Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094

• Type of Material: Article
• Authors: Maryam Ehteshami, Emory University; Sijia Tao, Emory University; Tugba Ozturk, Emory University; Longhu Zhou, Emory University; Jong Cho, Emory University; HongWang Zhang, Emory University; Sheida Amiralaei, Emory University; Jadd R. Shelton, Emory University; Xiao Lu, Emory University; Ahmed Khalil, Emory University; Robert Domaoal, Emory University; Robert A. Stanton, Emory University; Justin E. Suesserman, Emory University; Biing Lin, Cocrystal Pharma, Inc.; Sam S. Lee, Cocrystal Pharma, Inc.; Franck Amblard, Emory University; Tony Whitaker, Cocrystal Pharma, Inc.; Steven J. Coats, Cocrystal Pharma, Inc.; Raymond Schinazi, Emory University
• Language: English
• Date: 2016-08-01
• Publisher: American Society for Microbiology
• Publication Version: Final Published Version
• Copyright Statement: © 2016, American Society for Microbiology. All Rights Reserved.
• Final Published Version (URL): http://dx.doi.org/10.1128/AAC.00318-16
• Title of Journal or Parent Work: Antimicrobial Agents and Chemotherapy
• ISSN: 0066-4804
• Volume: 60
• Issue: 8
• Start Page: 4659
• End Page: 4669
• Grant/Funding Information: Emory received no funding from Cocrystal Pharma, Inc., to perform this work and vice versa.; National Institute of Allergy and Infectious Diseases (NIAID) http://dx.doi.org/10.13039/1000000605P30-AI-50409 to Raymond F. Schinazi, American Liver Foundation (ALF) http://dx.doi.org/10.13039/100001424 to Maryam Ehteshami; NIH; This paper was supported by the following grant(s): CFAR to Raymond F. Schinazi, NCRTP to Maryam Ehteshami, HHS
• Keywords: Pharmacology & Pharmacy; Science & Technology; RESISTANCE; UNITED-STATES; Microbiology; HCV INFECTION; Life Sciences & Biomedicine; MUTATION-RATE; INHIBITORS; POTENT; REPLICATION; ERADICATION; DEPENDENT RNA-POLYMERASE; IN-VITRO
• Research Categories: Health Sciences, Immunology; Biology, Microbiology

Abstract

Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on β-D-2′-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2′-C-methyl-GTP, is a wellcharacterized inhibitor of NS5B polymerase, whereas the second metabolite, 2′-C-methyl-DAPN-TP, behaves as an adenosine base analog. In vitro assays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolite in vivo. Finally, we found that although both 2′-C-methyl-GTP and 2′-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT) in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2′-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2′-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites.