1 Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA.
2 Department of Biochemistry and Molecular Biology, The Center for RNA Molecular Biology, The Pennsylvania State University, University Park, PA, USA.
SLAS Discov. 2017 Mar;22(3):287-297. doi: 10.1177/2472555216679994. Epub 2016 Dec 27.
Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (Rif) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant Rif RNAP (βS531L) containing a mutation (β'V408G) that compensates for the fitness defect of this Rif mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and Rif E. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.
利福平自问世以来一直是结核病（TB）治疗的基石。耐多药和广泛耐药结核病的出现使得开发针对这些菌株的新型治疗方法成为当务之急。利福霉素靶酶 RNA 聚合酶（RNAP）的靶位特异性突变构成了大多数（~97%）结核分枝杆菌（MTB）利福霉素耐药（Rif）菌株。为了鉴定新型细菌 RNAP 抑制剂，开发了一种基于质粒的体外转录测定法，该测定法使用孔雀绿（MG）检测含有 MG 适体的转录 RNA。该测定法在 384 孔板格式中进行了优化，并用于筛选 150,000 种化合物，以针对含有突变（β'V408G）的最具临床相关性 Rif RNAP 的大肠杆菌同源物进行筛选，该突变补偿了 Rif 突变体的适应性缺陷。经过确认和浓度反应研究，发现了 10 种在野生型和 Rif 大肠杆菌和 MTB RNAP 中具有相似体外抑制值的化合物。从筛选中鉴定出的四种化合物在低于 200µM 的浓度下对 MTB 在培养物中具有活性。初步随访结果导致由于潜在的全分析干扰而淘汰了一种支架。
