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
  • 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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