Establishment And Application Of High - Throughput Screening Model For Mycobacterium Tuberculosis DXS And DXR Inhibitors

In MTB, isoprenoids play an important role in building the bacterial cell wall core mycolic acid-arabinogalactan-peptidoglycan complex(mAPG), and in other key metabolic functions such as the electron transport system.All isoprenoids are biologically synthesized through the repetitive condensation of the5-carbon (C5) isoprene building precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In MTB, the MEP pathway that comprises eight enzymatic steps is the only way to synthesis the essential isoprenoids and it is absent in mammalian cells.In this part we take the essential enzyme DXS and DXR of MEP pathway as our research objects. We have cloned the encoding gene dxs as well as dxr of MTB, and expressed MtDXS and MtDXR. The expressed products were purified by Ni2+chelating affinity chromatography. Based on their enzyme activity assays, we finally build a screening model both targeting MtDXS and MtDXR and a screening model targeting MtDXR. We evaluate the model’s feasibility after determining some impacts on the model. After screening66,400compounds, we get9positive compound candidates. Finally, we get1compound target MtDXS named IMB-DXS-B1and1compound target MtDXR named IMB-DXR-B1showing a good anti-tuberculosis activity. Inhibitor IMB-DXS-B1’s MIC on MTB is2～4μg/mL and IMB-DXR-Bl’s is32μg/mL. Both have a good activity on clinical drug-sensitive MTB and drug-resistant MTB while showing weak effect on other bacteria.The results suggest that the two compounds with different action mechanism from traditional anti-tuberculosis drugs can be potential anti-tuberculosis drugs. MtDXS and MtDXR suggest to be promising new anti-tuberculosis drug targets. More active and specific anti-tuberculosis lead compound with new mechanism will be found through screening MtDXS or MtDXR inhibitors.