核糖体蛋白L12-L10相互作用抑制剂的筛选及其抗结核活性的研究

摘要

Objective To screen potential small-molecular inhibitors of L12-L10 interaction with anti-tuberculosis activity. Methods A yeast two-hybrid system was used to identify small molecules that block the interaction between L12 and L10 proteins. The minimum inhibitory concentration (MIC) was measured in sterile 96-well microplates. Anti-tuberculosis activity of compound IBM-T275 was determined by microplate alamar blue assay (MABA). Theβ-gal activity was measured to detect the disruption of L12-L10 interaction by compound IBM-T275. Translation inhibition by compound IBM-T275 was assessed using an in vitro cell-free translation system. The MICs of compound IBM-T275 against ATCC strains and clinical isolates were determined using the agar dilution method. Results Four compounds were found to inhibit the growth of AH109 (pAD-L12+pBD-L10) more dramatically than that of AH109 (pAD-T + pBD-53), and the MICs range was 3.125-12.5 μg/ml on mc2155. Among the hit compounds, IBM-T275 showed anti-tuberculosis activity on standard strain and clinical strains with MICs range at 5 - 10 μg/ml, but showed high MICs on other bacterial strains (MICs>64μg/ml). Compound IBM-T275 could inhibitβ-gal activity in the model as well as the in vitro protein translation with IC50 of 12.57μg/ml. Conclusion A new compound was found to be an inhibitor of L12-L10 interaction with anti-tuberculosis activity through yeast two-hybrid screening system.%目的以 L12-L10相互作用为靶点筛选具有抗结核活性的先导化合物。　　方法应用酵母双杂交模型 AH109（pAD-L12+pBD-L10）通过生长抑制方法筛选阳性化合物，以 AH109（pAD-T +pBD-53）作为对照；通过96孔板法检测阳性化合物对耻垢分枝杆菌的抑制活性；应用定量微孔板快速显色法（MABA）检测抗结核杆菌活性；通过β-半乳糖苷酶活性定量检测判断阳性化合物在模型上对 L12-L10相互作用的阻断活性；应用体外蛋白表达系统检测阳性化合物对蛋白表达的抑制作用；应用平皿二倍稀释法检测阳性化合物的药敏作用。　　结果筛选到4个在模型上具有活性的阳性化合物，其对耻垢分枝杆菌具有比较好的抑制活性，其最小抑制浓度（MIC）在3.125~12.5μg/ml 之间；其中 IBM-T275对结核分枝杆菌标准株和临床分离株均具有比较好的抑制活性， MIC 在5~10μg/ml 之间，而对细菌抑制活性较低，其MIC 均在64μg/ml 以上；IBM-T275能够抑制酵母模型内β-半乳糖苷酶的表达，并且能够体外抑制蛋白表达，其 IC50为12.57μg/ml。　　结论筛选到1个具有抗结核杆菌活性的阳性化合物，其抗结核活性可能与阻断 L12-L10蛋白相互作用相关。