{"doi":"10.1101/2021.12.18.473303","title":"A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors","abstract":"Abstract Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC 50 values in the 4-21 µM range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":216232,"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":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9589,"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":812120,"name":"Tim Krischuns","orcid":"0000-0003-2693-4701","position":1,"is_corresponding":false},{"id":812121,"name":"Laura Ortega Varga","orcid":"0000-0002-9373-6337","position":2,"is_corresponding":false},{"id":812122,"name":"Emna Harigua‐Souiai","orcid":"0000-0003-2974-9157","position":3,"is_corresponding":false},{"id":812123,"name":"Sylvain Paisant","orcid":"0000-0001-8880-9047","position":4,"is_corresponding":false},{"id":812124,"name":"Agnès Zettor","orcid":"0000-0002-0592-1963","position":5,"is_corresponding":false},{"id":616384,"name":"Jeanne Chiaravalli","orcid":"0000-0001-9135-4565","position":6,"is_corresponding":false},{"id":812125,"name":"D. Mark Courtney","orcid":"0000-0002-1656-5503","position":7,"is_corresponding":false},{"id":374706,"name":"Alison D. O’Brien","orcid":"0000-0002-1315-3204","position":8,"is_corresponding":false},{"id":77531,"name":"Susan C. Baker","orcid":"0000-0001-6485-8143","position":9,"is_corresponding":false},{"id":812126,"name":"Catherine Isel","orcid":"0000-0002-8014-7525","position":10,"is_corresponding":false},{"id":812127,"name":"Fabrice Agou","orcid":"0000-0001-6280-239X","position":11,"is_corresponding":false},{"id":106246,"name":"Yves Jacob","orcid":"0000-0001-9125-2374","position":12,"is_corresponding":false},{"id":812128,"name":"Arnaud Blondel","orcid":"0000-0001-9586-3025","position":13,"is_corresponding":false},{"id":812129,"name":"Nadia Naffakh","orcid":"0000-0002-0424-0277","position":14,"is_corresponding":false},{"id":812676,"name":"Chen Ky","orcid":null,"position":0,"is_corresponding":true}],"reference_count":71,"raw_metadata":null,"created_at":"2026-07-18T23:53:11.245932Z","pmid":"34981051","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":[]}