RESUMO
HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.
Assuntos
Southern Blotting , DNA Circular , DNA Viral , Vírus da Hepatite B , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Humanos , DNA Viral/genética , DNA Viral/isolamento & purificação , DNA Circular/isolamento & purificação , DNA Circular/análise , DNA Circular/genética , Southern Blotting/métodos , Hepatite B/virologia , Hepatite B/diagnóstico , Fígado/virologiaRESUMO
Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure. A series of dihydropyridoisoquinolizinone (DHQ) inhibitors of human poly(A) polymerases PAPD5/7 were reported to suppress HBsAg in vitro. An example from this class, RG7834, briefly entered the clinic. We set out to identify a potent, orally bioavailable, and safe PAPD5/7 inhibitor as a potential component of a functional cure regimen. Our efforts led to the identification of a dihydropyridophthalazinone (DPP) core with improved pharmacokinetic properties. A conformational restriction strategy and optimization of core substitution led to GS-8873, which was projected to provide deep HBsAg suppression with once-daily dosing.
RESUMO
The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs ("antiviral intensification"). Here, we describe GS-SBA-1, a capsid assembly modulator (CAM) belonging to class CAM-E, that demonstrates potent inhibition of extracellular HBV DNA in vitro (EC50 [50% effective concentration] = 19 nM) in HBV-infected primary human hepatocytes (PHHs) as well as in vivo in an HBV-infected immunodeficient mouse model. GS-SBA-1 has comparable activities across HBV genotypes and nucleos(t)ide-resistant mutants in HBV-infected PHHs. In addition, GS-SBA-1 demonstrated in vitro additivity in combination with tenofovir alafenamide (TAF). The administration of GS-SBA-1 to PHHs at the time of infection prevents covalently closed circular DNA (cccDNA) formation and, hence, decreases HBV RNA and antigen levels (EC50 = 80 to 200 nM). Furthermore, GS-SBA-1 prevents the production of extracellular HBV RNA-containing viral particles in vitro. Collectively, these data demonstrate that GS-SBA-1 is a potent CAM that has the potential to enhance viral suppression in combination with an NA.
Assuntos
Hepatite B Crônica , Hepatite B , Animais , Camundongos , Humanos , Hepatite B Crônica/tratamento farmacológico , Capsídeo , Vírus da Hepatite B , Antivirais/farmacologia , Antivirais/uso terapêutico , Proteínas do Capsídeo/genética , RNA , DNA Viral/genética , DNA Circular , Hepatite B/tratamento farmacológicoRESUMO
Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases.
Assuntos
Vírus da Hepatite B , Hepatite B Crônica , Humanos , Animais , Camundongos , Antivirais/farmacologia , Hepatite B Crônica/tratamento farmacológico , EpigenômicaRESUMO
Globally, 296 million people are infected with hepatitis B virus (HBV), and approximately one million people die annually from HBV-related causes, including liver cancer. Although there is a preventative vaccine and antiviral therapies suppressing HBV replication, there is no cure. Intensive efforts are under way to develop curative HBV therapies. Currently, only a few biomarkers are available for monitoring or predicting HBV disease progression and treatment response. As new therapies become available, new biomarkers to monitor viral and host responses are urgently needed. In October 2020, the International Coalition to Eliminate Hepatitis B Virus (ICE-HBV) held a virtual and interactive workshop on HBV biomarkers endorsed by the International HBV Meeting. Various stakeholders from academia, clinical practice and the pharmaceutical industry, with complementary expertise, presented and participated in panel discussions. The clinical utility of both classic and emerging viral and immunological serum biomarkers with respect to the course of infection, disease progression, and response to current and emerging treatments was appraised. The latest advances were discussed, and knowledge gaps in understanding and interpretation of HBV biomarkers were identified. This Roadmap summarizes the strengths, weaknesses, opportunities and challenges of HBV biomarkers.
Assuntos
Hepatite B Crônica , Hepatite B , Humanos , Vírus da Hepatite B , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/tratamento farmacológico , Antivirais/uso terapêutico , Replicação Viral , Biomarcadores , Progressão da Doença , Hepatite B/diagnóstico , Hepatite B/tratamento farmacológicoRESUMO
Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden, and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5, and Hep3B. To generate high-coverage, long-read sequencing data, a custom panel of HBV-targeting biotinylated oligonucleotide probes was designed. Targeted long-read DNA sequencing captured entire HBV integration events within individual reads, revealing that integrations may include deletions and inversions of viral sequences. Surprisingly, all three HCC cell lines contain integrations that are associated with host chromosomal translocations. In addition, targeted long-read RNA sequencing allowed for the assignment of transcriptional activity to specific integrations and resolved the contribution of overlapping HBV transcripts. HBV transcripts chimeric with host sequences were resolved in their entirety and often included >1,000 bp of host sequence. This study provides the first comprehensive description of HBV integrations and associated transcriptional activity in three commonly utilized HCC-derived cell lines. The application of novel methods sheds new light on the complexity of these integrations, including HBV bidirectional transcription, nested transcripts, silent integrations, and host genomic rearrangements. The observation of multiple HBV-associated chromosomal translocations gives rise to the hypothesis that HBV is a driver of genetic instability and provides a potential new mechanism for HCC development. IMPORTANCE HCC-derived cell lines have served as practical models to study HBV biology for decades. These cell lines harbor multiple HBV integrations and express only HBV surface antigen (HBsAg). To date, an accurate description of the integration burden, architecture, and transcriptional profile of these cell lines has been limited due to technical constraints. We have developed a targeted long-read sequencing assay that reveals the entire architecture of integrations in these cell lines. In addition, we identified five chromosomal translocations with integrated HBV DNA at the interchromosomal junctions. Incorporation of long-read transcriptome sequencing (RNA-Seq) data indicated that many integrations and translocations were transcriptionally silent. The observation of multiple HBV-associated translocations has strong implications regarding the potential mechanisms for the development of HBV-associated HCC.
Assuntos
Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , DNA Viral/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Transcrição Gênica , Translocação Genética , Integração Viral , Humanos , Análise de Sequência de DNA , Análise de Sequência de RNARESUMO
BACKGROUND AND AIMS: GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 in clinical development for the treatment of chronic hepatitis B. In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus. APPROACH AND RESULTS: WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688, or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells and various other immune cell subsets may play a role in the antiviral response to GS-9688. CONCLUSIONS: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks; the identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect.
Assuntos
Antivirais/uso terapêutico , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Vírus da Hepatite B da Marmota/genética , Hepatite B Crônica/tratamento farmacológico , Hexanóis/uso terapêutico , Pirimidinas/uso terapêutico , Receptor 8 Toll-Like/agonistas , Animais , Antivirais/farmacologia , DNA Viral/sangue , Modelos Animais de Doenças , Anticorpos Anti-Hepatite/sangue , Antígenos de Hepatite/sangue , Vírus da Hepatite B da Marmota/imunologia , Hepatite B Crônica/complicações , Hepatite B Crônica/imunologia , Hexanóis/farmacologia , Humanos , Marmota , Pirimidinas/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Toll-like receptor 8 (TLR8) recognizes pathogen-derived single-stranded RNA fragments to trigger innate and adaptive immune responses. Chronic hepatitis B (CHB) is associated with a dysfunctional immune response, and therefore a selective TLR8 agonist may be an effective treatment option. Structure-based optimization of a dual TLR7/8 agonist led to the identification of the selective TLR8 clinical candidate (R)-2-((2-amino-7-fluoropyrido[3,2-d]pyrimidin-4-yl)amino)-2-methylhexan-1-ol (GS-9688, (R)-7). Potent TLR8 agonism (IL-12p40 EC50 = 220 nM) and >100-fold TLR7 selectivity (IFN-α EC50 > 50 µM) was observed in human peripheral blood mononuclear cells (PBMCs). The TLR8-ectodomain:(R)-7 complex confirmed TLR8 binding and a direct ligand interaction with TLR8 residue Asp545. Oral (R)-7 had good absorption and high first pass clearance in preclinical species. A reduction in viral markers was observed in HBV-infected primary human hepatocytes treated with media from PBMCs stimulated with (R)-7, supporting the clinical development of (R)-7 for the treatment of CHB.
Assuntos
Antivirais/farmacologia , Hepatite B Crônica/tratamento farmacológico , Hexanóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptor 8 Toll-Like/agonistas , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/síntese química , Antivirais/metabolismo , Cristalografia por Raios X , Cães , Descoberta de Drogas , Vírus da Hepatite B/efeitos dos fármacos , Hexanóis/administração & dosagem , Hexanóis/síntese química , Hexanóis/metabolismo , Humanos , Macaca fascicularis , Estrutura Molecular , Domínios Proteicos , Piridinas/administração & dosagem , Piridinas/síntese química , Piridinas/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Pirimidinas/metabolismo , Ratos , Relação Estrutura-Atividade , Receptor 8 Toll-Like/metabolismoRESUMO
Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high-sequence homology of the HBV receptor, Na+/taurocholate co-transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP. Immunodeficient Fah -/- , NOD, Rag1 -/- , Il2Rg null (FNRG) mice engrafted with human or squirrel monkey hepatocytes were challenged with HBV or Woolly Monkey HBV (WMHBV). In addition, adult squirrel monkeys were inoculated with HBV, WMHBV, adeno-associated virus containing an infectious genome of HBV (AAV-HBV), and AAV-WMHBV. Finally, neonate squirrel monkeys were assessed for the potential of chronic infection with WMHBV. PreS1 peptide efficiently bound to human and squirrel monkey NTCP but not to mouse or capuchin NTCP. FNRG mice engrafted with squirrel monkey hepatocytes were susceptible to infection by WMHBV but not human HBV. Similarly, adult squirrel monkeys could be infected with WMHBV but not human HBV, whereas chimeric mice engrafted with human hepatocytes were susceptible to HBV but not WMHBV. Infection of squirrel monkeys with AAV-WMHBV yielded maximum viremia of 108 genomes/mL with detectable virus for up to 8 months. Notably, covalently closed circular DNA was detected in the liver of these animals. Infection of neonates with WMHBV led to detectable viremia for up to 6 months. Conclusions: Adult and neonate squirrel monkeys exhibited prolonged WMHBV viremia lasting 6-8 months. This is greater than twice the duration of viremia achieved in other nonhuman primates and suggests that squirrel monkeys may be a suitable model for testing HBV therapeutics.
RESUMO
BACKGROUND & AIMS: GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. METHODS: Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). RESULTS: GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8+ T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8+ T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8+ T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b+ myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. CONCLUSION: The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. LAY SUMMARY: New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection.
Assuntos
Antivirais/farmacologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/imunologia , Pteridinas/farmacologia , Receptor 7 Toll-Like/agonistas , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Agregação Celular/efeitos dos fármacos , Agregação Celular/imunologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hepatite B Crônica/virologia , Humanos , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Pan troglodytesRESUMO
BACKGROUND & AIMS: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. METHODS: Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. RESULTS: GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. CONCLUSIONS: Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. LAY SUMMARY: GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Interferon Tipo I/imunologia , Pteridinas/farmacologia , Receptor 7 Toll-Like/agonistas , Animais , Apresentação de Antígeno , Células Cultivadas , Citocinas/biossíntese , DNA Circular/genética , DNA Circular/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Antígenos da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Imunidade Inata , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Receptor 7 Toll-Like/genéticaRESUMO
The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (<58 copies/mL) or target detected (TD) (between 58 and 169 copies/mL). Here we have developed a more sensitive digital droplet PCR (ddPCR) assay to evaluate the impact of long-term tenofovir disoproxil fumarate (TDF) treatment in patients that did or did not achieve HBsAg seroconversion. A ddPCR assay was developed to detect HBV DNA to 8 copies/mL. HBV DNA levels in plasma from patients with or without HBsAg seroconversion were assessed by ddPCR. For patients who did not achieve HBsAg seroconversion, the majority of TD samples (33/58, 57%) were HBV DNA positive by ddPCR while (10/37, 27%) of TND samples were positive. In contrast, for patients who achieved HBsAg seroconversion, HBV DNA was rarely detected by ddPCR after HBsAg seroconversion (1/28, 3.6%). ddPCR is a sensitive method to evaluate low-level viral replication in plasma samples. Frequent detection of HBV DNA by ddPCR among patients who did not achieve HBsAg seroconversion suggests new agents may be needed to suppress low levels of replicating HBV.
Assuntos
Antivirais/uso terapêutico , DNA Viral/sangue , Vírus da Hepatite B/isolamento & purificação , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Reação em Cadeia da Polimerase/métodos , Tenofovir/uso terapêutico , Ensaios Clínicos Fase III como Assunto , Hepatite B/diagnóstico , Hepatite B/imunologia , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Humanos , Lamivudina/uso terapêutico , Limite de Detecção , Sensibilidade e Especificidade , Soroconversão , Carga Viral , Replicação ViralRESUMO
The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Imunidade Inata/imunologia , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cromossômicas não Histona , Citocinas/genética , Citocinas/metabolismo , Hepatite B/metabolismo , Hepatite B/virologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias , Replicação ViralRESUMO
BACKGROUND: GS-9256 is an inhibitor of HCV NS3 protease with a macrocyclic structure and novel phosphinic acid pharmacophore. METHODS: Key preclinical properties of GS-9256 including in vitro antiviral activity, cross-resistance and pharmacokinetic properties were investigated in non-human species. RESULTS: In genotype (GT) 1b Huh-luc cells with a replicon encoding luciferase, GS-9256 had a mean 50% effective concentration (EC50) value of 20.0 nM, with minimal cytotoxicity. Antiviral activity was similar in a number of additional GT1b and GT1a replicon cell lines. Similar potency was observed in chimeric replicons encoding the NS3 protease of GT1 clinical isolates. GS-9256 was less active in GT2a replicon cells (14.2-fold increase in EC50). Additive to synergistic in vitro antiviral activity was observed when GS-9256 was combined with other agents including interferon-α, ribavirin, NS5B polymerase inhibitors GS-6620 and tegobuvir, as well as the NS5A inhibitor ledipasvir. GS-9256 retained wild-type activity against all tested NS5B and NS5A inhibitor resistance mutations. GS-9256 was metabolically stable in microsomes and hepatocytes of tested species, including rodents, dogs and humans. GS-9256 had high bioavailability in mice (near 100%) and moderate bioavailability in rats (14%), dogs (21%) and monkeys (14%). Elimination half-lives were approximately 2 h in mice, 0.6 h in rats, 5 h in dogs and 4 h in monkey. A study in bile duct-cannulated rats indicated that the major route of elimination is through biliary excretion of unmetabolized GS-9256. CONCLUSIONS: GS-9256 showed a favourable preclinical profile supportive of clinical development for the treatment of chronic HCV infection in GT1 patients.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Ácidos Fosfínicos/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/farmacocinética , Disponibilidade Biológica , Linhagem Celular , Células Cultivadas , Cães , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Macaca fascicularis , Camundongos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Ácidos Fosfínicos/química , Ácidos Fosfínicos/farmacocinética , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Ratos , Replicação Viral/efeitos dos fármacosRESUMO
UNLABELLED: Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors. IMPORTANCE: Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Proteínas do Core Viral/química , Montagem de Vírus , Animais , Capsídeo/química , Proteínas do Capsídeo/química , Sistema Livre de Células , Regulação Viral da Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B/química , Interações Hospedeiro-Patógeno , Humanos , Fosforilação , Domínios Proteicos , RNA Viral/metabolismo , Coelhos , Reticulócitos , Proteínas do Core Viral/genética , Replicação ViralRESUMO
BACKGROUND: GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays. METHODS: Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods. RESULTS: GS-9256 and vedroprevir had measured Ki values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; Ki values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was >10,000-fold against all tested off-target proteases. Association rate constants of 4×10(5)M(-1)s(-1) and 1×10(6)M(-1)s(-1), respectively, were measured, and dissociation rate constants of 4.8×10(-5)s(-1) and 2.6×10(-4)s(-1) were determined. CONCLUSIONS: GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics. GENERAL SIGNIFICANCE: The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.
RESUMO
One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough. Currently, most DAAs inhibit HCV replication. We recently reported that the combination of two distinct classes of HCV inhibitors, entry inhibitors and replication inhibitors, prolonged reductions in extracellular HCV in persistently infected cells. We therefore sought to identify new inhibitors targeting aspects of the HCV replication cycle other than RNA replication. We report here the discovery of the first small-molecule HCV infectivity inhibitor, GS-563253, also called HCV infectivity inhibitor 1 (HCV II-1). HCV II-1 is a substituted tetrahydroquinoline that selectively inhibits genotype 1 and 2 HCVs with low-nanomolar 50% effective concentrations. It was identified through a high-throughput screen and subsequent chemical optimization. HCV II-1 only permits the production and release of noninfectious HCV particles from cells. Moreover, infectious HCV is rapidly inactivated in its presence. HCV II-1 resistance mutations map to HCV E2. In addition, HCV-II prevents HCV endosomal fusion, suggesting that it either locks the viral envelope in its prefusion state or promotes a viral envelope conformation change incapable of fusion. Importantly, the discovery of HCV II-1 opens up a new class of HCV inhibitors that prolong viral suppression by HCV replication inhibitors in persistently infected cell cultures.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Antivirais/química , Linhagem Celular , Farmacorresistência Viral , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Humanos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
GS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, including in vitro antiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50 = 316 nM). Additive to synergistic in vitro antiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were â¼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results of in vitro cross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Inibidores de Proteases/farmacologia , Quinolinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Benzimidazóis/farmacologia , Cães , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Fluorenos/farmacologia , Haplorrinos , Hepacivirus/fisiologia , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Concentração Inibidora 50 , Interferon-alfa/farmacologia , Inibidores de Proteases/farmacocinética , Purinas/farmacologia , Piridazinas/farmacologia , Quinolinas/farmacocinética , Ratos , Replicon/efeitos dos fármacos , Ribavirina/farmacologia , Proteínas não Estruturais Virais/metabolismoRESUMO
Efforts to treat HCV patients are focused on developing antiviral combinations that lead to the eradication of infection. Thus, it is important to identify optimal combinations from the various viral inhibitor classes. Based on viral dynamic models, HCV entry inhibitors are predicted to reduce viral load in a monophasic manner reflecting the slow death rate of infected hepatocytes (t1/2 = 2-70 days) and the protection of naïve, un-infected cells from HCV infection. In contrast, replication inhibitors are predicted to reduce viral load in a biphasic manner. The initial rapid reduction phase is due to the inhibition of virus production and elimination of plasma virus (t1/2â¼3 hours). The second, slower reduction phase results from the elimination of infected hepatocytes. Here we sought to compare the ability of HCV entry and replication inhibitors as well as combinations thereof to reduce HCV infection in persistently-infected Huh7 cells. Treatment with 5 × EC50 of entry inhibitors anti-CD81 Ab or EI-1 resulted in modest (≤ 1 log10 RNA copies/ml), monophasic declines in viral levels during 3 weeks of treatment. In contrast, treatment with 5 × EC50 of the replication inhibitors BILN-2016 or BMS-790052 reduced extracellular virus levels more potently (~2 log10 RNA copies/ml) over time in a biphasic manner. However, this was followed by a slow rise to steady-state virus levels due to the emergence of resistance mutations. Combining an entry inhibitor with a replication inhibitor did not substantially enhance the rate of virus reduction. However, entry/replication inhibitor and replication/replication inhibitor combinations reduced viral levels further than monotherapies (up to 3 log10 RNA copies/ml) and prolonged this reduction relative to monotherapies. Our results demonstrated that HCV entry inhibitors combined with replication inhibitors can prolong antiviral suppression, likely due to the delay of viral resistance emergence.
Assuntos
Antivirais/farmacologia , Carbamatos/farmacologia , Hepacivirus/efeitos dos fármacos , Imidazóis/farmacologia , Compostos Macrocíclicos/farmacologia , Quinolinas/farmacologia , Tiazóis/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Farmacorresistência Viral , Sinergismo Farmacológico , Hepacivirus/crescimento & desenvolvimento , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Pirrolidinas , Fatores de Tempo , Valina/análogos & derivados , Carga Viral/efeitos dos fármacosRESUMO
Chronic hepatitis B virus (CHB) is managed effectively with either nucleoside/nucleotide-based or interferon-based therapies. However, most patients receiving these therapies do not establish long-term, durable control of infection after treatment withdrawal. In particular, rates of hepatitis B surface-antigen loss and seroconversion to antisurface-antigen antibody are very low. Thus, novel therapies and treatment modalities are necessary to achieve either elimination of the virus from the liver or durable immune control of hepatitis B virus (HBV) infection in the absence of chronic therapy ("functional cure"). Here the authors review new targets and approaches for treating CHB. These approaches can be divided into two broad categories-those targeting the virus or host factors required by the virus and those targeting the innate or adaptive immune systems. Unfortunately, although a variety of promising strategies have been identified and several new approaches have achieved preclinical validation, relatively few novel drug candidates are in active clinical studies to treat CHB. Thus, functional cure of CHB infection remains an important therapeutic challenge.