Mechanism Study Of MtSK Inhibitor IMB-5147 And Virtual Screening Of MtSK Inhibitor

Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis(MTB).It is one of the major public health problems faced by all mankind.Although there are two kinds of anti-tuberculosis drugs approved to market since the 1990s,there are two kinds of anti-tuberculosis drugs:Dramani and betaquinoline.so far,there have been reports of side effects and drug-resistant strains.Therefore,the development of new anti-tuberculosis drugs is still of great significance.Shikimate pathway is an important metabolic pathway in plants,bacteria and fungi,which can synthesize folic acid,naphthoquinone,ubiquinone,mycobacterial acid,aromatic amino acids and other essential substances.The growth of MTB is inhibited after blocking shikimic pathway.In addition,the coding genes of each enzyme in shikimic pathway do not exist in human body,and there are no reports of clinical application of anti-tuberculosis drugs targeting this pathway.Therefore,anti-tuberculosis drugs targeting key enzymes in shikimic pathway may have the advantages of effective,low toxicity and no cross drug resistance.In this study,we targeted shikimate kinase(MtSK),a key enzyme in shikimic pathway,to screen anti-tuberculosis lead compounds and study the related mechanism.In the previous work,the positive compound IMB-T5147,was obtained by phenotypic screening and enzyme activity screening.In this study,the enzyme inhibitory activity and anti-tuberculosis activity of the compound were further tested.Firstly,MtSK protein was expressed and purified in E.coli prokaryotic system,and the evaluation method of MtSK enzyme inhibition based on ATP fluorescence was established.The inhibitory activity of compound IMB-T5147 was studied.The results showed that the enzyme inhibitory activity of the compound was high,and the IC50 value was 4.489 ?g/mL.Then,an enzyme activity detection method based on HPLC technique was established,and the inhibition mechanism of IMB-T5147 activity was deeply studied.The results showed that the enzyme inhibition activity decreased with the increase of shikimic acid substrate concentration,which proved that the compound was a competitive enzyme inhibitor of shikimic acid substrate.SPR detection showed that the affinity KD value of IMB-T5147 and MtSK protein was 1.27×10-5 M.Compound IMB-T5147 had low to icity to mammalian cells and had no growth inhibitory activity on HepG2 cells at 100 ?g/mL.The MIC of IMB-T5147 to MTB standard strain H37Rv was 31.25 ?g/mL.In this study,virtual screening of inhibitors was also carried out with the 2IYQ protein of MtSK as the target.First of all,the large commercial database was virtual docked by molecular docking software AutoDock Vina,and then the first 1000 compounds were re-docked by Dock6.7,and finally the first 100 compounds were selected as the first batch of candidate compounds,the second batch of candidate compounds were obtained by molecular similarity screening method,two batches of compounds were analyzed by cluster analysis,and 38 compounds were selected for follow-up research.The MtSK enzyme inhibitory activity of the candidate compounds was evaluated,and it was found that at 20 ?g/mL,there were 5 compounds with an inhibition rate of more than 50%,and their MIC to the standard strain H37Rv of MTB was 62.5 ?g/mL.In a word,the enzyme inhibitory activity,cytotoxicity and anti-tuberculosis activity of compound IMB-T5147 were evaluated for the first time in this study.The virtual screening of inhibitors in large commercial libraries ChemDiv and TargetMol was carried out by AutoDock Vina and UCSF Dock 6.7 software,and five compounds with MtSK inhibition rate of more than 50%were found,which promoted the research of anti-tuberculosis drugs targeting MtSK.