Study On TPP And Lead Compounds Of New Anti - Tuberculosis Drug Targets

Tuberculosis, caused by Mycobacterium tuberculosis, is one of leading killer of human beings, and do the most harm to human health. During the middle of twentieth century, anti-tuberculosis drugs were discovered and used to cure patient with tuberculosis, so the disease was once under control. However, in the last decades the widespread occurrence of drug-resistant strains, leading to resurgence of tuberculosis worldwide, pose a serious threat to mankind and give a big challenge to ours. Therefore, it is urgent for us to research and develop new anti-tuberculosis drug.The mycobacterial cell wall is a complex mixture of unique components that sets mycobacteria apart from other typical 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. The works of other researchers showed that trehalose phosphate phosphatase (TPP) is an important factor required for the growth of Mycobacterium tuberculosis in laboratory culture, and the expression of TPP was upregulated in isoniazid-resistant M. tuberculosis.This work of study aim to trehalose phosphate phosphatase of M. tuberculosis as a drug target, and based on TPP we design lead compounds to combat with tuberculosis by virtual screening method, and carry out research of crystallization of TPP.1) The gene, Rv3372, was cloned from M. tuberculosis H37Rv strain using molecular biology technique, then was linked into the prokaryotic expression vector for expression in E. coli and TPP protein was purified, the result of biochemical analysis showed that recombinant protein have TPP enzymatic activity;2) We obtained the three-dimensional structure of TPP by homology modeling, and further analysed the active site of protein;3) The result of site-directed mutagenesis demonstrated that the mutant proteins almost lost enzymatic activity, and verify the correctness of the active site;4) By structure-based virtual screening technique we obtained67 small molecule compounds from LeadQuest database, further research of ligand-based technique we got41small molecule compounds, and finally we obtained21small molecule compounds after optimization;5) Small molecule compounds were tested against M. tuberculosis H37Ra, H37Rv and clinical isolate drug-resistant strain in vitro, and the results showed that there are5novel compounds effectively against the growth of H37Ra strain and MIC is less than0.39μg/ml. Furthermore, there is one compound among five compounds which can inhibit H37Rv and clinical drug-resistant strains with MIC=0.14μg/ml;6) Four compounds can effectively reduce the enzymatic activity through analysis of interaction between11small molecule compounds and TPP protein, and there were three compounds effectively inhibiting the growth of different strains of M. tuberculosis;7) We carried out the expression of recombinant TPP protein and exploration of protein purification methods, finally establish the method of protein purification;8) In order to get crystals suitable for data collection, TPP samples that meet the requirements of condition of protein crystal growth were cultured and do extensive screening and optimization of growth condition, and crystals were analyzed by X-ray diffraction.The lead compounds based on our research lay a firm foundation for the development of novel anti-tuberculosis agents.