结核分枝杆菌1-脱氧-D-木酮糖-5-磷酸还原异构酶抑制剂高通量筛选模型的建立与应用

Establishment and application of a high-throughput screening model targeting to 1-deoxy-d-xylulose-5-phosphate reductoisomerase from Mycobacterium tuberculosis H37Rv

目的建立1-脱氧-D-木酮糖-5-磷酸还原异构酶抑制剂的高通量筛选模型,从不同来源的样品中筛选该酶抑制剂,以期获得作用于该靶点的全新作用机制的抗结核药物先导物。方法以结核分枝杆菌H37Rv基因组为模板,克隆基因dxr构建重组表达质粒pET28a(+)::dxr;IPTG诱导表达并经Ni2+亲和层析柱纯化得到具有天然酶活性的1-脱氧-D-木酮糖-5-磷酸还原异构酶;基于该酶促反应底物NADPH在340nm波长下具有光吸收的原理,建立和优化酶活测定体系,构建并评价酶抑制剂的高通量筛选模型;应用此模型进行酶抑制剂筛选并进行抑酶活性及抗菌活性评价。结果成功得到具有天然酶活性的1-脱氧-D-木酮糖-5-磷酸还原异构酶,构建了1-脱氧-D-木酮糖-5-磷酸还原异构酶抑制剂的高通量筛选模型并得到3个抑酶活性较高的阳性化合物,其中化合物IMB-DXR-B1为该酶的反竞争性抑制剂,其Ki值为6.2μmol/L,IC50为29.5μmol/L,该化合物对于结核分枝杆菌H37Rv的MIC为32μg/mL。结论成功构建了稳定性好、灵敏度高的结核分枝杆菌1-脱氧-D-木酮糖-5-磷酸还原异构酶抑制剂的高通量筛选模型,筛选得到具有抗结核活性的1-脱氧-D-木酮糖-5-磷酸还原异构酶抑制剂,为获得全新作用靶点的新型抗结核药物奠定了基础。

Objective To establish a high-throughput screening （HTS） model targeting Mycobacterium tuberculosis 1-deoxy-d-xylulose-5-phosphate reductoisomerase（MtDXR） for the discovery of novel antituberculosis drugs. The model was used to screen inhibitors targeting MtDXR and to explore the effects of compounds on the activity of DXR. Methods The H37Rv DXR coding gene dxr was amplified and cloned into pET28a（＋） expression vector. The recombinant MtDXR protein was expressed in Escherichia coli BL2 I（DE3）pLysS induced by IPTG and purified through immobilized metal affinity chromatography with NiE＋-NTA agarose and confirmed by SDS-PAGE.MtDXR catalyze 1-deoxy-d-xylulose-5-phosphate （DXP） to form 2-C-methyl-D-erythritol-4-phosphate（MEP） with oxidation of NADPH and a decrease in absorbance signal at 340nm wavelength. MtDXR activity was measured by detecting the change of NADPH absorbance at 340nm wavelength. To improve enzymatic assays and establish model for HTS, a series of experiments were proceeded to study enzymatic properties ofMtDXR. A HTS model for MtDXR inbibitors was established based on the activity of MtDXR and Z-factors were used to evaluate the quality of the HTS model. A total of 30,000 compounds were screened by using this model and IC5o as well as antitubercular activity of inhibitors were determined. Besides, the enzyme kinetics of inhibitor IMB-DXR-B1 was investigated. Results Recombinant MtDXR was successfully purified and has the optimal activity of 9800U/mg. A HTS model for MtDXR inhibitors was successfully established with the parameter Z factor of 0.79. It was suggested that the model is highly feasible and stable for HTS drug screening. 3 compounds were found to inhibit MtDXR using the HTS model, and the inhibitory effect of inhibitor IMB-DXR-B 1 on the activity of MtDXR was reversible with a Ki value of 6.2ktmol/L and an IC50 value of 29.5μmol/L. The MIC of inhibitor IMB-DXR-B 1 was 32μg/mL for Mycobacterium tuberculosis. Conclusion A steady and sensitive HTS model for potential MtDXR inhibitors was established. One of the MtDXR inhibitors was evaluated a potential to become an active and specific anti-TB lead compound.