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

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

Research output: Contribution to journalArticlepeer-review

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 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.

Original languageEnglish (US)
Pages (from-to)14054-14066
Number of pages13
JournalJournal of Medicinal Chemistry
Volume63
Issue number22
DOIs
StatePublished - Nov 25 2020

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

Fingerprint Dive into the research topics of 'A Multipronged Approach Establishes Covalent Modification of β-Tubulin as the Mode of Action of Benzamide Anti-cancer Toxins'. Together they form a unique fingerprint.

Cite this