An orally available P1’-5-fluorinated Mpro inhibitor blocks SARS-CoV-2 replication without booster and exhibits high genetic barrier
Nobuyo Higashi-Kuwata, Haydar Bulut, Hironori Hayashi, Kohei Tsuji, Hiromi Ogata-Aoki, Maki Kiso, Nobutoki Takamune, Naoki Kishimoto, Shin-ichiro Hattori, Takahiro Ishii, Takuya Kobayakawa, Kenta Nakano, Yukiko Shimizu, Debananda Das, Junji Saruwatari, Kazuya Hasegawa, Kazutaka Murayama, Yoshikazu Sukenaga, Yuki Takamatsu, Kazuhisa Yoshimura, Manabu Aoki, Yuri Furusawa, Tadashi Okamura, Seiya Yamayoshi, Yoshihiro Kawaoka, Shogo Misumi, Hirokazu Tamamura, Hiroaki MitsuyaAbstract
We identified a 5-fluoro-benzothiazole-containing small molecule, TKB272, through fluorine-scanning of the benzothiazole moiety, which more potently inhibits the enzymatic activity of SARS-CoV-2’s main protease (Mpro) and more effectively blocks the infectivity and replication of all SARS-CoV-2 strains examined including Omicron variants such as SARS-CoV-2XBB1.5 and SARS-CoV-2EG.5.1 than two Mpro inhibitors, nirmatrelvir and ensitrelvir. Notably, the administration of ritonavir-boosted nirmatrelvir and ensitrelvir causes drug-drug interactions warranting cautions due to their CYP3A4 inhibition, thereby limiting their clinical utility. When orally administered, TKB272 blocked SARS-CoV-2XBB1.5 replication without ritonavir in B6.Cg-Tg(K18-hACE2)2-Prlmn/J-transgenic mice, comparably as did ritonavir-boosted nirmatrelvir. When the ancestral SARCoV-2 was propagated with nirmatrelvir in vitro, a highly-nirmatrelvir-resistant E166V-carrying variant (SARS-CoV-2E166V-P14) readily emerged by passage 14; however, when propagated with TKB272, no variants emerged by passage 25. SARS-CoV-2E166V showed some cross-resistance to TKB272 but was substantially sensitive to the compound. X-ray structural analyses and mass-spectrometric data showed that the E166V substitution disrupts the critical dimerization-initiating Ser1’-E166 interactions, thereby limiting nirmatrelvir’s Mpro inhibition but that TKB272 nevertheless forms a tight binding with Mpro’s catalytic active sight even in the presence of the E166V substitution. TKB272 shows no apparent genotoxicity as tested in the micro-Ames test. Highly potent TKB272 may serve as a COVID-19 therapeutic, overcome resistance to existing Mpro inhibitors.