以丙氨酸消旋酶为靶点的高通量抗结核药物筛选模型的建立及应用

目的建立以结核分枝杆菌丙氨酸消旋酶为靶点的新型高通量抗结核药物筛选模型,筛选丙氨酸消旋酶的抑制剂,获得以丙氨酸消旋酶为靶点的新型抗结核药物先导物。方法以结核分枝杆菌H37Rv基因组为模板,pET28a表达质粒为载体,将alr基因克隆至pET28a,构建pET28a::alr重组表达质粒,表达并纯化得到重组结核分枝杆菌丙氨酸消旋酶;通过测定反应产物NADH在340 nm处光密度变化速率,检测酶反应活性,构建并优化该酶抑制剂的高通量筛选模型;应用该模型对化合物库进行筛选;测定活性化合物IC50以及对结核分枝杆菌的MIC。结果成功构建了结核分枝杆菌alr基因的表达载体;得到了纯度较高的重组丙氨酸消旋酶,测得该酶的比活力为13.53 kU/mg;所建立的丙氨酸消旋酶高通量筛选模型稳定性高,符合高通量筛选的要求;通过对70 000个化合物进行筛选,得到了5个活性较高的化合物,其中,IMB-XZ5对结核分枝杆菌的MIC为4~8μg/ml,且对结核分枝杆菌的作用具有较高的特异性。结论建立了稳定性好、灵敏度较高的结核分枝杆菌丙氨酸消旋酶抑制剂高通量筛选模型,应用该模型筛选得到了具有较好抗结核活性的丙氨酸消旋酶抑制剂。

Establishment and application of a novel high throughput screening model targeting alanine racemase from mycobacterium tuberculosis H37Rv

Objective To establish a high throughput screening （HTS） model targeting mycobacterium tuberculosis （MTB） alanine racemase （ALR） for the discovery of novel antitubercular drugs and to determine inhibitory activities against MTB of the inhibitors.
Methods The H37Rv ALR coding gene alr was amplified and cloned into pET28a expression vector. The recombinant ALR was expressed in Escherichia coli BL21（DE3）pLysS and its activity was measured by detecting the absorption at 340 nm wavelength. HTS model was established based on the activity of ALR and Z＇ factor was used to evaluate the quality of HTS model. About 70 000 compounds were screened using this model and the IC50 of inhibitors were determined. Inhibitory activities against H37Rv and some other bacteria strains of the inhibitors were also evaluated.
Results Recombinant H37Rv alr vector was successfully constructed and expressed, with the optimal enzymatic activity of ALR being 13.53 kU/mg. The parameters suggested that this HTS model was highly stable and feasible for HTS drug screening. Of the 70 000 compounds, 5 compounds appeared inhibitory activities to ALR in this HTS model. The inhibitor IMB-XZ5 was determined active against H37Rv with the MIC 4-8μg/ml and the specificity was obvious.
Conclusion A steady and sensitive HTS model for MTB ALR inhibitors was established. One of the ALR inhibitors was evaluated and has potential to become an active and specific anti-TB lead compounds.