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

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.