Higashi-Kuwata Nobuyo, Bulut Haydar, Hayashi Hironori, Tsuji Kohei, Ogata-Aoki Hiromi, Kiso Maki, Takamune Nobutoki, Kishimoto Naoki, Hattori Shin-Ichiro, Ishii Takahiro, Kobayakawa Takuya, Nakano Kenta, Shimizu Yukiko, Das Debananda, Saruwatari Junji, Hasegawa Kazuya, Murayama Kazutaka, Sukenaga Yoshikazu, Takamatsu Yuki, Yoshimura Kazuhisa, Aoki Manabu, Furusawa Yuri, Okamura Tadashi, Yamayoshi Seiya, Kawaoka Yoshihiro, Misumi Shogo, Tamamura Hirokazu, Mitsuya Hiroaki
Department of Refractory Viral Diseases, National Center for Global Health and Medicine Research Institute, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
PNAS Nexus. 2025 Jan 7;4(1):pgae578. doi: 10.1093/pnasnexus/pgae578. eCollection 2025 Jan.
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) and more effectively blocks the infectivity and replication of all SARS-CoV-2 strains examined including Omicron variants such as SARS-CoV-2 and SARS-CoV-2 than two M 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 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) readily emerged by passage 14; however, when propagated with TKB272, no variants emerged by passage 25. SARS-CoV-2 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 inhibition but that TKB272 nevertheless forms a tight binding with M'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 inhibitors.
我们通过对苯并噻唑部分进行氟扫描，鉴定出一种含5-氟苯并噻唑的小分子TKB272，它比两种主要蛋白酶（M）抑制剂奈玛特韦和恩赛特韦更有效地抑制严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的酶活性，更有效地阻断所有检测的SARS-CoV-2毒株（包括奥密克戎变种如SARS-CoV-2和SARS-CoV-2）的感染性和复制。值得注意的是，服用利托那韦增强的奈玛特韦和恩赛特韦会引起药物相互作用，由于它们对细胞色素P450 3A4（CYP3A4）的抑制作用而需要谨慎使用，从而限制了它们的临床应用。口服给药时，TKB272在B6.Cg-Tg(K18-hACE2)2-Prlmn/J转基因小鼠中无需利托那韦即可阻断SARS-CoV-2复制，与利托那韦增强的奈玛特韦相当。当原始SARS-CoV-2在体外与奈玛特韦传代培养时，到第14代时很容易出现携带高度耐奈玛特韦的E166V变体（SARS-CoV-2）；然而，当与TKB272传代培养时，到第25代时没有变体出现。SARS-CoV-2对TKB272表现出一些交叉耐药性，但对该化合物基本敏感。X射线结构分析和质谱数据表明，E166V取代破坏了关键的二聚化起始Ser1'-E166相互作用，从而限制了奈玛特韦对M的抑制作用，但即使在存在E166V取代的情况下，TKB272仍与M的催化活性位点紧密结合。在微Ames试验中测试，TKB272没有明显的遗传毒性。高效的TKB272可作为治疗冠状病毒病（COVID-19）的药物，克服对现有M抑制剂的耐药性。
