CAJ | 학술논문

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70000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。
목적건립이결핵분지간균병안산소선매위파점적신형고통량항결핵약물사선모형，사선병안산소선매적억제제，획득이병안산소선매위파점적신형항결핵약물선도물。 　　방법이결핵분지간균 H37Rv 기인조위모판，pET28a표체질립위재체，장 alr 기인극륭지 pET28a ，구건pET28a：alr 중조표체질립，표체병순화득도중조결핵분지간균병안산소선매；통과측정반응산물 NADH 재340 nm처광밀도변화속솔，검측매반응활성，구건병우화해매억제제적고통량사선모형；응용해모형대화합물고진행사선；측정활성화합물 IC50이급대결핵분지간균적 MIC。 　　결과성공구건료결핵분지간균 alr 기인적표체재체；득도료순도교고적중조병안산소선매，측득해매적비활력위13.53 kU/mg；소건립적병안산소선매고통량사선모형은정성고，부합고통량사선적요구；통과대70000개화합물진행사선，득도료5개활성교고적화합물，기중，IMB-XZ5대결핵분지간균적 MIC 위4~8μg/ml，차대결핵분지간균적작용구유교고적특이성。 　　결론건립료은정성호、령민도교고적결핵분지간균병안산소선매억제제고통량사선모형，응용해모형사선득도료구유교호항결핵활성적병안산소선매억제제。
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.