Screening Of New Anti - Tuberculosis Drugs Targeted With IspD

With drug resistance of Mycobacterium tuberculosis(MTB) getting more and more severe, developing novel anti-TB drugs with new mechanism of action based on new targets is an effective way to tackle this problem.The unique cell wall structure of TB is vital to its viablity,proliferation and pathogenesis.Thus,the biosyn-thetic pathway of MTB cell wall contains a great number of new potential anti-TB targets.Isopentenyl diphosphate(IPP) and its isomer dimethylallyl diphosphate(DMAPP) are essential premers of peptidoglycan and arabinogalactan layer in MTB cell wall. Pathway 2-C-methyl-D-erythritol-4-phosphate is the only way for MTB to biosyn-thesize IPP and DMAPP. Eight enzymes are involved in this pathway and 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase(IspD) is a key and veloci-ty-limiting one in it. This paper is to obtain new anti-TB drugs targeting IspD, and to verify its druggability by utilizing it as a probe.This paper comprises of three major parts. The first part is to screen anti-TB compounds from a 40,000 compounds library through whole cell screening model of H37R.v, and 159 anti-TB compounds were acquired at a concentration of 20 μg/ml with a total positive ratio of 0.4%. In the second, we amplified the ispD gene by polymer-ase chain reaction with H37Rv genome as a template, then ligated the ispD gene into the expression vector pET28a(+), transformed the vector into E. coli BL21(DE3)pLysS to construct Mycobacterium tuberculosis 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase(MtIspD) recombinant ex-pression strain, induced the E. coli BL21(DE3)pLysS to express MtIspD, After purifying MtIspD, we established and optimized the activity assay method of MtIspD, construted the MtIspD inhibitors screening model, and applied the model to screen in-hibitors of MtIspD from the previously obtained anti-TB compounds, and got 5 inhibitors of MtIspD. Among them, IMB-4901 has potent MtIspD and MTB inhibitory activities (IC50 of 19.3 μg/ml and MIC for H37Rv of 1.6 μg/ml); Enzymatic dynam-ics(Enzyme kinetics) indicated IMB-4901 were competitive inhibitors of substrate MEP and CTP, with Ki of 24.9 μg/ml and 15.1 μg/ml, respectively. The results of anti-bacterial spectrum suggested that IMB-4901 specifically inhibited gram-positive bacteria, especially posing a strong and specific inhibition on MTB; Unfortunately, IMB-4901 showed cytotoxic activity on Vero and HepG2 with with TC50 of 1.8μg/ml and 1.9 μg/ml; In the third part, we cloned the upstream 320bp of ispD gene by using pET28a(+)::ispD as a template, and constructed a MtIspD regulatable vector which could be used in MTB, thus laying a foundation to finally obtain a MtIspD regulatable MTB stain and to verify the target of IMB-4901.