Functional Analysis Of Novel Antituberculosis Drug Targets TPP And LipF, And Primary Screening Of Lead Compounds

Tuberculosis represents one of the world' s greatest sources ofmortality and morbidity, with approximately eight million new infectionsand 2.5-3 million deaths per year. Mycobacterium tuberculosis is mainpathogen that lead to tuberculosis in human. Compounding the problem,strains of M. tuberculosis that are resistant to the major drugs used totreat tuberculosis are rapidly emerging worldwide. The eradication oftuberculosis requires the development of novel drugs for therapeutictreatment of M. tuberculosis.The mycobaeterial eell wall is a complex mixture of unique componentsthat sets myeobaeteria apart from other typical bacterial species and isthe target of many first-line antitubereulosis agents. Thus thebiosynthetic pathways leading to formation of key mycobaeterial cell wallare attractive targets for the design of new antituberculosis agents.Trehalosephosphate phosphatase (TPP), which involved in the mycobacteriacell wall biosynthesis, was aimed in this thesis.①The condingsequence of TPP: otsB2/Rv3372was cloned from M. tuberculosis H37Rv, thenexpressed in E. coli and purified to homogeneity. The recombinant proteinwas enzymatic.②The TPP specific antiserum was prepared from rabbit.Different cellular components of M. tuberculosis H37Rv and Mycobacteriumboris bacillus Calmette-Guerin (BCG) were separated by differentialvelocity eentrifugation, and TPP was subeellular localized in the cellwall and cell membrane of M. tuberculosis H37Rv and BCG.③The rabbitanti-TPP serum could inhibite the proliferation of M. tuberculosis.④TPP was detected to enhance the uptake of dextran by monocyte-derivedmacrophages, stimulate the proliferation of macrophages, and induce anincreased Yh1 type immune response in mice.⑤The mycobacterial TPP wasan immunodominant antigen, while it increased the colony forming unitesof BCG in the spleen and lung of C57BL/6 mice after BCG challenge. Thereis no homologous TPP in human and mammalia, and TPP is a key enzyme involvedin mycobacteria growth, which represent excellent potential target sitefor chemotherapy against tuberculosis.⑥The 3D structure of TPP wasmodeled, and Asp147, Asp330, Asp331, and Asp334 are potential active sites of TPP,⑦which was verified by site-directed mutagenesis.⑧55compounds that "hit" the target TPP were screened from the compoundlibrariy Tripos.⑨The compounds that inbibite the enzyme activity ofTPP were screened in vitro.⑩The active compounds that could inhibitethe proliferation of M. tuberculosis were screened in vitro, and theminimal inhibitory concentrations were evaluated. 3 lead compounds couldinhibit the proliferation of M. tuberculosis H37Rv, two of which couldinhibit the proliferation of multi-drug resistant tuberculosis.Another candidate drug target of M. tuberculosis is Esterase F (LipF,Rv3487c).①LipF was induced at low pH.②The recombinant LipF wasenzymatic to ester with short chain. Because LipF shows low homology tohuman/mammalia and the Rv3487c mutant strain exhibits an attenuatedphenotype in the lung of mice in comparison with the virulent M.tuberculosis strain, LipF was selected as the candidate target forscreening of antituberculosis drugs.②The structure of LipF was modeledby homologous modeling, and the active sites were predicted as Ser90,Glu189, and His219.③The active amino acids were mutated to Alanine bysite-directed mutagenesis, and the mutant proteins were malfunctioned.④50 compounds were screened from the database Tripos,⑤and one ofwhich could inhibite the proliferation of M. tuberculosis H37Rv in vitro.The achievement of 4 lead compounds against M. tuberculosis paves theway for the development of new antituberculosis drugs.