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 - 17 . (canceled)  
   
   
       18 . A method of treating a condition characterized by abnormal smooth muscle contraction, the method comprising administering a compound that modulates smooth muscle myosin contractility to a patient having or susceptible to the condition, wherein the compound is characterized by: 
 a) adding a test compound to an in vitro 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 the 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 the level between the presence and absence of the test compound indicates that the test compound is suitable for use as the compound in treating a condition characterized by abnormal smooth muscle contraction.    
   
   
       19 . The method of  claim 18 , wherein said compound increases myosin ATPase rate by at least 40% over control at an in vitro compound concentration of 10 μM.  
   
   
       20 . The method of  claim 18 , wherein said determining comprises detecting fluorescence, luminescence, radioactivity, or absorbance associated with the enzymatic reaction.  
   
   
       21 . The method of  claim 18 , wherein said level of activity of said enzymatic reaction is determined at multiple time points.  
   
   
       22 . The method of  claim 18 , wherein a plurality of test compounds are added to the in vitro mixture.  
   
   
       23 . The method of  claim 18 , wherein said target protein complex directly produces phosphate or ADP.  
   
   
       24 . The method of  claim 18 , wherein the enzymatic reaction occurs at a calcium concentration below pCa equal to 4.  
   
   
       25 . The method of  claim 18 , wherein the enzymatic reaction occurs at a calcium concentration of about pCa equal to 4.  
   
   
       26 . The method of  claim 18 , wherein the enzymatic reaction occurs at a calcium concentration of about pCa equal to 6.5.  
   
   
       27 . The method of  claim 18 , wherein the enzymatic reaction occurs in the presence of ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA).  
   
   
       28 . The method of  claim 18 , wherein said compound does not activate skeletal myosin.  
   
   
       29 . The method of  claim 18 , wherein the condition characterized by abnormal smooth muscle contraction is selected from asthma, glaucoma, hypertension, and pre-term uterine labor.