Synthesis and Antiviral Activity of a Series of 2′-C-Methyl-4′-thionucleoside Monophosphate Prodrugs

The NS5B RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is a validated target for nucleoside antiviral drug therapy. We endeavored to synthesize and test a series of 4′-thionucleosides with a monophosphate prodrug moiety for their antiviral activity against HCV and other related viruses in the Flaviviridae family. Nucleoside analogs were prepared via the stereoselective Vorbrüggen glycosylation of various nucleobases with per-acetylated 2-C-methyl-4-thio-d-ribose built in a 10-step synthetic sequence from the corresponding ribonolactone. Conjugation of the thionucleoside to a ProTide phosphoramidate allowed for evaluation of the prodrugs in the cellular HCV replicon assay with anti-HCV activities ranging from single-digit micromolar (μM) to >200 μM. The diminished anti-HCV potency of our best compound compared to its 4′-oxo congener is the subject of ongoing research in our lab and is proposed to stem from changes in sugar geometry imparted by the larger sulfur atom.

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Hepatitis C Virus RNA-dependent RNA Polymerase (NS5B) as a Mediator of the Antiviral Activity of Ribavirin

Journal of Biological Chemistry ◽

10.1074/jbc.c100349200 ◽

2001 ◽

Vol 276 (49) ◽

pp. 46094-46098 ◽

Cited By ~ 200

Author(s):

David Maag ◽

Christian Castro ◽

Zhi Hong ◽

Craig E. Cameron

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Rna Polymerase ◽

Hepatitis C Virus Rna ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

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Hindered nucleoside analogs as antiflaviviridae agents

Pure and Applied Chemistry ◽

10.1351/pac200476051007 ◽

2004 ◽

Vol 76 (5) ◽

pp. 1007-1015 ◽

Cited By ~ 8

Author(s):

Stefano Manfredini ◽

Angela Angusti ◽

A. C. Veronese ◽

Elisa Durini ◽

S. Vertuani ◽

...

Keyword(s):

Hepatitis C ◽

Molecular Modeling ◽

Rna Polymerase ◽

Nucleoside Analogs ◽

Binding Pocket ◽

West Nile Fever ◽

Rna Dependent Rna Polymerase ◽

West Nile ◽

Important Family ◽

Allosteric Binding Pocket

Flaviviridae are an important family of viruses, responsible for widely spread diseases such as dengue and West Nile fever and hepatitis C. Despite the severity of the related diseases, no effective antiviral treatments for infection are available. Following our discovery of adenosine-hindered analogs as potent antiflaviviridae agents, we have continued our investigation on guanosine and inosine derivatives, which were evaluated for activity against BVDV, YFV, DENV, and WNV viruses in cell-based assays. The present study allowed us to identify some newer features that led to improve the antiviral potency (down to the µM range) and to selectively inhibit BVDV and YFV viruses. The molecular modeling results were consistent with the hypothesis that test analogs act as RNA-dependent RNA polymerase (RdRp) inhibitors by interacting with a surface allosteric binding pocket.

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Resistance to excision determines efficiency of hepatitis C virus RNA-dependent RNA polymerase inhibition by nucleotide analogs

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013422 ◽

2020 ◽

Vol 295 (30) ◽

pp. 10112-10124 ◽

Cited By ~ 2

Author(s):

Brian Villalba ◽

Jiawen Li ◽

Kenneth A. Johnson

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

Transient State ◽

Nucleoside Analogs ◽

Clinical Effectiveness ◽

Virus Genome ◽

Rna Dependent Rna Polymerase ◽

Chain Terminators

NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the hepatitis C virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs mericitabine and sofosbuvir, which get metabolized to 2′-fluoro-2′C-methylcytidine-5′-triphosphate and 2′-fluoro-2′C-methyluridine-5′-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, it has been shown that NS5B can efficiently remove chain terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study, we use transient-state kinetics to understand the efficiency of inhibition for five nucleoside analogs. We show that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised. In contrast, although UMP analogs are more slowly incorporated, the excision of UMP is slow and inefficient, and modifications to the 2′-carbon of the UTP ribose ring further decreased rates of excision to an undetectable level. Taken together, these data suggest that the clinical effectiveness of sofosbuvir is largely a function of being intractable to nucleotide-mediated excision compared with similar nucleoside analogs.

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PSI-7851, a Pronucleotide of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine Monophosphate, Is a Potent and Pan-Genotype Inhibitor of Hepatitis C Virus Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00399-10 ◽

2010 ◽

Vol 54 (8) ◽

pp. 3187-3196 ◽

Cited By ~ 103

Author(s):

Angela M. Lam ◽

Eisuke Murakami ◽

Christine Espiritu ◽

Holly M. Micolochick Steuer ◽

Congrong Niu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Nucleoside Analogs ◽

Cross Resistance ◽

Bone Marrow Toxicity ◽

Significant Activity ◽

Dna And Rna ◽

Highly Active

ABSTRACT The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine-5′-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075 ± 0.050 μM (mean ± standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 μM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.

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Preclinical Characterization of JTK-853, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00312-12 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4250-4256 ◽

Cited By ~ 12

Author(s):

Izuru Ando ◽

Tsuyoshi Adachi ◽

Naoki Ogura ◽

Yukiyo Toyonaga ◽

Kazuyuki Sugimoto ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Rna Polymerase ◽

Antiviral Agents ◽

Hcv Rna ◽

Resistance Mutations ◽

Rna Dependent Rna Polymerase ◽

Virus Rna ◽

Polymerase Inhibitor

ABSTRACTJTK-853 is a novel piperazine derivative nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase. JTK-853 showed potent inhibitory activity against genotype 1 HCV polymerase, with a 50% inhibitory concentration in the nanomolar range, and showed potent antiviral activity against the genotype 1b replicon, with a 50% effective concentration of 0.035 μM. The presence of human serum at up to 40% had little effect on the antiviral activity of JTK-853. Structure analysis of HCV polymerase with JTK-853 revealed that JTK-853 associates with the palm site and β-hairpin region of HCV polymerase, and JTK-853 showed decreased antiviral activity against HCV replicons bearing the resistance mutations C316Y, M414T, Y452H, and L466V in the palm site region of HCV polymerase. JTK-853 showed an additive combination effect with other DAAs (direct antiviral agents), such as nucleoside polymerase inhibitor, thumb pocket-binding nonnucleoside polymerase inhibitor, NS5A inhibitor, and protease inhibitor. Collectively, these data demonstrate that JTK-853 is a potent and novel nonnucleoside palm site-binding HCV polymerase inhibitor, suggesting JTK-853 as a potentially useful agent in combination with other DAAs for treatment of HCV infections.

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