Ribonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent-RNA polymerase non-structural protein 5B (NS5B) and cause RNA chain-termination. Herein, 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 well-characterized 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 has previously been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells lead to 87-fold higher levels of intracellular 2’ –C-methyl-GTP when compared to DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites.
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Date of publication: 17 May 2016; American Society for Microbiology: Antimicrobial Agents and Chemotherapy
Author information: Maryam Ehteshami (1), Sijia Tao (1), Tugba Ozturk (1), Longhu Zhou (1), Jong Hyun Cho (1), Hongwang Zhang (1), Sheida Amiralaei (1), Jadd R. Shelton (1), Xiao Lu (1), Ahmed Khalil (1), Robert A. Domaoal (1), Richard A. Stanton (1), Justin E. Suesserman (1), Biing Lin (2), Sam S. Lee (2), Franck Amblard (1), Tony Whitaker (2), Steven J. Coats (2), & Raymond F. Schinazia(*)
(1) Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
(2) Cocrystal Pharma, Inc. Tucker, GA, USA
(*) Emory University School of Medicine, Department of Pedicatrics [Corresponding author]