{"doi":"10.1101/2020.12.19.423537","title":"An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity","abstract":"Abstract The main protease (M pro ) of SARS-CoV-2 is a validated antiviral drug target. Several M pro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent M pro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV M pro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 M pro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 M pro . Lead optimization led to the discovery of 23R , which inhibits SARS-CoV-2 M pro and SARS-CoV-2 viral replication with an IC 50 of 0.31 μM and EC 50 of 1.27 μM, respectively. The binding and specificity of 23R to SARS-CoV-2 M pro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 M pro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the α-methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 M pro inhibitors reported to date and a novel binding pocket that can be explored for M pro inhibitor design.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":125753,"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":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9517,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":107687,"name":"M. Sacco","orcid":"0000-0001-5930-7363","position":1,"is_corresponding":false},{"id":107686,"name":"Chunlong Ma","orcid":"0000-0002-5766-8189","position":2,"is_corresponding":false},{"id":107690,"name":"Yanmei Hu","orcid":"0000-0003-0525-7342","position":3,"is_corresponding":false},{"id":107689,"name":"Julia A. Townsend","orcid":"0000-0002-2132-1052","position":4,"is_corresponding":false},{"id":230204,"name":"Xiangzhi Meng","orcid":"0000-0002-3828-5609","position":5,"is_corresponding":false},{"id":567348,"name":"Fushun Zhang","orcid":"0000-0002-0696-8907","position":6,"is_corresponding":false},{"id":572587,"name":"Xiujun Zhang","orcid":"0000-0002-6704-031X","position":7,"is_corresponding":false},{"id":573187,"name":"Adis Kukuljac","orcid":null,"position":8,"is_corresponding":false},{"id":107694,"name":"Michael T. Marty","orcid":"0000-0001-8115-1772","position":9,"is_corresponding":false},{"id":78937,"name":"D. Schultz","orcid":"0000-0002-7890-8815","position":10,"is_corresponding":false},{"id":105475,"name":"Sara Cherry","orcid":"0000-0003-3956-6610","position":11,"is_corresponding":false},{"id":230207,"name":"Yan Xiang","orcid":"0000-0002-7633-1629","position":12,"is_corresponding":false},{"id":107695,"name":"Yu Chen","orcid":"0000-0002-5115-3600","position":13,"is_corresponding":false},{"id":107696,"name":"Jun Wang","orcid":"0000-0002-4845-4621","position":14,"is_corresponding":false},{"id":230206,"name":"Naoya Kitamura","orcid":"0000-0001-9683-6002","position":0,"is_corresponding":true}],"reference_count":26,"raw_metadata":null,"created_at":"2026-07-18T23:15:19.482428Z","pmid":null,"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":[]}