The activation cascade of the broad-spectrum antiviral bemnifosbuvir characterized at atomic resolution

Bemnifosbuvir (AT-527) and AT-752 are guanosine analogues currently undergoing clinical trials against various RNA viruses. In this study, we demonstrate that these drugs require a minimal set of five cellular enzymes to be activated into their common 5′-triphosphate form, AT-9010, following a specific order of reactions. AT-9010 selectively inhibits crucial viral enzymes, contributing to its antiviral potency.

We provide functional and structural data at atomic resolution that reveal how N6-purine deamination is compatible with the drug’s metabolic activation. The crystal structures of human histidine triad nucleotide binding protein 1, adenosine deaminase-like protein 1, guanylate kinase 1, and nucleoside diphosphate kinase, resolved to 2.09, 2.44, 1.76, and 1.9 Å respectively, with the relevant precursors of AT-9010, clarify the activation pathway from the orally administered bemnifosbuvir to AT-9010. This highlights critical drug-protein interactions along the activation pathway. Our findings establish a framework for designing antiviral nucleotide analogues by addressing the requirements and constraints posed by the activation enzymes involved in the assembly of 5′-triphosphates.