US2003235899A1PendingUtilityA1

Catalytic domain of the human effector cell cycle checkpoint protein kinase Chk1 materials and methods for identification of inhibitors thereof

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Priority: Sep 1, 1999Filed: Jan 28, 2003Published: Dec 25, 2003
Est. expirySep 1, 2019(expired)· nominal 20-yr term from priority
Inventors:Ping ChenMark B. AndersonYa-Li DengSmita GaurChen-Chen KanChun LuoKaren LundgrenStephen MargosiakBinh NguyenPatrick O'ConnorJames ReqisterAnna RussellJay Sarup
C12N 9/1205C12Q 1/34C12N 15/52
47
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Claims

Abstract

The present invention relates to the identification, isolation and purification of the catalytic domain of the human effector checkpoint protein kinase (hChk1). A 1.7 Å crystal structure of the hChk1 kinase domain in the active conformation is reported herein. The kinase domain of hChk1 and its associated crystal structure is described for use in the discovery, identification and characterization of inhibitors of hChk1. This structure provides a three-dimensional description of the binding site of the hChk1 for structure-based design of small molecule inhibitors thereof as therapeutic agents. Inhibitors of hChk1 find utility in the treatment of hyperproliferative disorders such as HIV and cancer.

Claims

exact text as granted — not AI-modified
What is claimed:  
     
         1 . A composition comprising an isolated, purified polynucleotide which encodes the active form of the human Chk1 kinase or a functional, active human Chk1 kinase analog thereof.  
     
     
         2 . The composition of  claim 1 , wherein the nucleotide sequence of said polynucleotide comprises bases 35 to 830 of SEQ ID NO. 1 or a functional, active mutant or variant thereof.  
     
     
         3 . A polypeptide in a crystallized form comprising the catalytically active form of the human Chk1 kinase and the inhibitor binding site thereof.  
     
     
         4 . The polypeptide of  claim 3  wherein the crystal is solved to a resolution of at least 2.5 Å.  
     
     
         5 . The polypeptide of  claim 3  wherein the crystal is solved to a resolution of at least 2.0 Å.  
     
     
         6 . The polypeptide of  claim 3  wherein the crystal is solved to a resolution of about 1.7 Å.  
     
     
         7 . The polypeptide of  claim 3  wherein the amino acid sequence of said polypeptide comprises amino acids 16 to 265 of SEQ ID NO. 2 or an active mutant or variant thereof.  
     
     
         8 . The polypeptide of  claim 3  wherein the amino acid sequence of said polypeptide comprises amino acids 16 to 289 of SEQ ID NO. 2 or an active mutant or variant thereof.  
     
     
         9 . The polypeptide of  claim 3  wherein the amino acid sequence of said polypeptide comprises amino acids 16 to 291 of SEQ ID NO. 2 or an active mutant or variant thereof.  
     
     
         10 . The polypeptide of  claim 3  wherein the amino acid sequence of said polypeptide comprises amino acids 1 to 265 of SEQ ID NO. 2 or an active mutant or variant thereof.  
     
     
         11 . The polypeptide of  claim 3  wherein the amino acid sequence of said polypeptide comprises amino acids 1 to 289 of SEQ ID NO. 2 or an active mutant or variant thereof.  
     
     
         12 . The polypeptide of  claim 3  wherein the amino acid sequence of said polypeptide comprises amino acids 1 to 291 of SEQ ID NO. 2 or an active mutant or variant thereof.  
     
     
         13 . The polypeptide of  claim 3  wherein said polypeptide further comprises a six histidine tag on the C-terminal thereof.  
     
     
         14 . An isolated, soluble, catalytically active polypeptide comprising the active form of the human Chk1 kinase or a functional, active human Chk1 kinase analog thereof.  
     
     
         15 . The polypeptide of  claim 14  comprising the full length human Chk1 protein having the C-terminal portion thereof deleted so as yield the human Chk1 kinase domain in its active configuration.  
     
     
         16 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 16 to 265 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         17 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 16 to 289 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         18 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 16 to 291 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         19 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 1 to 265 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         20 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 1 to 289 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         21 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 1 to 291 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         22 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 5 to 265 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         23 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 5 to 289 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         24 . The polypeptide of  claim 14  wherein said polypeptide comprises amino acids 5 to 291 of the sequence as set forth in SEQ ID NO. 2 or a conservatively substituted variant thereof.  
     
     
         25 . An expression vector for producing active human Chk1 kinase in a host cell, which vector comprises: a polynucleotide encoding active form of the human Chk1 kinase or an active human Chk1 kinase analog thereof; transcriptional and translational regulatory sequences functional in said host cell operably linked to said human Chk1 kinase-encoding polynucleotide; and a selectable marker  
     
     
         26 . The vector of  claim 25  wherein said polynucleotide encodes the active human Chk1 kinase, said active kinase comprising bases 35 to 830 of SEQ ID NO. 1.  
     
     
         27 . The vector of  claim 25  wherein said vector is selected from the group consisting of pET28a, pAcSG2, and pFastBac.  
     
     
         28 . The vector of  claim 25  wherein said vector is pFastBac-Nde.  
     
     
         29 . The vector of  claim 25  wherein said selectable marker is selected from the group consisting of beta galactosidase green fluorescent protein, and luciferase.  
     
     
         30 . A host cell stably transformed and transfected with a polynucleotide encoding active form of the human Chk1 kinase or an active human Chk1 kinase analog thereof in a manner allowing the expression in said host cell of the human Chk1 kinase.  
     
     
         31 . The host cell of  claim 30 , wherein said polynucleotide encodes the active hChk1 kinase, said active kinase comprising bases 35 to 830 of SEQ ID NO. 1.  
     
     
         32 . The host cell of  claim 30  wherein said host is  E. coli.    
     
     
         33 . The host cell of  claim 30  wherein said host is a recombinant baculovirus.  
     
     
         34 . The host cell of  claim 30  wherein said host is an insect cell.  
     
     
         35 . The host cell of  claim 34  wherein said insect cell is Sf9.  
     
     
         36 . The host cell of  claim 30  wherein said host cell is transformed and transfected with said polynucleotide via an expression vector comprising said polynucleotide; a transcriptional and translational regulatory sequences functional in said host cell operably linked to said hChk1 kinase-encoding polynucleotide; and a selectable marker.  
     
     
         37 . The host cell of  claim 36  wherein said expression vector is selected from the group consisting of pET28a, pAcSG2, and pFastBac.  
     
     
         38 . The host cell of  claim 36  wherein said expression vector is pFastBac-Nde.  
     
     
         39 . The host cell of  claim 36  wherein said selectable marker is selected from the group consisting of beta galactosidase, green fluorescent protein, and luciferase.  
     
     
         40 . A method for assaying a candidate compound for its ability to interact with the human Chk1 comprising: 
 (a) expressing an isolated DNA sequence or variants thereof encoding the kinase domain of said human Chk1 in a host capable of producing said kinase in the catalytically active configuration, said kinase in a form which may be assayed for interaction of said kinase with said candidate compound;    (b) exposing said kinase to said candidate compound; and    (c) evaluating the interaction of said kinase with said candidate compound.    
     
     
         41 . A method of identifying a Chk1 kinase inhibitor by determining the binding interactions between an organic compound and the binding site of the Chk1 kinase in the active conformation, said binding sites being defined by the crystal coordinates of provided in FIG. 11, said method comprising: 
 (a) generating the binding cavity defined by the binding site on a computer screen;    (b) generating compounds with their spatial structure; and    (c) testing to see whether the compounds bind to at the Chk1 binding site;    wherein those compounds that do bind to the Chk1 binding site can be identified as Chk1 inhibitors.

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