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A Multipronged Approach Establishes Covalent Modification of β‑Tubulin as the Mode of Action of Benzamide Anti-cancer Toxins

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posted on 12.11.2020, 17:11 by Juan Manuel Povedano, Rameshu Rallabandi, Xin Bai, Xuecheng Ye, Joel Liou, Hong Chen, Jiwoong Kim, Yang Xie, Bruce Posner, Luke Rice, Jef K. De Brabander, David G. McFadden
A phenotypic high-throughput screen identified a benzamide small molecule with activity against small cell lung cancer cells. A “clickable” benzamide probe was designed that irreversibly bound a single 50 kDa cellular protein, identified by mass spectrometry as β-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that β-tubulin was the functional target. Additional evidence suggested that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides impaired growth of microtubules formed with β-tubulin harboring Cys239, but not β3 tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in β3 tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in β-tubulin sufficient to induce compound resistance. The disclosed chemical probes are useful to identify other colchicine site binders, a frequent target of structurally diverse small molecules.