NS5B

About: NS5B is a research topic. Over the lifetime, 1314 publications have been published within this topic receiving 59534 citations.

The design, synthesis, and antiviral activity of 4'-azidocytidine analogues against hepatitis C virus replication: the discovery of 4'-azidoarabinocytidine

Abstract: 4'-Azidocytidine 3 (R1479) has been previously discovered as a potent and selective inhibitor of HCV replication targeting the RNA-dependent RNA polymerase of hepatitis C virus, NS5B. Here we describe the synthesis and biological evaluation of several derivatives of 4'-azidocytidine by varying the substituents at the ribose 2' and 3'-positions. The most potent compound in this series is 4'-azidoarabinocytidine with an IC(50) of 0.17 microM in the genotype 1b subgenomic replicon system. The structure-activity relationships within this series of nucleoside analogues are discussed.

Novel Nonnucleoside Inhibitor of Hepatitis C Virus RNA-Dependent RNA Polymerase

Abstract: A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 μM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.

Comprehensive Screening for Naturally Occurring Hepatitis C Virus Resistance to Direct-Acting Antivirals in the NS3, NS5A, and NS5B Genes in Worldwide Isolates of Viral Genotypes 1 to 6

Abstract: There is no comprehensive study available on the natural hepatitis C virus (HCV) polymorphism in sites associated with resistance including all viral genotypes which may present variable susceptibilities to particular direct-acting antivirals (DAAs). This study aimed to analyze the frequencies, genetic barriers, and evolutionary histories of naturally occurring resistance-associated variants (RAVs) in the six main HCV genotypes. A comprehensive analysis of up to 103 RAVs was performed in 2,901, 2,216, and 1,344 HCV isolates for the NS3, NS5A, and NS5B genes, respectively. We report significant intergenotypic differences in the frequencies of natural RAVs for these three HCV genes. In addition, we found a low genetic barrier for the generation of new RAVs, irrespective of the viral genotype. Furthermore, in 1,126 HCV genomes, including sequences spanning the three genes, haplotype analysis revealed a remarkably high frequency of viruses carrying more than one natural RAV to DAAs (53% of HCV-1a, 28.5% of HCV-1b, 67.1% of HCV-6, and 100% of genotype 2, 3, 4, and 5 haplotypes). With the exception of HCV-1a, the most prevalent haplotypes showed RAVs in at least two different viral genes. Finally, evolutionary analyses revealed that, while most natural RAVs appeared recently, others have been efficiently transmitted over time and cluster in well-supported clades. In summary, and despite the observed high efficacy of DAA-based regimens, we show that naturally occurring RAVs are common in all HCV genotypes and that there is an overall low genetic barrier for the selection of resistance mutations. There is a need for natural DAA resistance profiling specific for each HCV genotype.

Zebrafish as a Potential Model Organism for Drug Test Against Hepatitis C Virus

Abstract: Screening and evaluating anti- hepatitis C virus (HCV) drugs in vivo is difficult worldwide, mainly because of the lack of suitable small animal models. We investigate whether zebrafish could be a model organism for HCV replication. To achieve NS5B-dependent replication an HCV sub-replicon was designed and created with two vectors, one with HCV ns5b and fluorescent rfp genes, and the other containing HCV's 5′UTR, core, 3′UTR and fluorescent gfp genes. The vectors containing sub-replicons were co-injected into zebrafish zygotes. The sub-replicon amplified in liver showing a significant expression of HCV core RNA and protein. The sub-replicon amplification caused no abnormality in development and growth of zebrafish larvae, but induced gene expression change similar to that in human hepatocytes. As the amplified core fluorescence in live zebrafish was detectable microscopically, it rendered us an advantage to select those with replicating sub-replicon for drug experiments. Ribavirin and oxymatrine, two known anti-HCV drugs, inhibited sub-replicon amplification in this model showing reduced levels of HCV core RNA and protein. Technically, this method had a good reproducibility and is easy to operate. Thus, zebrafish might be a model organism to host HCV, and this zebrafish/HCV (sub-replicon) system could be an animal model for anti-HCV drug screening and evaluation.