High throughput sarcomeric assay

Abstract

The present invention provides high throughput screening systems for identifying compounds that modulate the biological activity of a biochemically functional sarcomere. The method can be performed in plurality simultaneously with fluorescence or absorbance readouts.

What is claimed is:  
     
         1 . A method of identifying a candidate agent as a modulator of function of a target protein complex wherein said target protein complex comprises a biochemically functional sarcomere and said method comprises: 
 a) adding a candidate agent to a mixture comprising a target protein complex that directly or indirectly produces ADP or phosphate under conditions which normally allow the production of ADP or phosphate;    b) subjecting the mixture to an enzymatic reaction that uses said ADP or phosphate as a substrate under conditions which normally allow the ADP or phosphate to be utilized; and    c) determining the level of activity of the enzymatic reaction wherein a change in said level between the presence and absence of said candidate agent indicates a modulator of said target protein function.    
     
     
         2 . The method of  claim 1 , wherein said determining occurs by a fluorescent, luminescent, radioactive, or absorbance readout.  
     
     
         3 . The method of  claim 1 , wherein said level of activity of said enzymatic reaction is determined at multiple time points.  
     
     
         4 . The method of  claim 1 , wherein a plurality of candidate agents are added.  
     
     
         5 . The method of  claim 1 , wherein said target protein complex directly produces phosphate or ADP.  
     
     
         6 . The method of  claim 1 , wherein said target protein complex comprises a mixture of myosin, actin, and cardiac regulatory proteins.  
     
     
         7 . The method of  claim 6 , wherein said candidate agent activates myosin.  
     
     
         8 . The method of  claim 6 , wherein said candidate agent modulates a cardiac regulatory protein.  
     
     
         9 . The method of  claim 1 , wherein the enzymatic reaction occurs at a calcium concentration below pCa equal to 4.  
     
     
         10 . The method of  claim 1 , wherein the enzymatic reaction occurs at a calcium concentration of about pCa equal to 4.  
     
     
         11 . The method of  claim 1 , wherein the enzymatic reaction occurs at a calcium concentration of about pCa equal to 6.5.  
     
     
         12 . The method of  claim 1 , wherein the enzymatic reactions occurs in the presence of EGTA.  
     
     
         13 . The method of  claim 1 , wherein the target protein complex comprises actin and myosin.  
     
     
         14 . A composition comprising an isolated biochemically functional sarcomere, wherein said sarcomere exhibits calcium regulated myosin ATPase activity  
     
     
         15 . The composition of  claim 12 , wherein said sarcomere comprises myosin, actin, and regulatory proteins.  
     
     
         16 . The composition of  claim 13 , wherein said actin is bovine.  
     
     
         17 . The composition of  claim 13 , wherein said regulatory proteins are from a human.