Burke Christopher, Jankute Monika, Moynihan Patrick, Gonzalez Del Rio Ruben, Li Xiaojun, Esquivias Jorge, Lelièvre Joël, Cox Jonathan A G, Sacchettini James, Besra Gurdyal S
Institute of Microbiology and Infection School of Biosciences University of Birmingham Birmingham UK.
Diseases of the Developing World GlaxoSmithKline Tres Cantos Madrid Spain.
FASEB Bioadv. 2020 Aug 20;2(10):600-612. doi: 10.1096/fba.2020-00022. eCollection 2020 Oct.
Whole-cell phenotypic screening is the driving force behind modern anti-tubercular drug discovery efforts. Focus has shifted from screening for bactericidal scaffolds to screens incorporating target deconvolution. Target-based screening aims to direct drug discovery toward known effective targets and avoid investing resources into unproductive lines of enquiry. The protein synthesis pipeline, including RNA polymerase and the ribosome, is a clinically proven target in . Screening for new hits of this effective target pathway is an invaluable tool in the drug discovery arsenal.
Using . H37Rv augmented with anhydrotetracycline-inducible expression of mCherry, a phenotypic screen was developed for the identification of protein synthesis inhibitors in a medium throughput screening format.
The assay was validated using known inhibitors of protein synthesis to show a dose-dependent reduction in mCherry fluorescence. This was expanded to a proprietary screen of hypothetical protein synthesis hits and modified to include quantitative viability measurement of cells using resazurin.
Following the success of the proprietary screen, a larger scale screen of the GlaxoSmithKline anti-tubercular library containing 2799 compounds was conducted. Combined single shot and dose-response screening yielded 18 hits, 0.64% of all screened compounds.
全细胞表型筛选是现代抗结核药物研发工作的驱动力。重点已从筛选杀菌性骨架转向纳入靶点反卷积的筛选。基于靶点的筛选旨在将药物研发导向已知的有效靶点，避免将资源投入到无成效的研究方向。包括RNA聚合酶和核糖体在内的蛋白质合成途径是临床上已证实的靶点。筛选该有效靶点途径的新命中物是药物研发武器库中的一项宝贵工具。
利用经脱水四环素诱导表达mCherry增强的H37Rv，开发了一种表型筛选方法，用于以中等通量筛选形式鉴定蛋白质合成抑制剂。
使用已知的蛋白质合成抑制剂对该测定法进行验证，结果显示mCherry荧光呈剂量依赖性降低。这一方法扩展到对假设的蛋白质合成命中物进行专有筛选，并进行修改以包括使用刃天青对细胞进行定量活力测定。
在专有筛选取得成功之后，对包含2799种化合物的葛兰素史克抗结核文库进行了更大规模的筛选。单次筛选和剂量反应筛选相结合产生了18个命中物，占所有筛选化合物的0.64%。
