Identification of key metabolic enzymes involved in the activation of obeldesivir and remdesivir to the active triphosphate metabolite

Obeldesivir (GS-5245, ODV), an orally administered 5′-isobutyryl ester prodrug of GS-441524, has demonstrated anti-SARS-CoV-2 activity in preclinical and clinical settings. ODV is hydrolyzed in plasma to provide high systemic exposures of GS-441524, enabling the efficient intracellular formation of the bioactive 5′-triphosphate GS-443902, an ATP analog inhibiting the SARS-CoV-2 RNA-dependent RNA polymerases. Remdesivir (RDV), the first US FDA-approved antiviral treatment for COVID-19, is also activated to GS-443902. In this paper, we systematically profiled the human enzymes potentially involved in ODV and RDV activation using multiple approaches: (i) biochemical studies on the catalytic efficiency ( k _cat / K _m ); (ii) analysis of activation in single-gene knockout cells; and (iii) the antiviral activity assessment in single-gene knockout cells. Our results demonstrated that ODV hydrolysis to GS-441524 is catalyzed by carboxylesterase 1 and 2 with high efficiency. For the enzymes involved in the consecutive formation of 5′-mono-, di-, and tri-phosphates (MP, DP, and TP), adenosine kinase (ADK) likely plays a minor role in forming GS-441524-MP, suggesting the involvement of other phosphotransferases. This was further supported by cell-based single-gene ADK-knockout studies. In contrast, both biochemical and cell-based assay data strongly support adenylate kinase 2 as the key enzyme for the phosphorylation of GS-441524-MP, while nucleoside diphosphate kinase NM23-H2, phosphoglycerate kinase, and pyruvate kinase likely all contribute to the formation of GS-441524-TP. This work leads to a deeper understanding of ODV and RDV metabolism, and suggests further studies are needed to identify the enzyme responsible for the activation of GS-441524 to its 5′-monophosphate.