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 (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.