TY - JOUR
T1 - A Multipronged Approach Establishes Covalent Modification of β-Tubulin as the Mode of Action of Benzamide Anti-cancer Toxins
AU - Povedano, Juan Manuel
AU - Rallabandi, Rameshu
AU - Bai, Xin
AU - Ye, Xuecheng
AU - Liou, Joel
AU - Chen, Hong
AU - Kim, Jiwoong
AU - Xie, Yang
AU - Posner, Bruce
AU - Rice, Luke
AU - De Brabander, Jef K.
AU - McFadden, David G.
N1 - Publisher Copyright:
©
PY - 2020/11/25
Y1 - 2020/11/25
N2 - 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.
AB - 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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U2 - 10.1021/acs.jmedchem.0c01482
DO - 10.1021/acs.jmedchem.0c01482
M3 - Article
C2 - 33180487
AN - SCOPUS:85096525764
SN - 0022-2623
VL - 63
SP - 14054
EP - 14066
JO - Journal of Medicinal Chemistry
JF - Journal of Medicinal Chemistry
IS - 22
ER -