Université Paris Cité, Inserm, System Engineering and Evolution Dynamics, Paris, France.
CRI, Paris, France.
Nat Commun. 2022 Jul 7;13(1):3905. doi: 10.1038/s41467-022-31570-3.
Whole-cell screening for Mycobacterium tuberculosis (Mtb) inhibitors is complicated by the pathogen's slow growth and biocontainment requirements. Here we present a synthetic biology framework for assaying Mtb drug targets in engineered E. coli. We construct Target Essential Surrogate E. coli (TESEC) in which an essential metabolic enzyme is deleted and replaced with an Mtb-derived functional analog, linking bacterial growth to the activity of the target enzyme. High throughput screening of a TESEC model for Mtb alanine racemase (Alr) revealed benazepril as a targeted inhibitor, a result validated in whole-cell Mtb. In vitro biochemical assays indicated a noncompetitive mechanism unlike that of clinical Alr inhibitors. We establish the scalability of TESEC for drug discovery by characterizing TESEC strains for four additional targets.
全细胞筛选结核分枝杆菌 (Mtb) 的抑制剂比较复杂，因为病原体的生长缓慢，而且需要生物控制。在这里，我们提出了一个用于在工程大肠杆菌中检测 Mtb 药物靶点的合成生物学框架。我们构建了靶向必需替代大肠杆菌（TESEC），其中一个必需的代谢酶被删除，并被 Mtb 衍生的功能类似物取代，将细菌生长与靶酶的活性联系起来。对 TESEC 模型进行 Mtb 丙氨酸消旋酶（Alr）的高通量筛选显示贝那普利是一种靶向抑制剂，这一结果在全细胞 Mtb 中得到了验证。体外生化分析表明，其作用机制与临床 Alr 抑制剂不同，是非竞争性的。我们通过对另外四个靶点的 TESEC 菌株进行特征描述，证明了 TESEC 在药物发现方面的可扩展性。
