Anti-tb Drug Targets Based On Bioinformatics Screening And Verification

Tuberculosis,caused by Mycobacterium tuberculosis.is one of the leading infectious diseases worldwide.Globally,9.2 million new cases and 1.7 million deaths from TB occurred in 2006.The persistent infection of M.tuberculosis and the emergence of drug-resistant strains present two major obstacles in gaining control over tuberculosis worldwide.It is urgent to develop new anti-tuberculosis drugs.Modern drug development is largely target-driven.Bioinformatics has certainly come to stay and is now ubiquitous with drug discovery.In present work,we firstly analyzed the unknown function genes in the tuberculosis H37Rv genome and identified two novel protein domains,DIM and MSTF.The functions of these novel domains were discussed based on the homology sequences.To facilitate novel drug target identification, we started with 332genes which are involved in persistent infection and cell wall synthesis and metabolism of M.tuberculosis.Using bioinformatics methods,we finally identified 66 genes which fill the basic discipline of potential drug target.The thick and complex cell envelope has been implicated in many aspects of the pathogenicity and drug resistance of M.tuberculosis.Based on the drug target screening result,we cloned galU/Rv0993 and glmU/Rv1018c which involved in the cell wall synthesis.M.tuberculosis galU/Rv0993 which coded UDP-glucose pyrophosphorylase(MtGalU) was used to synthesis important sugar donor UDP-glucose.M.tuberculosis glmU/Rv1018c coded a bifunctional enzyme which inhabits both GleN-1-P acetyltransferase and UDP-N-GlcNAc pyrophosphorylase activities(MtGlmU).MtGlmU was used to synthesis another essential sugar donor UDP-N-GlcNAc.In current work, we purified and characterized the recombinant MtGalU and MtGlmU proteins. MtGalU exists as a mixture of monomer and different oligomers and MtGlmU exists as a stable hexamer.The specific activity,enzyme kinetic properties of MtGalU and MtGlmU were determined.The subcellular localization study shows that MtGalU located on cell wall and MtGlmU located in cell cytoplasm.The 3D structures of MtGalU and MtGlmU were modeled and the models could be further explored for in silico docking studies with suitable inhibitors.Ic1/Rv0467 which is related with persistence infection of M. tuberculosis was subjected to further characterization.We cloned the icl/Rv0467 gene and purified the recombinant functional isocitrate lyase (ICL) protein.The crystal structure of M.tuberculosis ICL was determined formerly and eighty compounds that "hits" the target ICL were screened in silico from the compound library Specs.The enzymatic inhibition effects of these compounds were confirmed by enzymatic activity assay. By testing the inhibition effect of the compounds on the growth of bacteria in vitro,we finally got two compounds which inhibit the proliferation of analogue persistent M.tuberculosis H37Ra(cultured in the defined minimal medium with acetate as only carbon source) and M.tuberculosis multi-drug resistant stains.One compound,FD20,has low cytotoxicity and can inhibit the proliferation of M.tuberculosisH37Ra in human macrophage THP-1.In summary,two novel protein domains,DIM and MSTF,were identified in M.tuberculosis proteome by bioinformatics analysis in this work. Combined with bioinformatics analysis,we identified 66 potential anti-tuberculosis drug target genes,three of which were subjected to further biochemical analysis.Finally,the compound FD20 targeted against ICL represents an attractive anti-tuberculosis lead compound.