Samukawa Noriaki, Yamaguchi Takehiro, Ozeki Yuriko, Matsumoto Sohkichi, Igarashi Masayuki, Kinoshita Naoko, Hatano Masaki, Tokudome Kentaro, Matsunaga Shinji, Tomita Shuhei
Department of Pharmacology, Osaka Metropolitan University Graduate School of Medicine, Osaka, Japan.
Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
Microbiology (Reading). 2022 Dec;168(12). doi: 10.1099/mic.0.001285.
Tuberculosis (TB) is treated by chemotherapy with multiple anti-TB drugs for a long period, spanning 6 months even in a standard course. In perspective, to prevent the emergence of antimicrobial resistance, novel drugs that act synergistically or additively in combination with major anti-TB drugs and, if possible, shorten the duration of TB therapy are needed. However, their combinatorial effect cannot be predicted until the lead identification phase of the drug development. Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a powerful genetic tool that enables high-throughput screening of novel drug targets. The development of anti-TB drugs promises to be accelerated by CRISPRi. This study determined whether CRISPRi could be applicable for predictive screening of the combinatorial effect between major anti-TB drugs and an inhibitor of a novel target. In the checkerboard assay, isoniazid killed synergistically or additively in combinations with rifampicin or ethambutol, respectively. The susceptibility to rifampicin and ethambutol was increased by knockdown of , which encodes a target molecule of isoniazid. Additionally, knockdown of , which encodes a target molecule of rifampicin, increased the susceptibility to isoniazid and ethambutol, which act synergistically with rifampicin in the checkerboard assay. Moreover, CRISPRi could successfully predict the synergistic action of cyclomarin A, a novel TB drug candidate, with isoniazid or rifampicin. These results demonstrate that CRISPRi is a useful tool not only for drug target exploration but also for screening the combinatorial effects of novel combinations of anti-TB drugs. This study provides a rationale for anti-TB drug development using CRISPRi.
结核病（TB）通过使用多种抗结核药物进行长期化疗来治疗，即使在标准疗程中也需要6个月。从长远来看，为了防止抗菌药物耐药性的出现，需要与主要抗结核药物协同或相加作用且有可能缩短结核病治疗疗程的新型药物。然而，在药物开发的先导化合物鉴定阶段之前，它们的联合效应是无法预测的。成簇规律间隔短回文重复序列干扰（CRISPRi）是一种强大的基因工具，能够对新型药物靶点进行高通量筛选。CRISPRi有望加速抗结核药物的开发。本研究确定了CRISPRi是否可用于预测主要抗结核药物与新型靶点抑制剂之间的联合效应。在棋盘法试验中，异烟肼分别与利福平或乙胺丁醇联合使用时具有协同或相加杀菌作用。编码异烟肼靶分子的基因敲低会增加对利福平和乙胺丁醇的敏感性。此外，编码利福平靶分子的基因敲低会增加对异烟肼和乙胺丁醇的敏感性，在棋盘法试验中它们与利福平协同作用。此外，CRISPRi能够成功预测新型结核病候选药物环马林A与异烟肼或利福平的协同作用。这些结果表明，CRISPRi不仅是药物靶点探索的有用工具，也是筛选抗结核药物新组合联合效应的有用工具。本研究为使用CRISPRi进行抗结核药物开发提供了理论依据。
