{"doi":"10.1101/2021.08.28.458041","title":"Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays","abstract":"Abstract SARS-CoV-2 main protease (M pro ) is one of the most extensive exploited drug targets for COVID-19. Structurally disparate compounds have been reported as M pro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed M pro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based Flip-GFP assay. Collectively, our results have shown that majority of the M pro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to M pro , while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit M pro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery. Graphical abstract Flip-GFP and Protease-Glo luciferase assays, coupled with the FRET and thermal shift binding assays, were applied to validate the reported SARS-CoV-2 M pro inhibitors.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":214886,"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":8,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9519,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":642332,"name":"Haozhou Tan","orcid":"0000-0002-6640-8354","position":1,"is_corresponding":false},{"id":642333,"name":"Juliana Choza","orcid":"0000-0001-7038-9750","position":2,"is_corresponding":false},{"id":809001,"name":"Yuying Wang","orcid":"0009-0007-9924-4409","position":3,"is_corresponding":false},{"id":107696,"name":"Jun Wang","orcid":"0000-0002-4845-4621","position":4,"is_corresponding":false},{"id":107686,"name":"Chunlong Ma","orcid":"0000-0002-5766-8189","position":0,"is_corresponding":true}],"reference_count":61,"raw_metadata":null,"created_at":"2026-07-18T23:52:51.201033Z","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":[]}