The First Part Of The Screening Of Anti-TB Drugs For Shikimate Dehydrogenase As A Target For Preliminary Studies Of Mycobacterium Tuberculosis Acid Mechanisms Of Drug Screening Model Targeting The Second Part

This thesis includes two parts:The first partThe shikimate dehydrogenase of Mycobacterium tuberculosis （MtSD）, as a potential anti-tuberculosis drug targets, is closely related to the growth and reproduction. It is very important to get anti-tuberculosis activity compounds for the target validation. For this reason, we cloned and expressed the MtSD using repeated freeze-thawing and Ni2+affinity chromatography, and obtained high-purity MtSD. After detecting the nature activity of MtSD and optimization the assay system of enzyme, we built a high-throughput drug screening model for anti-tuberculosis targeting on MtSD. we screened50000compounds using the model, of which11compounds can inhibit MtSD activity. By detecting the antibacterial activity, we founded10inhibitors have anti-tuberculosis activity, in which the MIC of7957118is0.5μg/ml. Therefore the research provides practical methods for the high throughput screening of MtSD inhibitor.In this work, we also used the method of molecular docking to evaluate the interaction mode of anti-tuberculosis compounds with MtSD protein, and founded7957118has a high scores which has better anti-tuberculosis activity. Then through the study of enzyme kinetics, we elucidated7957118was a non-competitive inhibitor whose inhibition constant is70.55μM.Finally, we evaluated9anti-tuberculosis activities compounds on the toxicity of mouse macrophage, founded3compounds toxicity was low, and7957118maybe become anti-tuberculosis lead compounds because of its higher inhibit activity and lower toxicity, as well as providing probe molecules to velidate that the MtSD can be used as anti-tuberculosis drug targets.The second partMycobacterium tuberculosis which has ability to long-term survival in the host body is closely related with its acid-resistant ability. It is possible to obtain a new mode of action of anti-tuberculosis drugs from destruction of Mycobacterium tuberculosis acid-resistant. For this purpose, this thesis identified Mycobacterium smegmatis as a model for research on tuberculosis acid resistant mechanisms, by comparing of mycobacterium acid resistance. Then the MSMEG₆183of Mycobacterium smegmatis was knocked out by homologous recombination, which is homolog with Rv3671c of Mycobacterium tuberculosis. And we got the mutant of MSMEG₆183whose morphology changes and acid resistance descent. The obtained results lay a foundation for further related research.