Methods for identifying and using MarR family polypeptide binding compounds

Abstract

Methods for identifying MarR family inhibiting compounds are described. The methods include the use of computer aided rational based drug design programs and three dimensional structures of MarR family polypeptides.

1 . A method of identifying a MarR family modulating compound comprising: 
 selecting a candidate MarR family modulating compound by performing rational drug design with the set of atomic coordinates in  FIG. 1  or  2 , such that a MarR modulating compound is identified.    
     
     
         2 . The method of  claim 1 , further comprising the contacting the candidate MarR family modulating compound with MarR family polypeptide; and determining the ability of said candidate MarR family modulating compound to modulate a MarR family polypeptide.  
     
     
         3 . The method of  claim 1 , wherein said MarR family polypeptide is MarR.  
     
     
         4 . The method of  claim 3 , wherein MarR has the polypeptide sequence given in SEQ ID NO. 1.  
     
     
         5 . The method of  claim 1 , wherein MarR family modulating compound interacts with one or more amino acid residues, selected from the group consisting of: amino acid residues corresponding to the amino acid Ala at position 70 of SEQ ID NO:1, corresponding to the amio acid Arg at position 73 of SEQ ID NO:1, corresponding to the amino aicd Arg at position 77 of SEQ ID NO:1, corresponding to the amino aicd Asp at position 67 of SEQ ID NO:1, corresponding to the amino acid Thr at position 72 of SEQ ID NO:1, or corresponding to the amino acid Arg at position 86 of SEQ ID NO:1.  
     
     
         6 . The method of  claim 1 , wherein said MarR family modulating compound is a MarR family polypeptide inhibitor.  
     
     
         7 . The method of  claim 6 , wherein said MarR family modulating compound modulates the expression of MarA.  
     
     
         8 . The method of  claim 1 , wherein said rational drug design is performed using a computer program.  
     
     
         9 . The method of  claim 8 , wherein said computer program is LigBuilder, DOCK, GLIDE, AUTO_DOCK, CoMFA, GRID and/or FLEXX.  
     
     
         10 . A method of identifying a MarR family modulating compound comprising: 
 determining the structure of a MarR family polypeptide using the structure of MarR, such that a MarR family modulating compound is identified.    
     
     
         11 . The method of  claim 10 , wherein said method further comprises identifying a candidate MarR family modulating compound by performing rational drug design based on the structure.  
     
     
         12 . The method of  claim 11 , wherein said method further comprises contacting the candidate MarR family modulating compound with a MarR family peptide, and a nucleic acid molecule.  
     
     
         13 . The method of  claim 12 , wherein said method further comprises measuring the binding affinity of the MarR family polypeptide peptide with the nucleic acid molecule.  
     
     
         14 . The method of  claim 10 , wherein said MarR family polypeptide is selected from the group consisting of: SlyA, EmrR (MprA), PapX, PrsX, HpcR, Ec17 kD of  Escherichia coli ; MarR, SlyA, EmrR of  Salmonella typhimurium ; MexR of  Pseudomonas aeruginosa ; PecS of  Erwinia chrysanthemi ; BadR of  Rhodopseudomonas palustris ; OrfE of  Burkholderia pseudomallei ; YdcH, YhbI, YkmA, YkoM, Orf7, YfiV, YetL, YdgJ, YwoH, YwaE, YwhA, Hpr, YybA, YxaD, YsmB, YusO, YpoP, YkvE of  Bacillus subtilus ; Orf7 of  Bacillus firmus ; Orf145, Orf141 of  Staphylococcus sciuri ; CinR of  Butyrivibrio fibrisolvens ; Orf158 of  Sphingomonas aromaticivorans ; NhhD of  Rhodococcus rhodochrous ; Orf1 of  Streptomyces peucetius ; 14.7 kD, Rv1404, Rv0737, Rv0042c, Yz08 (15.6 kD) of  Mycobacterium tuberculosis ; Yz08 (15.6 kD) of  Mycobacterium leprae ; MTH313 of  Methanobacterium thermoautotrophicum ; Lrs14 of  Sulfolobus solfataricus ; CinR of  Archaeoglobus fulgidus ; PetP of  Rhodobacter capsulatus ; and SlyA (E293909) of  Sinorhizobium meliloti.    
     
     
         15 . The method of  claim 10 , wherein said MarR family modulating compound enhances or decreases the activity of the MarR family polypeptide.  
     
     
         16 . The method of  claim 15 , wherein said MarR modulating compound inhibits the expression of a protein.  
     
     
         17 . The method of  claim 10 , wherein the structure of said MarR family polypeptide is determined using COMPOSER, MODELER, or HOMOLOGY.  
     
     
         18 . A method of identifying a MarR modulating compound comprising: 
 obtaining a set of atomic coordinates defining the three-dimensional structure of MarR, wherein MarR has the peptide sequence given in SEQ ID NO. 1;    selecting a candidate MarR modulating compound by performing rational drug design with said three dimensional structure of MarR;    contacting said candidate MarR modulating compound with MarR;    measuring the ability of the candidate MarR modulating compound to modulate the activity of MarR, thus identifying a MarR modulating compound.    
     
     
         19 . The method of  claim 18 , wherein said rational drug design is aided by a computer program.  
     
     
         20 . The method of  claim 19 , wherein the set of atomic coordinates for the three-dimensional structure of MarR is given in  FIG. 1  or  2 .  
     
     
         21 . The method of  claim 19 , wherein said computer program is LigBuilder, DOCK, AUTO_DOCK, GLIDE, CoMFA, GRID and/or FLEXX.  
     
     
         22 . A MarR modulating compound of the formula (I):  
         X—Y-Z  (I)  wherein 
 X is an interacting moiety;  
 Y is a hydrophobic moiety; and  
 Z is a polar moiety.  
   
     
     
         23 . The MarR modulating compound of  claim 22 , wherein said interacting moiety interacts with the amino acid Thr at position 72 of SEQ ID. 1.  
     
     
         24 . The MarR modulating compound of  claim 22 , wherein said interacting moiety interacts with the amino acid Ala at position 70 of SEQ ID No. 1.  
     
     
         25 . The MarR modulating compound of  claim 24 , wherein said interacting moiety is capable of hydrogen bonding.  
     
     
         26 . The MarR modulating compound of  claim 25 , wherein said interacting moiety is selected from the group consisting of hydroxyl, thiol, sulfonyl, amino, carbonyl, alkyl, and acyl moieties.  
     
     
         27 . The MarR modulating compound of  claim 26 , wherein said interacting moiety is hydroxy, thiol, or amino.  
     
     
         28 . The MarR modulating compound of  claim 27 , wherein said hydrophobic moiety is selected from the group consisting of substituted and unsubstituted alkyl, alkenyl, alkynyl, and aryl moieties.  
     
     
         29 . The MarR modulating compound of  claim 28 , wherein said hydrophobic moiety is aryl.  
     
     
         30 . The MarR modulating compound of  claim 22 , wherein said hydrophobic moiety is capable of interacting hydrophobically with the amino acid Pro at position 57 or the amino acid Met at position 74 of MarR.  
     
     
         31 . The MarR modulating compound of  claim 22 , wherein said polar moiety is capable of interacting with the amino acid Arg at position 86 of MarR.  
     
     
         32 . The MarR modulating compound of  claim 22 , wherein said polar moiety is capable of interacting with the amino aicd Arg at position 77 of MarR.  
     
     
         33 . The MarR modulating compound of  claim 22 , wherein said polar moiety is negatively charged.  
     
     
         34 . The MarR modulating compound of  claim 22 , wherein said polar moiety is selected from the group consisting of: carboxylate, phosphate, phosphite, sulfate, sulfite, nitrate, nitrite, nitro, hydroxy, oxalate, and perchlororate.  
     
     
         35 . The MarR modulating compound of  claim 34 , wherein said polar moiety is carboxylate.  
     
     
         36 . The MarR modulating compound of  claim 22 , wherein said MarR modulating compound is a MarR inhibitor.  
     
     
         37 . The method of  claim 22 , wherein said MarR modulating compound is of the formula:  
       
         
           
           
               
               
           
         
         wherein  
         Y is a substituted or unsubstituted cyclic or bicyclic moiety, and pharmaceutically acceptable salts and esters thereof.  
       
     
     
         38 . The method of  claim 37 , wherein X is hydroxyl.  
     
     
         39 . The method of  claim 37 , wherein Y is monocyclic.  
     
     
         40 . The method of  claim 37 , wherein Y is bicyclic.  
     
     
         41 . The method of  claim 37 , wherein said MarR modulating compound is selected from the group consisting of:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         42 . A method for inhibiting expression of MarA, comprising contacting MarR with a MarR modulating compound, wherein said MarR modulating compound is of the formula (I):  
         X—Y-Z  (I)  wherein 
 X is an interacting moiety;  
 Y is a hydrophobic moiety; and  
 Z is a polar moiety.  
   
     
     
         43 . The method of  claim 42 , wherein said compound modulates the binding of DNA to MarR.  
     
     
         44 . A method for decreasing multidrug resistance in a microbe, comprising contacting the microbe with a MarR modulating compound, such that said multidrug resistance of the microbe is decreased, wherein said MarR modulating compound is of the formula (I):  
         X—Y-Z  (I)  wherein 
 X is an interacting moiety;  
 Y is a hydrophobic moiety; and  
 Z is a polar moiety.  
   
     
     
         45 . The method of  claim 44 , wherein microbe is present in a subject.  
     
     
         46 . A method for modulating activity of a MarR family polypeptide, comprising contacting a MarR family polypeptide with a MarR family modulating compound identified by the method of  claim 1 .  
     
     
         47 . A MarR family modulating compound identified by the method of  claim 1.