2. Academic Validation
  3. A Multipronged Approach Establishes Covalent Modification of β-Tubulin as the Mode of Action of Benzamide Anti-cancer Toxins

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

  • J Med Chem. 2020 Nov 25;63(22):14054-14066. doi: 10.1021/acs.jmedchem.0c01482.
Juan Manuel Povedano 1 2,  Rameshu Rallabandi 2,  Xin Bai 1 2,  Xuecheng Ye 3,  Joel Liou 1 2,  Hong Chen 2 4,  Jiwoong Kim 5,  Yang Xie 5,  Bruce Posner 2 4,  Luke Rice 3,  Jef K De Brabander 2 4,  David G McFadden 1 2 4 6
Affiliations

Affiliations

  • 1 Department of Internal Medicine, Division of Endocrinology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
  • 2 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
  • 3 Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
  • 4 Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
  • 5 Quantitative Biomedical Research Center, Department of Population and Data Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
  • 6 Program in Molecular Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
PMID: 33180487 DOI: 10.1021/acs.jmedchem.0c01482
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.

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