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

Abstract

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.

A Mini-Review on the Common Antiviral Drug Targets of Coronavirus

“…Unlike most RNA viruses, coronaviruses have the ability to edit mistakes during copying of their genetic information. The exoribonuclease domain of Nsp14 interacts with its cofactor Nsp10 to correct viral RNA mispairing by removing misincorporated nucleotides or nucleotide analogues from the 3′-end of the nascent RNA strand, a proofreading mechanism that is essential for the maintenance of the replicative fidelity of the coronavirus genome and is a candidate for antiviral drug targets for antiviral drugs [ 66 ]. Based on molecular docking technology, a number of Nsp14 inhibitors targeting the active site of exoribonuclease have been identified [ 67 ], such as nitrocatechol [ 68 ], SGC0946, and SGC8158 [ 69 ].…”