Bacterial small-molecule three-hybrid system

Abstract

A transgenic bacterial cell comprising (a) a dimeric small molecule which comprises a first moiety known to bind a first receptor domain covalently linked to a second moiety known to bind a second receptor domain; (b) nucleotide sequences which upon transcription encode i) a first fusion protein comprising the first receptor domain, and ii) a second fusion protein comprising the second receptor domain; and (c) a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein. The cell is also adapted for use in a method for identifying a molecule that binds to a known target in a bacterial cell from a pool of candidate molecules, and a method for identifying an unknown target receptor to which a molecule is capable of binding in a bacterial cell. Also described are compounds and kits for carrying out the methods.

1 . A transgenic bacterial cell comprising 
 (a) a dimeric small molecule which comprises a first moiety known to bind a first receptor domain covalently linked to a second moiety capable of binding a second receptor domain, wherein the first and second moieties are different;    (b) nucleotide sequences which upon transcription encode 
 i) a first fusion protein comprising the first receptor domain, and  
 ii) a second fusion protein comprising the second receptor domain; and  
   (c) a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein:    
     
     
         2 . The bacterial cell of  claim 1 , wherein the dimeric small molecule has the formula:  
         H1-Y-H2  wherein each of H1 and H2 may be the same or different and capable of binding to a receptor which is the same or different with a IC 50  of less than 100 μM; and    wherein Y is a linker which may be present or absent.    
     
     
         3 . The bacterial cell of  claim 2 , wherein H1 or H2 is capable of binding to a receptor with a IC 50  of less than 10 μM.  
     
     
         4 . The bacterial cell of  claim 2 , wherein H1 or H2 is capable of binding to a receptor with a IC 50  of less than 1 μM.  
     
     
         5 . The bacterial cell of  claim 2 , wherein H1 or H2 is capable of binding to a receptor with a IC 50  of less than 100 nM.  
     
     
         6 . The bacterial cell of  claim 2 , wherein H1 or H2 is capable of binding to a receptor with a IC 50  of less than 10 nM.  
     
     
         7 . The bacterial cell of  claim 2 , wherein H1 or H2 is capable of binding to a receptor with a IC 50  of less than 1 nM.  
     
     
         8 . The bacterial cell of  claim 2 , wherein H1 or H2 is a methotrexate moiety, FK506 moiety, an FK506 analog, a tetracycline moiety, or a cephem moiety.  
     
     
         9 . The bacterial cell of  claim 8 , wherein H1 or H2 is a methotrexate moiety.  
     
     
         10 . The bacterial cell of  claim 8 , wherein H1 or H2 is an FK506 analog.  
     
     
         11 . The bacterial cell of  claim 10 , wherein the FK506 analog has the structure:  
       
         
           
           
               
               
           
         
       
     
     
         12 . The bacterial cell of  claim 2 , wherein the dimeric small molecule has the structure:  
       
         
           
           
               
               
           
         
       
       wherein n is an integer from 1 to 20.  
     
     
         13 . The bacterial cell of  claim 12 , wherein n is an integer from 2 to 12.  
     
     
         14 . The bacterial cell of  claim 12 , wherein n is an integer from 3 to 9.  
     
     
         15 . The bacterial cell of  claim 12 , wherein n is 8.  
     
     
         16 . The bacterial cell of  claim 1 , wherein the first fusion protein further comprises a DNA binding domain, and the second fusion protein further comprises a transcription activation domain.  
     
     
         17 . The bacterial cell of  claim 1 , wherein the first fusion protein further comprises a transcription activation domain, and the second fusion protein further comprises a DNA binding domain.  
     
     
         18 . The bacterial cell of  claim 16 , wherein the transcription activation domain is αNTD.  
     
     
         19 . The bacterial cell of  claim 16 , wherein the DNA-binding domain is λcI, AraC, LexA, Gal4, or zinc fingers.  
     
     
         20 . The bacterial cell of  claim 1 , wherein the first or the second receptor domain is that of dihydrofolate reductase (“DHFR”), glucocorticoid receptor, FKBP12, FKBP mutants, tetracycline repressor, or a penicillin binding protein.  
     
     
         21 . The bacterial cell of  claim 20 , wherein the DHFR is the  E. coli  DHFR (“eDHFR”).  
     
     
         22 . The bacterial cell of  claim 1 , wherein the first fusion protein is DHFR-λcI or FKBP12-λcI.  
     
     
         23 . The bacterial cell of  claim 1 , wherein the second fusion protein is DHFR-αNTD or FKBP12-αNTD.  
     
     
         24 . The bacterial cell of  claim 1 , wherein the reporter gene is Lac Z, araBAD, aadA (spectinomycin resistance), his3, β-lactamase, GFP, luciferase, TetR (tetracyclin resistance), KanR (kanamycin resistance), Cm (chloramphenicol resistance).  
     
     
         25 . The bacterial cell of  claim 24 , wherein the reporter gene is Lac Z.  
     
     
         26 . A transgenic bacterial cell comprising 
 (a) a dimeric small molecule which comprises a methotrexate moiety covalently linked to a moiety capable of binding a receptor domain;    (b) nucleotide sequences which upon transcription encode 
 i) a first fusion protein comprising a DHFR domain, and  
 ii) a second fusion protein comprising the receptor domain; and  
   (c) a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein.    
     
     
         27 . The bacterial cell of  claim 26  wherein the moiety known to bind a receptor domain is capable of binding to a receptor with a IC 50  of less than 100 nM.  
     
     
         28 . The bacterial cell of  claim 26 , wherein the moiety known to bind a receptor domain is capable of binding to a receptor with a IC 50  of less than 10 nM.  
     
     
         29 . The bacterial cell of  claim 26 , wherein the moiety known to bind a receptor domain is capable of binding to a receptor with a IC 50  of less than 1 nM.  
     
     
         30 - 76 . (canceled)  
     
     
         77 . A transgenic bacterial cell comprising 
 (a) a dimeric small molecule which comprises a methotrexate moiety covalently linked to a moiety capable of binding a receptor domain;    (b) nucleotide sequences which upon transcription encode 
 i) a first fusion protein comprising a DHFR domain and a first fragment of an enzyme, and  
 ii) a second fusion protein comprising the receptor domain and a second fragment of the enzyme, wherein activity of the enzyme is conditioned on the proximity of the first fragment of the enzyme to the second fragment of the enzyme.  
   
     
     
         78 . The bacterial cell of  claim 77 , wherein the moiety capable of binding the receptor domain is capable of binding with a IC 50  of less than 100 μM.  
     
     
         79 . The bacterial cell of  claim 77 , wherein the moiety capable of binding the receptor domain is capable of binding with a IC 50  of less than 100 nM.  
     
     
         80 . The bacterial cell of  claim 77 , wherein the moiety capable of binding the receptor domain is capable of binding with a IC 50  of less than 1 nM.  
     
     
         81 - 82 . (canceled)  
     
     
         83 . The bacterial cell of  claim 17 , wherein the transcription activation domain is αNTD.  
     
     
         84 . The bacterial cell of  claim 17 , wherein the DNA-binding domain is λcI, AraC, LexA, Gal4, or zinc fingers.