Screen for CDC7 inhibitors

Abstract

Disclosed is a yeast cell whose genetic complement includes an inactive allele of the yeast CDC7 gene, a first nucleic acid that encodes a mammalian Cdc7 protein, and a second nucleic acid that encodes a mammalian Dbf4 protein. The yeast cell is dependent on the mammalian Cdc7 and Dbf4 proteins for viability. The yeast cell can be used to identify potential anti-proliferative agents by virtue of their inhibition of the mammalian Cdc7 and Dbf4 proteins. In some embodiments, a control yeast cell which does not depend on Cdc7 for viability is used in a secondary screen.

1 . A yeast cell, the genetic complement of which comprises: 
 a) an inactive allele of the yeast CDC7 gene;    b) a first nucleic acid molecule that encodes a mammalian Cdc7 polypeptide; and    c) a second nucleic acid molecule that encodes a mammalian Dbf4 polypeptide.    
     
     
         2 . The yeast cell of  claim 1 , wherein the mammalian Cdc7 polypeptide comprises SEQ ID NO: 19 with at least one conservative amino acid substitution.  
     
     
         3 . The yeast cell of  claim 1 , wherein the first nucleic acid molecule comprises SEQ ID NO: 18.  
     
     
         4 . The yeast cell of  claim 1 , wherein the mammalian Dbf4 polypeptide comprises SEQ ID NO:21 with at least one conservative amino acid substitution.  
     
     
         5 . The yeast cell of  claim 1 , wherein the second nucleic acid molecule comprises SEQ ID NO:20.  
     
     
         6 . A method for determining whether a test compound is an inhibitor of mammalian Cdc7.4 activity, the method comprising: 
 (i) obtaining a yeast cell of  claim 1;     (ii) contacting the yeast cell with a test compound; and    (iii) assessing growth or cell cycle progression of the yeast cell, wherein a decrease in growth or cell cycle progression indicates that the test compound is an inhibitor of mammalian Cdc7.4 activity.    
     
     
         7 . The method of  claim 6 , wherein the Cdc7 polypeptide is human Cdc7 polypeptide.  
     
     
         8 . The method of  claim 6 , wherein the Ddf4 polypeptide is human Ddf4 polypeptide.  
     
     
         9 . The method of  claim 6 , wherein assessing comprises culturing the yeast cell in a container.  
     
     
         10 . The method of  claim 9 , wherein assessing comprises culturing the yeast cell on a solid medium within the container and monitoring the size of a colony formed by the yeast cell.  
     
     
         11 . The method of  claim 9 , wherein assessing comprises culturing the yeast cell in a liquid medium within the container and determining the number of yeast cells in the liquid medium.  
     
     
         12 . The method of  claim 9 , wherein assessing comprises culturing the yeast cell in a liquid medium within the container and measuring turbidity of the liquid medium.  
     
     
         13 . The method of  claim 6 , wherein assessing comprises measuring endogenous ATP levels of the yeast cell.  
     
     
         14 . The method of  claim 6 , wherein assessing comprises fluorescent-activated cell sorting based on DNA content.  
     
     
         15 . The method of  claim 6 , further comprising assaying in vitro mammalian Cdc7.4 kinase activity.  
     
     
         16 . The method of  claim 15 , wherein assaying comprises determining mammalian Cdc7.4 kinase activity for a minichromosome maintenance protein substrate.  
     
     
         17 . A method for determining whether a test compound is an inhibitor of mammalian Cdc7.4 activity, the method comprising: 
 (i) contacting a test compound to a first yeast cell, the genetic complement of which comprises: 
 (a) an inactive allele of the yeast CDC7 gene;  
 (b) a first nucleic acid molecule that encodes a mammalian Cdc7 polypeptide; and  
 (c) a second nucleic acid molecule that encodes a mammalian Dbf4 polypeptide;  
   (ii) contacting the test compound to a second yeast cell, the viability of which is independent of a mammalian Cdc7 or Dbf4; and    (iii) assessing growth or cell cycle progression of the first and second yeast cells, wherein a decrease in growth or cell cycle progression in the first yeast cell relative to the second yeast strain indicates that the test compound is an inhibitor of mammalian Cdc7.4 activity.    
     
     
         18 . The method of  claim 17 , wherein the second yeast cell comprises a mutant allele of CDC46.  
     
     
         19 . The method of  claim 17 , wherein the Cdc7 polypeptide is human Cdc7 polypeptide.  
     
     
         20 . The method of  claim 17 , wherein the Dbf4 polypeptide is human Dbf4 polypeptide.  
     
     
         21 . The method of  claim 6 , further comprising: 
 (iii) repeating (i) and (ii) for multiple test compounds;    (iv) identifying candidate compounds that are inhibitors of mammalian Cdc7.4 activity;    (v) identifying and selecting a lead compound from the candidate compounds, the lead compound being an inhibitor of Cdc7.4 activity; and    (vi) formulating the selected lead compound as an anti-proliferative agent.    
     
     
         22 . The method of  claim 6 , further comprising: 
 (iii) repeating (i) and (ii) for multiple test compounds;    (iv) identifying candidate compounds that are inhibitors of mammalian Cdc7.4 activity;    (v) isolating one or more lead compounds from the candidate compounds;    (vi) derivatizing the one or more lead compounds, thereby producing derivatives of the lead compounds;    (vii) identifying one or more derivatives that are indicated as inhibitors of mammalian Cdc7.4 activity; and    (viii) formulating one or more derivatives as an anti-proliferative agent.    
     
     
         23 . A composition comprising an anti-proliferative agent identified by the method of  claim 6  and a pharmaceutically acceptable carrier.  
     
     
         24 . A method of treating a subject having a proliferative disorder, the method comprising administering to the subject the composition of  claim 23  in an amount effective to reduce cell proliferation associated with the proliferative disorder.  
     
     
         25 . A kit comprising: 
 (1) a first yeast cell, the genetic complement of which comprises a) an inactive allele of the yeast CDC7 gene, b) a first nucleic acid that encodes a mammalian Cdc7 polypeptide, and c) a second nucleic acid that encodes a mammalian Dbf4 polypeptide; and    (2) a second yeast cell, the genetic complement of which comprises a) an inactive allele of the yeast CDC7 gene, and b) a mutant allele that bypasses the requirement for Cdc7.4 activity.    
     
     
         26 . The kit of  claim 25 , wherein the second yeast cell comprises a mutant allele of CDC46.  
     
     
         27 . The kit of  claim 25 , wherein the Cdc7 polypeptide is human Cdc7 polypeptide.  
     
     
         28 . The kit of  claim 25 , wherein the Dbf4 polypeptide is human Dbf4 polypeptide.