2. Academic Validation
  3. An orally available P1'-5-fluorinated Mpro inhibitor blocks SARS-CoV-2 replication without booster and exhibits high genetic barrier

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

  • PNAS Nexus. 2025 Jan 7;4(1):pgae578. doi: 10.1093/pnasnexus/pgae578.
Nobuyo Higashi-Kuwata 1,  Haydar Bulut 2,  Hironori Hayashi 3,  Kohei Tsuji 4,  Hiromi Ogata-Aoki 1 5 6,  Maki Kiso 7,  Nobutoki Takamune 8,  Naoki Kishimoto 8,  Shin-Ichiro Hattori 1,  Takahiro Ishii 4,  Takuya Kobayakawa 4,  Kenta Nakano 9,  Yukiko Shimizu 9,  Debananda Das 2,  Junji Saruwatari 10,  Kazuya Hasegawa 11,  Kazutaka Murayama 12,  Yoshikazu Sukenaga 1,  Yuki Takamatsu 1,  Kazuhisa Yoshimura 13,  Manabu Aoki 1 14,  Yuri Furusawa 7 15,  Tadashi Okamura 9,  Seiya Yamayoshi 7 15,  Yoshihiro Kawaoka 7 15 16,  Shogo Misumi 8,  Hirokazu Tamamura 4,  Hiroaki Mitsuya 1 2 5
Affiliations

Affiliations

  • 1 Department of Refractory Viral Diseases, National Center for Global Health and Medicine Research Institute, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
  • 2 Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
  • 3 Department of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, Aoba-ku, Sendai 980-8575, Japan.
  • 4 Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan.
  • 5 Department of Clinical Sciences, Kumamoto University Hospital, Chuo-ku, Kumamoto 860-8556, Japan.
  • 6 Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection & Graduate School of Medical Sciences, Kumamoto University, Chuo-ku, Kumamoto 860-0811, Japan.
  • 7 Division of Virology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
  • 8 Department of Environmental and Molecular Health Sciences, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
  • 9 Department of Laboratory Animal Medicine, National Center for Global Health and Medicine Research Institute, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
  • 10 Division of Pharmacology and Therapeutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Chuo-ku, Kumamoto 862-0973, Japan.
  • 11 Structural Biology Division, Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan.
  • 12 Graduate School of Biomedical Engineering, Tohoku University, Miyagi 980-8579, Japan.
  • 13 Tokyo Metropolitan Institute of Public Health, Shinjuku-ku, Tokyo 169-0073, Japan.
  • 14 Department of Medical Technology, Kumamoto Health Science University, 325 Izumimachi, Kita-ku, Kumamoto 861-5598, Japan.
  • 15 The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
  • 16 Department of Pathobiological Sciences, School of Veterinary Medicine, Influenza Research Institute, University of Wisconsin-Madison, Madison, WI 53711, USA.
PMID: 39831159 DOI: 10.1093/pnasnexus/pgae578
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 (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 SARS-CoV-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.

Keywords

COVID-19; Mpro inhibitor; SARS-CoV-2; fluorine-scanning; genetic barrier.

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