Assay for non-peptide agonists to peptide hormone receptors

Abstract

The invention features a method for determining whether a candidate compound is a non-peptide agonist of a peptide hormone receptor. In this method, a candidate compound is exposed to a form of the peptide hormone receptor which has an enhanced ability to amplify the intrinsic activity of a non-peptide agonist. The second messenger signaling activity of the enhanced receptor is measured in the presence of the candidate compound, and compared to the second messenger signaling activity of the enhanced receptor measured in the absence of the candidate compound. A change in second messenger signaling activity indicates that the candidate compound is an agonist. An increase in second messenger signaling activity indicates that the compound is either a full or partial positive agonist; a decrease in second messenger signaling activity indicates that the compound is an inverse (also termed a ‘negative’) agonist.

What is claimed is:  
     
         1 . A method for determining whether a candidate compound is a non-peptide agonist of a peptide hormone receptor, said method comprising the steps of: 
 (a) exposing said candidate compound to a form of said peptide hormone receptor that has the ability to amplify the activity of a non-peptide agonist as compared to the corresponding wild-type receptor; and    (b) measuring the activity of said form in the presence of said candidate compound relative to the activity of said form in the absence of said compound, a change in said activity indicating that said candidate compound is an agonist.    
     
     
         2 . The method of  claim 1 , wherein said form of said receptor is a mutant eceptor.  
     
     
         3 . The method of  claim 1 , wherein said form of said receptor has a higher basal activity than the basal activity of a corresponding human wild-type receptor.  
     
     
         4 . The method of  claim 1 , wherein said form of said receptor is a constitutively active receptor.  
     
     
         5 . The method of  claim 1 , wherein said form of said receptor is a non-human receptor.  
     
     
         6 . The method of  claim 5 , wherein said form of said receptor is a non-human wild-type receptor.  
     
     
         7 . The method of  claim 1 , wherein said form of said receptor is a naturally-occurring mutant receptor.  
     
     
         8 . The method of  claim 1 , wherein an increase in said activity indicates that said candidate compound is a positive agonist.  
     
     
         9 . The method of  claim 1 , wherein said positive agonist is a partial agonist.  
     
     
         10 . The method of  claim 1 , wherein a decrease in said activity indicates that said candidate compound is an inverse agonist.  
     
     
         11 . A method of isolating a form of a peptide hormone receptor suitable for detecting agonist activity in a non-peptide ligand, said method comprising: 
 (a) exchanging a region of a functional domain of a first peptide hormone receptor with a corresponding region of a functional domain of a second peptide hormone receptor, said functional domain being selected from the group consisting of an intracellular loop and a transmembrane domain; and    (b) measuring the ability of said first peptide hormone receptor to amplify an agonist signal relative to the ability of a corresponding wild-type human receptor to amplify said agonist signal, a greater amplification in said first peptide hormone receptor indicating that said first peptide hormone receptor is suitable for detecting agonist activity in a ligand.    
     
     
         12 . The method of  claim 11 , wherein said second peptide hormone receptor is linked to a different second messenger pathway than said first peptide hormone receptor.  
     
     
         13 . A method of isolating a form of a peptide hormone receptor that amplifies the intrinsic activity of an agonist, said method comprising: 
 (a) constructing a series of mutant forms of said receptor by replacing an original amino acid with a replacement amino acid, said replacement amino acid; and    (b) measuring the ability of said first peptide hormone receptor to amplify an agonist signal relative to the ability of a corresponding wild-type human receptor to amplify said agonist signal, a greater amplification in said first peptide hormone receptor indicating that said first peptide hormone receptor is suitable for detecting agonist activity in a non-peptide ligand.    
     
     
         14 . The method of  claim 13 , wherein said replacing comprises replacing an amino acid in an intracellular domain of said receptor.  
     
     
         15 . The method of  claim 13 , wherein said replacing comprises replacing an amino acid in a transmembrane domain flanking an intracellular portion of said receptor.  
     
     
         16 . The method of  claim 13 , wherein said replacement amino acid is a different charge from said original amino acid.  
     
     
         17 . The method of  claim 13 , wherein said replacement amino acid is glutamic acid.