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

Authors

Yili Xu, Albert Liclican, Xiaofeng Zhao, Chen Chen, Joseph T Ortega, Hsiu-Chun Chuang, Cameron Soulette, Subhra Chaudhuri, Jingyu Zhang, Ting Wang, Darius Babusis, Bruno Marchand, Kai-Hui Sun, Ricardo Ramirez, Bin Ma, Arabinda Nayak, Liya Wang, Saurabh Menon, Jean-Philippe Belzile, Yurong Lai, Li Li, Richard L Mackman, John P Bilello, Tomas Cihlar, Joy Y Feng, Latesh Lad

Published in

Antimicrobial agents and chemotherapy. Pages e0005526. Jun 22, 2026. Epub Jun 22, 2026.

Abstract

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.

PMID:
42363837
Bibliographic data and abstract were imported from PubMed on 27 Jun 2026.

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