Method of screening anti-mycobacterial molecules

Abstract

This invention relates to a novel mycobacterial protein named DES, which appears to share significant amino acid sequence homology with soluble stearoyl-ACP desaturases. The results of allelic exchange experiments, indicate that the des gene may be essential to the survival of mycobacteria. These results coupled with the surface localization, the unique structure of DES, and the fact this antigen is expressed in vivo, and DES protein induces a humoral response in human patients, indicate that the DES protein provides a new target for the design of anti-mycobacterial drugs. This invention provides methods of screening molecules that can inhibit the DES enzyme activity of purified DES protein, in order to identify antibiotic molecules that are capable of inhibiting the growth or survival of mycobacteria. These methods may be practiced by using recombinant DES protein obtained from a recombinant mycobacterium host cell that was transformed with a vector containing the des gene, whose expression is controlled by regulatory or promoter sequences that function in mycobacteria. Another aspect of this invention relates to the molecules that have been identified according to the screening methods as having antibiotic activity against mycobacteria.

What is claimed is:  
     
         1 . A method for screening a molecule capable of inhibiting the growth or survival of a mycobacteria species, said method comprising: 
 a) contacting the molecule with a strain of mycobacteria species comprising an active DES protein, or a DES like protein, or a vector comprising a first polynucleotide sequence encoding an active DES protein, or a vector comprising a second polynucleotide sequence encoding an active site of the DES protein;    b) measuring the growth of said mycobacteria strain; and    c) identifying the molecule that inhibits the growth of said mycobacteria strain.    
     
     
         2 . The method of  claim 1 , wherein the mycobacteria species is  Mycobacterium tuberculosis.    
     
     
         3 . The method of  claim 2 , wherein the DES protein is a recombinant DES protein.  
     
     
         4 . The method of  claim 3 , wherein the recombinant DES protein is obtained from a recombinant mycobacterium host cell.  
     
     
         5 . The method of  claim 4 , wherein the recombinant DES protein is prepared by a process comprising: 
 a) transforming a mycobacterium host cell with a recombinant expression vector, wherein the recombinant expression vector comprises a polynucleotide encoding said recombinant DES protein and a regulatory or promoter sequence that functions in mycobacteria; and    b) purifying said recombinant DES protein from said transformed mycobacterium host cell.    
     
     
         6 . The method of  claim 5 , wherein the recombinant expression vector has been constructed from the pJAM2 plasmid.  
     
     
         7 . The method of  claim 6 , wherein the recombinant expression vector is pJAM21.  
     
     
         8 . A plasmid selected from the group consisting of pJAM2 and pJAM21.  
     
     
         9 . A recombinant mycobacterium host cell, comprising a plasmid according to  claim 8 .  
     
     
         10 . The recombinant mycobacterium host cell according to  claim 9 , wherein the plasmid is pJAM2.  
     
     
         11 . The recombinant mycobacterium host cell according to  claim 9 , wherein the plasmid is pJAM21.  
     
     
         12 . A molecule capable of inhibiting the growth or survival of  Mycobacterium tuberculosis , wherein said molecule has been screened according to the method of  claim 2  or wherein said molecule blocks the active site of the DES protein or modulates the expression of the des gene in vivo or in vitro.  
     
     
         13 . A molecule capable of inhibiting the growth or survival of  Mycobacterium tuberculosis , wherein said molecule has been screened according to the method of  claim 3 .  
     
     
         14 . A molecule capable of inhibiting the growth or survival of  Mycobacterium tuberculosis , wherein said molecule has been screened according to the method of  claim 4 .  
     
     
         15 . A molecule capable of inhibiting the growth or survival of  Mycobacterium tuberculosis , wherein said molecule has been screened according to the method of  claim 5 .  
     
     
         16 . A molecule capable of inhibiting the growth or survival of  Mycobacterium tuberculosis , wherein said molecule has been screened according to the method of  claim 6 .  
     
     
         17 . A molecule capable of inhibiting the growth or survival of  Mycobacterium tuberculosis , wherein said molecule has been screened according to the method of  claim 7 .  
     
     
         18 . A method according to  claim 17 , wherein the active site of the DES protein comprises a first amino acid sequence DEXXH (SEQ ID NO:4) or a second amino acid sequence EEXXH (SEQ ID NO:3), wherein X can represent any amino acid.  
     
     
         19 . A purified DES polypeptide in monomeric or dimeric form, comprising an amino acid sequence DEXXHEEXXH (SEQ ID NO:5), wherein X can represent any amino acid.  
     
     
         20 . A purified DES polypeptide in monomeric or dimeric form, comprising an amino acid sequence DEXXH (SEQ ID NO:4), wherein X can represent any amino acid.  
     
     
         21 . A purified polypeptide according to  claim 20 , further comprising a second amino acid sequence EEXXH (SEQ ID NO:3), wherein the amino acid sequence DEXXH (SEQ ID NO:4) and the second amino acid sequence EEXXH (SEQ ID NO:3) are separated by at least 10 to 100 amino acids.  
     
     
         22 . A purified polypeptide according to  claim 21 , wherein the amino acid sequence DEXXH (SEQ ID NO:4)and the second amino acid sequence EEXXH (SEQ ID NO:3) are separated by 85 amino acids.  
     
     
         23 . A purified polypeptide according to  claim 22 , further comprising a second amino acid sequence EEXXH (SEQ ID NO:3), wherein the polypeptide has between 10 to 100 amino acids.  
     
     
         24 . A composition for the treatment of mycobacteria infection, comprising a molecule capable of inhibiting the growth of the survival of mycobacteria strain by inhibition of DES protein activity, wherein said molecule has been screened according to  claim 1 .  
     
     
         25 . A method for screening a molecule capable of inhibiting the growth of mycobacterial species, said method comprising: 
 a) contacting the molecule with a purified stearoyl or acyl ACP desaturase; and    b) measuring the inhibition of the enzyme activity compared to an untreated enzyme.    
     
     
         26 . A method for screening a molecule capable of inhibiting the growth or survival of a mycobacteria species selected from the group consisting of  Mycobacterium tuberculosis  and  Mycobacterium leprae , said method comprising: 
 a) contacting the molecule with a purified desaturase protein;    b) detecting the catalytic activity of the desaturase protein; and    c) identifying the molecule that reduces the catalytic activity of the desaturase protein, as compared to the catalytic activity of the purified desaturase protein that is not contacted with the molecule.    
     
     
         27 . The method of  claim 26 , wherein the mycobacteria species is  Mycobacterium tuberculosis.    
     
     
         28 . The method of  claim 27 , wherein the purified desaturase protein is a recombinant desaturase protein.  
     
     
         29 . The method of  claim 28 , wherein the purified recombinant desaturase protein is obtained from a recombinant mycobacterium host cell.  
     
     
         30 . A molecule capable of inhibiting or decreasing the growth or survival of a mycobacteria species, wherein said molecule has been screened according to  claim 26.