{"doi":"10.1101/2024.04.22.590607","title":"Mechanism and spectrum of inhibition of a 4’-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses","abstract":"ABSTRACT The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRp) of prototypic respiratory viruses. GS-646939 is the active 5′-triphosphate (TP) metabolite of a 4ʹ-cyano modified C -adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 (EV-71) incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase (h-mtRNAP) does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain-terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4ʹ-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1′-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1ʹ-cyano and 4ʹ-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":496972,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9475,"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":404004,"name":"Simon Walker","orcid":"0000-0002-5750-3691","position":1,"is_corresponding":false},{"id":619088,"name":"Egor P. Tchesnokov","orcid":"0000-0003-1698-2961","position":2,"is_corresponding":false},{"id":1229879,"name":"Dana Kocíncová","orcid":null,"position":3,"is_corresponding":false},{"id":226334,"name":"Jared Pitts","orcid":"0000-0003-4080-4321","position":4,"is_corresponding":false},{"id":547002,"name":"Dustin S. Siegel","orcid":"0009-0008-1365-0287","position":5,"is_corresponding":false},{"id":97929,"name":"Jason K. Perry","orcid":"0000-0001-5492-0652","position":6,"is_corresponding":false},{"id":105123,"name":"Joy Y. Feng","orcid":"0000-0003-4837-1911","position":7,"is_corresponding":false},{"id":105311,"name":"John P. Bilello","orcid":"0000-0003-4327-1727","position":8,"is_corresponding":false},{"id":619089,"name":"Matthias Götte","orcid":"0000-0001-6213-5806","position":9,"is_corresponding":false},{"id":619087,"name":"Calvin J. Gordon","orcid":"0000-0002-8259-6201","position":0,"is_corresponding":true}],"reference_count":76,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:09:30.779495Z","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":[]}