RESUMO
The protein that forms the inner shell of the HBV virus, known as the capsid core protein, plays a crucial role in allowing chronic HBV infections to persist. Studies have shown that disrupting the assembly of the capsid can effectively combat the virus, and small molecule drugs that target the HBV capsid assembly modulator (CAM) process have been successful in clinical trials. Herein is described a distinct series of di-fluoro azepane CAMs with exceptional potency, pharmacokinetic, and solubility properties.
Assuntos
Capsídeo , Vírus da Hepatite B , Capsídeo/metabolismo , Montagem de Vírus , Antivirais/metabolismo , Proteínas do Capsídeo/metabolismo , Replicação ViralRESUMO
The HBV capsid core protein serves a number of important functions in the viral life cycle enabling chronic HBV infection to persist, and therefore is a promising drug target. Interfering with capsid assembly has shown efficacy in clinical trials with small molecule capsid assembly modulators (CAMs). Herein is described the further optimization of a progressive series of diazepinone HBV CAMs.
Assuntos
Capsídeo , Vírus da Hepatite B , Antivirais/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Vírus da Hepatite B/metabolismo , Montagem de VírusRESUMO
The HBV core protein serves multiple essential functions in the viral life cycle that enable chronic HBV infection to persist, and as such, represents a promising drug target. Modulation of the HBV capsid assembly has shown efficacy in early clinical trials through use of small molecule capsid assembly modulators (CAMs). Herein is described the evolution and SAR of a novel pyrazolo piperidine lead series into advanced oxadiazepinone HBV CAMs.
Assuntos
Antivirais/farmacologia , Azepinas/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Antivirais/química , Azepinas/química , Proteínas do Capsídeo/metabolismo , Relação Dose-Resposta a Droga , Vírus da Hepatite B/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The HBV core protein is a druggable target of interest due to the multiple essential functions in the HBV life cycle to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the HBV capsid assembly has shown efficacy in clinical trials. Herein is described the identification and hit to lead SAR of a novel series of pyrazolo piperidine HBV capsid assembly modulators.
Assuntos
Antivirais/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Piperidinas/farmacologia , Pirazóis/farmacologia , Antivirais/química , Proteínas do Capsídeo/metabolismo , Relação Dose-Resposta a Droga , Vírus da Hepatite B/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piperidinas/química , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
The HBV core protein has multiple essential functions in the HBV life cycle to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the HBV capsid assembly process has shown clinical efficacy in early clinical trials. Herein is described the SAR exploration of NVR 3-778, the first clinical compound in the sulfonyl carboxamide class.
Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Piperidinas/farmacologia , Antivirais/síntese química , Antivirais/química , Benzamidas/síntese química , Benzamidas/química , Proteínas do Capsídeo/metabolismo , Relação Dose-Resposta a Droga , Vírus da Hepatite B/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade , Montagem de Vírus/efeitos dos fármacosRESUMO
NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.
Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Capsídeo/efeitos dos fármacos , DNA Viral/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Piperidinas/farmacologia , RNA Viral/antagonistas & inibidores , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Antivirais/sangue , Antivirais/química , Antivirais/farmacocinética , Benzamidas/sangue , Benzamidas/química , Benzamidas/farmacocinética , Capsídeo/química , Capsídeo/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Piperidinas/sangue , Piperidinas/química , Piperidinas/farmacocinética , Cultura Primária de Células , RNA Viral/genética , RNA Viral/metabolismo , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND & AIMS: NVR3-778 is a capsid assembly modulator in clinical development. We determined the in vivo antiviral efficacy and effects on innate and endoplasmic reticulum (ER) stress responses of NVR3-778 alone or in combination with pegylated interferon alpha (peg-IFN) and compared with entecavir. METHODS: We performed 2 studies, with a total of 61 uPA/SCID mice with humanized livers. Mice were infected with a hepatitis B virus (HBV) genotype C preparation; we waited 8 weeks for persistent infection of the human hepatocytes in livers of mice. Mice were then randomly assigned to groups (5 or 6 per group) given vehicle (control), NVR3-778, entecavir, peg-IFN, NVR3-778 + entecavir, or NVR3-778 + peg-IFN for 6 weeks. We measured levels of HB surface antigen, HB e antigen, HBV RNA, alanine aminotransferase, and human serum albumin at different time points. Livers were collected and analyzed by immunohistochemistry; levels of HBV DNA, covalently closed circular DNA, and HBV RNA, along with markers of ER stress and IFN response, were quantified. RESULTS: Mice given NVR3-778 or entecavir alone for 6 weeks had reduced serum levels of HBV DNA compared with controls or mice given peg-IFN. The largest reduction was observed in mice given NVR3-778 + peg-IFN; in all mice in this group, the serum level of HBV DNA was below the limit of quantification. NVR3-778 and peg-IFN, but not entecavir, also reduced serum level of HBV RNA. The largest effect was obtained in the NVR3-778 + peg-IFN group, in which serum level of HBV RNA was below the limit of quantification. Levels of HB surface antigen and HB e antigen were reduced significantly in only the groups that received peg-IFN. Levels of covalently closed circular DNA did not differ significantly among groups. NVR3-778 was not associated with any significant changes in level of alanine aminotransferase, the ER stress response, or IFN-stimulated genes. CONCLUSIONS: NVR3-778 has high antiviral activity in mice with humanized livers and stable HBV infection, reducing levels of serum HBV DNA and HBV RNA. Entecavir reduced levels of serum HBV DNA, but had no effect on HBV RNA. The combination of NVR3-778 and peg-IFN prevented viral replication and HBV RNA particle production to a greater extent than each compound alone or entecavir.
Assuntos
Antivirais/farmacologia , Guanina/análogos & derivados , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Interferon-alfa/farmacologia , Polietilenoglicóis/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/genética , Alanina Transaminase/sangue , Animais , DNA Viral/genética , Modelos Animais de Doenças , Quimioterapia Combinada , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Genótipo , Guanina/farmacologia , Hepatite B/diagnóstico , Hepatite B/virologia , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos SCID , Camundongos Transgênicos , Fenótipo , RNA Viral/genética , Proteínas Recombinantes/farmacologia , Albumina Sérica Humana/metabolismo , Fatores de Tempo , Carga ViralRESUMO
The hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle, and antiviral agents that target the core protein are being developed. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with the treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. The results showed that CAMs blocked extracellular HBV RNA with efficiencies similar to those with which they blocked pregenomic RNA (pgRNA) encapsidation, HBV DNA replication, and Dane particle production. Nucleos(t)ide analogs inhibited viral replication and virion production but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both culture supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with an internal deletion(s) but still retained the sequences at both the 5' and 3' ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrate that HBV CAMs represent direct antiviral agents with a profile differentiated from that of nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA.
Assuntos
Antivirais/farmacologia , Proteínas do Capsídeo/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Proteínas do Nucleocapsídeo/metabolismo , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Linhagem Celular , DNA Viral/sangue , DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatócitos/virologia , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Viral/sangue , Sulfonamidas/farmacologia , Proteínas do Core Viral/metabolismoRESUMO
The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein-protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.
Assuntos
Antivirais/química , Vírus da Hepatite B/fisiologia , Proteínas do Core Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Antivirais/metabolismo , Antivirais/farmacologia , Cristalografia por Raios X , Conformação ProteicaRESUMO
The blood pressure (BP)-lowering effects of mineralocorticoid receptor (MR) antagonists in salt-sensitive rat models of hypertension are well understood. However, studies in salt-independent models have yielded mixed results, and therefore, we measured the hemodynamic effects of MR blockade in spontaneously hypertensive rats. We treated spontaneously hypertensive rats for 8 weeks with 30-300 mg.kg.d eplerenone or 20 mg.kg.d losartan and monitored BP using radiotelemetry and performed histopathological analyses of the hearts. Eplerenone, in contrast to losartan, caused only a small reduction in systolic BP at the highest dose tested. Both reduced left ventricular wall thickness, although eplerenone was less effective than losartan. Only losartan decreased heart weight. We observed foci of cardiomyopathy characterized by combinations of infiltrating monocytes, necrotic myocytes, and interstitial fibrosis in hearts of control animals. The number of foci seemed to be decreased in hearts of losartan- and eplerenone-treated animals. In a second study, using quantitative histomorphometry, the number of foci was significantly reduced by 20 mg.kg.d losartan (by 68%) or by 300 mg.kg.d eplerenone (by 50%). Our data support the hypothesis that a direct BP-independent effect on the progression of cardiomyopathy in the heart may be one basis for the cardiac protection afforded by MR antagonism.
Assuntos
Coração/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Eplerenona , Coração/fisiopatologia , Hipertensão/sangue , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Losartan/farmacologia , Losartan/uso terapêutico , Masculino , Ratos , Ratos Endogâmicos SHR , Cloreto de Sódio na Dieta/farmacologia , Cloreto de Sódio na Dieta/uso terapêutico , Espironolactona/análogos & derivadosRESUMO
The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Farmacorresistência Viral , Inibidores Enzimáticos/farmacologia , Genótipo , Hepatite C/virologia , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Pan troglodytes , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Replicon , beta-Lactamases/metabolismoRESUMO
This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter/genética , Hepacivirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , beta-Lactamases/genética , beta-Lactamases/metabolismo , Linhagem Celular , Ácido Clavulânico/farmacologia , Replicação do DNA , Avaliação Pré-Clínica de Medicamentos/instrumentação , Inibidores Enzimáticos/farmacologia , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Concentração Inibidora 50 , RNA Viral/genética , RNA Viral/metabolismo , Replicon/genética , Sensibilidade e Especificidade , Transfecção , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , beta-Lactamases/biossínteseRESUMO
Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility.
Assuntos
Antivirais/síntese química , Hepacivirus/genética , RNA Viral/antagonistas & inibidores , Ribonucleosídeos/síntese química , Adenosina Desaminase/química , Administração Oral , Animais , Antivirais/química , Antivirais/farmacocinética , Disponibilidade Biológica , Linhagem Celular , Estabilidade de Medicamentos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fosforilação , Purina-Núcleosídeo Fosforilase/química , RNA Viral/biossíntese , Ratos , Ribonucleosídeos/química , Ribonucleosídeos/farmacocinética , Relação Estrutura-AtividadeRESUMO
Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2'-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5'-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2'-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2'-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2'-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.
Assuntos
Antivirais , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/metabolismo , Tubercidina/farmacologia , Tubercidina/farmacocinética , Animais , Técnicas de Cultura , Farmacorresistência Viral , Feminino , Genótipo , Hepacivirus/enzimologia , Hepatite C/enzimologia , Humanos , Células Jurkat , Dose Letal Mediana , Camundongos , Polinucleotídeo Adenililtransferase/metabolismo , RNA/biossíntese , RNA Polimerase II/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Timidina/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2'-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-alpha results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2'-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2'-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections.
Assuntos
Antivirais/farmacologia , Hepacivirus/fisiologia , Ribonucleosídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Farmacorresistência Viral , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Ribonucleosídeos/químicaRESUMO
Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates.
Assuntos
Hepacivirus/fisiologia , Proteínas não Estruturais Virais/fisiologia , Replicação Viral , Células Cultivadas , Humanos , Mutação , RNA Helicases/fisiologia , Replicação Viral/genéticaRESUMO
Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than naïve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among naïve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.