A multipronged approach establishes covalent modification of beta-tubulin as the mode of action of benzamide anti-cancer toxins

Anti-cancer small molecule discovery remains a challenge for cancers that do not harbor recurrent mutations in chemically tractable proteins. Through a phenotypic high-throughput screen, we identified a promising benzamide small molecule 1 with activity against small cell lung cancer cell lines. Structure-activity relationship studies identified a clickable benzamide probe that irreversibly bound to a single dominant 50 kDa protein in cells (p50) through nucleophilic aromatic substitution (SNAr). The anti-cancer potency of benzamide analogs strongly correlated with p50-probe competition, indicating p50 was the benzamide target. Mass spectrometry identified p50 as beta-tubulin and provided evidence that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides induced cell cycle arrest at the G2-M transition and impaired microtubule dynamics. Because covalent modification of benzamides only occurred with Cys239-containing tubulin isoforms, we designed an aldehyde-containing analog capable of trapping Ser239 isoforms, presumably as a hemiacetal with Ser239. Using a forward genetics strategy, compound-resistant cell lines were identified that harbored a Thr238Ala mutation in beta-tubulin. This single point mutation was sufficient to induce resistance to benzamides. The disclosed chemical probes were further used to identify other small molecules that bound the colchicine site of beta-tubulin, a frequent target of structurally diverse small molecules.