Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2′-Deoxy-2′-Fluorocytidine

Stuyver, Lieven; McBrayer, Tamara R.; Whitaker, Tony; Tharnish, Phillip M.; Ramesh, Mangala; Lostia, Stefania; Cartee, Leanne; Shi, Jiyong; Hobbs, Ann; Schinazi, Raymond F.; Watanabe, Kyoichi A.; Otto, Michael

doi:10.1128/aac.48.2.651-654.2004

Cited by 63 publications

(47 citation statements)

References 18 publications

(27 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…7 and 8). The fact that 2=F-UTP did not cause any chain termination was surprising, given that 2=F-cytidine had previously been shown to inhibit HCV replication (34). In our hands, 2=F-CTP did not act as an inhibitor against HCV polymerase (see Fig.…”

Section: Discussionmentioning

confidence: 62%

Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

Fung¹,

Zhang²,

Dyatkina³

et al. 2014

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2=F-2=C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2=F-2=C-Me-UTP was efficiently incorporated by HCV polymerase with apparent K d (equilibrium constant) and k pol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ؎ 28 M and 0.67 ؎ 0.05 s ؊1 , respectively, giving an overall substrate efficiency (k pol /K d ) of 0.0059 ؎ 0.0015 M ؊1 s ؊1 . We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2= position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH 2 > F-C-Me > C-Me > N 3 > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2=F-2=C-Me-, 2=C-Me-, and 2=ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2=-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2=-modified NTP analogs against HCV polymerase. . The primary mode of transmission for HCV is via exposure to infected blood, including transfusions from infected donors, and through intravenous use of illicit drugs. Although a minority of all HCV infections will spontaneously resolve without any clinical outcome, an estimated 80% of cases will progress into chronic hepatitis, leading to a significant proportion of cirrhosis and cases of hepatocellular carcinoma (2). This makes HCV the leading cause of liver transplantation in the United States.Hepatitis C virus is a member of the Flaviviridae family (3). It contains a single, positive-strand RNA genome of about 9.5 kb. The viral genome encodes only one open reading frame translated to a polyprotein of approximately 3,000 amino acids. The NS5B protein is composed of 591 amino acids that are cleaved at the C-terminal end of the polyprotein. NS5B acts as the RNA-dependent RNA polymerase (RdRp), with critical functions in RNA replication and transcription. Similar to other known RdRps, NS5B contains six conserved motifs, designated A through F. The amino acids involved in the catalytic activity of NS5B are located within motif A (aspartate at position 220) and in the catalytic triad GDD at positions 318 to ...

show abstract

Section: Discussionmentioning

confidence: 62%

Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

Fung¹,

Zhang²,

Dyatkina³

et al. 2014

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…3A). Each titration was performed in triplicate, and the average IC 50 was calculated for each compound/ enzyme combination (Fig. 3B).…”

Section: Resultsmentioning

confidence: 99%

“…Other NTP analogs have also been shown to inhibit HCV replication via the NS5B protein and are in various stages of development. They include 2Ј-deoxy-2Ј-fluorocytidine, which inhibits replication of subgenomic replicons in Huh-7 cells (50). Similarly, R7128, a prodrug of ␤-D-2Ј-deoxy-2Ј-fluoro-2Ј-C-methyl-cytidine, is being tested in combination with interferon and ribavirin (41), and 4Ј-azidocytidine is in preclinical trials (28).…”

mentioning

confidence: 99%

Effects of Mutagenic and Chain-Terminating Nucleotide Analogs on Enzymes Isolated from Hepatitis C Virus Strains of Various Genotypes

Heck

Lam

Narayanan

et al. 2008

Antimicrob Agents Chemother

View full text Add to dashboard Cite

The development of effective therapies for hepatitis C virus (HCV) must take into account genetic variation among HCV strains. Response rates to interferon-based treatments, including the current preferred treatment of pegylated alpha interferon administered with ribavirin, are genotype specific. Of the numerous HCV inhibitors currently in development as antiviral drugs, nucleoside analogs that target the conserved NS5B active site seem to be quite effective against diverse HCV strains. To test this hypothesis, we examined the effects of a panel of nucleotide analogs, including ribavirin triphosphate (RTP) and several chain-terminating nucleoside triphosphates, on the activities of purified HCV NS5B polymerases derived from genotype 1a, 1b, and 2a strains. Unlike the genotype-specific effects on NS5B activity reported previously for nonnucleoside inhibitors ( , only minor differences in inhibition were observed among the various genotypes; thus, nucleoside analogs that are current drug candidates may be more promising for treatment of a broader variety of HCV strains. We also examined the effects of RTP on the HCV NS3 helicase/ATPase. As with the polymerase, only minor differences were observed among 1a-, 1b-, and 2a-derived enzymes. RTP did not inhibit the rate of NS3 helicase-catalyzed DNA unwinding but served instead as a substrate to fuel unwinding. NS3 added to RNA synthesis reactions relieved inhibition of the polymerase by RTP, presumably due to RTP hydrolysis. These results suggest that NS3 can limit the incorporation of ribavirin into viral RNA, thus reducing its inhibitory or mutagenic effects.Hepatitis C virus (HCV) infects more than 170 million people worldwide, causing chronic hepatitis, cirrhosis, hepatocellular carcinoma, and/or liver failure (12). Clinical HCV isolates are categorized into different genotypes that display up to 30% sequence variation. Response rates to all interferon-based HCV therapies are genotype dependent, with patients infected with HCV genotype 1 having about one-half the chance of a sustained virological response as patients infected with other genotypes (37). Current HCV therapies combine pegylated alpha interferon and ribavirin (1-␤-D-ribofuranosyl-1,2,4-triazole-3-carboxamide). A number of viral factors have been proposed to contribute to the different response rates to interferon (reviewed in reference 59), but less is known about the effects of viral variation on sensitivity to ribavirin. Here, we employ biochemical methods to examine the simple hypothesis that as a nucleoside analog, ribavirin may affect the enzymes encoded by HCV, and that variation among the enzymes from different genotypes could affect ribavirin response rates. We also examine whether HCV genetic variation influences the sensitivities of HCV enzymes to other nucleoside analogs similar to those currently in development as HCV-specific drugs.The ϳ9,600-nucleotide HCV genome harbors a single open reading frame that encodes a polyprotein, which is processed into 10 distinct proteins by both host and v...

show abstract

“…2Ј-␣-FluoroNTPs showed antiviral potencies against influenza virus and were substrates for the RNA-dependent RNA polymerase of influenza virus (35,36). 2Ј-␣-Fluorocytidine was also assessed as an inhibitor of HCV replication but was a potent inhibitor of cell proliferation in the HCV replicon system (7,37). The addition of a 2Ј-␤-methyl substituent resulted in the discovery of PSI-6130 (2Ј-␣-fluoro-2Ј-␤-methylcytidine), a potent inhibitor of HCV replication (38).…”

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

View full text Add to dashboard Cite

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...

show abstract

Inhibition of the Subgenomic Hepatitis C Virus Replicon in Huh-7 Cells by 2′-Deoxy-2′-Fluorocytidine

Cited by 63 publications

References 18 publications

Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

Effects of Mutagenic and Chain-Terminating Nucleotide Analogs on Enzymes Isolated from Hepatitis C Virus Strains of Various Genotypes

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

Contact Info

Product

Resources

About