In vivo screen using chemical inducers of dimerization

Abstract

A method for identifying a molecule that binds a known target in a cell from a pool of candidate molecules, comprising: (a) covalently bonding each molecule in the pool of candidate molecules to a methotrexate moiety to form a screening molecule; (b) introducing the screening molecule into a cell which expresses a first fusion-protein comprising a binding domain capable of binding methotrexate, a second fusion protein comprising the known target, and a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein; (c) permitting the screening molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; (d) selecting which cell expresses the reporter gene; and (e) identifying the small molecule that binds the known target.

1 - 157 . (canceled) 
     
     
         158 . A method for determining if a protein interacts with a ligand in vivo, wherein the method comprises activating a reporter gene by contacting a cell expressing two fusion proteins, the first comprising a ligand-binding domain fused to a DNA-binding domain and the second comprising a transcription activation domain fused to the protein, with a covalently linked hybrid-ligand so as to activate the reporter gene, characterized in that the covalently linked hybrid-ligand comprises a methotrexate moiety. 
     
     
         159 . The method of  claim 158 , wherein the DNA-binding domain is a LexA DNA-binding domain. 
     
     
         160 . The method of  claim 158 , wherein the transcription activation domain is B42. 
     
     
         161 . The method of  claim 158 , wherein the reporter gene is LacZ or Gal4. 
     
     
         162 . A method for identifying a receptor as being able to bind a ligand comprising:
 (a) providing a hybrid ligand comprising a methotrexate moiety which binds to dihydrofolate reductase covalently bonded to the ligand;   (b) introducing the molecule into a cell which i) expresses a first fusion protein comprising a dihydrofolate reductase and a DNA-binding domain, ii) a second fusion protein comprising the receptor and a transcription activation domain, and iii) has a reporter gene, wherein activation of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein;   (c) permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the reporter gene; and   (d) selecting the cell having an activated reporter gene,   so as to thereby identify the receptor as being able to bind the ligand.   
     
     
         163 . The method of  claim 162 , wherein the first fusion protein is (dihydrofolate reductase)-(LexA). 
     
     
         164 . The method of  claim 162 , wherein the second fusion protein comprises B42. 
     
     
         165 . The method of  claim 162 , wherein the reporter gene is LacZ or Gal4. 
     
     
         166 . A method for identifying a protein target as being able to bind a ligand comprising:
 (a) providing a molecule comprising a methotrexate moiety which binds to dihydrofolate reductase, covalently bonded to the ligand;   (b) introducing the molecule into a cell which i) expresses a first fusion protein comprising a dihydrofolate reductase capable of binding methotrexate, ii) a second fusion protein comprising the protein target, wherein one of the first and second fusion proteins also comprises a transcription activator domain and the other comprises a DNA-binding domain, and iii) has a reporter gene, wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein;   (c) permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and   (d) selecting the cell if it expresses the reporter gene,   so as to thereby identify the protein target as being able to bind the ligand.   
     
     
         167 . The method of  claim 166 , wherein the protein target is encoded by a DNA from the group consisting of genomic DNA, cDNA and synthetic DNA. 
     
     
         168 . The method of  claim 166 , wherein the ligand has a known biological function. 
     
     
         169 . The method of  claim 166 , wherein the first fusion protein is (dihdrofolate reductase)-(DNA-binding domain). 
     
     
         170 . The method of  claim 166 , wherein the first fusion protein is (dihdrofolate reductase)-(LexA). 
     
     
         171 . The method of  claim 166 , wherein the first fusion protein is (dihdrofolate reductase)-(transcription activation domain). 
     
     
         172 . The method of  claim 166 , wherein the first fusion protein is (dihdrofolate reductase)-(B42). 
     
     
         173 . The method of  claim 166 , wherein the second fusion comprises a DNA-binding domain. 
     
     
         174 . The method of  claim 166 , wherein the second fusion protein comprises LexA. 
     
     
         175 . The method of  claim 166 , wherein the second fusion protein comprises a transcription activation domain. 
     
     
         176 . The method of  claim 166 , wherein the second fusion protein comprises B42. 
     
     
         177 . The method of  claim 166 , wherein the cell is  S. cerevisiae  or  E. coli.    
     
     
         178 . The method of  claim 166 , wherein the reporter gene is lacZ, Gal4 or Ura-3. 
     
     
         179 . The method of  claim 166 , wherein the cell is a bacterial cell, the molecule comprises a methotrexate moiety bound to the ligand, the first fusion protein comprises a dihydrofolate reductase and a LexA, the second fusion protein comprises the protein target and B42, and the reporter gene is LacZ. 
     
     
         180 . The method of  claim 166 , wherein the cell is a yeast cell, the molecule comprises a methotrexate moiety bound to the ligand, the first fusion protein comprises a dihydrofolate reductase and a LexA, the second fusion protein comprises the protein target and B42, and the reporter gene is Gal4. 
     
     
         181 . In a method for determining if a protein interacts with a ligand in vivo, wherein the method comprises activating a reporter gene by contacting a cell expressing two fusion proteins, the first comprising a ligand-binding domain fused to a DNA-binding domain and the second comprising a transcription activation domain fused to the protein, with a covalently linked hybrid-ligand so as to activate the reporter gene, the improvement comprising a covalently linked hybrid-ligand having a methotrexate moiety. 
     
     
         182 . The method of  claim 181 , wherein the DNA-binding domain is a LexA DNA-binding domain. 
     
     
         183 . The method of  claim 181 , wherein the transcription activation domain is B42. 
     
     
         184 . The method of  claim 181 , wherein the reporter gene is LacZ or Gal4.