RESUMO
We describe the structure-activity relationship of the C7-position of pyrano[3,4-b]indole-based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compounds 13 and 14.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Indóis , Piranos , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítio Alostérico , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Piranos/síntese química , Piranos/química , Piranos/farmacologia , Relação Estrutura-AtividadeRESUMO
We describe the structure-activity relationship of the C1-group of pyrano[3,4-b]indole based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compound 12.
Assuntos
Antivirais/química , Inibidores Enzimáticos/química , Indóis/química , Piranos/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/síntese química , Antivirais/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/síntese química , Indóis/farmacologia , Piranos/síntese química , Piranos/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log(10) HCV copies/mug total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 A of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.
Assuntos
Antivirais/farmacologia , Benzofuranos/antagonistas & inibidores , Farmacorresistência Viral , Inibidores Enzimáticos/farmacologia , Variação Genética , Hepacivirus/efeitos dos fármacos , Replicon/efeitos dos fármacos , Antivirais/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Inibidores Enzimáticos/metabolismo , Genótipo , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Modelos Moleculares , Mutação , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Replicon/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA-dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695-residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical "core," although overall sequence identity is low. Eighty-four expression clones of the BVDV polymerase were designed by fine-sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high-throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96-well format, and two of these proteins were successfully crystallized.
Assuntos
Vírus da Diarreia Viral Bovina/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Cristalização , Cristalografia , Vírus da Diarreia Viral Bovina/genética , Escherichia coli/enzimologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Tripsina/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
Biochemical characterization of hepatitis C virus (HCV) replication using purified, membrane-associated replication complexes is hampered by the presence of endogenous nuclease activity that copurifies with the replication complex. In this study, pulse-chase analyses were used to demonstrate that newly synthesized replicon RNA was protected from nuclease activity by a factor(s) that was sensitive to 0.5% NP-40 or protease treatment. Nuclease susceptibility was not related to disruption of lipid membranes, since NP-40 did not significantly affect the buoyant density of HCV replication complexes or protease susceptibility of HCV NS3 and NS5A proteins. These results suggest that a protease-sensitive factor(s) protects newly synthesized RNA from nuclease degradation.
Assuntos
Hepacivirus/genética , RNA Viral/genética , RNA Viral/metabolismo , Linhagem Celular , Endopeptidases/metabolismo , Hepacivirus/fisiologia , Humanos , Octoxinol , Polietilenoglicóis/farmacologia , Replicon , Ribonucleases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
The bovine viral diarrhea virus (BVDV) RNA-dependent RNA polymerase can initiate RNA replication by a de novo mechanism without a primer. The structure of BVDV polymerase, determined to 2.9-A resolution, contains a unique N-terminal domain, in addition to the fingers, palm, and thumb domains common to other polymerases. The structure of BVDV polymerase complexed with GTP, which is required for de novo (primer-independent) initiation, shows that GTP binds adjacent to the initiation NTP, suggesting that the GTP mimics a vestigial RNA product. Comparison of five monomers in two different crystal forms showed conformational changes in the fingertip region and in the thumb domain that may help to translocate the RNA template and product strands during elongation. The putative binding sites of previously reported BVDV inhibitors are also discussed.
Assuntos
Vírus da Diarreia Viral Bovina/enzimologia , Guanosina Trifosfato/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Guanosina Trifosfato/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
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 microM 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.