An orally available P1′-5-fluorinated Mpro inhibitor blocks SARS-CoV-2 replication without booster and exhibits high genetic barrier

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 (M<sup>pro</sup>) and more effectively blocks the infectivity and replication of all SARS-CoV-2 strains examined including Omicron variants such as SARS-CoV-2<sup>XBB1.5</sup> and SARS-CoV-2<sup>EG.5.1</sup> than two M<sup>pro</sup> 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-2<sup>XBB1.5</sup> replication without ritonavir in B6.Cg-Tg(K18-hACE2)2-Prlmn/J-transgenic mice, comparably as did ritonavir-boosted nirmatrelvir. When the ancestral SARS-CoV-2 was propagated with nirmatrelvir in vitro, a highly nirmatrelvir-resistant E166V-carrying variant (SARS-CoV-2<sup>E166V-P14</sup>) readily emerged by passage 14; however, when propagated with TKB272, no variants emerged by passage 25. SARS-CoV-2<sub>E166V</sub> 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 M<sup>pro</sup> inhibition but that TKB272 nevertheless forms a tight binding with M<sup>pro</sup>'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 M<sup>pro</sup> inhibitors.

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Publication Details

Journal
PNAS Nexus
Published
2024-12-23
DOI
https://doi.org/10.1093/pnasnexus/pgae578
Citations
8
Primary Topic
interferon and immune responses
Type
article
Field-Weighted Citation Impact
1.92

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article

An orally available P1′-5-fluorinated Mpro inhibitor blocks SARS-CoV-2 replication without booster and exhibits high genetic barrier

Nobuyo Higashi‐Kuwata, Kenta Nakano, Junji Saruwatari, Takahiro Ishii et al.
8 citations
PNAS Nexus
interferon and immune responses
1.92
article

An orally available P1′-5-fluorinated Mpro inhibitor blocks SARS-CoV-2 replication without booster and exhibits high genetic barrier

Nobuyo Higashi‐Kuwata, Kenta Nakano, Junji Saruwatari, Takahiro Ishii, Haydar Bulut, Manabu Aoki, Kazuya Hasegawa, Hiroaki Mitsuya, Shin-ichiro Hattori, Takuya Kobayakawa, Seiya Yamayoshi, Hironori Hayashi, Nobutoki Takamune, Naoki Kishimoto, Yukiko Shimizu, Yuki Takamatsu, Maki Kiso, Hirokazu Tamamura, Debananda Das, Yoshihiro Kawaoka, Kazutaka Murayama, Yuri Furusawa, Tadashi Okamura, Kohei Tsuji, Shogo Misumi, Hiromi Ogata-Aoki, Yoshikazu Sukenaga, Kazuhisa Yoshimura
article en
8 citations

Abstract

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 (M<sup>pro</sup>) and more effectively blocks the infectivity and replication of all SARS-CoV-2 strains examined including Omicron variants such as SARS-CoV-2<sup>XBB1.5</sup> and SARS-CoV-2<sup>EG.5.1</sup> than two M<sup>pro</sup> 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-2<sup>XBB1.5</sup> replication without ritonavir in B6.Cg-Tg(K18-hACE2)2-Prlmn/J-transgenic mice, comparably as did ritonavir-boosted nirmatrelvir. When the ancestral SARS-CoV-2 was propagated with nirmatrelvir in vitro, a highly nirmatrelvir-resistant E166V-carrying variant (SARS-CoV-2<sup>E166V-P14</sup>) readily emerged by passage 14; however, when propagated with TKB272, no variants emerged by passage 25. SARS-CoV-2<sub>E166V</sub> 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 M<sup>pro</sup> inhibition but that TKB272 nevertheless forms a tight binding with M<sup>pro</sup>'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 M<sup>pro</sup> inhibitors.

PNAS NexusVol. 4(1)
Tokyo Medical and Dental University (JP), University of Wisconsin–Madison (US), Tokyo University of Science (JP), Kumamoto Health Science University (JP), Tohoku University (JP), Global Viral (US), National Center for Global Health and Medicine (JP), Tokyo Metropolitan Institute of Public Health (JP), Japan Synchrotron Radiation Research Institute (JP), Kumamoto University Hospital (JP), Center for Global Health (US), National Cancer Institute (US), The University of Tokyo (JP), Kumamoto University (JP)
U.S. Department of Health and Human Services, Japan Agency for Medical Research and Development, National Center for Global Health and Medicine, National Institutes of Health, Japan Society for the Promotion of Science, National Cancer Institute
Good health and well-being
Openalex Percentile: Top 13%
interferon and immune responses
1.92
DOIOpen Access PDFOpenAlex
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