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

ABSTRACT: 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 ?-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that ?-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 ?-tubulin harboring Cys239, but not ?3 tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in ?3 tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in ?-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.

SUBMITTER: Povedano JM 

PROVIDER: S-EPMC7707623 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature