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.

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.