An efficient CRISPR interference-based prediction method for synergistic/additive effects of novel combinations of anti-tuberculosis drugs
An efficient CRISPR interference-based prediction method for synergistic/additive effects of novel combinations of anti-tuberculosis drugs
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一种基于 CRISPR 干扰的高效抗结核药物组合的协同/相加效应预测方法
DOI：
10.1099/mic.0.001285
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发表时间：
2022
期刊：
影响因子：
1.5
通讯作者：
Tomita Shuhei
中科院分区：
文献类型：
--
作者：
Samukawa Noriaki;Yamaguchi Takehiro;Ozeki Yuriko;Matsumoto Sohkichi;Igarashi Masayuki;Kinoshita Naoko;Hatano Masaki;Tokudome Kentaro;Matsunaga Shinji;Tomita Shuhei
结核病（TB）通过多种抗结核药物的化疗进行长期治疗，即使在标准疗程中也持续6个月。从长远来看，为了防止抗生素耐药性的出现，需要与主要抗结核药物联合发挥协同或相加作用的新药，如果可能的话，还需要缩短结核病治疗的持续时间。然而，它们的组合效应直到药物开发的先导鉴定阶段才能预测。规则间隔短回文重复序列干扰（CRISPRi）是一种强大的遗传工具，可以高通量筛选新的药物靶点。CRISPRi有望加速抗结核药物的开发。这项研究确定了CRISPRi是否适用于预测性筛选主要抗结核药物和新靶点抑制剂之间的组合效应。在棋盘格试验中，异烟肼分别与利福平或乙胺丁醇联合使用，可协同或相加地杀死耻垢分枝杆菌。通过敲低hA（编码异烟肼的靶分子）增加了对利福平和乙胺丁醇的敏感性。此外，敲低编码利福平靶分子的rpoB增加了对异烟肼和乙胺丁醇的敏感性，异烟肼和乙胺丁醇在棋盘测定中与利福平协同作用。此外，CRISPRi可以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的组合效应的有用工具。这项研究为使用CRISPRi开发抗结核药物提供了理论基础。
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 killedMycobacterium smegmatissynergistically or additively in combinations with rifampicin or ethambutol, respectively. The susceptibility to rifampicin and ethambutol was increased by knockdown ofinhA, which encodes a target molecule of isoniazid. Additionally, knockdown ofrpoB, 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.