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

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 alpha-phosphate of Bemnifosbuvir 5 '-triphosphate form (AT-9010) by an alpha-thiophosphate renders it resistant to excision. The resulting alpha-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. Graphical Abstract