Compound screening methods

Abstract

The invention provides methods of screening for compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA), using the nematode worm C. elegans.

1 . A method of identifying compounds which modulate the interaction between a sarco/endoplasmic reticulum calcium ATPase and phospholamban, which method comprises: 
 exposing transgenic  C. elegans  which contains a first transgene comprising nucleic acid encoding a vertebrate phospholamban protein and which expresses a SERCA protein to a compound under test; and    detecting a phenotypic, biochemical or behavioural change in the transgenic  C. elegans  indicating an increase in the activity of the SERCA protein.    
     
     
         2 . A method as claimed in  claim 1  wherein the vertebrate phospholamban protein is pig phospholamban or human phospholamban.  
     
     
         3 . A method as claimed in  claim 1  or  claim 2  wherein the first transgene comprises nucleic acid encoding a vertebrate phospholamban protein operatively linked to a tissue-specific promoter.  
     
     
         4 . A method as claimed in  claim 3  wherein the tissue-specific promoter is capable of directing gene expression in cells of the  C. elegans  pharynx, preferably pharynx muscle cells or capable of directing gene expression in cells of the  C. elegans  vulva, preferably vulva muscle cells or capable of directing gene expression in the cells of the  C. elegans  body wall muscles.  
     
     
         5 . A method as claimed in any one of  claims 1  to  4  wherein the transgenic  C. elegans  contains a second transgene comprising nucleic acid encoding the said SERCA protein.  
     
     
         6 . A method as claimed in  claim 5  wherein the second transgene comprises nucleic acid encoding the said SERCA protein operatively linked to the promoter region of a SERCA gene.  
     
     
         7 . A method as claimed in  claim 6  wherein said promoter region is the promoter region of the  C. elegans  SERCA gene.  
     
     
         8 . A method as claimed in any one of  claims 5  to  7  wherein the SERCA protein is a vertebrate SERCA protein.  
     
     
         9 . A method as claimed in  claim 8  wherein the vertebrate SERCA protein is pig SERCA1a, pig SERCA1b, pig SERCA2a, pig SERCA2b, Pig SERCA3, human SERCA1a, human SERCA1b, human SERCA2a, human SERCA2b or human SERCA3.  
     
     
         10 . A method as claimed in any one of  claims 5  to  7  wherein the SERCA protein is a fusion between a  C. elegans  SERCA protein and a vertebrate SERCA protein.  
     
     
         11 . A method as claimed in any one of  claims 1  to  10  wherein the vertebrate phospholamban protein is expressed in at least cells of the pharynx and preferably the pharynx muscle cells of said transgenic  C. elegans.    
     
     
         12 . A method as claimed in  claim 11  wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic  C. elegans  indicating an increase in the activity of the vertebrate SERCA protein comprises detecting a change in the pharynx pumping efficiency of the  C. elegans.    
     
     
         13 . A method as claimed in  claim 11  wherein the transgenic  C. elegans  contains a further transgene comprising a promoter which is capable of directing gene expression in the muscles of the  C. elegans  pharynx operatively linked to nucleic acid encoding an apoaequorin protein.  
     
     
         14 . A method as claimed in  claim 13  wherein the promoter is the  C. elegans  myo-2 promoter or the  C. elegans  SERCA promoter.  
     
     
         15 . A method as claimed in  claim 13  or  claim 14  wherein the step of detecting a phenotypic, biochemical or behavioural change indicating an increase in the activity of the vertebrate SERCA protein comprises comparing the level of apoaequorin luminescence in the absence of the compound under test and the level of apoaequorin luminescence in the presence of the compound under test.  
     
     
         16 . A method as claimed in any one of  claims 1  to  10  wherein the vertebrate phospholamban protein is expressed in at least cells of the  C. elegans  vulva and preferably vulva muscle cells of said transgenic  C. elegans.    
     
     
         17 . A method as claimed in  claim 16  wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic  C. elegans  indicating a change in the activity of the vertebrate SERCA protein comprises comparing the growth rate of the  C. elegans  in the absence of the compound under test and the growth rate of the  C. elegans  in the presence of the compound under test.  
     
     
         18 . A method as claimed in  claim 16  wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic  C. elegans  indicating a change in the activity of the vertebrate SERCA protein comprises comparing the turbidity of the  C. elegans  in the absence of the compound under test and the turbidity of the  C. elegans  in the presence of the compound under test.  
     
     
         19 . A method as claimed in  claim 16  wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic  C. elegans  indicating a change in the activity of the vertebrate SERCA protein comprises detecting a change in the egg laying behaviour of the  C. elegans.    
     
     
         20 . A method as claimed in  claim 16  wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic  C. elegans  indicating a change in the activity of the vertebrate SERCA protein comprises detecting a change in the number of progeny produced by the  C. elegans.