HADHs as Modifiers of the p21 Pathway and Methods of Use

Abstract

Human HADH genes are identified as modulators of the p21 pathway, and thus are therapeutic targets for disorders associated with defective p21 function. Methods for identifying modulators of p21, comprising screening for agents that modulate the activity of HADH are provided.

1 . A method of identifying a candidate p21 pathway modulating agent, said method comprising the steps of:
 (a) providing an assay system comprising a purified HADH polypeptide or nucleic acid or a functionally active fragment or derivative thereof;   (b) contacting the assay system with a test agent under conditions whereby, but for the presence of the test agent, the system provides a reference activity; and   (c) detecting a test agent-biased activity of the assay system, wherein a difference between the test agent-biased activity and the reference activity identifies the test agent as a candidate p21 pathway modulating agent.   
     
     
         2 . The method of  claim 1  wherein the assay system comprises cultured cells that express the HADH polypeptide. 
     
     
         3 . The method of  claim 2  wherein the cultured cells additionally have defective p21 function. 
     
     
         4 . The method of  claim 1  wherein the assay system includes a screening assay comprising a HADH polypeptide, and the candidate test agent is a small molecule modulator. 
     
     
         5 . The method of  claim 4  wherein the assay is a binding assay. 
     
     
         6 . The method of  claim 1  wherein the assay system is selected from the group consisting of an apoptosis assay system, a cell proliferation assay system, an angiogenesis assay system, and a hypoxic induction assay system. 
     
     
         7 . The method of  claim 1  wherein the assay system includes a binding assay comprising a HADH polypeptide and the candidate test agent is an antibody. 
     
     
         8 . The method of  claim 1  wherein the assay system includes an expression assay comprising a HADH nucleic acid and the candidate test agent is a nucleic acid modulator. 
     
     
         9 . The method of  claim 8  wherein the nucleic acid modulator is an antisense oligomer. 
     
     
         10 . The method of  claim 8  wherein the nucleic acid modulator is a PMO. 
     
     
         11 . The method of  claim 1  additionally comprising:
 (d) administering the candidate p21 pathway modulating agent identified in (c) to a model system comprising cells defective in p21 function and, detecting a phenotypic change in the model system that indicates that the p21 function is restored.   
     
     
         12 . The method of  claim 11  wherein the model system is a mouse model with defective p21 function. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 1 , comprising the additional steps of:
 (d) providing a secondary assay system comprising cultured cells or a nonhuman animal expressing HADH, contacting the secondary assay system with the test agent of (b) or an agent derived therefrom under conditions whereby, but for the presence of the test agent or agent derived therefrom, the system provides a reference activity; and   (e) detecting an agent-biased activity of the second assay system, wherein a difference between the agent-biased activity and the reference activity of the second assay system confirms the test agent or agent derived therefrom as a candidate p21 pathway modulating agent, and wherein the second assay detects an agent-biased change in the p21 pathway.   
     
     
         17 . The method of  claim 16  wherein the secondary assay system comprises cultured cells. 
     
     
         18 . The method of  claim 16  wherein the secondary assay system comprises a nonhuman animal 
     
     
         19 . The method of  claim 18  wherein the non-human animal mis-expresses a p21 pathway gene. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . A method for diagnosing a disease in a patient comprising:
 (a) obtaining a biological sample from the patient;   (b) contacting the sample with a probe for HADH expression;   (c) comparing results from step (b) with a control;   (d) determining whether step (c) indicates a likelihood of disease.   
     
     
         24 . The method of  claim 23  wherein said disease is cancer. 
     
     
         25 . The method according to  claim 24 , wherein said cancer is colon or ovarian cancer.