Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent.
Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent.
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DOI：
10.1016/j.molcel.2017.09.012
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发表时间：
2017-10-05
期刊：
影响因子：
16
通讯作者：
Weissman JS
中科院分区：
文献类型：
--
作者：
Jost M;Chen Y;Gilbert LA;Horlbeck MA;Krenning L;Menchon G;Rai A;Cho MY;Stern JJ;Prota AE;Kampmann M;Akhmanova A;Steinmetz MO;Tanenbaum ME;Weissman JS
与表型筛选配对的化学文库现在可以很容易地识别具有治疗潜力的化合物。然而，开发这些化合物的一个主要限制是鉴定它们的相关细胞靶点。在这里，我们提出了一种双层CRISPR介导的化学遗传学策略用于靶点识别：组合的全基因组敲除和过表达筛选以及集中的比较化学遗传学分析。这些策略应用于rigosertib，一种用于高危骨髓增生异常综合征的3期临床试验的药物，其分子靶点仍然存在争议，特别指出微管是rigosertib的靶点。我们发现，rigosertib确实直接结合和不稳定的微管使用细胞生物学，体外和结构的方法。最后，在rigosertib结合口袋中具有结构指导突变的微管蛋白的表达赋予了对rigosertib的耐药性，确定rigosertib通过破坏微管的稳定来杀死癌细胞。这些结果证明了我们的化学遗传筛选策略的力量，用于精确定位化学试剂的生理相关目标。结合CRISPRi/a化学遗传筛选揭示治疗药物的靶点聚焦化学遗传分析通过作用机制快速分类药物使用rigosertib的化学遗传筛选揭示微管去稳定化特征靶向体内和体外方法证实了rigosertib的作用机制Jost et al.提出了一种双层策略，使用CRISPRi/a介导的化学遗传筛选来鉴定生物活性化合物的分子靶标。对作用机制尚不清楚的抗癌药物rigosertib的应用表明rigosertib是一种微管去稳定剂。靶向细胞生物学、生物化学和结构方法证实了这种作用机制。
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib’s target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents. Combined CRISPRi/a chemical-genetic screening reveals targets of therapeutic agents Focused chemical-genetic profiling rapidly classifies agents by mechanism of action Chemical-genetic screens with rigosertib reveal a microtubule-destabilizing signature Targeted in vivo and in vitro approaches confirm rigosertib’s mechanism of action Jost et al. present a two-tiered strategy to identify molecular targets of bioactive compounds using CRISPRi/a-mediated chemical-genetic screens. Application to rigosertib, an anti-cancer drug with an unclear mechanism of action, points to rigosertib being a microtubule-destabilizing agent. Targeted cell biological, biochemical, and structural approaches confirm this mechanism of action.
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影响因子：
14.8
作者：
Arrowsmith CH;Audia JE;Austin C;Baell J;Bennett J;Blagg J;Bountra C;Brennan PE;Brown PJ;Bunnage ME;Buser-Doepner C;Campbell RM;Carter AJ;Cohen P;Copeland RA;Cravatt B;Dahlin JL;Dhanak D;Edwards AM;Frederiksen M;Frye SV;Gray N;Grimshaw CE;Hepworth D;Howe T;Huber KV;Jin J;Knapp S;Kotz JD;Kruger RG;Lowe D;Mader MM;Marsden B;Mueller-Fahrnow A;Müller S;O'Hagan RC;Overington JP;Owen DR;Rosenberg SH;Roth B;Ross R;Schapira M;Schreiber SL;Shoichet B;Sundström M;Superti-Furga G;Taunton J;Toledo-Sherman L;Walpole C;Walters MA;Willson TM;Workman P;Young RN;Zuercher WJ
通讯作者：
Zuercher WJ
影响因子：
3.6
作者：
Lu, Tiangong;Laughton, Charles A.;Bradshaw, Tracey D.
通讯作者：
Bradshaw, Tracey D.
影响因子：
50.3
作者：
Gumireddy, K;Reddy, MVR;Reddy, EP
通讯作者：
Reddy, EP
DOI：
10.1073/pnas.1307002110
发表时间：
2013-06-18
影响因子：
11.1
作者：
Kampmann, Martin;Bassik, Michael C.;Weissman, Jonathan S.
通讯作者：
Weissman, Jonathan S.
影响因子：
14.9
作者：
Huang, Da Wei;Sherman, Brad T.;Lempicki, Richard A.
通讯作者：
Lempicki, Richard A.