A 7-Deaza-Adenosine Analog Is a Potent and Selective Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic Properties

Olsen, David B.; Eldrup, Anne B.; Bartholomew, Linda; Bhat, Balkrishen; Bosserman, Michele; Ceccacci, Alessandra; Colwell, Lawrence F.; Fay, John F.; Flores, Osvaldo; Getty, Krista; Grobler, Jay A.; LaFemina, Robert L.; Markel, Eric; Migliaccio, Giovanni; Prhavc, Marija; Stahlhut, Mark W.; Tomassini, Joanne E.; MacCoss, Malcolm; Hazuda, Daria J.; Carroll, Steven S.

doi:10.1128/aac.48.10.3944-3953.2004

Cited by 221 publications

(181 citation statements)

References 33 publications

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“…In vivo, other factors, such as metabolic activation of nucleoside analogue prodrugs and rates of incorporation of the monophosphate, are important parameters that determine the potency of these compounds (40). Based on our results, we propose that the phosphorolytic attack at the ultimate phosphodiester bond of the primer is perhaps an additional parameter that could account for the increased antiviral activity of 2Ј-C-Me-adenosine over that of 2Ј-C-Me-cytidine in cell-based replicon systems (26,40). Overall, these findings warrant further investigation towards the development of novel purine analogues, despite the fact that these compounds compete with relatively high intracellular concentrations of ATP and GTP.…”

Section: Discussionmentioning

confidence: 85%

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Deval¹,

Powdrill²,

D’Abramo³

et al. 2007

Antimicrob Agents Chemother

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Nonobligate chain terminators, such as 2-C-methylated nucleotides, block RNA synthesis by the RNAdependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3-hydroxyl group can be excised in the presence of pyrophosphate (PP i ), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.Hepatitis C virus (HCV) infection is a serious public health concern that affects 170 million people worldwide (33, 42). Among those infected, approximately 20 to 30% develop severe liver disease, such as chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (2). The combined use of the nucleoside analogue ribavirin and pegylated alpha interferon is the current treatment standard; however, success in treatment depends largely on the viral genotype, and this drug combination has also been associated with severe side effects (30,43). Thus, the development of novel, more potent, specific drugs is urgent.HCV belongs to the Flaviviridae family. The HCV RNA genome consists of approximately 10 kb, encoding a polyprotein which is processed into several smaller polypeptides, including the capsid protein (C), the envelope proteins (E1 and E2), and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Initial cis cleavage through NS2-NS3 releases the NS3 protein, which in turn continues to process the precursor. Promising compounds with the ability to inhibit the viral protease (NS3) and the polymerase (NS5B) have been identified.NS5B is a 65-kDa RNA-dependent RNA polymerase capable of initiating RNA synthesis de novo in the absence of a primer (16,17,25,27,45). Three classes of inhibitors of HCV NS5B have been developed, namely, nucleoside analogue inhibitors, nonnucleoside analogue inhibitors, and pyrophosphate (PP i ) analogues. Nonnucleoside inhibitors and PP i analogues are still under preclinical evaluation, while a 2Ј-modified nucleoside analogue has advanc...

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Section: Discussionmentioning

confidence: 85%

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Deval¹,

Powdrill²,

D’Abramo³

et al. 2007

Antimicrob Agents Chemother

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“…Derivatives of these ribonucleotide analogs may serve as potential antiviral molecules to combat ZIKV infection. It has been shown before that 2=-C-ethynyl-7-deaza-ATP is also a potent inhibitor of HCV and dengue virus RdRps, and its antiviral activity has been extensively studied (16,18,24).…”

Section: Resultsmentioning

confidence: 99%

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Bluemling

Collop

et al. 2017

Antimicrob Agents Chemother

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Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primertemplate pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn 2ϩ is required for enzymatic activity, while Mg 2ϩ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5=-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2=-C-methyl-and 2=-C-ethynyl-substituted analog 5=-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

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“…Similarly, 2Ј-␤-hydroxy-GTP was described as a moderate inhibitor of HCV NS5B, but 2Ј-␤-hydroxy-ATP was inactive (33). The increased base pairing stability of G:C base pairs may therefore be required to allow sufficient binding affinity of these compounds in the NS5B active site to confer measurable inhibitory potency.…”

Section: Discussionmentioning

confidence: 99%

2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

Klumpp

Kalayanov

et al. 2008

Journal of Biological Chemistry

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RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2-␣-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of ␣-hydroxy moieties. 2-Deoxy-2-␤-fluoro-4-azidocytidine (RO-0622) and 2-deoxy-2-␤-hydroxy-4-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC 50 ‫؍‬ 171 ؎ 12 nM and 24 ؎ 3 nM for RO-9187 and RO-0622, respectively; CC 50 >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4-azidocytidine) or 2-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC 50 values 8 -150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2-␣-deoxy-4-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection. Hepatitis C virus (HCV)3 infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation. Current treatment options available to HCV-infected persons have limitations with regard to efficacy and tolerability. Only about 50% of individuals infected with HCV genotype 1 achieve sustained virological response when treated with a combination of pegylated interferon ␣ and ribavirin (1, 2). In addition, high viral load, age, body weight, co-infection with human immunodeficiency virus, and cirrhosis negatively affect the probability of achieving sustained virological response (3, 4). Therefore, there is an urgent need to develop new and more effective therapies for the treatment of HCV infection. A number of new antiviral candidates are currently being evaluated in clinical studies, the majority targeting either the HCV protease or HCV polymerase enzymes, which are essential for viral replication (5). The HCV RNA-dependent RNA polymerase, NS5B, contains the active site responsible for viral RNA synthesis and functions as part of a membrane-associated replicase complex. Nucleoside and non-nucleoside inhibitors of HCV polymerase h...

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A 7-Deaza-Adenosine Analog Is a Potent and Selective Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic Properties

Cited by 221 publications

References 33 publications

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Pyrophosphorolytic Excision of Nonobligate Chain Terminators by Hepatitis C Virus NS5B Polymerase

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

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