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
  • 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.

Products