Assays for detecting modulators of cytoskeletal function

Abstract

Described herein are methods of identifying compounds which modulate the activity of the cytoskeletal system. The methods are rapid, convenient and sensitive. Preferably, the method is used to identify lead compounds that can be used as therapeutics, diagnostics and agricultural agents. Generally, test compounds are added to two cytoskeletal components which bind to one another, to determine whether the binding is affected by the test compound. Wherein the binding is affected, a compound which modulates the cytoskeletal system is identified.

1 - 69 . (canceled)  
     
     
         70 . A method of identifying a therapeutic lead compound that modulates activity of a cytoskeletal system, said method comprising: 
 i) providing an assay mixture comprising a first component of said cytoskeletal system and a second component of said cytoskeletal system, wherein said cytoskeletal system is a microtubule system, and wherein said first component comprises a kinesin motor protein and said second component comprises a tubulin protein that specifically bind to each other;    ii) contacting said assay mixture with a test compound to be screened for the ability to inhibit or enhance binding between said first component and said second component;    iii) detecting a difference in one or both of ATP hydrolysis and force generation; wherein said difference indicates that said test compound modulates activity said cytoskeletal system.    
     
     
         71 . The method of  claim 70 , further comprising step iv) testing said test compound on a variety of ATPases.  
     
     
         72 . The method of  claim 70 , wherein said assay mixture comprises a cell lysate.  
     
     
         73 . The method of  claim 70 , wherein at least 50 test compounds are screened.  
     
     
         74 . The method of  claim 70 , wherein said first component or said second component is attached to a solid support.  
     
     
         75 . The method of  claim 71 , wherein said variety of ATPases comprises CENP-E.  
     
     
         76 . The method of  claim 71 , wherein said variety of ATPases comprises kinesin.  
     
     
         77 . The method of  claim 71 , wherein said variety of ATPases comprises TI-gamma.  
     
     
         78 . The method of  claim 70 , wherein said step iii) comprises said detecting a difference in ATP hydrolysis, at a test concentration and a control concentration of said compound to be screened.  
     
     
         79 . The method of  claim 70 , wherein said step iii) comprises said detecting a difference in force generation, at a test concentration and a control concentration of said compound to be screened.  
     
     
         80 . The method of  claim 70 , wherein said step iii) comprises said detecting a difference in said ATP hydrolysis, and said detecting a difference in said force generation, at a test concentration and a control concentration of said compound to be screened.  
     
     
         81 . The method of  claim 80 , wherein said detecting a difference in said force generation comprises detecting a difference in binding of said kinesin motor protein to said tubulin protein.  
     
     
         82 . The method of  claim 80 , wherein said detecting a difference in said force generation comprises detecting a difference in motility.