We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

我们最近使用基于 CRISPRi/a 的化学遗传筛选和细胞生物学、生化和结构分析来确定 rigosertib，一种处于 III 期临床试验中的抗癌剂，通过破坏微管的稳定性来杀死癌细胞。Reddy 和同事（Baker 等人，2020 年，本期Molecular Cell) 表明 rigosertib 商业制剂中的污染降解产物是造成微管不稳定活性的原因。在这里，我们证明了用药物级 rigosertib（> 99.9% 纯度）或商业获得的 rigosertib 处理的细胞在多个检测中具有定性无法区分的表型。这两种制剂与调节微管稳定性的基因具有难以区分的化学-遗传相互作用，这两种基因都破坏了细胞内和体外微管的稳定性，并且在rigosertib 结合位点 (L240F TUBB ) 中具有突变的合理设计的微管蛋白突变体的表达允许细胞在任一配方的存在。重要的是，L240F TUBB 的特异性微管不稳定剂的突变体已被独立证实。因此，rigosertib 通过破坏微管的稳定性来杀死癌细胞，这与我们最初的发现一致。