An exonuclease-resistant chain-terminating nucleotide analogue targeting the SARS-CoV-2 replicase complex.

Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE

Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.

Antiviral Agents*/pharmacology ; Antiviral Agents*/chemistry ; COVID-19*/virology ; Polyphosphates* ; SARS-CoV-2*/genetics ; SARS-CoV-2*/metabolism ; COVID-19 Drug Treatment*; Humans ; Exonucleases ; Nucleotides/metabolism ; Nucleotidyltransferases ; RNA, Viral/genetics ; Viral Nonstructural Proteins/metabolism ; Virus Replication/genetics ; Coronavirus RNA-Dependent RNA Polymerase/metabolism

Nucleotide analogues (NA) are currently employed for treatment of several viral diseases, including COVID-19. NA prodrugs are intracellularly activated to the 5'-triphosphate form. They are incorporated into the viral RNA by the viral polymerase (SARS-CoV-2 nsp12), terminating or corrupting RNA synthesis. For Coronaviruses, natural resistance to NAs is provided by a viral 3'-to-5' exonuclease heterodimer nsp14/nsp10, which can remove terminal analogues. Here, we show that the replacement of the α-phosphate of Bemnifosbuvir 5'-triphosphate form (AT-9010) by an α-thiophosphate renders it resistant to excision. The resulting α-thiotriphosphate, AT-9052, exists as two epimers (RP/SP). Through co-crystallization and activity assays, we show that the Sp isomer is preferentially used as a substrate by nucleotide diphosphate kinase (NDPK), and by SARS-CoV-2 nsp12, where its incorporation causes immediate chain-termination. The same -Sp isomer, once incorporated by nsp12, is also totally resistant to the excision by nsp10/nsp14 complex. However, unlike AT-9010, AT-9052-RP/SP no longer inhibits the N-terminal nucleotidylation domain of nsp12. We conclude that AT-9052-Sp exhibits a unique mechanism of action against SARS-CoV-2. Moreover, the thio modification provides a general approach to rescue existing NAs whose activity is hampered by coronavirus proofreading capacity.
(© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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Fondation pour la Recherche Médicale; 101003627 H2020 SC1-PHE-Coronavirus-2020; 101005077 Innovative Medicines Initiative; ANR-10-INSB-05-01 French Infrastructure for Integrated Structural Biology

0 (Antiviral Agents)
EC 3.1.- (Exonucleases)
0 (Nucleotides)
EC 2.7.7.- (Nucleotidyltransferases)
0 (Polyphosphates)
0 (RNA, Viral)
NU43IAG5BC (triphosphoric acid)
0 (Viral Nonstructural Proteins)
EC 2.7.7.48 (NSP12 protein, SARS-CoV-2)
EC 2.7.7.48 (Coronavirus RNA-Dependent RNA Polymerase)

Date Created: 20231214 Date Completed: 20240215 Latest Revision: 20240215

20240215

PMC10853775

10.1093/nar/gkad1194

38096103