Selective and reversible modification of kinase cysteines with chlorofluoroacetamides

Naoya Shindo, Hirokazu Fuchida, Mami Sato, Kosuke Watari, Tomohiro Shibata, Keiko Kuwata, Chizuru Miura, Kei Okamoto, Yuji Hatsuyama, Keisuke Tokunaga, Seiichi Sakamoto, Satoshi Morimoto, Yoshito Abe, Mitsunori Shiroishi, Jose M. M. Caaveiro, Tadashi Ueda, Tomonori Tamura, Naoya Matsunaga, Takaharu Nakao, Satoru Koyanagi, Shigehiro Ohdo, Yasuchika Yamaguchi, Itaru Hamachi, Mayumi Ono & Akio Ojida

Nature Chemical Biology, 2019
DOI: https://doi.org/10.1038/s41589-018-0204-3

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton’s tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.