{"doi":"10.1073/pnas.2404175121","title":"Structural and virologic mechanism of the emergence of resistance to M <sup>pro</sup> inhibitors in SARS-CoV-2","abstract":"We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (M pro ) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2 WK521 WT in VeroE6 TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2 WK521 E166V ), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2 E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of M pro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1’-Glu166 interactions. TKB245 stayed bound to M pro E166V , whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of M pro , and compromised the enzymatic activity; the Ki values of recombinant M pro E166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to M pro E166V than nirmatrelvir. SARS-CoV-2 WK521 WT selected with 5h acquired A191T substitution in M pro (SARS-CoV-2 WK521 A191T ) and better replicated in the presence of 5h, than SARS-CoV-2 WK521 WT . However, no significant enzymatic or structural changes in M pro A191T were observed. The replicability of SARS-CoV-2 WK521 E166V proved to be compromised compared to SARS-CoV-2 WK521 WT but predominated over SARS-CoV-2 WK521 WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2 WK521 A191T surpassed that of SARS-CoV-2 WK521 WT in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2’s drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.","journal":"Proceedings of the National Academy of Sciences","year":2024,"id":427495,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":15,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9524,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":295827,"name":"Haydar Bulut","orcid":"0000-0003-0262-6296","position":1,"is_corresponding":false},{"id":295828,"name":"Hironori Hayashi","orcid":"0000-0002-5892-0040","position":2,"is_corresponding":false},{"id":590174,"name":"Naoki Kishimoto","orcid":"0000-0003-2988-456X","position":3,"is_corresponding":false},{"id":299089,"name":"Nobutoki Takamune","orcid":"0000-0001-7173-240X","position":4,"is_corresponding":false},{"id":623764,"name":"Kazuya Hasegawa","orcid":"0000-0003-0331-1624","position":5,"is_corresponding":false},{"id":641902,"name":"Yuri Furusawa","orcid":"0000-0002-7489-7303","position":6,"is_corresponding":false},{"id":615260,"name":"Seiya Yamayoshi","orcid":"0000-0001-7768-5157","position":7,"is_corresponding":false},{"id":623763,"name":"Kazutaka Murayama","orcid":"0000-0003-1749-4038","position":8,"is_corresponding":false},{"id":588041,"name":"Hirokazu Tamamura","orcid":"0000-0003-2788-2579","position":9,"is_corresponding":false},{"id":623765,"name":"Mi Li","orcid":"0000-0002-9304-4146","position":10,"is_corresponding":false},{"id":376430,"name":"Alexander Wlodawer","orcid":"0000-0002-5510-9703","position":11,"is_corresponding":false},{"id":107110,"name":"Yoshihiro Kawaoka","orcid":"0000-0001-5061-8296","position":12,"is_corresponding":false},{"id":299092,"name":"Shogo Misumi","orcid":"0000-0002-9465-4720","position":13,"is_corresponding":false},{"id":295833,"name":"Hiroaki Mitsuya","orcid":"0000-0001-9274-3853","position":14,"is_corresponding":false},{"id":137994,"name":"Shin-ichiro Hattori","orcid":null,"position":0,"is_corresponding":true}],"reference_count":38,"raw_metadata":null,"created_at":"2026-07-19T01:58:47.805440Z","pmid":"39236245","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}