Activity Evaluation And Mechanism Of New Anti - Tuberculosis Lead Compounds

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the leading infectious diseases worldwide. Globally,8.8million new cases,1.1million deaths from TB and0.35million deaths from TB-HIV co-infection occurred in2010according to the WHO Global TB Control Report2011. The persistent infection of M. tuberculosis and emergence of drug-resistant strains present two major obstacles in gaining control over tuberculosis worldwide. It is so urgent to develop new anti-tuberculosis drugs.Mycobacterial cell wall is a complex mixture of unique components that distinguish mycobacteria from other bacterial species.Thus the key factors of biosynthetic pathways leading to formation of mycobacterial cell wall are attractive targets for the design of novel anti-tuberculosis agents. Trehalose phosphate phosphatase(TPP) is an important factor required for the growth of Mycobacterium tuberculosis in laboratory culture but absent from mammals, which synthesize an essential formation of mycobacterial cell wall----trehaiose-6-phosphate. Accordingly, TPP is a very potential target for the development of anti-tuberculosis agents.This previous work of study aim to trehalose phosphate phosphatase of M. tuberculosis as a drug target, and based on TPP lead compounds have been designed to combat with tuberculosis by virtual screening method. Several lead compounds have good performance on the of anti-TB activity. Now we choose one of them to do the further evaluation of the anti-TB activity.The Minimum inhibitory concentrations (MIC) of compound E for standard H37Ra, H37Rv, Mycobacterium smegmatis, Mycobacterium marinum,Mycobacterium fortuitum, Mycobacterium avium and Mycobacterium kansasii strains were0.078μg/ml,0.4μg/ml,12.5μg/ml,50μg/ml,32μg/ml,32μg/ml and32μg/ml respectively. The Minimum bactericidal concentration (MBC) of compound E for H37Ra strain was equal to4fold of MIC(0.312μg/ml). After three days’drug exposure, E showed better antituberculous activity against the activity growth phase H37Ra (10days old) than the late phase strains (6weeks old).The H37Ra strain was killed100%under1MBC concentration of compound E at the eighth day. The growth curve of H37Ra strain changed significantly under sub-inhibitory concentrations of compound E (0.02μg/ml). E showed synergy with Rifampicin (RIF) against H37Ra (FICI=0.27) and was additive with Isoniazid (INH)(FICI=0.6). The compound E had no toxicity to THP-1cell. Furthermore, the compound E had bactericidal activity against M.tuberculosis in macrophages. Most important of all, the compound E had bactericidal activity against nongrowing persister Mycobacterium tuberculosis. H37Ra was not easily resistant against E after three independent repeats and the isoniazid-resistant frequency was3.4×10-6. These data fully illustrated that compound E had antibacterial and bactericidal activity against Mycobacterium tuberculosis in vitro and lay a firm foundation for the development of novel anti-tuberculosis agents.To identify the hypothesis that TPP is the target of compound E, TPP protein was expressed and purified. The result of biochemical analysis showed that recombinant protein had enzymatic activity. Isothermal titration microcalorimeter (ITC) was used to find out if compound E can bind to TPP protein in vitro; Enzyme assay was carried out to identify if compound E could inhibit the enzymatic activity of TPP; Over-expression and low-expression TPP in H37Ra strain were constructed and MIC of compound E for them were tested to find out if MIC values were increased or decreased compared with the MIC value of compound E for standard H37Ra strain. Although these results showed TPP may not the main target of E, its good performance of antituberculosis activity against H37Ra and the new technology of target identification still demonstate the promising future.