPSMWENL
1397.7
−
(SEQ ID NO: 5)


7
LYRSPSFPENL
1324.5
−
(SEQ ID NO: 6)


8
LYRSPSFFENL
1374.5
−
(SEQ ID NO: 7)


9
LYRSPSFYENL
1390.5
−
(SEQ ID NO: 8)


10
LYRSPSFWENL
1413.6
−
(SEQ ID NO: 9)


12
LYRSPSYPENL
1340.5
−
(SEQ ID NO: 10)


13
LYRSPSYFENL
1390.5
+
(SEQ ID NO: 11)


14
LYRSPSYYENL
1406.5
+
(SEQ ID NO: 12)


15
LYRSPSYWENL
1429.6
+
(SEQ ID NO: 13)


17
LYRSPSDPENL
1292.4
−
(SEQ ID NO: 14)


18
LYRSPSDFENL
1342.4
−
(SEQ ID NO: 15)


19
LYRSPSDYENL
1358.4
−
(SEQ ID NO: 16)


20
LYRSPSDWENL
1381.5
−
(SEQ ID NO: 17)


22
LYRSPSEPENL
1306.4
−
(SEQ ID NO: 18)


23
LYRSPSEFENL
1356.4
−
(SEQ ID NO: 19)


24
LYRSPSEYENL
1372.4
−
(SEQ ID NO: 20)


25
LYRSPSEWENL
1395.5
−
(SEQ ID NO: 21)


27
LYRSPSNPENL
1291.5
+
(SEQ ID NO: 22)


28
LYRSPSNFENL
1341.5
−
(SEQ ID NO: 23)


29
LYRSPSNYENL
1357.5
+
(SEQ ID NO: 24)


30
LYRSPSNWENL
1380.6
+
(SEQ ID NO: 25)


32
LYRSPSQPENL
1305.5
−
(SEQ ID NO: 26)


33
LYRSPSQFENL
1355.5
−
(SEQ ID NO: 27)


34
LYRSPSQYENL
1371.5
−
(SEQ ID NO: 28)


35
LYRSPSQWENL
1394.6
−
(SEQ ID NO: 29)


37
LYRSPSHPENL
1314.5
+
(SEQ ID NO: 30)


38
LYRSPSHFENL
1364.5
+
(SEQ ID NO: 31)


39
LYRSPSHYENL
1380.5
+
(SEQ ID NO: 32)


40
LYRSPSHWENL
1403.6
+
(SEQ ID NO: 33)


42
LYSSPSMPENL
1240.3
+
(SEQ ID NO: 34)


43
LYSSPSMFENL
1290.3
+
(SEQ ID NO: 35)


44
LYSSPSMYENL
1306.3
+
(SEQ ID NO: 36)


45
LYSSPSMWENL
1329.4
+
(SEQ ID NO: 37)


47
LYSSPSFPENL
1256.2
+
(SEQ ID NO: 38)


48
LYSSPSFFENL
1306.2
+
(SEQ ID NO: 39)


49
LYSSPSFYENL
1322.2
+
(SEQ ID NO: 40)


50
LYSSPSFWENL
1345.3
+
(SEQ ID NO: 41)


52
LYSSPSYPENL
1272.2
+
(SEQ ID NO: 42)


53
LYSSPSYFENL
1322.2
+
(SEQ ID NO: 43)


54
LYSSPSYYENL
1338.2
+
(SEQ ID NO: 44)


55
LYSSPSYWENL
1361.3
+
(SEQ ID NO: 45)


57
LYSSPSDPENL
1224.1
−
(SEQ ID NO: 46)


58
LYSSPSDFENL
1274.1
−
(SEQ ID NO: 47)


59
LYSSPSDYENL
1290.1
−
(SEQ ID NO: 48)


60
LYSSPSDWENL
1313.2
−
(SEQ ID NO: 49)


62
LYSSPSEPENL
1238.1
−
(SEQ ID NO: 50)


63
LYSSPSEFENL
1288.1
−
(SEQ ID NO: 51)


64
LYSSPSEYENL
1304.1
−
(SEQ ID NO: 52)


65
LYSSPSEWENL
1327.2
−
(SEQ ID NO: 53)


67
LYSSPSNPENL
1223.2
−
(SEQ ID NO: 54)


68
LYSSPSNFENL
1273.2
−
(SEQ ID NO: 55)


69
LYSSPSNYENL
1289.2
−
(SEQ ID NO: 56)


70
LYSSPSNWENL
1312.3
−
(SEQ ID NO: 57)


72
LYSSPSQPENL
1237.2
+
(SEQ ID NO: 58)


73
LYSSPSQFENL
1287.2
−
(SEQ ID NO: 59)


74
LYSSPSQYENL
1303.2
−
(SEQ ID NO: 60)


75
LYSSPSQWENL
1326.3
+
(SEQ ID NO: 61)


77
LYSSPSHPENL
1246.2
++
(SEQ ID NO: 62)


78
LYSSPSHFENL
1296.2
++
(SEQ ID NO: 63)


79
LYSSPSHYENL
1312.2
++
(SEQ ID NO: 64)


80
LYSSPSHWENL
1335.3
++
(SEQ ID NO: 65)


82
LYTSPSMPENL
1253.5
++
(SEQ ID NO: 66)


83
LYTSPSMFENL
1303.5
++
(SEQ ID NO: 67)


84
LYTSPSMYENL
1319.5
++
(SEQ ID NO: 68)


85
LYTSPSMWENL
1342.6
++
(SEQ ID NO: 69)


87
LYTSPSFPENL
1269.4
++
(SEQ ID NO: 70)


88
LYTSPSFFENL
1319.4
++
(SEQ ID NO: 71)


89
LYTSPSFYENL
1335.4
++
(SEQ ID NO: 72)


90
LYTSPSFWENL
1358.5
++
(SEQ ID NO: 73)


92
LYTSPSYPENL
1285.4
++
(SEQ ID NO: 74)


93
LYTSPSYFENL
1335.4
++
(SEQ ID NO: 75)


94
LYTSPSYYENL
1351.4
+++
(SEQ ID NO: 76)


95
LYTSPSYWENL
1374.5
++
(SEQ ID NO: 77)


97
LYTSPSDPENL
1237.3
−
(SEQ ID NO: 78)


98
LYTSPSDFENL
1287.3
−
(SEQ ID NO: 79)


99
LYTSPSDYENL
1303.3
−
(SEQ ID NO: 80)


100
LYTSPSDWENL
1326.4
−
(SEQ ID NO: 81)


102
LYTSPSEPENL
1251.3
−
(SEQ ID NO: 82)


103
LYTSPSEFENL
1301.3
−
(SEQ ID NO: 83)


104
LYTSPSEYENL
1317.3
−
(SEQ ID NO: 84)


105
LYTSPSEWENL
1340.4
−
(SEQ ID NO: 85)


107
LYTSPSNPENL
1236.4
+
(SEQ ID NO: 86)


108
LYTSPSNFENL
1286.4
+
(SEQ ID NO: 87)


109
LYTSPSNYENL
1302.4
+
(SEQ ID NO: 88)


110
LYTSPSNWENL
1325.5
+
(SEQ ID NO: 89)


112
LYTSPSQPENL
1250.4
−
(SEQ ID NO: 90)


113
LYTSPSQFENL
1300.4
−
(SEQ ID NO: 91)


114
LYTSPSQYENL
1316.4
−
(SEQ ID NO: 92)


115
LYTSPSQWENL
1339.5
−
(SEQ ID NO: 93)


117
LYTSPSHPENL
1259.4
+
(SEQ ID NO: 94)


118
LYTSPSHFENL
1309.4
+
(SEQ ID NO: 95)


119
LYTSPSHYENL
1325.4
+
(SEQ ID NO: 96)


120
LYTSPSHWENL
1348.5
+
(SEQ ID NO: 97)


122
LYHSPSMPENL
1289.6
−
(SEQ ID NO: 98)


123
LYHSPSMFENL
1339.6
−
(SEQ ID NO: 99)


124
LYHSPSMYENL
1355.6
−
(SEQ ID NO: 100)


125
LYHSPSMWENL
1378.7
−
(SEQ ID NO: 101)


127
LYHSPSFPENL
1305.5
−
(SEQ ID NO: 102)


128
LYHSPSFFENL
1355.5
−
(SEQ ID NO: 103)


129
LYHSPSFYENL
1371.5
−
(SEQ ID NO: 104)


130
LYHSPSFWENL
1394.6
−
(SEQ ID NO: 105)


132
LYHSPSYPENL
1321.5
+
(SEQ ID NO: 106)


133
LYHSPSYFENL
1371.5
+
(SEQ ID NO: 107)


134
LYHSPSYYENL
1387.5
+++
(SEQ ID NO: 108)


135
LYHSPSYWENL
1410.6
+
(SEQ ID NO: 109)


137
LYHSPSDPENL
1273.4
−
(SEQ ID NO: 110)


138
LYHSPSDFENL
1323.4
−
(SEQ ID NO: 111)


139
LYHSPSDYENL
1339.4
−
(SEQ ID NO: 112)


140
LYHSPSDWENL
1362.5
−
(SEQ ID NO: 113)


142
LYHSPSEPENL
1287.4
−
(SEQ ID NO: 114)


143
LYHSPSEFENL
1337.4
−
(SEQ ID NO: 115)


144
LYHSPSEYENL
1353.4
−
(SEQ ID NO: 116)


145
LYHSPSEWENL
1376.5
−
(SEQ ID NO: 117)


147
LYHSPSNPENL
1272.5
−
(SEQ ID NO: 118)


148
LYHSPSNFENL
1322.5
−
(SEQ ID NO: 119)


149
LYHSPSNYENL
1338.5
−
(SEQ ID NO: 120)


150
LYHSPSNWENL
1361.6
−
(SEQ ID NO: 121)


152
LYHSPSQPENL
1286.5
−
(SEQ ID NO: 122)


153
LYHSPSQFENL
1336.5
−
(SEQ ID NO: 123)


154
LYHSPSQYENL
1352.5
−
(SEQ ID NO: 124)


155
LYHSPSQWENL
1375.6
−
(SEQ ID NO: 125)


157
LYHSPSHPENL
1295.5
−
(SEQ ID NO: 126)


158
LYHSPSHFENL
1345.5
−
(SEQ ID NO: 127)


159
LYHSPSHYENL
1361.5
−
(SEQ ID NO: 128)


160
LYHSPSHWENL
1384.6
−
(SEQ ID NO: 129)


162
LYNSPSMPENL
1266.6
−
(SEQ ID NO: 130)


163
LYNSPSMFENL
1316.6
−
(SEQ ID NO: 131)


164
LYNSPSMYENL
1332.6
−
(SEQ ID NO: 132)


165
LYNSPSMWENL
1355.7
−
(SEQ ID NO: 133)


167
LYNSPSFPENL
1282.5
−
(SEQ ID NO: 134)


168
LYNSPSFFENL
1332.5
−
(SEQ ID NO: 135)


169
LYNSPSFYENL
1348.5
−
(SEQ ID NO: 136)


170
LYNSPSFWENL
1371.6
−
(SEQ ID NO: 137)


172
LYNSPSYPENL
1298.5
−
(SEQ ID NO: 138)


173
LYNSPSYFENL
1348.5
−
(SEQ ID NO: 139)


174
LYNSPSYYENL
1364.5
−
(SEQ ID NO: 140)


175
LYNSPSYWENL
1387.6
−
(SEQ ID NO: 141)


177
LYNSPSDPENL
1250.4
−
(SEQ ID NO: 142)


178
LYNSPSDFENL
1300.4
−
(SEQ ID NO: 143)


179
LYNSPSDYENL
1316.4
−
(SEQ ID NO: 144)


180
LYNSPSDWENL
1339.5
−
(SEQ ID NO: 145)


182
LYNSPSEPENL
1264.4
−
(SEQ ID NO: 146)


183
LYNSFSEFENL
1314.4
−
(SEQ ID NO: 147)


184
LYNSPSEYENL
1330.4
−
(SEQ ID NO: 148)


185
LYNSPSEWENL
1353.5
−
(SEQ ID NO: 149)


187
LYNSPSNPENL
1249.5
−
(SEQ ID NO: 150)


188
LYNSPSNFENL
1299.5
−
(SEQ ID NO: 151)


189
LYNSPSNYENL
1315.5
−
(SEQ ID NO: 152)


190
LYNSPSNWENL
1338.6
−
(SEQ ID NO: 153)


192
LYNSPSQPENL
1263.5
−
(SEQ ID NO: 154)


193
LYNSPSQFENL
1313.5
−
(SEQ ID NO: 155)


194
LYNSPSQYENL
1329.5
−
(SEQ ID NO: 156)


195
LYNSPSQWENL
1352.6
−
(SEQ ID NO: 157)


197
LYNSPSHPENL
1272.5
−
(SEQ ID NO: 158)


198
LYNSPSHFENL
1322.5
−
(SEQ ID NO: 159)


199
LYNSPSHYENL
1338.5
−
(SEQ ID NO: 160)


200
LYNSFSHWENL
1361.6
−
(SEQ ID NO: 161)


202
LYGSPSMPENL
1209.5
−
(SEQ ID NO: 162)


203
LYGSPSMFENL
1259.5
−
(SEQ ID NO: 163)


204
LYGSPSMYENL
1275.5
−
(SEQ ID NO: 164)


205
LYGSPSMWENL
1298.6
−
(SEQ ID NO: 165)


207
LYGSPSFPENL
1225.4
−
(SEQ ID NO: 166)


208
LYGSPSFFENL
1275.4
−
(SEQ ID NO: 167)


209
LYGSPSFYENL
1291.4
−
(SEQ ID NO: 168)


210
LYGSPSFWENL
1314.5
−
(SEQ ID NO: 169)


212
LYGSPSYPENL
1241.4
−
(SEQ ID NO: 170)


213
LYGSPSYFENL
1291.4
−
(SEQ ID NO: 171)


214
LYGSPSYYENL
1307.4
−
(SEQ ID NO: 172)


215
LYGSPSYWENL
1330.5
−
(SEQ ID NO: 173)


217
LYGSPSDPENL
1193.3
−
(SEQ ID NO: 174)


218
LYGSPSDFENL
1243.3
−
(SEQ ID NO: 175)


219
LYGSPSDYENL
1259.3
−
(SEQ ID NO: 176)


220
LYGSPSDWENL
1282.4
−
(SEQ ID NO: 177)


222
LYGSPSEPENL
1207.3
−
(SEQ ID NO: 178)


223
LYGSPSEFENL
1257.3
−
(SEQ ID NO: 179)


224
LYGSPSEYENL
1273.3
−
(SEQ ID NO: 180)


225
LYGSPSEWENL
1296.4
−
(SEQ ID NO: 181)


227
LYGSPSNPENL
1192.4
−
(SEQ ID NO: 182)


228
LYGSPSNFENL
1242.4
−
(SEQ ID NO: 183)


229
LYGSPSNYENL
1258.4
−
(SEQ ID NO: 184)


230
LYGSPSNWENL
1281.5
−
(SEQ ID NO: 185)


232
LYGSPSQPENL
1206.4
−
(SEQ ID NO: 186)


233
LYGSPSQFENL
1256.4
−
(SEQ ID NO: 187)


234
LYGSPSQYENL
1272.4
−
(SEQ ID NO: 188)


235
LYGSPSQWENL
1295.5
−
(SEQ ID NO: 189)


237
LYGSPSHPENL
1215.4
−
(SEQ ID NO: 190)


238
LYGSPSHFENL
1265.4
−
(SEQ ID NO: 191)


239
LYGSPSHYENL
1281.4
−
(SEQ ID NO: 192)


240
LYGSPSHWENL
1304.5
−
(SEQ ID NO: 193)


242
LYASPSMPENL
1223.5
−
(SEQ ID NO: 194)


243
LYASPSMFENL
1273.5
−
(SEQ ID NO: 195)


244
LYASPSMYENL
1289.5
−
(SEQ ID NO: 196)


245
LYASPSMWENL
1312.6
−
(SEQ ID NO: 197)


247
LYASPSFPENL
1239.4
−
(SEQ ID NO: 198)


248
LYASPSFFENL
1289.4
−
(SEQ ID NO: 199)


249
LYASPSFYENL
1305.4
−
(SEQ ID NO: 200)


250
LYASPSFWENL
1328.5
−
(SEQ ID NO: 201)


252
LYASPSYPENL
1255.4
−
(SEQ ID NO: 202)


253
LYASPSYFENL
1305.4
−
(SEQ ID NO: 203)


254
LYASPSYYENL
1321.4
−
(SEQ ID NO: 204)


255
LYASPSYWENL
1344.5
−
(SEQ ID NO: 205)


257
LYASPSDPENL
1207.3
−
(SEQ ID NO: 206)


258
LYASPSDFENL
1257.3
−
(SEQ ID NO: 207)


259
LYASPSDYENL
1273.3
−
(SEQ ID NO: 208)


260
LYASPSDWENL
1296.4
−
(SEQ ID NO: 209)


262
LYASPSEPENL
1221.3
−
(SEQ ID NO: 210)


263
LYASPSEFENL
1271.3
−
(SEQ ID NO: 211)


264
LYASPSEYENL
1287.3
−
(SEQ ID NO: 212)


265
LYASPSEWENL
1310.4
−
(SEQ ID NO: 213)


267
LYASPSNPENL
1206.4
−
(SEQ ID NO: 214)


268
LYASPSNFENL
1256.4
−
(SEQ ID NO: 215)


269
LYASPSNYENL
1272.4
−
(SEQ ID NO: 216)


270
LYASPSNWENL
1295.5
−
(SEQ ID NO: 217)


272
LYASPSQPENL
1220.4
−
(SEQ ID NO: 218)


273
LYASPSQFENL
1270.4
−
(SEQ ID NO: 219)


274
LYASPSQYENL
1286.4
−
(SEQ ID NO: 220)


275
LYASPSQWENL
1309.5
−
(SEQ ID NO: 221)


277
LYASPSHPENL
1229.4
−
(SEQ ID NO: 222)


278
LYASPSHFENL
1279.4
−
(SEQ ID NO: 223)


279
LYASPSHYENL
1295.4
−
(SEQ ID NO: 224)


280
LYASPSHWENL
1318.5
−
(SEQ ID NO: 225)


282
LFRSPSMPENL
1292.6
−
(SEQ ID NO: 226)


283
LFRSPSMFENL
1342.6
−
(SEQ ID NO: 227)


284
LFRSPSMYENL
1358.6
−
(SEQ ID NO: 228)


285
LFRSPSMWENL
1381.7
−
(SEQ ID NO: 229)


287
LFRSPSFPENL
1308.5
−
(SEQ ID NO: 230)


288
LFRSPSFFENL
1358.5
−
(SEQ ID NO: 231)


289
LFRSPSFYENL
1374.5
−
(SEQ ID NO: 232)


290
LFRSPSFWENL
1397.6
−
(SEQ ID NO: 233)


292
LFRSPSYPENL
1324.5
−
(SEQ ID NO: 234)


293
LFRSPSYFENL
1374.5
−
(SEQ ID NO: 235)


294
LFRSPSYYENL
1390.5
−
(SEQ ID NO: 236)


295
LFRSPSYWENL
1413.6
−
(SEQ ID NO: 237)


297
LFRSPSDPENL
1276.4
−
(SEQ ID NO: 238)


298
LFRSPSDFENL
1326.4
−
(SEQ ID NO: 239)


299
LFRSPSDYENL
1342.4
−
(SEQ ID NO: 240)


300
LFRSPSDWENL
1365.5
−
(SEQ ID NO: 241)


302
LFRSPSEPENL
1290.4
−
(SEQ ID NO: 242)


303
LFRSPSEFENL
1340.4
−
(SEQ ID NO: 243)


304
LFRSPSEYENL
1356.4
−
(SEQ ID NO: 244)


305
LFRSPSEWENL
1379.5
−
(SEQ ID NO: 245)


307
LFRSPSNPENL
1275.5
−
(SEQ ID NO: 246)


308
LFRSPSNFENL
1325.5
−
(SEQ ID NO: 247)


309
LFRSPSNYENL
1341.5
−
(SEQ ID NO: 248)


310
LFRSPSNWENL
1364.6
−
(SEQ ID NO: 249)


312
LFRSPSQPENL
1289.5
−
(SEQ ID NO: 250)


313
LFRSPSQFENL
1339.5
−
(SEQ ID NO: 251)


314
LFRSPSQYENL
1355.5
−
(SEQ ID NO: 252)


315
LFRSPSQWENL
1378.6
−
(SEQ ID NO: 253)


317
LFRSPSHPENL
1298.5
−
(SEQ ID NO: 254)


318
LFRSPSHFENL
1348.5
−
(SEQ ID NO: 255)


319
LFRSPSHYENL
1364.5
−
(SEQ ID NO: 256)


320
LFRSPSHWENL
1387.6
−
(SEQ ID NO: 257)


322
LFSSPSMPENL
1224.3
−
(SEQ ID NO: 258)


323
LFSSPSMFENL
1274.3
−
(SEQ ID NO: 259)


324
LFSSPSMYENL
1290.3
−
(SEQ ID NO: 260)


325
LFSSPSMWENL
1313.4
−
(SEQ ID NO: 261)


327
LFSSPSFPENL
1240.2
−
(SEQ ID NO: 262)


328
LFSSPSFFENL
1290.2
−
(SEQ ID NO: 263)


329
LFSSPSFYENL
1306.2
−
(SEQ ID NO: 264)


330
LFSSPSFWENL
1329.3
−
(SEQ ID NO: 265)


332
LFSSPSYPENL
1256.2
−
(SEQ ID NO: 266)


333
LFSSPSYFENL
1306.2
−
(SEQ ID NO: 267)


334
LFSSPSYYENL
1322.2
−
(SEQ ID NO: 268)


335
LFSSPSYWENL
1345.3
−
(SEQ ID NO: 269)


337
LFSSPSDPENL
1208.1
−
(SEQ ID NO: 270)


338
LFSSPSDFENL
1258.1
−
(SEQ ID NO: 271)


339
LFSSPSDYENL
1274.1
−
(SEQ ID NO: 272)


340
LFSSPSDWENL
1297.2
−
(SEQ ID NO: 273)


342
LFSSPSEPENL
1222.1
−
(SEQ ID NO: 274)


343
LFSSPSEFENL
1272.1
−
(SEQ ID NO: 275)


344
LFSSPSEYENL
1288.1
−
(SEQ ID NO: 276)


345
LFSSPSEWENL
1311.2
−
(SEQ ID NO: 277)


347
LFSSPSNPENL
1207.2
−
(SEQ ID NO: 278)


348
LFSSPSNFENL
1257.2
−
(SEQ ID NO: 279)


349
LFSSPSNYENL
1273.2
−
(SEQ ID NO: 280)


350
LFSSPSNWENL
1296.3
−
(SEQ ID NO: 281)


352
LFSSPSQPENL
1221.2
−
(SEQ ID NO: 282)


353
LFSSPSQFENL
1271.2
−
(SEQ ID NO: 283)


354
LFSSPSQYENL
1287.2
−
(SEQ ID NO: 284)


355
LFSSPSQWENL
1310.3
−
(SEQ ID NO: 285)


357
LFSSPSHPENL
1230.2
−
(SEQ ID NO: 286)


358
LFSSPSHFENL
1280.2
−
(SEQ ID NO: 287)


359
LFSSPSHYENL
1296.2
−
(SEQ ID NO: 288)


360
LFSSPSHWENL
1319.3
−
(SEQ ID NO: 289)


362
LFTSPSMPENL
1237.5
−
(SEQ ID NO: 290)


363
LFTSPSMFENL
1287.5
−
(SEQ ID NO: 291)


364
LFTSPSMYENL
1303.5
−
(SEQ ID NO: 292)


365
LFTSPSMWENL
1326.6
−
(SEQ ID NO: 293)


367
LFTSPSFPENL
1253.4
−
(SEQ ID NO: 294)


368
LFTSPSFFENL
1303.4
−
(SEQ ID NO: 295)


369
LFTSPSFYENL
1319.4
−
(SEQ ID NO: 296)


370
LFTSPSFWENL
1342.5
−
(SEQ ID NO: 297)


372
LFTSPSYPENL
1269.4
+
(SEQ ID NO: 298)


373
LFTSPSYFENL
1319.4
+
(SEQ ID NO: 299)


374
LFTSPSYYENL
1335.4
+++
(SEQ ID NO: 300)


375
LFTSPSYWENL
1358.5
+
(SEQ ID NO: 301)


377
LFTSPSDPENL
1221.3
−
(SEQ ID NO: 302)


378
LFTSPSDFENL
1271.3
−
(SEQ ID NO: 303)


379
LFTSPSDYENL
1287.3
−
(SEQ ID NO: 304)


380
LFTSPSDWENL
1310.4
−
(SEQ ID NO: 305)


382
LFTSPSEPENL
1235.3
−
(SEQ ID NO: 306)


383
LFTSPSEFENL
1285.3
−
(SEQ ID NO: 307)


384
LFTSPSEYENL
1301.3
−
(SEQ ID NO: 308)


385
LFTSPSEWENL
1324.4
−
(SEQ ID NO: 309)


387
LFTSPSNPENL
1220.4
−
(SEQ ID NO: 310)


388
LFTSPSNFENL
1270.4
−
(SEQ ID NO: 311)


389
LFTSPSNYENL
1286.4
−
(SEQ ID NO: 312)


390
LFTSPSNWENL
1309.5
−
(SEQ ID NO: 313)


392
LFTSPSQPENL
1234.4
−
(SEQ ID NO: 314)


393
LFTSPSQFENL
1284.4
−
(SEQ ID NO: 315)


394
LFTSPSQYENL
1300.4
−
(SEQ ID NO: 316)


395
LFTSPSQWENL
1323.5
−
(SEQ ID NO: 317)


397
LFTSPSHPENL
1243.4
−
(SEQ ID NO: 318)


398
LFTSPSHFENL
1293.4
−
(SEQ ID NO: 319)


399
LFTSPSHYENL
1309.4
−
(SEQ ID NO: 320)


400
LFTSPSHWENL
1332.5
−
(SEQ ID NO: 321)


402
LFHSPSMPENL
1273.6
−
(SEQ ID NO: 322)


403
LFHSPSMFENL
1323.6
−
(SEQ ID NO: 323)


404
LFHSPSMYENL
1339.6
−
(SEQ ID NO: 324)


405
LFHSPSMWENL
1362.7
−
(SEQ ID NO: 325)


407
LFHSPSFPENL
1289.5
−
(SEQ ID NO: 326)


408
LFHSPSFFENL
1339.5
−
(SEQ ID NO: 327)


409
LFHSPSFYENL
1355.5
−
(SEQ ID NO: 328)


410
LFHSPSFWENL
1378.6
−
(SEQ ID NO: 329)


412
LFHSPSYPENL
1305.5
−
(SEQ ID NO: 330)


413
LFHSPSYFENL
1355.5
−
(SEQ ID NO: 331)


414
LFHSPSYYENL
1371.5
−
(SEQ ID NO: 332)


415
LFHSPSYWENL
1394.6
−
(SEQ ID NO: 333)


417
LFHSPSDPENL
1257.4
−
(SEQ ID NO: 334)


418
LFHSPSDFENL
1307.4
−
(SEQ ID NO: 335)


419
LFHSPSDYENL
1323.4
−
(SEQ ID NO: 336)


420
LFHSPSDWENL
1346.5
−
(SEQ ID NO: 337)


422
LFHSPSEPENL
1271.4
−
(SEQ ID NO: 338)


423
LFHSPSEFENL
1321.4
−
(SEQ ID NO: 339)


424
LFHSPSEYENL
1337.4
−
(SEQ ID NO: 340)


425
LFHSPSEWENL
1360.5
−
(SEQ ID NO: 341)


427
LFHSPSNPENL
1256.5
−
(SEQ ID NO: 342)


428
LFHSPSNFENL
1306.5
−
(SEQ ID NO: 343)


429
LFHSPSNYENL
1322.5
−
(SEQ ID NO: 344)


430
LFHSPSNWENL
1345.6
−
(SEQ ID NO: 345)


432
LFHSPSQPENL
1270.5
−
(SEQ ID NO: 346)


433
LFHSPSQFENL
1320.5
−
(SEQ ID NO: 347)


434
LFHSPSQYENL
1336.5
−
(SEQ ID NO: 348)


435
LFHSPSQWENL
1359.6
−
(SEQ ID NO: 349)


437
LFHSPSHPENL
1279.5
−
(SEQ ID NO: 350)


438
LFHSPSHFENL
1329.5
−
(SEQ ID NO: 351)


439
LFHSPSHYENL
1345.5
−
(SEQ ID NO: 352)


440
LFHSPSHWENL
1368.6
−
(SEQ ID NO: 353)


442
LFNSPSMPENL
1250.6
−
(SEQ ID NO: 354)


443
LFNSPSMFENL
1300.6
−
(SEQ ID NO: 355)


444
LFNSPSMYENL
1316.6
−
(SEQ ID NO: 356)


445
LFNSPSMWENL
1339.7
−
(SEQ ID NO: 357)


447
LFNSPSFPENL
1266.5
−
(SEQ ID NO: 358)


448
LFNSPSFFENL
1316.5
−
(SEQ ID NO: 359)


449
LFNSPSFYENL
1332.5
−
(SEQ ID NO: 360)


450
LFNSPSFWENL
1355.6
−
(SEQ ID NO: 361)


452
LFNSPSYPENL
1282.5
−
(SEQ ID NO: 362)


453
LFNSPSYFENL
1332.5
−
(SEQ ID NO: 363)


454
LFNSPSYYENL
1348.5
−
(SEQ ID NO: 364)


455
LFNSPSYWENL
1371.6
−
(SEQ ID NO: 365)


457
LFNSPSDPENL
1234.4
−
(SEQ ID NO: 366)


458
LFNSPSDFENL
1284.4
−
(SEQ ID NO: 367)


459
LFNSPSDYENL
1300.4
−
(SEQ ID NO: 368)


460
LFNSPSDWENL
1323.5
−
(SEQ ID NO: 369)


462
LFNSPSEPENL
1248.4
−
(SEQ ID NO: 370)


463
LFNSPSEFENL
1298.4
−
(SEQ ID NO: 371)


464
LFNSPSEYENL
1314.4
−
(SEQ ID NO: 372)


465
LFNSPSEWENL
1337.5
−
(SEQ ID NO: 373)


467
LFNSPSNPENL
1233.5
−
(SEQ ID NO: 374)


468
LFNSPSNFENL
1283.5
−
(SEQ ID NO: 375)


469
LFNSPSNYENL
1299.5
−
(SEQ ID NO: 376)


470
LFNSPSNWENL
1322.6
−
(SEQ ID NO: 377)


472
LFNSPSQPENL
1247.5
−
(SEQ ID NO: 378)


473
LFNSPSQFENL
1297.5
−
(SEQ ID NO: 379)


474
LFNSPSQYENL
1313.5
−
(SEQ ID NO: 380)


475
LFNSPSQWENL
1336.6
−
(SEQ ID NO: 381)


477
LFNSPSHPENL
1256.5
−
(SEQ ID NO: 382)


478
LFNSPSHFENL
1306.5
−
(SEQ ID NO: 383)


479
LFNSPSHYENL
1322.5
−
(SEQ ID NO: 384)


480
LFNSPSHWENL
1345.6
−
(SEQ ID NO: 385)


482
LFGSPSMPENL
1193.5
−
(SEQ ID NO: 386)


483
LFGSPSMFENL
1243.5
−
(SEQ ID NO: 387)


484
LFGSPSMYENL
1259.5
−
(SEQ ID NO: 388)


485
LFGSPSMWENL
1282.6
−
(SEQ ID NO: 389)


487
LFGSPSFPENL
1209.4
−
(SEQ ID NO: 390)


488
LFGSPSFFENL
1259.4
−
(SEQ ID NO: 391)


489
LFGSPSFYENL
1275.4
−
(SEQ ID NO: 392)


490
LFGSPSFWENL
1298.5
−
(SEQ ID NO: 393)


492
LFGSPSYPENL
1225.4
−
(SEQ ID NO: 394)


493
LFGSPSYFENL
1275.4
−
(SEQ ID NO: 395)


494
LFGSPSYYENL
1291.4
−
(SEQ ID NO: 396)


495
LFGSPSYWENL
1314.5
−
(SEQ ID NO: 397)


497
LFGSPSDPENL
1177.3
−
(SEQ ID NO: 398)


498
LFGSPSDFENL
1227.3
−
(SEQ ID NO: 399)


499
LFGSPSDYENL
1243.3
−
(SEQ ID NO: 400)


500
LFGSPSDWENL
1266.4
−
(SEQ ID NO: 401)


502
LFGSPSEPENL
1191.3
−
(SEQ ID NO: 402)


503
LFGSPSEFENL
1241.3
−
(SEQ ID NO: 403)


504
LFGSPSEYENL
1257.3
−
(SEQ ID NO: 404)


505
LFGSPSEWENL
1280.4
−
(SEQ ID NO: 405)


507
LFGSPSNPENL
1176.4
−
(SEQ ID NO: 406)


508
LFGSPSNFENL
1226.4
−
(SEQ ID NO: 407)


509
LFGSPSNYENL
1242.4
−
(SEQ ID NO: 408)


510
LFGSPSNWENL
1265.5
−
(SEQ ID NO: 409)


512
LFGSPSQPENL
1190.4
−
(SEQ ID NO: 410)


513
LFGSPSQFENL
1240.4
−
(SEQ ID NO: 411)


514
LFGSPSQYENL
1256.4
−
(SEQ ID NO: 412)


515
LFGSPSQWENL
1279.5
−
(SEQ ID NO: 413)


517
LFGSPSHPENL
1199.4
−
(SEQ ID NO: 414)


518
LFGSPSHFENL
1249.4
−
(SEQ ID NO: 415)


519
LFGSPSHYENL
1265.4
−
(SEQ ID NO: 416)


520
LFGSPSHWENL
1288.5
−
(SEQ ID NO: 417)


522
LFASPSMPENL
1207.5
−
(SEQ ID NO: 418)


523
LFASPSMFENL
1257.5
−
(SEQ ID NO: 419)


524
LFASPSMYENL
1273.5
−
(SEQ ID NO: 420)


525
LFASPSMWENL
1296.6
−
(SEQ ID NO: 421)


527
LFASPSFPENL
1223.4
−
(SEQ ID NO: 422)


528
LFASPSFFENL
1273.4
−
(SEQ ID NO: 423)


529
LFASPSFYENL
1289.4
−
(SEQ ID NO: 424)


530
LFASPSFWENL
1312.5
−
(SEQ ID NO: 425)


532
LFASPSYPENL
1239.4
−
(SEQ ID NO: 426)


533
LFASPSYFENL
1289.4
−
(SEQ ID NO: 427)


534
LFASPSYYENL
1305.4
−
(SEQ ID NO: 428)


535
LFASPSYWENL
1328.5
−
(SEQ ID NO: 429)


537
LFASPSDPENL
1191.3
−
(SEQ ID NO: 430)


538
LFASPSDFENL
1241.3
−
(SEQ ID NO: 431)


539
LFASPSDYENL
1257.3
−
(SEQ ID NO: 432)


540
LFASPSDWENL
1280.4
−
(SEQ ID NO: 433)


542
LFASPSEPENL
1205.3
−
(SEQ ID NO: 434)


543
LFASPSEFENL
1255.3
−
(SEQ ID NO: 435)


544
LFASPSEYENL
1271.3
−
(SEQ ID NO: 436)


545
LFASPSEWENL
1294.4
−
(SEQ ID NO: 437)


547
LFASPSNPENL
1190.4
−
(SEQ ID NO: 438)


548
LFASPSNFENL
1240.4
−
(SEQ ID NO: 439)


549
LFASPSNYENL
1256.4
−
(SEQ ID NO: 440)


550
LFASPSNWENL
1279.5
−
(SEQ ID NO: 441)


552
LFASPSQPENL
1204.4
−
(SEQ ID NO: 442)


553
LFASPSQFENL
1254.4
−
(SEQ ID NO: 443)


554
LFASPSQYENL
1270.4
−
(SEQ ID NO: 444)


555
LFASPSQWENL
1293.5
−
(SEQ ID NO: 445)


557
LFASPSHPENL
1213.4
−
(SEQ ID NO: 446)


558
LFASPSHFENL
1263.4
−
(SEQ ID NO: 447)


559
LFASPSHYENL
1279.4
−
(SEQ ID NO: 448)


560
LFASPSHWENL
1302.5
−
(SEQ ID NO: 449)


562
FYRSPSMPENL
1342.6
−
(SEQ ID NO: 450)


563
FYRSPSMFENL
1392.6
−
(SEQ ID NO: 451)


564
FYRSPSMYENL
1408.6
−
(SEQ ID NO: 452)


565
FYRSPSMWENL
1431.7
−
(SEQ ID NO: 453)


567
FYRSPSFPENL
1358.5
−
(SEQ ID NO: 454)


568
FYRSPSFFENL
1408.5
−
(SEQ ID NO: 455)


569
FYRSPSFYENL
1424.5
−
(SEQ ID NO: 456)


570
FYRSPSFWENL
1447.6
−
(SEQ ID NO: 457)


572
FYRSPSYPENL
1374.5
−
(SEQ ID NO: 458)


573
FYRSPSYFENL
1424.5
−
(SEQ ID NO: 459)


574
FYRSPSYYENL
1440.5
−
(SEQ ID NO: 460)


575
FYRSPSYWENL
1463.6
−
(SEQ ID NO: 461)


577
FYRSPSDPENL
1326.4
−
(SEQ ID NO: 462)


578
FYRSPSDFENL
1376.4
−
(SEQ ID NO: 463)


579
FYRSPSDYENL
1392.4
−
(SEQ ID NO: 464)


580
FYRSPSDWENL
1415.5
−
(SEQ ID NO: 465)


582
FYRSPSEPENL
1340.4
−
(SEQ ID NO: 466)


583
FYRSPSEFENL
1390.4
−
(SEQ ID NO: 467)


584
FYRSPSEYENL
1406.4
−
(SEQ ID NO: 468)


585
FYRSPSEWENL
1429.5
−
(SEQ ID NO: 469)


587
FYRSPSNPENL
1325.5
−
(SEQ ID NO: 470)


588
FYRSPSNFENL
1375.5
−
(SEQ ID NO: 471)


589
FYRSPSNYENL
1391.5
−
(SEQ ID NO: 472)


590
FYRSPSNWENL
1414.6
−
(SEQ ID NO: 473)


592
FYRSPSQPENL
1339.5
−
(SEQ ID NO: 474)


593
FYRSPSQFENL
1389.5
−
(SEQ ID NO: 475)


594
FYRSPSQYENL
1405.5
−
(SEQ ID NO: 476)


595
FYRSPSQWENL
1428.6
−
(SEQ ID NO: 477)


597
FYRSPSHPENL
1348.5
−
(SEQ ID NO: 478)


598
FYRSPSHFENL
1398.5
−
(SEQ ID NO: 479)


599
FYRSPSHYENL
1414.5
−
(SEQ ID NO: 480)


600
FYRSPSHWENL
1437.6
−
(SEQ ID NO: 481)


602
FYSSPSMPENL
1274.3
−
(SEQ ID NO: 482)


603
FYSSPSMFENL
1324.3
−
(SEQ ID NO: 483)


604
FYSSPSMYENL
1340.3
−
(SEQ ID NO: 484)


605
FYSSPSMWENL
1363.4
−
(SEQ ID NO: 485)


607
FYSSPSFPENL
1290.2
−
(SEQ ID NO: 486)


608
FYSSPSFFENL
1340.2
−
(SEQ ID NO: 487)


609
FYSSPSFYENL
1356.2
−
(SEQ ID NO: 488)


610
FYSSPSFWENL
1379.3
−
(SEQ ID NO: 489)


612
FYSSPSYPENL
1306.2
−
(SEQ ID NO: 490)


613
FYSSPSYFENL
1356.2
−
(SEQ ID NO: 491)


614
FYSSPSYYENL
1372.2
−
(SEQ ID NO: 492)


615
FYSSPSYWENL
1395.3
−
(SEQ ID NO: 493)


617
FYSSPSDPENL
1258.1
−
(SEQ ID NO: 494)


618
FYSSPSDFENL
1308.1
−
(SEQ ID NO: 495)


619
FYSSPSDYENL
1324.1
−
(SEQ ID NO: 496)


620
FYSSPSDWENL
1347.2
−
(SEQ ID NO: 497)


622
FYSSPSEPENL
1272.1
−
(SEQ ID NO: 498)


623
FYSSPSEFENL
1322.1
−
(SEQ ID NO: 499)


624
FYSSPSEYENL
1338.1
−
(SEQ ID NO: 500)


625
FYSSPSEWENL
1361.2
−
(SEQ ID NO: 501)


627
FYSSPSNPENL
1257.2
−
(SEQ ID NO: 502)


628
FYSSPSNFENL
1307.2
−
(SEQ ID NO: 503)


629
FYSSPSNYENL
1323.2
−
(SEQ ID NO: 504)


630
FYSSPSNWENL
1346.3
−
(SEQ ID NO: 505)


632
FYSSPSQPENL
1271.2
−
(SEQ ID NO: 506)


633
FYSSPSQFENL
1321.2
−
(SEQ ID NO: 507)


634
FYSSPSQYENL
1337.2
−
(SEQ ID NO: 508)


635
FYSSPSQWENL
1360.3
−
(SEQ ID NO: 509)


637
FYSSPSHPENL
1280.2
+
(SEQ ID NO: 510)


638
FYSSPSHFENL
1330.2
+
(SEQ ID NO: 511)


639
FYSSPSHYENL
1346.2
+
(SEQ ID NO: 512)


640
FYSSPSHWENL
1369.3
+
(SEQ ID NO: 513)


642
FYTSPSMPENL
1287.5
+
(SEQ ID NO: 514)


643
FYTSPSMFENL
1337.5
+
(SEQ ID NO: 515)


644
FYTSPSMYENL
1353.5
+
(SEQ ID NO: 516)


645
FYTSPSMWENL
1376.6
+
(SEQ ID NO: 517)


647
FYTSPSFPENL
1303.4
+
(SEQ ID No: 518)


648
FYTSPSFFENL
1353.4
+
(SEQ ID NO: 519)


649
FYTSPSFYENL
1369.4
+
(SEQ ID NO: 520)


650
FYTSPSFWENL
1392.5
+
(SEQ ID NO: 521)


652
FYTSPSYPENL
1319.4
+
(SEQ ID NO: 522)


653
FYTSPSYFENL
1369.4
+
(SEQ ID NO: 523)


654
FYTSPSYYENL
1385.4
+
(SEQ ID NO: 524)


655
FYTSPSYWENL
1408.5
+
(SEQ ID NO: 525)


657
FYTSPSDPENL
1271.3
−
(SEQ ID NO: 526)


658
FYTSPSDFENL
1321.3
−
(SEQ ID NO: 527)


659
FYTSPSDYENL
1337.3
−
(SEQ ID NO: 528)


660
FYTSPSDWENL
1360.4
−
(SEQ ID NO: 529)


662
FYTSPSEPENL
1285.3
−
(SEQ ID NO: 530)


663
FYTSPSEFENL
1335.3
−
(SEQ ID NO: 531)


664
FYTSPSEYENL
1351.3
−
(SEQ ID NO: 532)


665
FYTSPSEWENL
1374.4
−
(SEQ ID NO: 533)


667
FYTSPSNPENL
1270.4
−
(SEQ ID NO: 534)


668
FYTSPSNFENL
1320.4
−
(SEQ ID NO: 535)


669
FYTSPSNYENL
1336.4
−
(SEQ ID NO: 536)


670
FYTSPSNWENL
1359.5
−
(SEQ ID NO: 537)


672
FYTSPSQPENL
1284.4
−
(SEQ ID NO: 538)


673
FYTSPSQFENL
1334.4
−
(SEQ ID NO: 539)


674
FYTSPSQYENL
1350.4
−
(SEQ ID NO: 540)


675
FYTSPSQWENL
1373.5
−
(SEQ ID NO: 541)


677
FYTSPSHPENL
1293.4
−
(SEQ ID NO: 542)


678
FYTSPSHFENL
1343.4
−
(SEQ ID NO: 543)


679
FYTSPSHYENL
1359.4
−
(SEQ ID NO: 544)


680
FYTSPSHWENL
1382.5
−
(SEQ ID NO: 545)


682
FYHSPSMPENL
1323.6
−
(SEQ ID NO: 546)


683
FYHSPSMFENL
1373.6
−
(SEQ ID NO: 547)


684
FYHSPSMYENL
1389.6
−
(SEQ ID NO: 548)


685
FYHSPSMWENL
1412.7
−
(SEQ ID NO: 549)


687
FYHSPSFPENL
1339.5
−
(SEQ ID NO: 550)


688
FYHSPSFFENL
1389.5
−
(SEQ ID NO: 551)


689
FYHSPSFYENL
1405.5
−
(SEQ ID NO: 552)


690
FYHSPSFWENL
1428.6
−
(SEQ ID NO: 553)


692
FYHSPSYPENL
1355.5
−
(SEQ ID NO: 554)


693
FYHSPSYFENL
1405.5
−
(SEQ ID NO: 555)


694
FYHSPSYYENL
1421.5
−
(SEQ ID NO: 556)


695
FYHSPSYWENL
1444.6
−
(SEQ ID NO: 557)


697
FYHSPSDPENL
1307.4
−
(SEQ ID NO: 558)


698
FYHSPSDFENL
1357.4
−
(SEQ ID NO: 559)


699
FYHSPSDYENL
1373.4
−
(SEQ ID NO: 560)


700
FYHSPSDWENL
1396.5
−
(SEQ ID NO: 561)


702
FYHSPSEPENL
1321.4
−
(SEQ ID NO: 562)


703
FYHSPSEFENL
1371.4
−
(SEQ ID NO: 563)


704
FYHSPSEYENL
1387.4
−
(SEQ ID NO: 564)


705
FYHSPSEWENL
1410.5
−
(SEQ ID NO: 565)


707
FYHSPSNPENL
1306.5
−
(SEQ ID NO: 566)


708
FYHSPSNFENL
1356.5
−
(SEQ ID NO: 567)


709
FYHSPSNYENL
1372.5
−
(SEQ ID NO: 568)


710
FYHSPSNWENL
1395.6
−
(SEQ ID NO: 569)


712
FYHSPSQPENL
1320.5
−
(SEQ ID NO: 570)


713
FYHSPSQFENL
1370.5
−
(SEQ ID NO: 571)


714
FYHSPSQYENL
1386.5
−
(SEQ ID NO: 572)


715
FYHSPSQWENL
1409.6
−
(SEQ ID NO: 573)


717
FYHSPSHPENL
1329.5
−
(SEQ ID NO: 574)


718
FYHSPSHFENL
1379.5
−
(SEQ ID NO: 575)


719
FYHSPSHYENL
1395.5
−
(SEQ ID NO: 576)


720
FYHSPSHWENL
1418.6
−
(SEQ ID NO: 577)


722
FYNSPSMPENL
1300.6
−
(SEQ ID NO: 578)


723
FYNSPSMFENL
1350.6
−
(SEQ ID NO: 579)


724
FYNSPSMYENL
1366.6
−
(SEQ ID NO: 580)


725
FYNSPSMWENL
1389.7
−
(SEQ ID NO: 581)


727
FYNSPSFPENL
1316.5
−
(SEQ ID NO: 582)


728
FYNSPSFFENL
1366.5
−
(SEQ ID NO: 583)


729
FYNSPSFYENL
1382.5
−
(SEQ ID NO: 584)


730
FYNSPSFWENL
1405.6
−
(SEQ ID NO: 585)


732
FYNSPSYPENL
1332.5
−
(SEQ ID NO: 586)


733
FYNSPSYFENL
1382.5
−
(SEQ ID NO: 587)


734
FYNSPSYYENL
1398.5
−
(SEQ ID NO: 588)


735
FYNSPSYWENL
1421.6
−
(SEQ ID NO: 589)


737
FYNSPSDPENL
1284.4
−
(SEQ ID NO: 590)


738
FYNSPSDFENL
1334.4
−
(SEQ ID NO: 591)


739
FYNSPSDYENL
1350.4
−
(SEQ ID NO: 592)


740
FYNSPSDWENL
1373.5
−
(SEQ ID NO: 593)


742
FYNSPSEPENL
1298.4
−
(SEQ ID NO: 594)


743
FYNSPSEFENL
1348.4
−
(SEQ ID NO: 595)


744
FYNSPSEYENL
1364.4
−
(SEQ ID NO: 596)


745
FYNSPSEWENL
1387.5
−
(SEQ ID NO: 597)


747
FYNSPSNPENL
1283.5
−
(SEQ ID NO: 598)


748
FYNSPSNFENL
1333.5
−
(SEQ ID NO: 599)


749
FYNSPSNYENL
1349.5
−
(SEQ ID NO: 600)


750
FYNSPSNWENL
1372.6
−
(SEQ ID NO: 601)


752
FYNSPSQPENL
1297.5
−
(SEQ ID NO: 602)


753
FYNSPSQFENL
1347.5
−
(SEQ ID NO: 603)


754
FYNSPSQYENL
1363.5
−
(SEQ ID NO: 604)


755
FYNSPSQWENL
1386.6
−
(SEQ ID NO: 605)


757
FYNSPSHPENL
1306.5
−
(SEQ ID NO: 606)


758
FYNSPSHFENL
1356.5
−
(SEQ ID NO: 607)


759
FYNSPSHYENL
1372.5
−
(SEQ ID NO: 608)


760
FYNSPSHWENL
1395.6
−
(SEQ ID NO: 609)


762
FYGSPSMPENL
1243.5
−
(SEQ ID NO: 610)


763
FYGSPSMFENL
1293.5
−
(SEQ ID NO: 611)


764
FYGSPSMYENL
1309.5
−
(SEQ ID NO: 612)


765
FYGSPSMWENL
1332.6
−
(SEQ ID NO: 613)


767
FYGSPSFPENL
1259.4
−
(SEQ ID NO: 614)


768
FYGSPSFFENL
1309.4
−
(SEQ ID NO: 615)


769
FYGSPSFYENL
1325.4
−
(SEQ ID NO: 616)


770
FYGSPSFWENL
1348.5
−
(SEQ ID NO: 617)


772
FYGSPSYPENL
1275.4
−
(SEQ ID NO: 618)


773
FYGSPSYFENL
1325.4
−
(SEQ ID NO: 619)


774
FYGSPSYYENL
1341.4
−
(SEQ ID NO: 620)


775
FYGSPSYWENL
1364.5
−
(SEQ ID NO: 621)


777
FYGSPSDPENL
1227.3
−
(SEQ ID NO: 622)


778
FYGSPSDFENL
1277.3
−
(SEQ ID NO: 623)


779
FYGSPSDYENL
1293.3
−
(SEQ ID NO: 624)


780
FYGSPSDWENL
1316.4
−
(SEQ ID NO: 625)


782
FYGSPSEPENL
1241.3
−
(SEQ ID NO: 626)


783
FYGSPSEFENL
1291.3
−
(SEQ ID NO: 627)


784
FYGSPSEYENL
1307.3
−
(SEQ ID NO: 628)


785
FYGSPSEWENL
1330.4
−
(SEQ ID NO: 629)


787
FYGSPSNPENL
1226.4
−
(SEQ ID NO: 630)


788
FYGSPSNFENL
1276.4
−
(SEQ ID NO: 631)


789
FYGSPSNYENL
1292.4
−
(SEQ ID NO: 632)


790
FYGSPSNWENL
1315.5
−
(SEQ ID NO: 633)


792
FYGSPSQPENL
1240.4
−
(SEQ ID NO: 634)


793
FYGSPSQFENL
1290.4
−
(SEQ ID NO: 635)


794
FYGSPSQYENL
1306.4
−
(SEQ ID NO: 636)


795
FYGSPSQWENL
1329.5
−
(SEQ ID NO: 637)


797
FYGSPSHPENL
1249.4
−
(SEQ ID NO: 638)


798
FYGSPSHFENL
1299.4
−
(SEQ ID NO: 639)


799
FYGSPSHYENL
1315.4
−
(SEQ ID NO: 640)


800
FYGSPSHWENL
1338.5
−
(SEQ ID NO: 641)


802
FYASPSMPENL
1257.5
−
(SEQ ID NO: 642)


803
FYASPSMFENL
1307.5
−
(SEQ ID NO: 643)


804
FYASPSMYENL
1323.5
−
(SEQ ID NO: 644)


805
FYASPSMWENL
1346.6
−
(SEQ ID NO: 645)


807
FYASPSFPENL
1273.4
−
(SEQ ID NO: 646)


808
FYASPSFFENL
1323.4
−
(SEQ ID NO: 647)


809
FYASPSFYENL
1339.4
−
(SEQ ID NO: 648)


810
FYASPSFWENL
1362.5
−
(SEQ ID NO: 649)


812
FYASPSYPENL
1289.4
−
(SEQ ID NO: 650)


813
FYASPSYFENL
1339.4
−
(SEQ ID NO: 651)


814
FYASPSYYENL
1355.4
−
(SEQ ID NO: 652)


815
FYASPSYWENL
1378.5
−
(SEQ ID NO: 653)


817
FYASPSDPENL
1241.3
−
(SEQ ID NO: 654)


818
FYASPSDFENL
1291.3
−
(SEQ ID NO: 655)


819
FYASPSDYENL
1307.3
−
(SEQ ID NO: 656)


820
FYASPSDWENL
1330.4
−
(SEQ ID NO: 657)


822
FYASPSEPENL
1255.3
−
(SEQ ID NO: 658)


823
FYASPSEFENL
1305.3
−
(SEQ ID NO: 659)


824
FYASPSEYENL
1321.3
−
(SEQ ID NO: 660)


825
FYASPSEWENL
1344.4
−
(SEQ ID NO: 661)


827
FYASPSNPENL
1240.4
−
(SEQ ID NO: 662)


828
FYASPSNFENL
1290.4
−
(SEQ ID NO: 663)


829
FYASPSNYENL
1306.4
−
(SEQ ID NO: 664)


830
FYASPSNWENL
1329.5
−
(SEQ ID NO: 665)


832
FYASPSQPENL
1254.4
−
(SEQ ID NO: 666)


833
FYASPSQFENL
1304.4
−
(SEQ ID NO: 667)


834
FYASPSQYENL
1320.4
−
(SEQ ID NO: 668)


835
FYASPSQWENL
1343.5
−
(SEQ ID NO: 669)


837
FYASPSHPENL
1263.4
−
(SEQ ID NO: 670)


838
FYASPSHFENL
1313.4
−
(SEQ ID NO: 671)


839
FYASPSHYENL
1329.4
−
(SEQ ID NO: 672)


840
FYASFSHWENL
1352.5
−
(SEQ ID NO: 673)


842
FFRSPSMPENL
1326.6
−
(SEQ ID NO: 674)


843
FFRSPSMFENL
1376.6
−
(SEQ ID NO: 675)


844
FFRSPSMYENL
1392.6
−
(SEQ ID NO: 676)


845
FFRSPSMWENL
1415.7
−
(SEQ ID NO: 677)


847
FFRSPSFPENL
1342.5
−
(SEQ ID NO: 678)


848
FFRSPSFFENL
1392.5
−
(SEQ ID NO: 679)


849
FFRSPSFYENL
1408.5
−
(SEQ ID NO: 680)


850
FFRSPSFWENL
1431.6
−
(SEQ ID NO: 681)


852
FFRSPSYPENL
1358.5
−
(SEQ ID NO: 682)


853
FFRSPSYFENL
1408.5
−
(SEQ ID NO: 683)


854
FFRSPSYYENL
1424.5
−
(SEQ ID NO: 684)


855
FFRSPSYWENL
1447.6
−
(SEQ ID NO: 685)


857
FFRSPSDPENL
1310.4
−
(SEQ ID NO: 686)


858
FFRSPSDFENL
1360.4
−
(SEQ ID NO: 687)


859
FFRSPSDYENL
1376.4
−
(SEQ ID NO: 688)


860
FFRSPSDWENL
1399.5
−
(SEQ ID NO: 689)


862
FFRSPSEPENL
1324.4
−
(SEQ ID NO: 690)


863
FFRSPSEFENL
1374.4
−
(SEQ ID NO: 691)


864
FFRSPSEYENL
1390.4
−
(SEQ ID NO: 692)


865
FFRSPSEWENL
1413.5
−
(SEQ ID NO: 693)


867
FFRSPSNPENL
1309.5
−
(SEQ ID NO: 694)


868
FFRSPSNFENL
1359.5
−
(SEQ ID NO: 695)


869
FFRSPSNYENL
1375.5
−
(SEQ ID NO: 696)


870
FFRSPSNWENL
1398.6
−
(SEQ ID NO: 697)


872
FFRSPSQPENL
1323.5
−
(SEQ ID NO: 698)


873
FFRSPSQFENL
1373.5
−
(SEQ ID NO: 699)


874
FFRSPSQYENL
1389.5
−
(SEQ ID NO: 700)


875
FFRSPSQWENL
1412.6
−
(SEQ ID NO: 701)


877
FFRSPSHPENL
1332.5
−
(SEQ ID NO: 702)


878
FFRSPSHFENL
1382.5
−
(SEQ ID NO: 703)


879
FFRSPSHYENL
1398.5
−
(SEQ ID NO: 704)


880
FFRSPSHWENL
1421.6
−
(SEQ ID NO: 705)


882
FFSSPSMPENL
1258.3
−
(SEQ ID NO: 706)


883
FFSSPSMFENL
1308.3
−
(SEQ ID NO: 707)


884
FFSSPSMYENL
1324.3
−
(SEQ ID NO: 708)


885
FFSSPSMWENL
1347.4
−
(SEQ ID NO: 709)


887
FFSSPSFPENL
1274.2
−
(SEQ ID NO: 710)


888
FFSSPSFFENL
1324.2
−
(SEQ ID NO: 711)


889
FFSSPSFYENL
1340.2
−
(SEQ ID NO: 712)


890
FFSSPSFWENL
1363.3
−
(SEQ ID NO: 713)


892
FFSSPSYPENL
1290.2
−
(SEQ ID NO: 714)


893
FFSSPSYFENL
1340.2
−
(SEQ ID NO: 715)


894
FFSSPSYYENL
1356.2
−
(SEQ ID NO: 316)


895
FFSSPSYWENL
1379.3
−
(SEQ ID NO: 717)


897
FFSSPSDPENL
1242.1
−
(SEQ ID NO: 718)


898
FFSSPSDFENL
1292.1
−
(SEQ ID NO: 719)


899
FFSSPSDYENL
1308.1
−
(SEQ ID NO: 720)


900
FFSSPSDWENL
1331.2
−
(SEQ ID NO: 721)


902
FFSSPSEPENL
1256.1
−
(SEQ ID NO: 722)


903
FFSSPSEFENL
1306.1
−
(SEQ ID NO: 723)


904
FFSSPSEYENL
1322.1
−
(SEQ ID NO: 724)


905
FFSSPSEWENL
1345.2
−
(SEQ ID NO: 725)


907
FFSSPSNPENL
1241.2
−
(SEQ ID NO: 726)


908
FFSSPSNFENL
1291.2
−
(SEQ ID NO: 727)


909
FFSSPSNYENL
1307.2
−
(SEQ ID NO: 728)


910
FFSSPSNWENL
1330.3
−
(SEQ ID NO: 729)


912
FFSSFSQPENL
1255.2
−
(SEQ ID NO: 730)


913
FFSSPSQFENL
1305.2
−
(SEQ ID NO: 731)


914
FFSSPSQYENL
1321.2
−
(SEQ ID NO: 732)


915
FFSSPSQWENL
1344.3
−
(SEQ ID NO: 733)


917
FFSSPSHPENL
1264.2
−
(SEQ ID NO: 734)


918
FFSSPSHFENL
1314.2
−
(SEQ ID NO: 735)


919
FFSSPSHYENL
1330.2
−
(SEQ ID NO: 736)


920
FFSSPSHWENL
1353.3
−
(SEQ ID NO: 737)


922
FFTSPSMPENL
1271.5
−
(SEQ ID NO: 738)


923
FFTSPSMFENL
1321.5
−
(SEQ ID NO: 739)


924
FFTSPSMYENL
1337.5
−
(SEQ ID NO: 740)


925
FFTSPSMWENL
1360.6
−
(SEQ ID NO: 741)


927
FFTSPSFPENL
1287.4
−
(SEQ ID NO: 742)


928
FFTSPSFFENL
1337.4
−
(SEQ ID NO: 743)


929
FFTSPSFYENL
1353.4
−
(SEQ ID NO: 744)


930
FFTSPSFWENL
1376.5
−
(SEQ ID NO: 745)


932
FFTSPSYPENL
1303.4
−
(SEQ ID NO: 746)


933
FFTSPSYFENL
1353.4
−
(SEQ ID NO: 747)


934
FFTSPSYYENL
1369.4
−
(SEQ ID NO: 748)


935
FFTSPSYWENL
1392.5
−
(SEQ ID NO: 749)


937
FFTSPSDPENL
1255.3
−
(SEQ ID NO: 750)


938
FFTSPSDFENL
1305.3
−
(SEQ ID NO: 751)


939
FFTSPSDYENL
1321.3
−
(SEQ ID NO: 752)


940
FFTSPSDWENL
1344.4
−
(SEQ ID NO: 753)


942
FFTSPSEPENL
1269.3
−
(SEQ ID NO: 754)


943
FFTSPSEFENL
1319.3
−
(SEQ ID NO: 755)


944
FFTSPSEYENL
1335.3
−
(SEQ ID NO: 756)


945
FFTSPSEWENL
1358.4
−
(SEQ ID NO: 757)


947
FFTSPSNPENL
1254.4
−
(SEQ ID NO: 758)


948
FFTSPSNFENL
1304.4
−
(SEQ ID NO: 759)


949
FFTSPSNYENL
1320.4
−
(SEQ ID NO: 760)


950
FFTSPSNWENL
1343.5
−
(SEQ ID NO: 761)


952
FFTSPSQPENL
1268.4
−
(SEQ ID NO: 762)


953
FFTSPSQFENL
1318.4
−
(SEQ ID NO: 763)


954
FFTSPSQYENL
1334.4
−
(SEQ ID NO: 764)


955
FFTSPSQWENL
1357.5
−
(SEQ ID NO: 765)


957
FFTSPSHPENL
1277.4
−
(SEQ ID NO: 766)


958
FFTSPSHFENL
1327.4
−
(SEQ ID NO: 767)


959
FFTSPSHYENL
1343.4
−
(SEQ ID NO: 768)


960
FFTSPSHWENL
1366.5
−
(SEQ ID NO: 769)


962
FFHSPSMPENL
1307.6
−
(SEQ ID NO: 770)


963
FFHSPSMFENL
1357.6
−
(SEQ ID NO: 771)


964
FFHSPSMYENL
1373.6
−
(SEQ ID NO: 772)


965
FFHSPSMWENL
1396.7
−
(SEQ ID NO: 773)


967
FFHSPSFPENL
1323.5
−
(SEQ ID NO: 774)


968
FFHSPSFFENL
1373.5
−
(SEQ ID NO: 775)


969
FFHSPSFYENL
1389.5
−
(SEQ ID NO: 776)


970
FFHSPSFWENL
1412.6
−
(SEQ ID NO: 777)


972
FFHSPSYPENL
1339.5
−
(SEQ ID NO: 778)


973
FFHSPSYFENL
1389.5
−
(SEQ ID NO: 779)


974
FFHSPSYYENL
1405.5
−
(SEQ ID NO: 780)


975
FFHSPSYWENL
1428.6
−
(SEQ ID NO: 781)


977
FFHSPSDPENL
1291.4
−
(SEQ ID NO: 782)


978
FFHSPSDFENL
1341.4
−
(SEQ ID NO: 783)


979
FFHSPSDYENL
1357.4
−
(SEQ ID NO: 784)


980
FFHSPSDWENL
1380.5
−
(SEQ ID NO: 785)


982
FFHSPSEPENL
1305.4
−
(SEQ ID NO: 786)


983
FFHSPSEFENL
1355.4
−
(SEQ ID NO: 787)


984
FFHSPSEYENL
1371.4
−
(SEQ ID NO: 788)


985
FFHSPSEWENL
1394.5
−
(SEQ ID NO: 789)


987
FFHSPSNPENL
1290.5
−
(SEQ ID NO: 790)


988
FFHSPSNFENL
1340.5
−
(SEQ ID NO: 791)


989
FFHSPSNYENL
1356.5
−
(SEQ ID NO: 792)


990
FFHSPSNWENL
1379.6
−
(SEQ ID NO: 793)


992
FFHSPSQPENL
1304.5
−
(SEQ ID NO: 794)


993
FFHSPSQFENL
1354.5
−
(SEQ ID NO: 795)


994
FFHSPSQYENL
1370.5
−
(SEQ ID NO: 796)


995
FFHSPSQWENL
1393.6
−
(SEQ ID NO: 797)


997
FFHSPSHPENL
1313.5
−
(SEQ ID NO: 798)


998
FFHSPSHFENL
1363.5
−
(SEQ ID NO: 799)


999
FFHSPSHYENL
1379.5
−
(SEQ ID NO: 800)


1000
FFHSPSHWENL
1402.6
−
(SEQ ID NO: 801)


1002
FFNSPSMPENL
1284.6
−
(SEQ ID NO: 802)


1003
FFNSPSMFENL
1334.6
−
(SEQ ID NO: 803)


1004
FFNSPSMYENL
1350.6
−
(SEQ ID NO: 804)


1005
FFNSPSMWENL
1373.7
−
(SEQ ID NO: 805)


1007
FFNSPSFPENL
1300.5
−
(SEQ ID NO: 806)


1008
FFNSPSFFENL
1350.5
−
(SEQ ID NO: 807)


1009
FFNSPSFYENL
1366.5
−
(SEQ ID NO: 808)


1010
FFNSPSFWENL
1389.6
−
(SEQ ID NO: 809)


1012
FFNSPSYPENL
1316.5
−
(SEQ ID NO: 810)


1013
FFNSPSYFENL
1366.5
−
(SEQ ID NO: 811)


1014
FFNSPSYYENL
1382.5
−
(SEQ ID NO: 812)


1015
FFNSPSYWENL
1405.6
−
(SEQ ID NO: 813)


1017
FFNSPSDPENL
1268.4
−
(SEQ ID NO: 814)


1018
FFNSPSDFENL
1318.4
−
(SEQ ID NO: 815)


1019
FFNSPSDYENL
1334.4
−
(SEQ ID NO: 816)


1020
FFNSPSDWENL
1357.5
−
(SEQ ID NO: 817)


1022
FFNSPSEPENL
1282.4
−
(SEQ ID NO: 818)


1023
FFNSPSEFENL
1332.4
−
(SEQ ID NO: 819)


1024
FFNSPSEYENL
1348.4
−
(SEQ ID NO: 820)


1025
FFNSPSEWENL
1371.5
−
(SEQ ID NO: 821)


1027
FFNSPSNPENL
1267.5
−
(SEQ ID NO: 822)


1028
FFNSPSNFENL
1317.5
−
(SEQ ID NO: 823)


1029
FFNSPSNYENL
1333.5
−
(SEQ ID NO: 824)


1030
FFNSPSNWENL
1356.6
−
(SEQ ID NO: 825)


1032
FFNSPSQPENL
1281.5
−
(SEQ ID NO: 826)


1033
FFNSPSQFENL
1331.5
−
(SEQ ID NO: 827)


1034
FFNSPSQYENL
1347.5
−
(SEQ ID NO: 828)


1035
FFNSPSQWENL
1370.6
−
(SEQ ID NO: 829)


1037
FFNSPSHPENL
1290.5
−
(SEQ ID NO: 830)


1038
FFNSPSHFENL
1340.5
−
(SEQ ID NO: 831)


1039
FFNSPSHYENL
1356.5
−
(SEQ ID NO: 832)


1040
FFNSPSHWENL
1379.6
−
(SEQ ID NO: 833)


1042
FFGSPSMPENL
1227.5
−
(SEQ ID NO: 834)


1043
FFGSPSMFENL
1277.5
−
(SEQ ID NO: 835)


1044
FFGSPSMNENL
1293.5
−
(SEQ ID NO: 836)


1045
FFGSPSMWENL
1316.6
−
(SEQ ID NO: 837)


1047
FFGSPSFPENL
1243.4
−
(SEQ ID NO: 838)


1048
FFGSPSFFENL
1293.4
−
(SEQ ID NO: 839)


1049
FFGSPSFYENL
1309.4
−
(SEQ ID NO: 840)


1050
FFGSPSFWENL
1332.5
−
(SEQ ID NO: 841)


1052
FFGSPSYPENL
1259.4
−
(SEQ ID NO: 842)


1053
FFGSPSYFENL
1309.4
−
(SEQ ID NO: 843)


1054
FFGSPSYYENL
1325.4
−
(SEQ ID NO: 844)


1055
FFGSPSYWENL
1348.5
−
(SEQ ID NO: 845)


1057
FFGSPSDPENL
1211.3
−
(SEQ ID NO: 846)


1058
FFGSPSDFENL
1261.3
−
(SEQ ID NO: 847)


1059
FFGSPSDYENL
1277.3
−
(SEQ ID NO: 848)


1060
FFGSPSDWENL
1300.4
−
(SEQ ID NO: 849)


1062
FFGSPSEPENL
1225.3
−
(SEQ ID NO: 850)


1063
FFGSPSEFENL
1275.3
−
(SEQ ID NO: 851)


1064
FFGSPSEYENL
1291.3
−
(SEQ ID NO: 852)


1065
FFGSPSEWENL
1314.4
−
(SEQ ID NO: 853)


1067
FFGSPSNPENL
1210.4
−
(SEQ ID NO: 854)


1068
FFGSPSNFENL
1260.4
−
(SEQ ID NO: 855)


1069
FFGSPSNYENL
1276.4
−
(SEQ ID NO: 856)


1070
FFGSPSNWENL
1299.5
−
(SEQ ID NO: 857)


1072
FFGSPSQPENL
1224.4
−
(SEQ ID NO: 858)


1073
FFGSPSQFENL
1274.4
−
(SEQ ID NO: 859)


1074
FFGSPSQYENL
1290.4
−
(SEQ ID NO: 860)


1075
FFGSPSQWENL
1313.5
−
(SEQ ID NO: 861)


1077
FFGSPSHPENL
1233.4
−
(SEQ ID NO: 862)


1078
FFGSPSHFENL
1283.4
−
(SEQ ID NO: 863)


1079
FFGSPSHYENL
1299.4
−
(SEQ ID NO: 864)


1080
FFGSPSHWENL
1322.5
−
(SEQ ID NO: 865)


1082
FFASPSMPENL
1241.5
−
(SEQ ID NO: 866)


1083
FFASPSMFENL
1291.5
−
(SEQ ID NO: 867)


1084
FFASPSMYENL
1307.5
−
(SEQ ID NO: 868)


1085
FFASPSMWENL
1330.6
−
(SEQ ID NO: 869)


1087
FFASPSFPENL
1257.4
−
(SEQ ID NO: 870)


1088
FFASPSFFENL
1307.4
−
(SEQ ID NO: 871)


1089
FFASPSFYENL
1323.4
−
(SEQ ID NO: 872)


1090
FFASPSFWENL
1346.5
−
(SEQ ID NO: 873)


1092
FFASPSYPENL
1273.4
−
(SEQ ID NO: 874)


1093
FFASPSYFENL
1323.4
−
(SEQ ID NO: 875)


1094
FFASPSYYENL
1339.4
−
(SEQ ID NO: 876)


1095
FFASPSYWENL
1362.5
−
(SEQ ID NO: 877)


1097
FFASPSDPENL
1225.3
−
(SEQ ID NO: 878)


1098
FFASPSDFENL
1275.3
−
(SEQ ID NO: 879)


1099
FFASPSDYENL
1291.3
−
(SEQ ID NO: 880)


110O
FFASPSDWENL
1314.4
−
(SEQ ID NO: 881)


1102
FFASPSEPENL
1239.3
−
(SEQ ID NO: 882)


1103
FFASPSEFENL
1289.3
−
(SEQ ID NO: 883)


1104
FFASPSEYENL
1305.3
−
(SEQ ID NO: 884)


1105
FFASPSEWENL
1328.4
−
(SEQ ID NO: 885)


1107
FFASPSNPENL
1224.4
−
(SEQ ID NO: 886)


1108
FFASPSNFENL
1274.4
−
(SEQ ID NO: 887)


1109
FFASPSNYENL
1290.4
−
(SEQ ID NO: 888)


1110
FFASPSNWENL
1313.5
−
(SEQ ID NO: 889)


1112
FFASPSQPENL
1238.4
−
(SEQ ID NO: 890)


1113
FFASPSQFENL
1288.4
−
(SEQ ID NO: 891)


1114
FFASPSQYENL
1304.4
−
(SEQ ID NO: 892)


1115
FFASPSQWENL
1327.5
−
(SEQ ID NO: 893)


1117
FFASPSHPENL
1247.4
−
(SEQ ID NO: 894)


1118
FFASPSHFENL
1297.4
−
(SEQ ID NO: 895)


1119
FFASPSHYENL
1313.4
−
(SEQ ID NO: 896)


1120
FFASPSHWENL
1336.5
−
(SEQ ID NO: 897)


1122
WYRSPSMPENL
1381.7
+
(SEQ ID NO: 898)


1123
WYRSPSMFENL
1431.7
+
(SEQ ID NO: 899)


1124
WYRSPSMYENL
1447.7
++
(SEQ ID NO: 900)


1125
WYRSPSMWENL
1470.8
++
(SEQ ID NO: 901)


1127
WYRSPSFPENL
1397.6
++
(SEQ ID NO: 902)


1128
WYRSPSFFENL
1447.6
++
(SEQ ID NO: 903)


1129
WYRSPSFYENL
1463.6
+++
(SEQ ID NO: 904)


1130
WYRSPSFWENL
1486.7
++
(SEQ ID NO: 905)


1132
WYRSPSYPENL
1413.6
++
(SEQ ID NO: 906)


1133
WYRSPSYFENL
1463.6
+
(SEQ ID NO: 907)


1134
WYRSPSYYENL
1479.6
++
(SEQ ID NO: 908)


1135
WYRSPSYWENL
1502.7
+
(SEQ ID NO: 909)


1137
WYRSPSDPENL
1365.5
−
(SEQ ID NO: 910)


1138
WYRSPSDFENL
1415.5
−
(SEQ ID NO: 911)


1139
WYRSPSDYENL
1431.5
−
(SEQ ID NO: 912)


1140
WYRSPSDWENL
1454.6
−
(SEQ ID NO: 913)


1142
WYRSPSEPENL
1379.5
−
(SEQ ID NO: 914)


1143
WYRSPSEFENL
1429.5
−
(SEQ ID NO: 915)


1144
WYRSPSEYENL
1445.5
−
(SEQ ID NO: 916)


1145
WYRSPSEWENL
1468.6
−
(SEQ ID NO: 917)


1147
WYRSPSNPENL
1364.6
−
(SEQ ID NO: 918)


1148
WYRSPSNFENL
1414.6
−
(SEQ ID NO: 919)


1149
WYRSPSNYENL
1430.6
−
(SEQ ID NO: 920)


1150
WYRSPSNWENL
1453.7
−
(SEQ ID NO: 921)


1152
WYRSPSQPENL
1378.6
−
(SEQ ID NO: 922)


1153
WYRSPSQFENL
1428.6
−
(SEQ ID NO: 923)


1154
WYRSPSQYENL
1444.6
−
(SEQ ID NO: 924)


1155
WYRSPSQWENL
1467.7
−
(SEQ ID NO: 925)


1157
WYRSPSHPENL
1387.6
−
(SEQ ID NO: 926)


1158
WYRSPSHFENL
1437.6
−
(SEQ ID NO: 927)


1159
WYRSPSHYENL
1453.6
−
(SEQ ID NO: 928)


1160
WYRSPSHWENL
1476.7
−
(SEQ ID NO: 929)


1162
WYSSPSMPENL
1313.4
−
(SEQ ID NO: 930)


1163
WYSSPSMFENL
1363.4
−
(SEQ ID NO: 931)


1164
WYSSPSMYENL
1379.4
−
(SEQ ID NO: 932)


1165
WYSSPSMWENL
1402.5
−
(SEQ ID NO: 933)


1167
WYSSPSFPENL
1329.3
−
(SEQ ID NO: 934)


1168
WYSSPSFFENL
1379.3
−
(SEQ ID NO: 935)


1169
WYSSPSFYENL
1395.3
−
(SEQ ID NO: 936)


1170
WYSSPSFWENL
1418.4
−
(SEQ ID NO: 937)


1172
WYSSPSYPENL
1345.3
−
(SEQ ID NO: 938)


1173
WYSSPSYFENL
1395.3
−
(SEQ ID NO: 939)


1174
WYSSPSYYENL
1411.3
−
(SEQ ID NO: 940)


1175
WYSSPSYWENL
1434.4
−
(SEQ ID NO: 941)


1177
WYSSPSDPENL
1297.2
−
(SEQ ID NO: 942)


1178
WYSSPSDFENL
1347.2
−
(SEQ ID NO: 943)


1179
WYSSPSDYENL
1363.2
−
(SEQ ID NO: 944)


1180
WYSSPSDWENL
1386.3
−
(SEQ ID NO: 945)


1182
WYSSPSEPENL
1311.2
−
(SEQ ID NO: 946)


1183
WYSSPSEFENL
1361.2
−
(SEQ ID NO: 947)


1184
WYSSPSEYENL
1377.2
−
(SEQ ID NO: 948)


1185
WYSSPSEWENL
1400.3
−
(SEQ ID NO: 949)


1187
WYSSPSNPENL
1296.3
−
(SEQ ID NO: 950)


1188
WYSSPSNFENL
1346.3
−
(SEQ ID NO: 951)


1189
WYSSPSNYENL
1362.3
−
(SEQ ID NO: 952)


1190
WYSSPSNWENL
1385.4
−
(SEQ ID NO: 953)


1192
WYSSPSQPENL
1310.3
−
(SEQ ID NO: 954)


1193
WYSSPSQFENL
1360.3
−
(SEQ ID NO: 955)


1194
WYSSPSQYENL
1376.3
−
(SEQ ID NO: 956)


1195
WYSSPSQWENL
1399.4
−
(SEQ ID NO: 957)


1197
WYSSPSHPENL
1319.3
−
(SEQ ID NO: 958)


1198
WYSSPSHFENL
1369.3
−
(SEQ ID NO: 959)


1199
WYSSPSHYENL
1385.3
−
(SEQ ID NO: 960)


1200
WYSSPSHWENL
1408.4
−
(SEQ ID NO: 961)


1202
WYTSPSMPENL
1326.6
+
(SEQ ID NO: 962)


1203
WYTSPSMFENL
1376.6
+
(SEQ ID NO: 963)


1204
WYTSPSMYENL
1392.6
+
(SEQ ID NO: 964)


1205
WYTSPSMWENL
1415.7
+
(SEQ ID NO: 965)


1207
WYTSPSFPENL
1342.5
+
(SEQ ID NO: 966)


1208
WYTSPSFFENL
1392.5
+
(SEQ ID NO: 967)


1209
WYTSPSFYENL
1408.5
+
(SEQ ID NO: 968)


1210
WYTSPSFWENL
1431.6
+
(SEQ ID NO: 969)


1212
WYTSPSYPENL
1358.5
++
(SEQ ID NO: 970)


1213
WYTSPSYFENL
1408.5
+
(SEQ ID NO: 971)


1214
WYTSPSYYENL
1424.5
+
(SEQ ID NO: 972)


1215
WYTSPSYWENL
1447.6
+
(SEQ ID NO: 973)


1217
WYTSPSDPENL
1310.4
−
(SEQ ID NO: 974)


1218
WYTSPSDFENL
1360.4
−
(SEQ ID NO: 975)


1219
WYTSPSDYENL
1376.4
−
(SEQ ID NO: 976)


1220
WYTSPSDWENL
1399.5
−
(SEQ ID NO: 977)


1222
WYTSPSEPENL
1324.4
−
(SEQ ID NO: 978)


1223
WYTSPSEFENL
1374.4
−
(SEQ ID NO: 979)


1224
WYTSPSEYENL
1390.4
−
(SEQ ID NO: 980)


1225
WYTSPSEWENL
1413.5
−
(SEQ ID NO: 981)


1227
WYTSPSNPENL
1309.5
−
(SEQ ID NO: 982)


1228
WYTSPSNFENL
1359.5
−
(SEQ ID NO: 983)


1229
WYTSPSNYENL
1375.5
−
(SEQ ID NO: 984)


1230
WYTSPSNWENL
1398.6
−
(SEQ ID NO: 985)


1232
WYTSPSQPENL
1323.5
−
(SEQ ID NO: 986)


1233
WYTSPSQFENL
1373.5
−
(SEQ ID NO: 987)


1234
WYTSPSQYENL
1389.5
−
(SEQ ID NO: 988)


1235
WYTSPSQWENL
1412.6
−
(SEQ ID NO: 989)


1237
WYTSPSHPENL
1332.5
+
(SEQ ID NO: 990)


1238
WYTSPSHFENL
1382.5
+
(SEQ ID NO: 991)


1239
WYTSPSHYENL
1398.5
+
(SEQ ID NO: 992)


1240
WYTSPSHWENL
1421.6
+
(SEQ ID NO: 993)


1242
WYHSPSMPENL
1362.7
−
(SEQ ID NO: 994)


1243
WYHSPSMFENL
1412.7
−
(SEQ ID NO: 995)


1244
WYHSPSMYENL
1428.7
−
(SEQ ID NO: 996)


1245
WYHSPSMWENL
1451.8
−
(SEQ ID NO: 997)


1247
WYHSPSFPENL
1378.6
−
(SEQ ID NO: 998)


1248
WYHSPSFFENL
1428.6
−
(SEQ ID NO: 999)


1249
WYHSPSFYENL
1444.6
−
(SEQ ID NO: 1000)


1250
WYHSPSFWENL
1467.7
−
(SEQ ID NO: 1001)


1252
WYHSPSYPENL
1394.6
−
(SEQ ID NO: 1002)


1253
WYHSPSYFENL
1444.6
−
(SEQ ID NO: 1003)


1254
WYHSPSYYENL
1460.6
−
(SEQ ID NO: 1004)


1255
WYHSPSYWENL
1483.7
−
(SEQ ID NO: 1005)


1257
WYHSPSDPENL
1346.5
−
(SEQ ID NO: 1006)


1258
WYHSPSDFENL
1396.5
−
(SEQ ID NO: 1007)


1259
WYHSPSDYENL
1412.5
−
(SEQ ID NO: 1008)


1260
WYHSPSDWENL
1435.6
−
(SEQ ID NO: 1009)


1262
WYHSPSEPENL
1360.5
−
(SEQ ID NO: 1010)


1263
WYHSPSEFENL
1410.5
−
(SEQ ID NO: 1011)


1264
WYHSPSEYENL
1426.5
−
(SEQ ID NO: 1012)


1265
WYHSPSEWENL
1449.6
−
(SEQ ID NO: 1013)


1267
WYHSPSNPENL
1345.6
−
(SEQ ID NO: 1014)


1268
WYHSPSNFENL
1395.6
−
(SEQ ID NO: 1015)


1269
WYHSPSNYENL
1411.6
−
(SEQ ID NO: 1016)


1270
WYHSPSNWENL
1434.7
−
(SEQ ID NO: 1017)


1272
WYHSPSQPENL
1359.6
−
(SEQ ID NO: 1018)


1273
WYHSPSQFENL
1409.6
−
(SEQ ID NO: 1019)


1274
WYHSPSQYENL
1425.6
−
(SEQ ID NO: 1020)


1275
WYHSPSQWENL
1448.7
−
(SEQ ID NO: 1021)


1277
WYHSPSHPENL
1368.6
−
(SEQ ID NO: 1022)


1278
WYHSPSHFENL
1418.6
−
(SEQ ID NO: 1023)


1279
WYHSPSHYENL
1434.6
−
(SEQ ID NO: 1024)


1280
WYHSPSHWENL
1457.7
−
(SEQ ID NO: 1025)


1282
WYNSPSMPENL
1339.7
−
(SEQ ID NO: 1026)


1283
WYNSPSMEENL
1389.7
−
(SEQ ID NO: 1027)


1284
WYNSPSMYENL
1405.7
−
(SEQ ID NO: 1028)


1285
WYNSPSMWENL
1428.8
−
(SEQ ID NO: 1029)


1287
WYNSPSFPENL
1355.6
−
(SEQ ID NO: 1030)


1288
WYNSPSFFENL
1405.6
−
(SEQ ID NO: 1031)


1289
WYNSPSFYENL
1421.6
−
(SEQ ID NO: 1032)


1290
WYNSPSFWENL
1444.7
−
(SEQ ID NO: 1033)


1292
WYNSPSYPENL
1371.6
−
(SEQ ID NO: 1034)


1293
WYNSPSYFENL
1421.6
−
(SEQ ID NO: 1035)


1294
WYNSPSYYENL
1437.6
−
(SEQ ID NO: 1036)


1295
WYNSPSYWENL
1460.7
−
(SEQ ID NO: 1037)


1297
WYNSPSDPENL
1323.5
−
(SEQ ID NO: 1038)


1298
WYNSPSDFENL
1373.5
−
(SEQ ID NO: 1039)


1299
WYNSPSDYENL
1389.5
−
(SEQ ID NO: 1040)


1300
WYNSPSDWENL
1412.6
−
(SEQ ID NO: 1041)


1302
WYNSPSEPENL
1337.5
−
(SEQ ID NO: 1042)


1303
WYNSPSEFENL
1387.5
−
(SEQ ID NO: 1043)


1304
WYNSPSEYENL
1403.5
−
(SEQ ID NO: 1044)


1305
WYNSPSEWENL
1426.6
−
(SEQ ID NO: 1045)


1307
WYNSPSNPENL
1322.6
−
(SEQ ID NO: 1046)


1308
WYNSPSNFENL
1372.6
−
(SEQ ID NO: 1047)


1309
WYNSPSNYENL
1388.6
−
(SEQ ID NO: 1048)


1310
WYNSPSNWENL
1411.7
−
(SEQ ID NO: 1049)


1312
WYNSPSQPENL
1336.6
−
(SEQ ID NO: 1050)


1313
WYNSPSQFENL
1386.6
−
(SEQ ID NO: 1051)


1314
WYNSPSQYENL
1402.6
−
(SEQ ID NO: 1052)


1315
WYNSPSQWENL
1425.7
−
(SEQ ID NO: 1053)


1317
WYNSPSHPENL
1345.6
−
(SEQ ID NO: 1054)


1318
WYNSPSHFENL
1395.6
−
(SEQ ID NO: 1055)


1319
WYNSPSHYENL
1411.6
−
(SEQ ID NO: 1056)


1320
WYNSPSHWENL
1434.7
−
(SEQ ID NO: 1057)


1322
WYGSPSMPENL
1282.6
−
(SEQ ID NO: 1058)


1323
WYGSPSMFENL
1332.6
−
(SEQ ID NO: 1059)


1324
WYGSPSMYENL
1348.6
−
(SEQ ID NO: 1060)


1325
WYGSPSMWENL
1371.7
−
(SEQ ID NO: 1061)


1327
WYGSPSFPENL
1298.5
−
(SEQ ID NO: 1062)


1328
WYGSPSFFENL
1348.5
−
(SEQ ID NO: 1063)


1329
WYGSPSFYENL
1364.5
−
(SEQ ID NO: 1064)


1330
WYGSPSEWENL
1387.6
−
(SEQ ID NO: 1065)


1332
WYGSPSYPENL
1314.5
−
(SEQ ID NO: 1066)


1333
WYGSPSYFENL
1364.5
−
(SEQ ID NO: 1067)


1334
WYGSPSYYENL
1380.5
−
(SEQ ID NO: 1068)


1335
WYGSPSYWENL
1403.6
−
(SEQ ID NO: 1069)


1337
WYGSPSDPENL
1266.4
−
(SEQ ID NO: 1070)


1338
WYGSPSDFENL
1316.4
−
(SEQ ID NO: 1071)


1339
WYGSPSDYENL
1332.4
−
(SEQ ID NO: 1072)


1340
WYGSPSDWENL
1355.5
−
(SEQ ID NO: 1073)


1342
WYGSPSEPENL
1280.4
−
(SEQ ID NO: 1074)


1343
WYGSPSEFENL
1330.4
−
(SEQ ID NO: 1075)


1344
WYGSPSEYENL
1346.4
−
(SEQ ID NO: 1076)


1345
WYGSPSEWENL
1369.5
−
(SEQ ID NO: 1077)


1347
WYGSPSNPENL
1265.5
−
(SEQ ID NO: 1078)


1348
WYGSPSNFENL
1315.5
−
(SEQ ID NO: 1079)


1349
WYGSPSNYENL
1331.5
−
(SEQ ID NO: 1080)


1350
WYGSPSNWENL
1354.6
−
(SEQ ID NO: 1081)


1352
WYGSPSQPENL
1279.5
−
(SEQ ID NO: 1082)


1353
WYGSPSQFENL
1329.5
−
(SEQ ID NO: 1083)


1354
WYGSPSQYENL
1345.5
−
(SEQ ID NO: 1084)


1355
WYGSPSQWENL
1368.6
−
(SEQ ID NO: 1085)


1357
WYGSPSHPENL
1288.5
−
(SEQ ID NO: 1086)


1358
WYGSPSHFENL
1338.5
−
(SEQ ID NO: 1087)


1359
WYGSPSHYENL
1354.5
−
(SEQ ID NO: 1088)


1360
WYGSPSHWENL
1377.6
−
(SEQ ID NO: 1089)


1362
WYASPSMPENL
1296.6
−
(SEQ ID NO: 1090)


1363
WYASPSMFENL
1346.6
−
(SEQ ID NO: 1091)


1364
WYASPSMYENL
1362.6
−
(SEQ ID NO: 1092)


1365
WYASPSMWENL
1385.7
−
(SEQ ID NO: 1093)


1367
WYASPSFPENL
1312.5
−
(SEQ ID NO: 1094)


1368
WYASPSFFENL
1362.5
−
(SEQ ID NO: 1095)


1369
WYASPSFYENL
1378.5
−
(SEQ ID NO: 1096)


1370
WYASPSFWENL
1401.6
−
(SEQ ID NO: 1097)


1372
WYASPSYPENL
1328.5
−
(SEQ ID NO: 1098)


1373
WYASPSYFENL
1378.5
−
(SEQ ID NO: 1099)


1374
WYASPSYYENL
1394.5
−
(SEQ ID NO: 1100)


1375
WYASPSYWENL
1417.6
−
(SEQ ID NO: 1101)


1377
WYASPSDPENL
1280.4
−
(SEQ ID NO: 1102)


1378
WYASPSDFENL
1330.4
−
(SEQ ID NO: 1103)


1379
WYASPSDYENL
1346.4
−
(SEQ ID NO: 1104)


1380
WYASPSDWENL
1369.5
−
(SEQ ID NO: 1105)


1382
WYASPSEPENL
1294.4
−
(SEQ ID NO: 1106)


1383
WYASPSEFENL
1344.4
−
(SEQ ID NO: 1107)


1384
WYASPSEYENL
1360.4
−
(SEQ ID NO: 1108)


1385
WYASPSEWENL
1383.5
−
(SEQ ID NO: 1109)


1387
WYASPSNPENL
1279.5
−
(SEQ ID NO: 1110)


1388
WYASPSNFENL
1329.5
−
(SEQ ID NO: 1111)


1389
WYASPSNYENL
1345.5
−
(SEQ ID NO: 1112)


1390
WYASPSNWENL
1368.6
−
(SEQ ID NO: 1113)


1392
WYASPSQPENL
1293.5
−
(SEQ ID NO: 1114)


1393
WYASPSQFENL
1343.5
−
(SEQ ID NO: 1115)


1394
WYASPSQYENL
1359.5
−
(SEQ ID NO: 1116)


1395
WYASPSQWENL
1382.6
−
(SEQ ID NO: 1117)


1397
WYASPSHPENL
1302.5
−
(SEQ ID NO: 1118)


1398
WYASPSHFENL
1352.5
−
(SEQ ID NO: 1119)


1399
WYASPSHYENL
1368.5
−
(SEQ ID NO: 1120)


1400
WYASPSHWENL
1391.6
−
(SEQ ID NO: 1121)


1402
WFRSPSMPENL
1365.7
−
(SEQ ID NO: 1122)


1403
WFRSPSMFENL
1415.7
−
(SEQ ID NO: 1123)


1404
WFRSPSMYENL
1431.7
−
(SEQ ID NO: 1124)


1405
WFRSPSMWENL
1454.8
−
(SEQ ID NO: 1125)


1407
WFRSPSFPENL
1381.6
−
(SEQ ID NO: 1126)


1408
WFRSPSFFENL
1431.6
−
(SEQ ID NO: 1127)


1409
WFRSPSFYENL
1447.6
−
(SEQ ID NO: 1128)


1410
WFRSPSFWENL
1470.7
−
(SEQ ID NO: 1129)


1412
WFRSPSYPENL
1397.6
−
(SEQ ID NO: 1130)


1413
WFRSPSYFENL
1447.6
−
(SEQ ID NO: 1131)


1414
WFRSPSYYENL
1463.6
−
(SEQ ID NO: 1132)


1415
WFRSPSYWENL
1486.7
−
(SEQ ID NO: 1133)


1417
WFRSPSDPENL
1349.5
−
(SEQ ID NO: 1134)


1418
WFRSPSDFENL
1399.5
−
(SEQ ID NO: 1135)


1419
WFRSPSDYENL
1415.5
−
(SEQ ID NO: 1136)


1420
WFRSPSDWENL
1438.6
−
(SEQ ID NO: 1137)


1422
WFRSPSEPENL
1363.5
−
(SEQ ID NO: 1138)


1423
WFRSPSEFENL
1413.5
−
(SEQ ID NO: 1139)


1424
WFRSPSEYENL
1429.5
−
(SEQ ID NO: 1140)


1425
WFRSPSEWENL
1452.6
−
(SEQ ID NO: 1141)


1427
WFRSPSNPENL
1348.6
−
(SEQ ID NO: 1142)


1428
WFRSPSNFENL
1398.6
−
(SEQ ID NO: 1143)


1429
WFRSPSNYENL
1414.6
−
(SEQ ID NO: 1144)


1430
WFRSPSNWENL
1437.7
−
(SEQ ID NO: 1145)


1432
WFRSPSQPENL
1362.6
−
(SEQ ID NO: 1146)


1433
WFRSPSQFENL
1412.6
−
(SEQ ID NO: 1147)


1434
WFRSPSQYENL
1428.6
−
(SEQ ID NO: 1148)


1435
WFRSPSQWENL
1451.7
−
(SEQ ID NO: 1149)


1437
WFRSPSHPENL
1371.6
−
(SEQ ID NO: 1150)


1438
WFRSPSHFENL
1421.6
−
(SEQ ID NO: 1151)


1439
WFRSPSHYENL
1437.6
−
(SEQ ID NO: 1152)


1440
WFRSPSHWENL
1460.7
−
(SEQ ID NO: 1153)


1442
WFSSPSMPENL
1297.4
−
(SEQ ID NO: 1154)


1443
WFSSPSMFENL
1347.4
−
(SEQ ID NO: 1155)


1444
WFSSPSMYENL
1363.4
−
(SEQ ID NO: 1156)


1445
WFSSPSMWENL
1386.5
−
(SEQ ip NO: 1157)


1447
WFSSPSFPENL
1313.3
−
(SEQ ID NO: 1158)


1448
WFSSPSFFENL
1363.3
−
(SEQ ID NO: 1159)


1449
WFSSPSFYENL
1379.3
−
(SEQ ID NO: 1160)


1450
WFSSPSFWENL
1402.4
−
(SEQ ID NO: 1161)


1452
WFSSPSYPENL
1329.3
−
(SEQ ID NO: 1162)


1453
WFSSPSYFENL
1379.3
−
(SEQ ID NO: 1163)


1454
WFSSPSYYENL
1395.3
−
(SEQ ID NO: 1164)


1455
WFSSPSYWENL
1418.4
−
(SEQ ID NO: 1165)


1457
WFSSPSDPENL
1281.2
−
(SEQ ID NO: 1166)


1458
WFSSPSDFENL
1331.2
−
(SEQ ID NO: 1167)


1459
WFSSPSDYENL
1347.2
−
(SEQ ID NO: 1168)


1460
WFSSPSDWENL
1370.3
−
(SEQ ID NO: 1169)


1462
WFSSPSEPENL
1295.2
−
(SEQ ID NO: 1170)


1463
WFSSPSEFENL
1345.2
−
(SEQ ID NO: 1171)


1464
WFSSPSEYENL
1361.2
−
(SEQ ID NO: 1172)


1465
WFSSPSEWENL
1384.3
−
(SEQ ID NO: 1173)


1467
WFSSPSNPENL
1280.3
−
(SEQ ID NO: 1174)


1468
WFSSPSNFENL
1330.3
−
(SEQ ID NO: 1175)


1469
WFSSPSNYENL
1346.3
−
(SEQ ID NO: 1176)


1470
WFSSPSNWENL
1369.4
−
(SEQ ID NO: 1177)


1472
WFSSPSQPENL
1294.3
−
(SEQ ID NO: 1178)


1473
WFSSPSQFENL
1344.3
−
(SEQ ID NO: 1179)


1474
WFSSPSQYENL
1360.3
−
(SEQ ID NO: 1180)


1475
WFSSPSQWENL
1383.4
−
(SEQ ID NO: 1181)


1477
WFSSPSHPENL
1303.3
−
(SEQ ID NO: 1182)


1478
WFSSPSHFENL
1353.3
−
(SEQ ID NO: 1183)


1479
WFSSPSHYENL
1369.3
−
(SEQ ID NO: 1184)


1480
WFSSPSHWENL
1392.4
−
(SEQ ID NO: 1185)


1482
WFTSPSMPENL
1310.6
−
(SEQ ID NO: 1186)


1483
WFTSPSMFENL
1360.6
−
(SEQ ID NO: 1187)


1484
WFTSPSMYENL
1376.6
−
(SEQ ID NO: 1188)


1485
WFTSPSMWENL
1399.7
−
(SEQ ID NO: 1189)


1487
WFTSPSFPENL
1326.5
−
(SEQ ID NO: 1190)


1488
WFTSPSFFENL
1376.5
−
(SEQ ID NO: 1191)


1489
WFTSPSFYENL
1392.5
−
(SEQ ID NO: 1192)


1490
WFTSPSFWENL
1415.6
−
(SEQ ID NO: 1193)


1492
WFTSPSYPENL
1342.5
−
(SEQ ID NO: 1194)


1493
WFTSPSYFENL
1392.5
−
(SEQ ID NO: 1195)


1494
WFTSPSYYENL
1408.5
−
(SEQ ID NO: 1196)


1495
WFTSPSYWENL
1431.6
−
(SEQ ID NO: 1197)


1497
WFTSPSDPENL
1294.4
−
(SEQ ID NO: 1198)


1498
WFTSPSDFENL
1344.4
−
(SEQ ID NO: 1199)


1499
WFTSPSDYENL
1360.4
−
(SEQ ID NO: 1200)


1500
WFTSPSDWENL
1383.5
−
(SEQ ID NO: 1201)


1502
WFTSPSEPENL
1308.4
−
(SEQ ID NO: 1202)


1503
WFTSPSEFENL
1358.4
−
(SEQ ID NO: 1203)


1504
WFTSPSEYENL
1374.4
−
(SEQ ID NO: 1204)


1505
WFTSPSEWENL
1397.5
−
(SEQ ID NO: 1205)


1507
WFTSPSNPENL
1293.5
−
(SEQ ID NO: 1206)


1508
WFTSPSNFENL
1343.5
−
(SEQ ID NO: 1207)


1509
WFTSPSNYENL
1359.5
−
(SEQ ID NO: 1208)


1510
WFTSPSNWENL
1382.6
−
(SEQ ID NO: 1209)


1512
WFTSPSQPENL
1307.5
−
(SEQ ID NO: 1210)


1513
WFTSPSQFENL
1357.5
−
(SEQ ID NO: 1211)


1514
WFTSPSQYENL
1373.5
−
(SEQ ID NO: 1212)


1515
WFTSPSQWENL
1396.6
−
(SEQ ID NO: 1213)


1517
WFTSPSHPENL
1316.5
−
(SEQ ID NO: 1214)


1518
WFTSPSHFENL
1366.5
−
(SEQ ID NO: 1215)


1519
WFTSPSHYENL
1382.5
−
(SEQ ID NO: 1216)


1520
WFTSPSHWENL
1405.6
−
(SEQ ID NO: 1217)


1522
WFHSPSMPENL
1346.7
−
(SEQ ID NO: 1218)


1523
WFHSPSMFENL
1396.7
−
(SEQ ID NO: 1219)


1524
WFHSPSMYENL
1412.7
−
(SEQ ID NO: 1220)


1525
WFHSPSMWENL
1435.8
−
(SEQ ID NO: 1221)


1527
WFHSPSFPENL
1362.6
−
(SEQ ID NO: 1222)


1528
WFHSPSFFENL
1412.6
−
(SEQ ID NO: 1223)


1529
WFHSPSFYENL
1428.6
−
(SEQ ID NO: 1224)


1530
WFHSPSFWENL
1451.7
−
(SEQ ID NO: 1225)


1532
WFHSPSYPENL
1378.6
−
(SEQ ID NO: 1226)


1533
WFHSPSYFENL
1428.6
−
(SEQ ID NO: 1227)


1534
WFHSPSYYENL
1444.6
−
(SEQ ID NO: 1228)


1535
WFHSPSYWENL
1467.7
−
(SEQ ID NO: 1229)


1537
WFHSPSDPENL
1330.5
−
(SEQ ID NO: 1230)


1538
WFHSPSDFENL
1380.5
−
(SEQ ID NO: 1231)


1539
WFHSPSDYENL
1396.5
−
(SEQ ID NO: 1232)


1540
WFHSPSDWENL
1419.6
−
(SEQ ID NO: 1233)


1542
WFHSPSEPENL
1344.5
−
(SEQ ID NO: 1234)


1543
WFHSPSEFENL
1394.5
−
(SEQ ID NO: 1235)


1544
WFHSPSEYENL
1410.5
−
(SEQ ID NO: 1236)


1545
WFHSPSEWENL
1433.6
−
(SEQ ID NO: 1237)


1547
WFHSPSNPENL
1329.6
−
(SEQ ID NO: 1238)


1548
WFHSPSNFENL
1379.6
−
(SEQ ID NO: 1239)


1549
WFHSPSNYENL
1395.6
−
(SEQ ID NO: 1240)


1550
WFHSPSNWENL
1418.7
−
(SEQ ID NO: 1241)


1552
WFHSPSQPENL
1343.6
−
(SEQ ID NO: 1242)


1553
WFHSPSQFENL
1393.6
−
(SEQ ID NO: 1243)


1554
WFHSPSQYENL
1409.6
−
(SEQ ID NO: 1244)


1555
WFYSPSQWENL
1432.7
−
(SEQ ID NO: 1245)


1557
WFHSPSHPENL
1352.6
−
(SEQ ID NO: 1246)


1558
WFFSPSHFENL
1402.6
−
(SEQ ID NO: 1247)


1559
WFHSPSHYENL
1418.6
−
(SEQ ID NO: 1248)


1560
WFHSPSHWENL
1441.7
−
(SEQ ID NO: 1249)


1562
WFNSPSMPENL
1323.7
−
(SEQ ID NO: 1250)


1563
WFNSPSMFENL
1373.7
−
(SEQ ID NO: 1251)


1564
WFNSPSMYENL
1389.7
−
(SEQ ID NO: 1252)


1565
WFNSPSMWENL
1412.8
−
(SEQ ID NO: 1253)


1567
WFNSPSFPENL
1339.6
−
(SEQ ID NO: 1254)


1568
WFNSPSFFENL
1389.6
−
(SEQ ID NO: 1255)


1569
WFNSPSFYENL
1405.6
−
(SEQ ID NO: 1256)


1570
WFNSPSFWENL
1428.7
−
(SEQ ID NO: 1257)


1572
WFNSPSYPENL
1355.6
−
(SEQ ID NO: 1258)


1573
WFNSPSYFENL
1405.6
−
(SEQ ID NO: 1259)


1574
WFNSPSYYENL
1421.6
−
(SEQ ID NO: 1260)


1575
WFNSPSYWENL
1444.7
−
(SEQ ID NO: 1261)


1577
WFNSPSDPENL
1307.5
−
(SEQ ID NO: 1262)


1578
WFNSPSDFENL
1357.5
−
(SEQ ID NO: 1263)


1579
WFNSPSDYENL
1373.5
−
(SEQ ID NO: 1264)


1580
WFNSPSDWENL
1396.6
−
(SEQ ID NO: 1265)


1582
WFNSPSEPENL
1321.5
−
(SEQ ID NO: 1266)


1583
WFNSPSEFENL
1371.5
−
(SEQ ID NO: 1267)


1584
WFNSPSEYENL
1387.5
−
(SEQ ID NO: 1268)


1585
WENSPSEWENL
1410.6
−
(SEQ ID NO: 1269)


1587
WFNSPSNPENL
1306.6
−
(SEQ ID NO: 1270)


1588
WFNSPSNFENL
1356.6
−
(SEQ ID NO: 1271)


1589
WFNSPSNYENL
1372.6
−
(SEQ ID NO: 1272)


1590
WFNSPSNWENL
1395.7
−
(SEQ ID NO: 1273)


1592
WFNSPSQPENL
1320.6
−
(SEQ ID NO: 1274)


1593
WFNSPSQFENL
1370.6
−
(SEQ ID NO: 1275)


1594
WFNSPSQYENL
1386.6
−
(SEQ ID NO: 1276)


1595
WFNSPSQWENL
1409.7
−
(SEQ ID NO: 1277)


1597
WFNSPSHPENL
1329.6
−
(SEQ ID NO: 1278)


1598
WFNSPSHFENL
1379.6
−
(SEQ ID NO: 1279)


1599
WFNSPSHYENL
1395.6
−
(SEQ ID NO: 1280)


1600
WFNSPSHWENL
1418.7
−
(SEQ ID NO: 1281)


1602
WFGSPSMPENL
1266.6
−
(SEQ ID NO: 1282)


1603
WFGSPSMFENL
1316.6
−
(SEQ ID NO: 1283)


1604
WFGSPSMYENL
1332.6
−
(SEQ ID NO: 1284)


1605
WFGSPSMWENL
1355.7
−
(SEQ ID NO: 1285)


1607
WFGSPSFPENL
1282.5
−
(SEQ ID NO: 1286)


1608
WFGSPSFFENL
1332.5
−
(SEQ ID NO: 1287)


1609
WFGSPSFYENL
1348.5
−
(SEQ ID NO: 1288)


1610
WFGSPSFWENL
1371.6
−
(SEQ ID NO: 1289)


1612
WFGSPSYPENL
1298.5
−
(SEQ ID NO: 1290)


1613
WFGSPSYFENL
1348.5
−
(SEQ ID NO: 1291)


1614
WFGSPSYYENL
1364.5
−
(SEQ ID NO: 1292)


1615
WFGSPSYWENL
1387.6
−
(SEQ ID NO: 1293)


1617
WFGSPSDPENL
1250.4
−
(SEQ ID NO: 1294)


1618
WFGSPSDFENL
1300.4
−
(SEQ ID NO: 1295)


1619
WFGSPSDYENL
1316.4
−
(SEQ ID NO: 1296)


1620
WFGSPSDWENL
1339.5
−
(SEQ ID NO: 1297)


1622
WFGSPSEPENL
1264.4
−
(SEQ ID NO: 1298)


1623
WFGSPSEFENL
1314.4
−
(SEQ ID NO: 1299)


1624
WFGSPSEYENL
1330.4
−
(SEQ ID NO: 1300)


1625
WFGSPSEWENL
1353.5
−
(SEQ ID NO: 1301)


1627
WFGSPSNPENL
1249.5
−
(SEQ ID NO: 1302)


1628
WFGSPSNFENL
1299.5
−
(SEQ ID NO: 1303)


1629
WFGSPSNYENL
1315.5
−
(SEQ ID NO: 1304)


1630
WFOSPSNWENL
1338.6
−
(SEQ ID NO: 1305)


1632
WFGSPSQPENL
1263.5
−
(SEQ ID NO: 1306)


1633
WFGSPSQFENL
1313.5
−
(SEQ ID NO: 1307)


1634
WFGSPSQYENL
1329.5
−
(SEQ ID NO: 1308)


1635
WFGSPSQWENL
1352.6
−
(SEQ ID NO: 1309)


1637
WFGSPSHPENL
1272.5
−
(SEQ ID NO: 1310)


1638
WFGSPSHFENL
1322.5
−
(SEQ ID NO: 1311)


1639
WFGSPSHYENL
1338.5
−
(SEQ ID NO: 1312)


1640
WFGSPSHWENL
1361.6
−
(SEQ ID NO: 1313)


1642
WFASPSMPENL
1280.6
−
(SEQ ID NO: 1314)


1643
WFASPSMFENL
1330.6
−
(SEQ ID NO: 1315)


1644
WFASPSMYENL
1346.6
−
(SEQ ID NO: 1316)


1645
WFASPSMWENL
1369.7
−
(SEQ ID NO: 1317)


1647
WFASPSFPENL
1296.5
−
(SEQ ID NO: 1318)


1648
WFASPSFFENL
1346.5
−
(SEQ ID NO: 1319)


1649
WFASPSFYENL
1362.5
−
(SEQ ID NO: 1320)


1650
WFASPSFWENL
1385.6
−
(SEQ ID NO: 1321)


1652
WFASPSYPENL
1312.5
−
(SEQ ID NO: 1322)


1653
WFASPSYFENL
1362.5
−
(SEQ ID NO: 1323)


1654
WFASPSYYENL
1378.5
−
(SEQ ID NO: 1324)


1655
WFASPSYWENL
1401.6
−
(SEQ ID NO: 1325)


1657
WFASPSDPENL
1264.4
−
(SEQ ID NO: 1326)


1658
WFASPSDFENL
1314.4
−
(SEQ ID NO: 1327)


1659
WFASPSDYENL
1330.4
−
(SEQ ID NO: 1328)


1660
WFASPSDWENL
1353.5
−
(SEQ ID NO: 1329)


1662
WFASPSEPENL
1278.4
−
(SEQ ID NO: 1330)


1663
WFASPSEFENL
1328.4
−
(SEQ ID NO: 1331)


1664
WFASPSEYENL
1344.4
−
(SEQ ID NO: 1332)


1665
WFASPSEWENL
1367.5
−
(SEQ ID NO: 1333)


1667
WFASPSNPENL
1263.5
−
(SEQ ID NO: 1334)


1668
WFASPSNFENL
1313.5
−
(SEQ ID NO: 1335)


1669
WFASPSNYENL
1329.5
−
(SEQ ID NO: 1336)


1670
WFASPSNWENL
1352.6
−
(SEQ ID NO: 1337)


1672
WFASPSQPENL
1277.5
−
(SEQ ID NO: 1338)


1673
WFASPSQFENL
1327.5
−
(SEQ ID NO: 1339)


1674
WFASPSQYENL
1343.5
−
(SEQ ID NO: 1340)


1675
WFASPSQWENL
1366.6
−
(SEQ ID NO: 1341)


1677
WFASPSHPENL
1286.5
−
(SEQ ID NO: 1342)


1678
WFASPSHFENL
1336.5
−
(SEQ ID NO: 1343)


1679
WFASPSHYENL
1352.5
−
(SEQ ID NO: 1344)


1680
WFASPSHWENL
1375.6
−
(SEQ ID NO: 1345)


1682
MYRSPSMPENL
1326.7
−
(SEQ ID NO: 1346)


1683
MYRSPSMFENL
1376.7
−
(SEQ ID NO: 1347)


1684
MYRSPSMYENL
1392.7
−
(SEQ ID NO: 1348)


1685
MYRSPSMWENL
1415.8
−
(SEQ ID NO: 1349)


1687
MYRSPSFPENL
1342.6
−
(SEQ ID NO: 1350)


1688
MYRSPSFFENL
1392.6
−
(SEQ ID NO: 1351)


1689
MYRSPSFYENL
1408.6
−
(SEQ ID NO: 1352)


1690
MYRSPSFWENL
1431.7
−
(SEQ ID NO: 1353)


1692
MYRSPSYPENL
1358.6
−
(SEQ ID NO: 1354)


1693
MYRSPSYFENL
1408.6
−
(SEQ ID NO: 1355)


1694
MYRSPSYYENL
1424.6
−
(SEQ ID NO: 1356)


1695
MYRSPSYWENL
1447.7
−
(SEQ ID NO: 1357)


1697
MYRSPSDPENL
1310.5
−
(SEQ ID NO: 1358)


1698
MYRSPSDFENL
1360.5
−
(SEQ ID NO: 1359)


1699
MYRSPSDYENL
1376.3
−
(SEQ ID NO: 1360)


1700
MYRSPSDWENL
1399.6
−
(SEQ ID NO: 1361)


1702
MYRSPSEPENL
1324.5
−
(SEQ ID NO: 1362)


1703
MYRSPSEFENL
1374.5
−
(SEQ ID NO: 1363)


1704
MYRSPSEYENL
1390.5
−
(SEQ ID NO: 1364)


1705
MYRSSENPENL
1413.6
−
(SEQ ID NO: 1365)


1707
MYRSPSNPENL
1309.6
−
(SEQ ID NO: 1366)


1708
MYRSPSNFENL
1359.6
−
(SEQ ID NO: 1367)


1709
MYRSPSNYENL
1375.6
−
(SEQ 16 NO: 1368)


1710
MYRSPSNWENL
1398.7
−
(SEQ ID NO: 1369)


1712
MYRSPSQPENL
1323.6
−
(SEQ ID NO: 1370)


1713
MYRSPSQFENL
1373.6
−
(SEQ ID NO: 1371)


1714
MYRSPSQYENL
1389.6
−
(SEQ ID NO: 1372)


1715
MYRSPSQWENL
1412.7
−
(SEQ ID NO: 1373)


1717
MYRSPSHPENL
1332.6
−
(SEQ ID NO: 1374)


1718
MYRSPSFFENL
1382.6
−
(SEQ ID NO: 1375)


1719
MYRSPSHYENL
1398.6
−
(SEQ ID NO: 1376)


1720
MYRSPSHWENL
1421.7
−
(SEQ ID NO: 1377)


1722
MYSSPSMPENL
1258.4
−
(SEQ ID NO: 1378)


1723
MYSSPSMFENL
1308.4
−
(SEQ ID NO: 1379)


1724
MYSSPSMYENL
1324.4
−
(SEQ ID NO: 1380)


1725
MYSSPSMWENL
1347.5
−
(SEQ ID NO: 1381)


1727
MYSSPSFPENL
1274.3
−
(SEQ ID NO: 1382)


1728
MYSSPSFFENL
1324.3
−
(SEQ ID NO: 1383)


1729
MYSSPSFYENL
1340.3
−
(SEQ ID NO: 1384)


1730
MYSSPSFWENL
1363.4
−
(SEQ ID NO: 1385)


1732
MYSSPSYPENL
1290.3
−
(SEQ ID NO: 1386)


1733
MYSSPSYFENL
1340.3
−
(SEQ ID NO: 1387)


1734
MYSSPSYYENL
1356.3
−
(SEQ ID NO: 1388)


1735
MYSSPSYWENL
1379.4
−
(SEQ ID NO: 1389)


1737
MYSSPSDPENL
1242.2
−
(SEQ ID NO: 1390)


1738
MYSSPSDFENL
1292.2
−
(SEQ ID NO: 1391)


1739
MYSSPSDYENL
1308.2
−
(SEQ ID NO: 1392)


1740
MYSSPSDWENL
1331.3
−
(SEQ ID NO: 1393)


1742
MYSSPSEPENL
1256.2
−
(SEQ ID NO: 1394)


1743
MYSSPSEFENL
1306.2
−
(SEQ ID NO: 1395)


1744
MYSSPSEYENL
1322.2
−
(SEQ ID NO: 1396)


1745
MYSSPSEWENL
1345.3
−
(SEQ ID NO: 1397)


1747
MYSSPSNPENL
1241.3
−
(SEQ ID NO: 1398)


1748
MYSSPSNFENL
1291.3
−
(SEQ ID NO: 1399)


1749
MYSSPSNYENL
1307.3
−
(SEQ ID NO: 1400)


1750
MYSSPSNWENL
1330.4
−
(SEQ ID NO: 1401)


1752
MYSSPSQPENL
1255.3
−
(SEQ ID NO: 1402)


1753
MYSSPSQFENL
1305.3
−
(SEQ ID NO: 1403)


1754
MYSSPSQYENL
1321.3
−
(SEQ ID NO: 1404)


1755
MYSSPSQWENL
1344.4
−
(SEQ ID NO: 1405)


1757
MYSSPSHPENL
1264.3
−
(SEQ ID NO: 1406)


1758
MYSSPSHFENL
1314.3
−
(SEQ ID NO: 1407)


1759
MYSSPSHYENL
1330.3
−
(SEQ ID NO: 1408)


1760
MYSSPSHWENL
1353.4
−
(SEQ ID NO: 1409)


1762
MYTSPSMPENL
1271.6
−
(SEQ ID NO: 1410)


1763
MYTSPSMFENL
1321.6
−
(SEQ ID NO: 1411)


1764
MYTSPSMYENL
1337.6
−
(SEQ ID NO: 1412)


1765
MYTSPSMWENL
1360.7
−
(SEQ ID NO: 1413)


1767
MYTSPSFPENL
1287.5
−
(SEQ ID NO: 1414)


1768
MYTSPSFFENL
1337.5
−
(SEQ ID NO: 1415)


1769
MYTSPSFYENL
1353.5
−
(SEQ ID NO: 1416)


1770
MYTSPSFWENL
1376.6
−
(SEQ ID NO: 1417)


1772
MYTSPSYPENL
1303.5
−
(SEQ ID NO: 1418)


1773
MYTSPSYFENL
1353.5
−
(SEQ ID NO: 1419)


1774
MYTSPSYYENL
1369.5
−
(SEQ ID NO: 1420)


1775
MYTSPSYWENL
1392.6
−
(SEQ ID NO: 1421)


1777
MYTSPSDPENL
1255.4
−
(SEQ ID NO: 1422)


1778
MYTSPSDFENL
1305.4
−
(SEQ ID NO: 1423)


1779
MYTSPSDYENL
1321.4
−
(SEQ ID NO: 1424)


1780
MYTSPSDWENL
1344.5
−
(SEQ ID NO: 1425)


1782
MYTSPSEPENL
1269.4
−
(SEQ ID NO: 1426)


1783
MYTSPSEFENL
1319.4
−
(SEQ ID NO: 1427)


1784
MYTSPSEYENL
1335.4
−
(SEQ ID NO: 1428)


1785
MYTSPSEWENL
1358.5
−
(SEQ ID NO: 1429)


1787
MYTSPSNPENL
1254.5
−
(SEQ ID NO: 1430)


1788
MYTSPSNFENL
1304.5
−
(SEQ ID NO: 1431)


1789
MYTSPSNYENL
1320.5
−
(SEQ ID NO: 1432)


1790
MYTSPSNWENL
1343.6
−
(SEQ ID NO: 1433)


1792
MYTSPSQPENL
1268.5
−
(SEQ ID NO: 1434)


1793
MYTSPSQFENL
1318.5
−
(SEQ ID NO: 1435)


1794
MYTSPSQYENL
1334.5
−
(SEQ ID NO: 1436)


1795
MYTSPSQWENL
1357.6
−
(SEQ ID NO: 1437)


1797
MYTSPSEPENL
1277.5
−
(SEQ ID No: 1438)


1798
MYTSPSHFENL
1327.5
−
(SEQ ID NO: 1439)


1799
MYTSPSHYENL
1343.5
−
(SEQ ID NO: 1440)


1800
MYTSPSHWENL
1366.6
−
(SEQ ID NO: 1441)


1802
MYHSPSMPENL
1307.7
−
(SEQ ID NO: 1442)


1803
MYHSPSMFENL
1357.7
−
(SEQ ID NO: 1443)


1804
MYHSPSMYENL
1373.7
−
(SEQ ID No: 1444)


1805
MYHSPSMWENL
1396.8
−
(SEQ ID NO: 1445)


1807
MYHSPSFPENL
1323.6
−
(SEQ ID NO: 1446)


1808
MYHSPSFFENL
1373.6
−
(SEQ ID NO: 1447)


1809
MYHSTSFYENL
1389.6
−
(SEQ ID NO: 1448)


1810
MYHSPSFWENL
1412.7
−
(SEQ ID NO: 1449)


1812
MYHSPSYPENL
1339.6
−
(SEQ ID NO: 1450)


1813
MYHSPSYFENL
1389.6
−
(SEQ ID NO: 1451)


1814
MYHSPSYYENL
1405.6
−
(SEQ ID NO: 1452)


1815
MYHSPSYWENL
1428.7
−
(SEQ ID NO: 1453)


1817
MYHSPSDPENL
1291.5
−
(SEQ ID NO: 1454)


1818
MYHSPSDFENL
1341.5
−
(SEQ ID NO: 1455)


1819
MYHSPSDYENL
1357.5
−
(SEQ ID NO: 1456)


1820
MYHSPSDWENL
1380.6
−
(SEQ ID NO: 1457)


1822
MYHSPSEPENL
1305.5
−
(SEQ ID NO: 1458)


1823
MYHSPSEFENL
1355.5
−
(SEQ ID NO: 1459)


1824
MYHSPSEYENL
1371.5
−
(SEQ ID NO: 1460)


1825
MYHSPSEWENL
1394.6
−
(SEQ ID NO: 1461)


1827
MYHSPSNPENL
1290.6
−
(SEQ ID NO: 1462)


1828
MYHSPSNFENL
1340.6
−
(SEQ ID NO: 1463)


1829
MYHSPSNYENL
1356.6
−
(SEQ ID NO: 1464)


1830
MYHSPSNWENL
1379.7
−
(SEQ ID NO: 1465)


1832
MYHSPSQPENL
1304.6
−
(SEQ ID NO: 1466)


1833
MYHSPSQFENL
1354.6
−
(SEQ ID NO: 1467)


1834
MYHSPSQYENL
1370.6
−
(SEQ ID NO: 1468)


1835
MYHSPSQWENL
1393.7
−
(SEQ ID NO: 1469)


1837
MYHSPSHPENL
1313.6
−
(SEQ ID NO: 1470)


1838
MYHSPSHFENL
1363.6
−
(SEQ ID NO: 1471)


1839
MYHSPSHYENL
1379.6
−
(SEQ ID NO: 1472)


1840
MYHSPSHWENL
1402.7
−
(SEQ ID NO: 1473)


1842
MYNSPSMPENL
1284.7
−
(SEQ ID NO: 1474)


1843
MYNSPSMFENL
1334.7
−
(SEQ ID NO: 1475)


1844
MYNSPSMYENL
1350.7
−
(SEQ ID NO: 1476)


1845
MYNSPSMWENL
1373.8
−
(SEQ ID NO: 1477)


1847
MYNSPSFPENL
1300.6
−
(SEQ ID NO: 1478)


1848
MYNSPSFFENL
1350.6
−
(SEQ ID NO: 1479)


1849
MYNSPSFYENL
1366.6
−
(SEQ ID NO: 1480)


1850
MYNSPSFWENL
1389.7
−
(SEQ ID NO: 1481)


1852
MYNSPSYPENL
1316.6
−
(SEQ ID NO: 1482)


1853
MYNSPSYFENL
1366.6
−
(SEQ ID NO: 1483)


1854
MYNSPSYYENL
1382.6
−
(SEQ ID NO: 1484)


1855
MYNSPSYWENL
1405.7
−
(SEQ ID NO: 1485)


1857
MYNSPSDPENL
1268.5
−
(SEQ ID NO: 1486)


1858
MYNSPSDFENL
1318.5
−
(SEQ ID NO: 1487)


1859
MYNSPSDYENL
1334.5
−
(SEQ ID NO: 1488)


1860
MYNSPSDWENL
1357.6
−
(SEQ ID NO: 1489)


1862
MYNSPSEPENL
1282.5
−
(SEQ ID NO: 1490)


1863
MYNSPSEFENL
1332.5
−
(SEQ ID NO: 1491)


1864
MYNSPSEYENL
1348.5
−
(SEQ ID NO: 1492)


1865
MYNSPSEWENL
1371.6
−
(SEQ ID NO: 1493)


1867
MYNSPSNPENL
1267.6
−
(SEQ ID NO: 1494)


1868
MYNSPSNFENL
1317.6
−
(SEQ ID NO: 1495)


1869
MYNSPSNYENL
1333.6
−
(SEQ ID NO: 1496)


1870
MYNSPSNWENL
1356.7
−
(SEQ ID NO: 1497)


1872
MYNSPSQPENL
1281.6
−
(SEQ ID NO: 1498)


1873
MYNSPSQFENL
1331.6
−
(SEQ ID NO: 1499)


1874
MYNSPSQYENL
1347.6
−
(SEQ ID NO: 1500)


1875
MYNSPSQWENL
1370.7
−
(SEQ ID NO: 1501)


1877
MYNSPSHPENL
1290.6
−
(SEQ ID NO: 1502)


1878
MYNSPSHFENL
1340.6
−
(SEQ ID NO: 1503)


1879
MYNSPSHYENL
1356.6
−
(SEQ ID NO: 1504)


1880
MYNSPSHWENL
1379.7
−
(SEQ ID NO: 1505)


1882
MYGSPSMPENL
1227.6
−
(SEQ ID NO: 1506)


1883
MYGSPSMFENL
1277.6
−
(SEQ ID NO: 1507)


1884
MYGSPSMYENL
1293.6
−
(SEQ ID NO: 1508)


1885
MYGSPSMWENL
1316.7
−
(SEQ ID NO: 1509)


1887
MYGSPSFPENL
1243.5
−
(SEQ ID NO: 1510)


1888
MYGSPSFFENL
1293.5
−
(SEQ ID NO: 1511)


1889
MYGSPSFYENL
1309.5
−
(SEQ ID NO: 1512)


1890
MYGSPSFWENL
1332.6
−
(SEQ ID NO: 1513)


1892
MYGSPSYPENL
1259.5
−
(SEQ ID NO: 1514)


1893
MYGSPSYFENL
1309.5
−
(SEQ ID NO: 1515)


1894
MYGSPSYYENL
1325.5
−
(SEQ ID NO: 1516)


1895
MYGSPSYWENL
1348.6
−
(SEQ ID NO: 1517)


1897
MYGSPSDPENL
1211.4
−
(SEQ ID NO: 1518)


1898
MYGSPSDFENL
1261.4
−
(SEQ ID NO: 1519)


1899
MYGSPSDYENL
1277.4
−
(SEQ ID NO: 1520)


1900
MYGSPSDWENL
1300.5
−
(SEQ ID NO: 1521)


1902
MYGSPSEPENL
1225.4
−
(SEQ ID NO: 1522)


1903
MYGSPSEFENL
1275.4
−
(SEQ ID NO: 1523)


1904
MYGSPSEYENL
1291.4
−
(SEQ ID NO: 1524)


1905
MYGSPSEWENL
1314.5
−
(SEQ ID NO: 1525)


1907
MYGSPSNPENL
1210.5
−
(SEQ ID NO: 1526)


1908
MYGSPSNFENL
1260.5
−
(SEQ ID NO: 1527)


1909
MYGSPSNYENL
1276.5
−
(SEQ ID NO: 1528)


1910
MYGSPSNWENL
1299.6
−
(SEQ ID NO: 1529)


1912
MYGSPSQPENL
1224.5
−
(SEQ ID NO: 1530)


1913
MYGSPSQFENL
1274.5
−
(SEQ ID NO: 1531)


1914
MYGSPSQYENL
1290.5
−
(SEQ ID NO: 1532)


1915
MYGSPSQWENL
1313.6
−
(SEQ ID NO: 1533)


1917
MYGSPSHPENL
1233.5
−
(SEQ ID NO: 1534)


1918
MYGSPSHFENL
1283.5
−
(SEQ ID NO: 1535)


1919
MYGSPSHYENL
1299.5
−
(SEQ ID NO: 1536)


1920
MYGSPSHWENL
1322.6
−
(SEQ ID NO: 1537)


1922
MYASPSMPENL
1241.6
−
(SEQ ID NO: 1538)


1923
MYASPSMFENL
1291.6
−
(SEQ ID NO: 1539)


1924
MYASPSMYENL
1307.6
−
(SEQ ID NO: 1540)


1925
MYASPSMWENL
1330.7
−
(SEQ ID NO: 1541)


1927
MYASPSFPENL
1257.5
−
(SEQ ID NO: 1542)


1928
MYASPSFFENL
1307.5
−
(SEQ ID NO: 1543)


1929
MYASPSFYENL
1323.5
−
(SEQ ID NO: 1544)


1930
MYASPSFWENL
1346.6
−
(SEQ ID NO: 1545)


1932
MYASPSYPENL
1273.5
−
(SEQ ID NO: 1546)


1933
MYASPSYFENL
1323.5
−
(SEQ ID NO: 1547)


1934
MYASPSYYENL
1339.5
−
(SEQ ID NO: 1548)


1935
MYASPSYWENL
1362.6
−
(SEQ ID NO: 1549)


1937
MYASPSDPENL
1225.4
−
(SEQ ID NO: 1550)


1938
MYASPSDFENL
1275.4
−
(SEQ ID NO: 1551)


1939
MYASPSDYENL
1291.4
−
(SEQ ID NO: 1552)


1940
MYASPSDWENL
1314.5
−
(SEQ ID NO: 1553)


1942
MYASPSEPENL
1239.4
−
(SEQ ID NO: 1554)


1943
MYASPSEFENL
1289.4
−
(SEQ ID NO: 1555)


1944
MYASPSEYENL
1305.4
−
(SEQ ID NO: 1556)


1945
MYASPSEWENL
1328.5
−
(SEQ ID NO: 1557)


1947
MYASPSNPENL
1224.5
−
(SEQ ID NO: 1558)


1948
MYASPSNFENL
1274.5
−
(SEQ ID NO: 1559)


1949
MYASPSNYENL
1290.5
−
(SEQ ID NO: 1560)


1950
MYASPSNWENL
1313.6
−
(SEQ ID NO: 1561)


1952
MYASPSQPENL
1238.5
−
(SEQ ID NO: 1562)


1953
MYASPSQFENL
1288.5
−
(SEQ ID NO: 1563)


1954
MYASPSQYENL
1304.5
−
(SEQ ID NO: 1564)


1955
MYASPSQWENL
1327.6
−
(SEQ ID NO: 1565)


1957
MYASPSHPENL
1247.5
−
(SEQ ID NO: 1566)


1958
MYASPSHFENL
1297.5
−
(SEQ ID NO: 1567)


1959
MYASPSHYENL
1313.5
−
(SEQ ID NO: 1568)


1960
MYASPSHWENL
1336.6
−
(SEQ ID NO: 1569)


1962
MFRSPSMPENL
1310.7
−
(SEQ ID NO: 1570)


1963
MFRSPSMFENL
1360.7
−
(SEQ ID NO: 1571)


1964
MFRSPSMYENL
1376.7
−
(SEQ ID NO: 1572)


1965
MFRSPSMWENL
1399.8
−
(SEQ ID NO: 1573)


1967
MFRSPSFPENL
1326.6
−
(SEQ ID NO: 1574)


1968
MFRSPSFFENL
1376.6
−
(SEQ ID NO: 1575)


1969
MFRSPSFYENL
1392.6
−
(SEQ ID NO: 1576)


1970
MFRSPSFWENL
1415.7
−
(SEQ ID NO: 1577)


1972
MFRSPSYPENL
1342.6
−
(SEQ ID NO: 1578)


1973
MFRSPSYFENL
1392.6
−
(SEQ ID NO: 1579)


1974
MFRSPSYYENL
1408.6
−
(SEQ ID NO: 1580)


1975
MFRSPSYWENL
1431.7
−
(SEQ ID NO: 1581)


1977
MFRSPSDPENL
1294.5
−
(SEQ ID NO: 1582)


1978
MFRSPSDFENL
1344.5
−
(SEQ ID NO: 1583)


1979
MFRSPSDYENL
1360.5
−
(SEQ ID NO: 1584)


1980
MFRSPSDWENL
1383.6
−
(SEQ ID NO: 1585)


1982
MFRSPSEPENL
1308.5
−
(SEQ ID NO: 1586)


1983
MFRSPSEFENL
1358.5
−
(SEQ ID NO: 1587)


1984
MFRSPSEYENL
1374.5
−
(SEQ ID NO: 1588)


1985
MFRSPSEWENL
1397.6
−
(SEQ ID NO: 1589)


1987
MFRSPSNPENL
1293.6
−
(SEQ ID NO: 1590)


1988
MFRSPSNFENL
1343.6
−
(SEQ ID NO: 1591)


1989
MFRSPSNYENL
1359.6
−
(SEQ ID NO: 1592)


1990
MFRSPSNWENL
1382.7
−
(SEQ ID NO: 1593)


1992
MFRSPSQPENL
1307.6
−
(SEQ ID NO: 1594)


1993
MFRSPSQFENL
1357.6
−
(SEQ ID NO: 1595)


1994
MFRSPSQYETL
1373.6
−
(SEQ ID NO: 1596)


1995
MFRSPSQWENL
1396.7
−
(SEQ ID NO: 1597)


1997
MFRSPSHPENL
1316.6
−
(SEQ ID NO: 1598)


1998
MFRSPSHFENL
1366.6
−
(SEQ ID NO: 1599)


1999
MFRSPSHYENL
1382.6
−
(SEQ ID NO: 1600)


2000
MFRSPSHWENL
1405.7
−
(SEQ ID NO: 1601)


2002
MFSSPSMPENL
1242.4
−
(SEQ ID NO: 1602)


2003
MFSSPSMFENL
1292.4
−
(SEQ ID NO: 1603)


2004
MFSSPSMYENL
1308.4
−
(SEQ ID NO: 1604)


2005
MFSSPSMWENL
1331.5
−
(SEQ ID NO: 1605)


2007
MFSSPSFPENL
1258.3
−
(SEQ ID NO: 1606)


2008
MFSSPSFFENL
1308.3
−
(SEQ ID NO: 1607)


2009
MFSSPSFYENL
1324.3
−
(SEQ ID NO: 1608)


2010
MFSSPSFWENL
1347.4
−
(SEQ ID NO: 1609)


2012
MFSSPSYPENL
1274.3
−
(SEQ ID NO: 1610)


2013
MFSSPSYFENL
1324.3
−
(SEQ ID NO: 1611)


2014
MFSSPSYYENL
1340.3
−
(SEQ ID NO: 1612)


2015
MFSSPSYWENL
1363.4
−
(SEQ ID NO: 1613)


2017
MFSSPSDPENL
1226.2
−
(SEQ ID NO: 1614)


2018
MFSSPSDFENL
1276.2
−
(SEQ ID NO: 1615)


2019
MFSSPSDYENL
1292.2
−
(SEQ ID NO: 1616)


2020
MFSSPSDWENL
1315.3
−
(SEQ ID NO: 1617)


2022
MFSSPSEPENL
1240.2
−
(SEQ ID NO: 1618)


2023
MFSSPSEFENL
1290.2
−
(SEQ ID NO: 1619)


2024
MFSSPSEYENL
1306.2
−
(SEQ ID NO: 1620)


2025
MFSSPSEWENL
1329.3
−
(SEQ ID NO: 1621)


2027
MFSSPSNPENL
1225.3
−
(SEQ ID NO: 1622)


2028
MFSSPSNFENL
1275.3
−
(SEQ ID NO: 1623)


2029
MFSSPSNYENL
1291.3
−
(SEQ ID NO: 1624)


2030
MFSSPSNWENL
1314.4
−
(SEQ ID NO: 1625)


2032
MFSSPSQPENL
1239.3
−
(SEQ ID NO: 1626)


2033
MFSSPSQFENL
1289.3
−
(SEQ ID NO: 1627)


2034
MFSSPSQYENL
1305.3
−
(SEQ ID NO: 1628)


2035
MFSSPSQWENL
1328.4
−
(SEQ ID NO: 1629)


2037
MFSSPSHPENL
1248.3
−
(SEQ ID NO: 1630)


2038
MFSSPSHFENL
1298.3
−
(SEQ ID NO: 1631)


2039
MFSSPSHYENL
1314.3
−
(SEQ ID NO: 1632)


2040
MFSSPSHWENL
1337.4
−
(SEQ ID NO: 1633)


2042
MFTSPSMPENL
1255.6
−
(SEQ ID NO: 1634)


2043
MFTSPSMFENL
1305.6
−
(SEQ ID NO: 1635)


2044
MFTSPSMYENL
1321.6
−
(SEQ ID NO: 1636)


2045
MFTSPSMWENL
1344.7
−
(SEQ ID NO: 1637)


2047
MFTSPSFPENL
1271.5
−
(SEQ ID NO: 1638)


2048
MFTSPSFFENL
1321.5
−
(SEQ ID NO: 1639)


2049
MFTSPSFYENL
1337.5
−
(SEQ ID NO: 1640)


2050
MFTSPSFWENL
1360.6
−
(SEQ ID NO: 1641)


2052
MFTSPSYPENL
1287.5
−
(SEQ ID NO: 1642)


2053
MFTSPSYFENL
1337.5
−
(SEQ ID NO: 1643)


2054
MFTSPSYYENL
1353.5
−
(SEQ ID NO: 1644)


2055
MFTSPSYWENL
1376.6
−
(SEQ ID NO: 1645)


2057
MFTSPSDPENL
1239.4
−
(SEQ ID NO: 1646)


2058
MFTSPSDFENL
1289.4
−
(SEQ ID NO: 1647)


2059
MFTSPSDYENL
1305.4
−
(SEQ ID NO: 1648)


2060
MFTSPSDWENL
1328.5
−
(SEQ ID NO: 1649)


2062
MFTSPSEPENL
1253.4
−
(SEQ ID NO: 1650)


2063
MFTSPSEFENL
1303.4
−
(SEQ ID NO: 1651)


2064
MFTSPSEYENL
1319.4
−
(SEQ ID NO: 1652)


2065
MFTSPSEWENL
1342.5
−
(SEQ ID NO: 1653)


2067
MFTSPSNPENL
1238.5
−
(SEQ ID NO: 1654)


2068
MFTSPSNFENL
1288.5
−
(SEQ ID NO: 1655)


2069
MFTSPSNYENL
1304.5
−
(SEQ ID NO: 1656)


2070
MFTSPSNWENL
1327.6
−
(SEQ ID NO: 1657)


2072
MFTSPSQPENL
1252.5
−
(SEQ ID NO: 1658)


2073
MFTSPSQFENL
1302.5
−
(SEQ ID NO: 1659)


2074
MFTSPSQYENL
1318.5
−
(SEQ ID NO: 1660)


2075
MFTSPSQWENL
1341.6
−
(SEQ ID NO: 1661)


2077
MFTSPSHPENL
1261.5
−
(SEQ ID NO: 1662)


2078
MFTSPSHFENL
1311.5
−
(SEQ ID NO: 1663)


2079
MFTSPSHYENL
1327.5
−
(SEQ ID NO: 1664)


2080
MFTSPSHWENL
1350.6
−
(SEQ ID NO: 1665)


2082
MFHSPSMPENL
1291.7
−
(SEQ ID NO: 1666)


2083
MFHSPSMFENL
1341.7
−
(SEQ ID NO: 1667)


2084
MFHSPSMYENL
1357.7
−
(SEQ ID NO: 1668)


2085
MFHSPSMWENL
1380.8
−
(SEQ ID NO: 1669)


2087
MFHSPSFPENL
1307.6
−
(SEQ ID NO: 1670)


2088
MFHSPSFFENL
1357.6
−
(SEQ ID NO: 1671)


2089
MFHSPSFYENL
1373.6
−
(SEQ ID NO: 1672)


2090
MFHSPSFWENL
1396.7
−
(SEQ ID NO: 1673)


2092
MFHSPSYPENL
1323.6
−
(SEQ ID NO: 1674)


2093
MFHSPSYFENL
1373.6
−
(SEQ ID NO: 1675)


2094
MFHSPSYYENL
1389.6
−
(SEQ ID NO: 1676)


2095
MFHSPSYWENL
1412.7
−
(SEQ ID NO: 1677)


2097
MFHSPSDPENL
1275.5
−
(SEQ ID NO: 1678)


2098
MFHSPSDFENL
1325.5
−
(SEQ ID NO: 1679)


2099
MFHSPSDYENL
1341.5
−
(SEQ ID NO: 1680)


2100
MFHSPSDWENL
1364.6
−
(SEQ ID NO: 1681)


2102
MFHSPSEPENL
1289.5
−
(SEQ ID NO: 1682)


2103
MFHSPSEFENL
1339.5
−
(SEQ ID NO: 1683)


2104
MFHSPSEYENL
1355.5
−
(SEQ ID NO: 1684)


2105
MFHSPSEWENL
1378.6
−
(SEQ ID NO: 1685)


2107
MFHSPSNPENL
1274.6
−
(SEQ ID NO: 1686)


2108
MFHSPSNFENL
1324.6
−
(SEQ ID NO: 1687)


2109
MFHSPSNYENL
1340.6
−
(SEQ ID NO: 1688)


2110
MFHSPSNWENL
1363.7
−
(SEQ ID NO: 1689)


2112
MFHSPSQPENL
1288.6
−
(SEQ ID NO: 1690)


2113
MFHSPSQFENL
1338.6
−
(SEQ ID NO: 1691)


2114
MFHSPSQYENL
1354.6
−
(SEQ ID NO: 1692)


2115
MFHSPSQWENL
1377.7
−
(SEQ ID NO: 1693)


2117
MFHSPSHPENL
1297.6
−
(SEQ ID NO: 1694)


2118
MFHSPSHFENL
1347.6
−
(SEQ ID NO: 1695)


2119
MFHSPSHYENL
1363.6
−
(SEQ ID NO: 1696)


2120
MFHSPSHWENL
1386.7
−
(SEQ ID NO: 1697)


2122
MFNSPSMPENL
1268.7
−
(SEQ ID NO: 1698)


2123
MFNSPSMFENL
1318.7
−
(SEQ ID NO: 1699)


2124
MFNSPSMYENL
1334.7
−
(SEQ ID NO: 1700)


2125
MFNSPSMWENL
1357.8
−
(SEQ ID NO: 1701)


2127
MFNSPSFPENL
1284.6
−
(SEQ ID NO: 1702)


2128
MFNSPSFFENL
1334.6
−
(SEQ ID NO: 1703)


2129
MFNSPSFYENL
1350.6
−
(SEQ ID NO: 1704)


2130
MFNSPSFWENL
1373.7
−
(SEQ ID NO: 1705)


2132
MFNSPSYPENL
1300.6
−
(SEQ ID NO: 1706)


2133
MFNSPSYFENL
1350.6
−
(SEQ ID NO: 1707)


2134
MFNSPSYYENL
1366.6
−
(SEQ ID NO: 1708)


2135
MFNSPSYWENL
1389.7
−
(SEQ ID NO: 1709)


2137
MFNSPSDPENL
1252.5
−
(SEQ ID NO: 1710)


2138
MFNSPSDFENL
1302.5
−
(SEQ ID NO: 1711)


2139
MFNSPSDYENL
1318.5
−
(SEQ ID NO: 1712)


2140
MFNSPSDWENL
1341.6
−
(SEQ ID NO: 1713)


2142
MFNSPSEPENL
1266.5
−
(SEQ ID NO: 1714)


2143
MFNSPSEFENL
1316.5
−
(SEQ ID NO: 1715)


2144
MFNSPSEYENL
1332.5
−
(SEQ ID NO: 1716)


2145
MFNSPSEWENL
1355.6
−
(SEQ ID NO: 1717)


2147
MFNSPSNPENL
1251.6
−
(SEQ ID NO: 1718)


2148
MFNSPSNFENL
1301.6
−
(SEQ ID NO: 1719)


2149
MFNSPSNYENL
1317.6
−
(SEQ ID NO: 1720)


2150
MFNSPSNWENL
1340.7
−
(SEQ ID NO: 1721)


2152
MFNSPSQPENL
1265.6
−
(SEQ ID NO: 1722)


2153
MFNSPSQFENL
1315.6
−
(SEQ ID NO: 1723)


2154
MFNSPSQYENL
1331.6
−
(SEQ ID NO: 1724)


2155
MFNSPSQWENL
1354.7
−
(SEQ ID NO: 1725)


2157
MFNSPSHPENL
1274.6
−
(SEQ ID NO: 1726)


2158
MFNSPSHFENL
1324.6
−
(SEQ ID NO: 1727)


2159
MFNSPSHYENL
1340.6
−
(SEQ ID NO: 1728)


2160
MFNSPSHWENL
1363.7
−
(SEQ ID NO: 1729)


2162
MFGSPSMPENL
1211.6
−
(SEQ ID NO: 1730)


2163
MFGSPSMFENL
1261.6
−
(SEQ ID NO: 1731)


2164
MFGSPSMYENL
1277.6
−
(SEQ ID NO: 1732)


2165
MFGSPSMWENL
1300.7
−
(SEQ ID NO: 1733)


2167
MFGSPSFPENL
1227.5
−
(SEQ ID NO: 1734)


2168
MFGSPSFFENL
1277.5
−
(SEQ ID NO: 1735)


2169
MFGSPSFYENL
1293.5
−
(SEQ ID NO: 1736)


2170
MFGSPSFWENL
1316.6
−
(SEQ ID NO: 1737)


2172
MFGSPSYPENL
1243.5
−
(SEQ ID NO: 1738)


2173
MFGSPSYFENL
1293.5
−
(SEQ ID NO: 1739)


2174
MFGSPSYYENL
1309.5
−
(SEQ ID NO: 1740)


2175
MFGSPSYWENL
1332.6
−
(SEQ ID NO: 1741)


2177
MFGSPSDPENL
1195.4
−
(SEQ ID NO: 1742)


2178
MFGSPSDFENL
1245.4
−
(SEQ ID NO: 1743)


2179
MFGSPSDYENL
1261.4
−
(SEQ ID NO: 1744)


2180
MFGSPSDWENL
1284.5
−
(SEQ ID NO: 1745)


2182
MFGSPSEPENL
1209.4
−
(SEQ ID NO: 1746)


2183
MFGSPSEFENL
1259.4
−
(SEQ ID NO: 1747)


2184
MFGSPSEYENL
1275.4
−
(SEQ ID NO: 1748)


2185
MFGSPSEWENL
1298.5
−
(SEQ ID NO: 1749)


2187
MFGSPSNPENL
1194.5
−
(SEQ ID NO: 1750)


2188
MFGSPSNFENL
1244.5
−
(SEQ ID NO: 1751)


2189
MFGSPSNYENL
1260.5
−
(SEQ ID NO: 1752)


2190
MFGSPSNWENL
1283.6
−
(SEQ ID NO: 1753)


2192
MFGSPSQPENL
1208.5
−
(SEQ ID NO: 1754)


2193
MFGSPSQFENL
1258.5
−
(SEQ ID NO: 1755)


2194
MFGSPSQYENL
1274.5
−
(SEQ ID NO: 1756)


2195
MFGSPSQWENL
1297.6
−
(SEQ ID NO: 1757)


2197
MFGSPSHPENL
1217.5
−
(SEQ ID NO: 1758)


2198
MFGSPSHFENL
1267.5
−
(SEQ ID NO: 1759)


2199
MFGSPSHYENL
1283.5
−
(SEQ ID NO: 1760)


2200
MFGSPSHWENL
1306.6
−
(SEQ ID NO: 1761)


2202
MFASPSMPENL
1225.6
−
(SEQ ID NO: 1762)


2203
MFASPSMFENL
1275.6
−
(SEQ ID NO: 1763)


2204
MFASPSMYENL
1291.6
−
(SEQ ID NO: 1764)


2205
MFASPSMWENL
1314.7
−
(SEQ ID NO: 1765)


2207
MFASPSFPENL
1241.5
−
(SEQ ID NO: 1766)


2208
MFASPSFFENL
1291.5
−
(SEQ ID NO: 1767)


2209
MFASPSFYENL
1307.5
−
(SEQ ID NO: 1768)


2210
MFASPSFWENL
1330.6
−
(SEQ ID NO: 1769)


2212
MFASPSYPENL
1257.5
−
(SEQ ID NO: 1770)


2213
MFASPSYFENL
1307.5
−
(SEQ ID NO: 1771)


2214
MFASPSYYENL
1323.5
−
(SEQ ID NO: 1772)


2215
MFASPSYWENL
1346.6
−
(SEQ ID NO: 1773)


2217
MFASPSDPENL
1209.4
−
(SEQ ID NO: 1774)


2218
MFASPSDFENL
1259.4
−
(SEQ ID NO: 1775)


2219
MFASPSDYENL
1275.4
−
(SEQ ID NO: 1776)


2220
MFASPSDWENL
1298.5
−
(SEQ ID NO: 1777)


2222
MFASPSEPENL
1223.4
−
(SEQ ID NO: 1778)


2223
MFASPSEFENL
1273.4
−
(SEQ ID NO: 1779)


2224
MFASPSEYENL
1289.4
−
(SEQ ID NO: 1780)


2225
MFASPSEWENL
1312.5
−
(SEQ ID NO: 1781)


2227
MFASPSNPENL
1208.5
−
(SEQ ID NO: 1782)


2228
MFASPSNFENL
1258.5
−
(SEQ ID NO: 1783)


2229
MFASPSNYENL
1274.5
−
(SEQ ID NO: 1784)


2230
MFASPSNWENL
1297.6
−
(SEQ ID NO: 1785)


2232
MFASPSQPENL
1222.5
−
(SEQ ID NO: 1786)


2233
MFASPSQFENL
1272.5
−
(SEQ ID NO: 1787)


2234
MFASPSQYENL
1288.5
−
(SEQ ID NO: 1788)


2235
MFASPSQWENL
1311.6
−
(SEQ ID NO: 1789)


2237
MFASPSHPENL
1231.5
−
(SEQ ID NO: 1790)


2238
MFASPSHFENL
1281.5
−
(SEQ ID NO: 1791)


2239
MFASPSHYENL
1297.5
−
(SEQ ID NO: 1792)


2240
MFASPSHWENL
1320.6
−
(SEQ ID NO: 1793)


2242
RYSLPPELSNM
1308.6
−
(SEQ ID NO: 1794)


2243
AYRSPSMPENL
1266.5
−
(SEQ ID NO: 1795)


2244
RYRSPSMPENL
1351.6
−
(SEQ ID NO: 1796)


2245
NYRSPSMPENL
1309.6
−
(SEQ ID NO: 1797)


2246
DYRSPSMPENL
1310.5
−
(SEQ ID NO: 1798)


2247
CYRSPSMPENL
1298.6
−
(SEQ ID NO: 1799)


2248
QYRSPSMPENL
1323.6
−
(SEQ ID NO: 1800)


2249
EYRSPSMPENL
1324.5
−
(SEQ ID NO: 1801)


2250
GYRSPSMPENL
1252.5
−
(SEQ ID NO: 1802)


2251
HYRSPSMPENL
1332.6
−
(SEQ ID NO: 1803)


2252
IYRSPSMPENL
1308.6
−
(SEQ ID NO: 1804)


2253
LYRSPSMPENL
1308.6
−
(SEQ ID NO: 1805)


2254
KYRSPSMPENL
1323.6
−
(SEQ ID NO: 1806)


2255
MYRSPSMPENL
1326.7
−
(SEQ ID NO: 1807)


2256
FYRSPSMPENL
1342.6
−
(SEQ ID NO: 1808)


2257
PYRSPSMPENL
1292.6
−
(SEQ ID NO: 1809)


2258
SYRSPSMPENL
1283.3
−
(SEQ ID NO: 1810)


2259
TYRSPSMPENL
1296.5
−
(SEQ ID NO: 1811)


2260
WYRSPSMPENL
1381.7
−
(SEQ ID NO: 1812)


2261
YYRSPSMPENL
1358.6
−
(SEQ ID NO: 1813)


2262
VYRSPSMPENL
1294.6
−
(SEQ ID NO: 1814)


2263
LARSPSMPENL
1216.5
−
(SEQ ID NO: 1815)


2264
LRRSPSMPENL
1301.6
−
(SEQ ID NO: 1816)


2265
LNRSPSMPENL
1259.6
−
(SEQ ID NO: 1817)


2266
LDRSPSMPENL
1260.5
−
(SEQ ID NO: 1818)


2267
LCRSPSMPENL
1248.6
−
(SEQ ID NO: 1819)


2268
LQRSPSMPENL
1273.6
−
(SEQ ID NO: 1820)


2269
LERSPSMPENL
1274.5
−
(SEQ ID NO: 1821)


2270
LGRSPSMPENL
1202.5
−
(SEQ ID NO: 1822)


2271
LHRSPSMPENL
1282.6
−
(SEQ ID NO: 1823)


2272
LIRSPSMPENL
1258.6
−
(SEQ ID NO: 1824)


2273
LLRSPSMPENL
1258.6
−
(SEQ ID NO: 1825)


2274
LKRSPSMPENL
1273.6
+
(SEQ ID NO: 1826)


2275
LMRSPSMPENL
1276.7
−
(SEQ ID NO: 1827)


2276
LFRSPSMPENL
1292.6
−
(SEQ ID NO: 1828)


2277
LPRSPSMPENL
1242.6
−
(SEQ ID NO: 1829)


2278
LSRSPSMPENL
1233.3
−
(SEQ ID NO: 1830)


2279
LTRSPSMPENL
1246.5
−
(SEQ ID NO: 1831)


2280
LWRSPSMPENL
1331.7
−
(SEQ ID NO: 1832)


2281
LYRSPSMPENL
1308.6
−
(SEQ ID NO: 1833)


2282
LVRSPSMPENL
1244.6
−
(SEQ ID NO: 1834)


2283
LYASPSMPENL
1223.5
−
(SEQ ID NO: 1835)


2284
LYRSPSMPENL
1308.6
−
(SEQ ID NO: 1836)


2285
LYNSPSMPENL
1266.6
−
(SEQ ID NO: 1837)


2286
LYDSPSMPENL
1267.5
−
(SEQ ID NO: 1838)


2287
LYCSPSMPENL
1255.6
−
(SEQ ID NO: 1839)


2288
LYQSPSMPENL
1280.6
−
(SEQ ID NO: 1840)


2289
LYESPSMPENL
1281.5
−
(SEQ ID NO: 1841)


2290
LYGSPSMPENL
1209.5
−
(SEQ ID NO: 1842)


2291
LYHSPSMPENL
1289.6
−
(SEQ ID NO: 1843)


2292
LYISPSMPENL
1265.6
+
(SEQ ID NO: 1844)


2293
LYLSPSMPENL
1265.6
−
(SEQ ID NO: 1845)


2294
LYKSPSMPENL
1280.6
−
(SEQ ID NO: 1846)


2295
LYMSPSMPENL
1283.7
−
(SEQ ID NO: 1847)


2296
LYFSPSMPENL
1299.6
−
(SEQ ID NO: 1848)


2297
LYPSPSMPENL
1249.6
−
(SEQ ID NO: 1849)


2298
LYSSPSMPENL
1240.3
−
(SEQ ID NO: 1850)


2299
LYTSPSMPENL
1253.5
−
(SEQ ID NO: 1851)


2300
LYWSPSMPENL
1338.7
−
(SEQ ID NO: 1852)


2301
LYYSPSMPENL
1315.6
−
(SEQ ID NO: 1853)


2302
LYVSPSMPENL
1251.6
−
(SEQ ID NO: 1854)


2303
LYRSPSAPENL
1248.4
−
(SEQ ID NO: 1855)


2304
LYRSPSRPENL
1333.5
−
(SEQ ID NO: 1856)


2305
LYRSPSNPENL
1291.5
−
(SEQ ID NO: 1857)


2306
LYRSPSDPENL
1292.4
−
(SEQ ID NO: 1858)


2307
LYRSPSCPENL
1280.5
−
(SEQ ID NO: 1859)


2308
LYRSPSQPENL
1305.5
−
(SEQ ID NO: 1860)


2309
LYRSPSEPENL
1306.4
−
(SEQ ID NO: 1861)


2310
LYRSPSGPENL
1234.4
−
(SEQ ID NO: 1862)


2311
LYRSPSHPENL
1314.5
−
(SEQ ID NO: 1863)


2312
LYRSPSIPENL
1290.5
−
(SEQ ID NO: 1864)


2313
LYRSPSLPENL
1290.5
−
(SEQ ID NO: 1865)


2314
LYRSPSKPENL
1305.5
−
(SEQ ID NO: 1866)


2315
LYRSPSMPENL
1308.6
−
(SEQ ID NO: 1867)


2316
LYRSPSFPENL
1324.5
−
(SEQ ID NO: 1868)


2317
LYRSPSPPENL
1274.5
−
(SEQ ID NO: 1869)


2318
LYRSPSSPENL
1265.2
−
(SEQ ID NO: 1870)


2319
LYRSPSTPENL
1278.4
−
(SEQ ID NO: 1871)


2320
LYRSPSWPENL
1363.6
−
(SEQ ID NO: 1872)


2321
LYRSPSYPENL
1340.5
−
(SEQ ID NO: 1873)


2322
LYRSPSVPENL
1276.5
−
(SEQ ID NO: 1874)


2323
LYRSPSMAENL
1282.5
−
(SEQ ID NO: 1875)


2324
LYRSPSMRENL
1367.6
−
(SEQ ID NO: 1876)


2325
LYRSPSMNENL
1325.6
−
(SEQ ID NO: 1877)


2326
LYRSPSMDENL
1326.5
−
(SEQ ID NO: 1878)


2327
LYRSPSMCENL
1314.6
−
(SEQ ID NO: 1879)


2328
LYRSPSMQENL
1339.6
−
(SEQ ID NO: 1880)


2329
LYRSPSMEENL
1340.5
−
(SEQ ID NO: 1881)


2330
LYRSPSMGENL
1268.5
−
(SEQ ID NO: 1882)


2331
LYRSPSMHENL
1348.6
−
(SEQ ID NO: 1883)


2332
LYRSPSMIENL
1324.6
−
(SEQ ID NO: 1884)


2333
LYRSPSMLENL
1324.6
−
(SEQ ID NO: 1885)


2334
LYRSPSMKENL
1339.6
−
(SEQ ID NO: 1886)


2335
LYRSPSMMENL
1342.7
−
(SEQ ID NO: 1887)


2336
LYRSPSMFENL
1358.6
−
(SEQ ID NO: 1888)


2337
LYRSPSMPENL
1308.6
−
(SEQ ID NO: 1889)


2338
LYRSPSMSENL
1299.3
−
(SEQ ID NO: 1890)


2339
LYRSPSMTENL
1312.5
−
(SEQ ID NO: 1891)


2340
LYRSPSMWENL
1397.7
−
(SEQ ID NO: 1892)


2341
LYRSPSMYENL
1374.6
−
(SEQ ID NO: 1893)


2342
LYRSPSMVENL
1310.6
+
(SEQ ID NO: 1894)









Example 3
G2 Abrogating Peptides of the Invention
The following example describes studies which identified exemplary G2 checkpoint-abrogating peptides of the invention. The following peptides of the invention were synthesized directly on membranes and tested in in vitro phosphorylation (“kination” assays, as described above.







TABLE 2







(SEQ ID NOS 1922-929)


















PEPTIDE
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11





AAA
L
A
R
S
A
S
M
P
E
A
L


(SEQ ID NO 













1296)













 


RANDOMII
R
Y
S
L
P
P
E
L
S
N
M


(SEQ ID NO 1)













 


S216A
L
Y
R
S
P
A
M
P
E
N
L


(SEQ ID NO 













1897)













 


S216P
L
Y
R
S
P
S
M
P
E
N
L


(SEQ ID NO 2)













 


YPN

Y
G
G
P
G
G
G
G
N



(SEQ ID NO 













1895)













 


YG7N

Y
G
G
G
G
G
G
G
N



(SEQ ID NO 













1927)













 


YG6N

Y
G
G
G
G
G
G

N



(SEQ ID NO 













1928)













 


YG5N

Y
G
G
G
G
G


N



(SEQ ID NO 













1929)









These peptides were tested in in vitro kination reactions. The oligopeptides were used as phosphorylation substrates; added kinases are involved in the cell cycle G2 checkpoint. Thus, a substance that inhibits the kination reaction can be a cell cycle 62 checkpoint abrogator. For the detection of the phosphorylation status of substrates in this screening method, isotope-labeled ATP and anti-phospho-peptides antibody can be used.
hChk1; hChk1 fusion proteins (MBP-peptide, GST-peptide), HuCds1/Chk2; HuCds1/Chk2 fusion proteins (MBP-peptide, GST-peptide); or, the cell extract from DNA damaged cells, can be used as the kinases in the screening assay.
The oligopeptides tested as substrates are Y X2 X3 P S X6 X7 X8 N (SEQ ID NO: 1930) (X2 through X9, respectively; the first position (X1) “Y” in this abbreviated nine residue motif corresponds to position X2 in the eleven residue motif, described above) and variations thereof wherein amino acid residues at positions 2 (X2) and position 3 (X3) are Gly, Leu, Ser, or Arg; and the amino acid residue at position 6 through 8 are Gly, Leu, Ser, Met, Pro or Glu. Other tested oligopeptides sequence variations have amino acid residues at position 2 as Gly, Leu, Ser, or Arg; amino acid residues at position 3 as Gly, Leu or Ser; amino acid residues at position 6 as Gly, Met, Pro or Glu; amino acid residues at position 7 as Gly, Leu, or Pro; and, amino acid residues at position 8 as Gly, Met, Ser or Glu. In another variation the residue at position 2 was Arg; position 3 was Ser; position 6 was Met; position 7 was Pro; and, position 8 was Glu.
The cells with the deficient cell cycle G1 checkpoint (such as a human leukemia-derived cell line Jurkat) were treated with a DNA damaging treatment. As the DNA damaging treatment, the cells were treated with bleomycin or other anti-cancer drugs. These drugs were added to the cell culture medium. Alternatively, the cells were irradiated with gamma irradiation. Peptides were added to these cells and the amount of DNA was determined some 10 to 48 hours after the DNA damage. The harvested cells were re-suspended with the solution that includes propidium iodide, RNase and NP-40 and analyzed by flow cytometer. If the oligopeptide “candidate substance” induces cells not to accumulate DNA at G2/M by this analysis, the result is positive and the substance potentially abrogated G2/M checkpoint.
Other screening methods can be used to identify selective inhibitors of the G2 cell cycle checkpoint. For, the cells are simultaneously treated with an oligopeptide “candidate phosphorylation substrate” and an M phase checkpoint activator, such as colchicine or nocodazol. The DNA content of the cells are analyzed some 10 to 48 hours after the treatment as described above. The candidates that do not disturb the accumulation of the cells at G2/M will be the selected G2 checkpoint abrogators in this screening method.
In one embodiment, G2 checkpoint abrogators at positions 2 and 3 the have amino acid residues Gly, Leu, Ser or Arg, and at position 5 to 8 are amino acid residues Ser, Gly, Met, Pro or Glu.
In one embodiment of the invention the compositions are enhancers or augmenters of a DNA damaging anti-cancer treatment. By treating cancer cells simultaneously or sequentially with an anti-cancer treatment and a G2 checkpoint inhibiting composition of the invention, one can effectively kill the cancer cells. Since the most human cancer cells do not have an intact G1 checkpoint, the abrogation of the G2 checkpoint by a. G2 checkpoint inhibiting composition of the invention will effectively kill the cancer cells that are treated with a DNA damaging method. The compositions of the invention can be directly used as a drug (e.g., a pharmaceutical compositions) or these oligopeptides could be expressed recombinantly in vivo, e.g., from a virus vector or other expression vector, e.g., a plasmid, as an in vivo gene therapy.
Jurkat cells were cultured in 10% fetal calf serum with a medium (RPMI 1640) at 37° C./5% CO2 with: bleomycin at 20 μg/ml; bleomycin at 20 μg/ml and the peptide “4aa” (amino acid sequence is GGSPSM (SEQ ID NO: 1931)); bleomycin at 20 μg/ml and the peptide AAA (Table 1); bleomycin at 20 μg/ml and the peptide YNP (Table 1). The amount of DNA was analyzed at 0, 6, 12, 24 hours after the addition of ten microgram of bleomycin with or without the oligopeptides “4aa,” “YNP” and “AAA.” The DNA quantity was analyzed by a flow cytometer (FACS) after the addition of a solution comprising propidium iodide, RNase and NP-40.
The results are shown in FIG. 6. The left panels are actual results of flow cytometer (FACS) analysis. The right panel indicates the population of cells in each of the cell cycle phases (sub G1, G1, S, and G2/M). The results indicated that YNP peptide abrogated the G2 checkpoint because the cells do not accumulate at G2/M phases.
In another experiment, an M phase checkpoint activator, colchicine, was used instead of bleomycin: colchicine at 2.5 μg/ml; colchicine at 2.5 μg/ml and the peptide “4aa”; colchicine at 2.5 μg/ml and the peptide AAA (Table 1); colchicine at 2.5 μg/ml and the peptide YNP (Table 1), and no treatment. The results are shown in FIG. 7. None of the above tested oligopeptides (Table 1), including, YPN, affected the accumulation of the colchicine-treated cells at the G2/M phase. These data indicated that YPN specifically abrogated the cell cycle at the G2 checkpoint.
Peptides which were tested and the results of these experiments are further summarized in FIGS. 8 and 9.
Example 4
Peptides of the Invention Sensitize Cancer Cells in in vivo Animal Model
The following example describes studies in an art-accepted animal model which demonstrated that exemplary peptides of the invention are effective agents for selectively sensitizing cancer cells to DNA damaging agents. In particular, nude mouse studies demonstrated the in vivo efficacy of the compositions and methods of the invention.
Human colon cancer cell line SW620 were injected subcutaneously into 3 week old Balb/c nude mouse (1×108 cells per mouse). Some two weeks after the injection, the established subcutaneous tumors of diameter 2 to 4 mm were resected and transplanted to syngeneic mice. One week after the transplantation, the injection of cisplatin (CDDP) and peptides (TAT-control and TAT-S216, see Table 1) was started. The peptides were in the form of recombinant fusion proteins, with TAT being the protein transduction domain having the sequence YGRKKRRQRRR (SEQ ID NO: 1899).
Cisplatin (CDDP) at 6 mg/kg was injected once a week into peritoneum. Peptides (at 100 nM) were injected into tumor twice a week. Relative tumor weights were assessed at 3 and 5 weeks. The results are shown in FIG. 10, upper panel. Similar experiments were performed with 5-FU instead of cisplatin. The results are shown in FIG. 8, lower panel. As shown in FIG. 10, the S216-containing fusion protein effectively sensitized the cancer cells to a DNA damaging agent administered to the animal in vivo.
Similar experiments were performed with cisplatin (CDDP) and another exemplary peptide of the invention, “random II” or “R-II” (see Table 1). As with S216, RII peptide was in the form of a recombinant fusion protein with TAT. The relative volume of the transplanted subcutaneous tumor with or without cisplatin (“CDDP”), CDDP plus DMSO, CDDP plus TAT-FLAG or CDDP plus TAT-Random II peptide was determined. As shown in FIG. 11, the R-II containing fusion protein effectively sensitized the cancer cells to a DNA damaging agent administered to the animal in vivo.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

## Claims

1. An isolated or recombinant polypeptide comprising the amino acid sequence:


X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 

wherein X1 is L, F, W, M, R, I, V, Y, K, or absent,
X2 is Y, F, A, W, S or T,
X3 is R, S, G, T, or H,
X4 is S, L, P or G,
X5 is P, A, or G,
X6 is S, A, N, H, G, or P,
X7 is M, E, G, H, Q, Y, or F,
X8 is P, L, G, F, W or Y,
X9 is E, S, N, G, or absent,
X10 is N, G, L, S, M, P, N, A, or absent, and
X11 is L, M, or absent,
wherein the amino acid sequence is selected from the group consisting of R Y S L P P E L S N M (SEQ ID NO: 1), L Y T S P S Y Y E N L (SEQ ID NO: 76), L Y H S P S Y Y E N L (SEQ ID NO: 108), L F T S P S Y Y E N L (SEQ ID NO: 300), W Y R S P S F Y E N L (SEQ ID NO: 904), W Y T S P S H P E N L (SEQ ID NO: 990), Y G G P G G G G N (SEQ ID NO: 1895), and L A R S A S M P E A L (SEQ ID NO: 1896), and wherein the polypeptide can be a phosphorylation substrate of a human Chk2 kinase in vitro.

2. The isolated or recombinant polypeptide of claim 1, wherein the amino acid sequence comprises Y G G P G G G G N (SEQ ID NO: 1895).

3. The isolated or recombinant polypeptide of claim 1, wherein the amino acid sequence comprises R Y S L P P E L S N M (SEQ ID NO: 1).

4. The isolated or recombinant polypeptide of claim 1, comprising the amino acid sequence L A R S A S M P E A L (SEQ ID NO: 1896).

5. The isolated or recombinant polypeptide of claim 1, comprising the amino acid sequence W Y R SP S F Y E N L (SEQ ID NO: 904).

6. The isolated or recombinant polypeptide of claim 1, comprising the amino acid sequence L Y T S P S Y Y E N L (SEQ ID NO: 76).

7. The isolated or recombinant polypeptide of claim 1, comprising the amino acid sequence L Y H S P S Y Y E N L (SEQ ID NO: 108).

8. The isolated or recombinant polypeptide of claim 1, comprising the amino acid sequence L F T S P S Y Y E N L (SEQ ID NO: 300).

9. The isolated or recombinant polypeptide of claim 1, comprising the amino acid sequence W Y T S P S H P E N L (SEQ ID NO: 990).

10. The isolated or recombinant polypeptide of claim 1, further comprising a cell membrane permeant.

11. The isolated or recombinant polypeptide of claim 10, wherein the cell membrane permeant comprises a polypeptide.

12. The isolated or recombinant polypeptide of claim 11, wherein the polypeptide comprises a TAT protein transduction domain.

13. The isolated or recombinant polypeptide of claim 12, wherein the TAT protein transduction domain is Y G R K K R R Q R R R (SEQ ID NO: 1899).

14. The isolated or recombinant polypeptide of claim 10, wherein the cell membrane permeant comprises a lipid.

15. The isolated or recombinant polypeptide of claim 14, wherein the cell membrane permeant comprises a liposome.

16. A chimeric polypeptide comprising a first domain comprising a polypeptide as set forth in claim 1 and a second domain comprising a cell membrane permeant.

17. The chimeric polypeptide of claim 16, wherein the polypeptide is a recombinant fusion protein.

18. A pharmaceutical composition comprising a polypeptide as set forth in claim 1 or claim 16 and a pharmaceutically acceptable excipient.

19. The pharmaceutical composition of claim 18 comprising a liposome.