RNA dependent RNA polymerase of HCV: A potential target for the development of antiviral drugs

Waheed, Yasir; Bhatti, Attya; Ashraf, Muhammad Usman

doi:10.1016/j.meegid.2012.12.004

Cited by 23 publications

(14 citation statements)

References 107 publications

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“…S2). It has also been reported that the incorporation of 4=-C-azidocytidine into HCV RNA could block the synthesis of the complementary strand (14,22). This mechanism of inhibition still needs to be investigated.…”

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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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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“…In analysis of the reference sequence alignment at the level of the five important motifs (named A to E) within the palm region (62,63), the A motif (residues 213 to 228), which includes the catalytic pocket of the enzyme (D220 to D225) with two essential residues for metal binding (D220 and T221), was highly conserved among all the seven genotypes. The B motif (residues 282 to 302), taking part in the sugar selection, was also highly conserved within HCV genotype 1, with some variability found at a few positions (285-286, 289, 293, 296-297, and 300) in other, non-HCV-1 genotypes.…”

Section: Resultsmentioning

confidence: 99%

Hepatitis C Virus Genetic Variability and the Presence of NS5B Resistance-Associated Mutations as Natural Polymorphisms in Selected Genotypes Could Affect the Response to NS5B Inhibitors

Maio

Cento

Mirabelli

et al. 2014

Antimicrob Agents Chemother

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Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed the NS5B polymerase genetic variability in circulating HCV genotypes/subtypes and its impact on the genetic barrier for the development of resistance to clinically relevant nucleoside inhibitors (NIs)/nonnucleoside inhibitors (NNIs). The study included 1,145 NS5B polymerase sequences retrieved from the Los Alamos HCV database and GenBank. The genetic barrier was calculated for drug resistance emergence. Prevalence and genetic barrier were calculated for 1 major NI and 32 NNI resistance variants (13 major and 19 minor) at 21 total NS5B positions. Docking calculations were used to analyze sofosbuvir affinity toward the diverse HCV genotypes. Overall, NS5B polymerase was moderately conserved among all HCV genotypes, with 313/591 amino acid residues (53.0%) showing <1% variability and 83/591 residues (14.0%) showing high variability (>25.1%). Nine NNI resistance variants (2 major variants, 414L and 423I; 7 minor variants, 316N, 421V, 445F, 482L, 494A, 499A, and 556G) were found as natural polymorphisms in selected genotypes. In particular, 414L and 423I were found in HCV genotype 4 (HCV-4) (n ‫؍‬ 14/38, 36.8%) and in all HCV-5 sequences (n ‫؍‬ 17, 100%), respectively. Regardless of HCV genotype, the 282T major NI resistance variant and 10 major NNI resistance variants (316Y, 414L, 423I/T/V, 448H, 486V, 495L, 554D, and 559G) always required a single nucleotide substitution to be generated. Conversely, the other 3 major NNI resistance variants (414T, 419S, and 422K) were associated with a different genetic barrier score development among the six HCV genotypes. Sofosbuvir docking analysis highlighted a better ligand affinity toward HCV-2 than toward HCV-3, in agreement with the experimental observations. The genetic variability among HCV genotypes, particularly with the presence of polymorphisms at NNI resistance positions, could affect their responsiveness to NS5B inhibitors. A pretherapy HCV NS5B sequencing could help to provide patients with the full efficacy of NNI-containing regimens.T he advent of direct antiviral agents (DAAs) has opened a new era for the treatment of hepatitis C virus (HCV) infection. Since 2011, telaprevir and boceprevir, two linear protease inhibitors, have been approved by both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as a new standard of care for first-line treatment of HCV genotype 1 (HCV-1), in association with pegylated alpha interferon (PEG-IFN) and ribavirin. Both of these combination treatments have shown improved sustained virological response (SVR) rates along with reduced treatment duration (1-4). Despite the increase in SVR and their high potency, these two first approved protease inhibitors are still "fragile" drugs because of their low genetic barrier, extensive cross-resistance profile, and significant side effects. Therefore, additional DAAs that target different HCV proteins as well are currently in development (5-8) (http://www.pipelinereport.org; htt...

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“…The fact that mammalian cells lack a homologous enzyme also makes NS5B an attractive drug target due to the potential for selective inhibition of viral replication. Consequently, this enzyme has been the object of numerous studies in the last decade . Both nucleoside (of which sofosbuvir is an example) and non‐nucleoside inhibitors have demonstrated activity against the enzyme.…”

Section: Introductionmentioning

confidence: 99%

Molecular simulations to delineate functional conformational transitions in the HCV polymerase

2016

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Hepatitis C virus (HCV) is a global health concern for which there is no vaccine available. The HCV polymerase is responsible for the critical function of replicating the RNA genome of the virus. Transitions between at least two conformations (open and closed) are necessary to allow the enzyme to replicate RNA. In this study, molecular dynamic simulations were initiated from multiple crystal structures to understand the free energy landscape (FEL) explored by the enzyme as it interconverts between these conformations. Our studies reveal the location of distinct states within the FEL as well as the molecular interactions associated with these states. Specific hydrogen bonds appear to play a key role in modulating conformational transitions. This knowledge is essential to elucidate the role of these conformations in replication and may also be valuable in understanding the basis by which this enzyme is inhibited by small molecules. © 2016 Wiley Periodicals, Inc.

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RNA dependent RNA polymerase of HCV: A potential target for the development of antiviral drugs

Cited by 23 publications

References 107 publications

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

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

Hepatitis C Virus Genetic Variability and the Presence of NS5B Resistance-Associated Mutations as Natural Polymorphisms in Selected Genotypes Could Affect the Response to NS5B Inhibitors

Molecular simulations to delineate functional conformational transitions in the HCV polymerase

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