RESUMO
Streptococcus pneumoniae (pneumococcus) remains a serious cause of pulmonary and systemic infections globally, and host-directed therapies are lacking. The aim of this study was to test the therapeutic efficacy of asapiprant, an inhibitor of prostaglandin D2 signaling, against pneumococcal infection. Treatment of young mice with asapiprant after pulmonary infection with invasive pneumococci significantly reduced systemic spread, disease severity, and host death. Protection was specific against bacterial dissemination from the lung to the blood but had no effect on pulmonary bacterial burden. Asapiprant-treated mice had enhanced antimicrobial activity in circulating neutrophils, elevated levels of reactive oxygen species (ROS) in lung macrophages/monocytes, and improved pulmonary barrier integrity indicated by significantly reduced diffusion of fluorescein isothiocyanate (FITC)-dextran from lungs into the circulation. These findings suggest that asapiprant protects the host against pneumococcal dissemination by enhancing the antimicrobial activity of immune cells and maintaining epithelial/endothelial barrier integrity in the lungs.
Assuntos
Infecções Pneumocócicas , Animais , Feminino , Camundongos , Modelos Animais de Doenças , Pulmão/microbiologia , Pulmão/patologia , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E protein but did not inhibit endogenous currents or calcium-induced ion channel activity of TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 hours before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n = 12), while all vehicle-dosed animals reached a mortality endpoint by Day 9 across two studies (n = 12). When treatment started at 24 hours after infection, body weight loss, and mortality were also prevented (100% survival, n = 5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection.
Assuntos
COVID-19 , Hepatite C Crônica , Chlorocebus aethiops , Camundongos , Animais , Antivirais/farmacologia , Células Vero , SARS-CoV-2 , Camundongos Transgênicos , Proteínas Viroporinas , Fatores de Transcrição , Gravidade do Paciente , Redução de Peso , Canais Iônicos , Modelos Animais de DoençasRESUMO
Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Eicosanoides , Leucócitos Mononucleares , Camundongos , Compostos Orgânicos , Oxazóis , Piperazinas , Poliésteres , Prostaglandinas , Glicoproteína da Espícula de Coronavírus , SulfonamidasRESUMO
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
Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC)2. Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice3,4. Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD2 receptor, or PLA2G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA2G2D-PGD2/DP1 pathway is a useful target for therapeutic interventions.
RESUMO
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
BACKGROUND & AIMS: Liver macrophages can be involved in both pathogen clearance and/or pathogenesis. To get further insight on their role during chronic hepatitis B virus (HBV) infections, our aim was to phenotypically and functionally characterize in vivo and ex vivo the interplay between HBV, primary human liver macrophages (PLMs) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDMs or M2-MDMs, respectively). METHODS: PLMs or primary blood monocytes, either ex vivo differentiated into M1-MDMs or M2-MDMs, were exposed to HBV and their activation followed by ELISA or quantitative reverse transcription PCR (RT-qPCR). Liver biopsies from HBV-infected patients were analysed by RT-qPCR or immunohistochemistry. Viral parameters in HBV-infected primary human hepatocytes and differentiated HepaRG cells were followed by ELISA, qPCR and RT-qPCR analyses. RESULTS: HBc protein was present within the macrophages of liver biopsies taken from HBV-infected patients. Macrophages from HBV-infected patients also expressed higher levels of anti-inflammatory macrophage markers than those from non-infected patients. Ex vivo exposure of naive PLMs to HBV led to reduced secretion of pro-inflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDMs secreted less IL-6 and IL-1ß, whereas M2-MDMs secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDMs, but not those produced by HBV-exposed M1-MDMs, decreased HBV infection of hepatocytes. CONCLUSIONS: Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour the establishment of infection. LAY SUMMARY: Hepatitis B virus modulates liver macrophage function in order to favour the establishment and likely maintenance of infection. It impairs the production of the antiviral cytokine IL-1ß, while promoting that of IL-10 in the microenvironment. This phenotype can be recapitulated in naive liver macrophages or monocyte-derived-macrophages ex vivo by short exposure to the virus or cells replicating the virus, thus suggesting an "easy to implement" mechanism of inhibition.
Assuntos
Diferenciação Celular/imunologia , Vírus da Hepatite B/fisiologia , Hepatite B Crônica , Células de Kupffer , Ativação de Macrófagos/imunologia , Monócitos , Células Cultivadas , DNA Viral/isolamento & purificação , Hepatite B Crônica/imunologia , Hepatite B Crônica/patologia , Humanos , Imuno-Histoquímica , Imunomodulação , Interleucina-10 , Interleucina-1beta , Células de Kupffer/imunologia , Células de Kupffer/patologia , Monócitos/imunologia , Monócitos/patologia , Sistema Fagocitário Mononuclear/imunologiaRESUMO
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
BACKGROUND & AIMS: NVR 3-778 is a first-in-class hepatitis B virus (HBV) capsid assembly modulator that can inhibit HBV replication. We performed a proof-of-concept study to examine the safety, pharmacokinetics, and antiviral activity of NVR 3-778 in patients with chronic HBV infection. METHODS: We performed a phase 1 study in 73 hepatitis B envelope antigen (HBeAg)-positive patients with chronic HBV infection without cirrhosis. In a 2-part study (part 1 in New Zealand and part 2 in Hong Kong, Singapore, Taiwan, Korea, and the United States), patients were randomly assigned to groups that were given oral NVR 3-778 (100 mg, 200 mg, or 400 mg daily or 600 mg or 1000 mg twice daily) or placebo for 4 weeks. Additional groups received combination treatment with pegylated interferon (pegIFN) and NVR 3-778 (600 mg twice daily) or pegIFN with placebo. RESULTS: Reductions in serum levels of HBV DNA and HBV RNA were observed in patients receiving ≥1200 mg/d NVR 3-778. The largest mean reduction in HBV DNA was observed in the group given NVR 3-778 plus pegIFN (1.97 log10 IU/mL), compared with the groups given NVR 3-778 or pegIFN alone (1.43 log10 IU/mL and 1.06 log10 IU/mL, respectively). The mean reduction in HBV RNA was also greatest in the group given NVR 3-778 plus pegIFN (2.09 log10 copies/mL), compared with the groups given NVR 3-778 or pegIFN alone (1.42 log10 copies/mL and 0.89 log10 copies/mL, respectively). There was no significant mean reduction in HBsAg during the 4-week treatment period. There were no discontinuations and no pattern of dose-related adverse effects with NVR 3-778. CONCLUSIONS: In a phase 1 study of HBeAg-positive patients with chronic HBV infection without cirrhosis, NVR 3-778 was well tolerated and demonstrated antiviral activity. The agent reduced serum levels of HBV DNA and HBV RNA, to the greatest extent in combination with pegIFN. The observed reductions in HBV RNA confirmed the novel mechanism of NVR 3-778. Clinicaltrials.gov no. NCT02112799 (single-center) and NCT02401737 (multicenter).
Assuntos
Antivirais/farmacocinética , Benzamidas/farmacocinética , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Piperidinas/farmacocinética , Replicação Viral/efeitos dos fármacos , Administração Oral , Adulto , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Ásia , Benzamidas/administração & dosagem , Benzamidas/efeitos adversos , DNA Viral/sangue , DNA Viral/genética , Esquema de Medicação , Quimioterapia Combinada , Feminino , Antígenos E da Hepatite B/sangue , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/imunologia , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/virologia , Humanos , Interferon-alfa/administração & dosagem , Masculino , Pessoa de Meia-Idade , Nova Zelândia , Piperidinas/administração & dosagem , Piperidinas/efeitos adversos , Polietilenoglicóis/administração & dosagem , RNA Viral/sangue , RNA Viral/genética , Proteínas Recombinantes/administração & dosagem , Resultado do Tratamento , Estados Unidos , Carga Viral , Adulto JovemRESUMO
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
Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 large extracellular loop that equated to increased efficacy, while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies.
Assuntos
Hepacivirus/fisiologia , Anticorpos Anti-Hepatite C/química , Hepatite C/imunologia , Anticorpos de Cadeia Única/química , Tetraspanina 28/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/farmacologia , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Linhagem Celular , Células Hep G2 , Hepacivirus/efeitos dos fármacos , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/farmacologia , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Conformação Proteica , Anticorpos de Cadeia Única/farmacologia , Relação Estrutura-Atividade , Tetraspanina 28/química , Internalização do Vírus/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
Therapeutic targeting of membrane-associated viral proteins is complicated by the challenge of investigating their enzymatic activities in the native membrane-bound state. To permit functional characterization of these proteins, we hypothesized that the supported lipid bilayer (SLB) can support in situ reconstitution of membrane-associated viral protein complexes. As proof-of-principle, we selected the hepatitis C virus (HCV) NS5B polymerase which is essential for HCV genome replication, and determined that the SLB platform enables functional reconstitution of membrane protein activity. Quartz crystal microbalance with dissipation (QCM-D) monitoring enabled label-free detection of full-length NS5B membrane association, its interaction with replicase subunits NS3, NS5A, and template RNA, and most importantly its RNA synthesis activity. This latter activity could be inhibited by the addition of candidate small molecule drugs. Collectively, our results demonstrate that the SLB platform can support functional studies of membrane-associated viral proteins engaged in critical biological activities.
RESUMO
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
UNLABELLED: CD81 is a required receptor for hepatitis C virus (HCV) infection of human hepatocytes in vitro. We generated several high-affinity anti-human CD81 monoclonal antibodies (mAbs) that demonstrated potent, specific, and cross-genotype inhibition of HCV entry. One of these mAbs, K04, was administered to human liver chimeric mice before or after HCV infection to determine its ability to prevent HCV infection or spread of HCV infection, respectively. All vehicle control mice established HCV infection, reaching steady-state levels of serum HCV RNA by day 21. Pretreatment of mice with K04 prevented HCV infection in all mice (n = 5). Treatment of mice with mAb K04 every 3 days for 21 days, starting at 6 hours postinfection, resulted in effective inhibition of virus spread. In 3 mice that were sacrificed on day 24, serum HCV levels remained detectable, below the limit of quantification (LOQ), indicating that infection was established, but virus spread was blocked, by the anti-CD81 mAb. In 5 additional mice that were followed for a longer time, virus remained detectable, below LOQ, until days 24 and 30 in 4 of 5 mice. In the fifth mouse, viral load was quantifiable, but reduced to 64-fold below the mean viral load in vehicle control at day 24. In addition, 2 of 5 mice cleared the infection by day 30 and 1 mouse had undetectable virus load from day 6 onward. CONCLUSION: These results demonstrate that CD81 is required for HCV infection and virus spread in vivo, and that anti-CD81 antibodies such as K04 may have potential as broad-spectrum antiviral agents for prevention and treatment of HCV infection.
Assuntos
Anticorpos Anti-Idiotípicos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Hepatite C/prevenção & controle , Tetraspanina 28/imunologia , Animais , Quimera , Humanos , Fígado/virologia , Camundongos , Camundongos SCID , Carga ViralRESUMO
Mapping protein-protein interactions is essential to fully characterize the biological function of a protein and improve our understanding of diseases. Affinity purification coupled to mass spectrometry (AP-MS) using selective antibodies against a target protein has been commonly applied to study protein complexes. However, one major limitation is a lack of specificity as a substantial part of the proposed binders is due to nonspecific interactions. Here, we describe an innovative immuno-competitive capture mass spectrometry (ICC-MS) method to allow systematic investigation of protein-protein interactions. ICC-MS markedly increases the specificity of classical immunoprecipitation (IP) by introducing a competition step between free and capturing antibody prior to IP. Instead of comparing only one experimental sample with a control, the methodology generates a 12-concentration antibody competition profile. Label-free quantitation followed by a robust statistical analysis of the data is then used to extract the cellular interactome of a protein of interest and to filter out background proteins. We applied this new approach to specifically map the interactome of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) in a cellular HCV replication system and uncovered eight new NS5A-interacting protein candidates along with two previously validated binding partners. Follow-up biological validation experiments revealed that large tumor suppressor homolog 1 and 2 (LATS1 and LATS2, respectively), two closely related human protein kinases, are novel host kinases responsible for NS5A phosphorylation at a highly conserved position required for optimal HCV genome replication. These results are the first illustration of the value of ICC-MS for the analysis of endogenous protein complexes to identify biologically relevant protein-protein interactions with high specificity.
Assuntos
Hepacivirus/crescimento & desenvolvimento , Mapeamento de Interação de Proteínas/métodos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Replicação do DNA/genética , Genoma Viral/genética , Humanos , Espectrometria de Massas/métodos , Antígenos de Histocompatibilidade Menor , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteoma/análise , Interferência de RNA , RNA Interferente Pequeno , Proteínas Supressoras de Tumor/genética , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Viral proteins have enabled the design of selective and efficacious treatments for viral diseases. While focus in this area has been on viral enzymes, it appears that multifunctional viral proteins may be even more susceptible to small molecule interference. As exemplified by HIV capsid, small molecule inhibitors can bind to multiple binding sites on the capsid protein and induce or prevent protein interactions and conformational changes. Resistance selection is complicated by the fact that the capsid proteins have to engage in different protein interactions at different times of the life cycle. Viral capsid assembly and disassembly have therefore emerged as highly sensitive processes that could deliver a new generation of antiviral agents across viral diseases.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HumanosRESUMO
BACKGROUND & AIMS: Pegylated interferon-alpha (PegIFNα) remains an attractive treatment option for chronic hepatitis B virus (HBV) infection because it induces higher rates of antigen loss and seroconversion than treatment with polymerase inhibitors. Although early HBsAg decline is recognised as the best predictor of sustained response to IFN-based therapy, it is unclear whether immune cell functions are required to induce significant antigenemia reduction in the first weeks of treatment. Aim of the study was to investigate whether PegIFNα can induce sustained human hepatocyte responsiveness and substantial loss of circulating and intrahepatic viral antigen loads in a system lacking immune cell functions. METHODS: HBV-infected humanized uPA/SCID mice received either PegIFNα, entecavir (ETV), or both agents in combination. Serological and intrahepatic changes were determined by qRT-PCR and immunohistochemistry and compared to untreated mice. RESULTS: After 4 weeks of treatment, median viremia reduction was greater in mice treated with ETV (either with or without PegIFNα) than with PegIFNα. In contrast, levels of circulating HBeAg, HBsAg, and intrahepatic HBcAg were significantly reduced (p = 0.03) only in mice receiving PegIFNα alone or in combination, as compared to mice receiving ETV monotherapy. Progressive antigen reduction was also demonstrated in mice receiving PegIFNα for 12 weeks (HBeAg = Δ1log; HBsAg = Δ1.4log; p < 0.0001). Notably, repeated administrations of the longer-active PegIFNα could breach the impairment of HBV-infected hepatocyte responsiveness and induce sustained enhancement of human interferon stimulated genes (ISG). CONCLUSIONS: The antiviral effects of PegIFNα exerted on the human hepatocytes can induce sustained responsiveness and trigger substantial HBV antigen decline without claiming the involvement of immune cell responses.
Assuntos
Antivirais/administração & dosagem , Antígenos de Superfície da Hepatite B/sangue , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/administração & dosagem , Polietilenoglicóis/administração & dosagem , Animais , DNA Viral/sangue , Guanina/administração & dosagem , Guanina/análogos & derivados , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Hepatócitos/virologia , Humanos , Interferon alfa-2 , Camundongos , Proteínas Recombinantes/administração & dosagemRESUMO
UNLABELLED: We have recently shown that a cocktail of two short synthetic hairpin RNAs (sshRNAs), targeting the internal ribosome entry site of hepatitis C virus (HCV) formulated with lipid nanoparticles, was able to suppress viral replication in chimeric mice infected with HCV GT1a by up to 2.5 log10 (H. Ma et al., Gastroenterology 146:63-66.e5, http://dx.doi.org/10.1053/j.gastro.2013.09.049) Viral load remained about 1 log10 below pretreatment levels 21 days after the end of dosing. We have now sequenced the HCV viral RNA amplified from serum of treated mice after the 21-day follow-up period. Viral RNA from the HCV sshRNA-treated groups was altered in sequences complementary to the sshRNAs and nowhere else in the 500-nucleotide sequenced region, while the viruses from the control group that received an irrelevant sshRNA had no mutations in that region. The ability of the most commonly selected mutations to confer resistance to the sshRNAs was confirmed in vitro by introducing those mutations into HCV-luciferase reporters. The mutations most frequently selected by sshRNA treatment within the sshRNA target sequence occurred at the most polymorphic residues, as identified from an analysis of available clinical isolates. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNA interference (RNAi) mechanism of action. IMPORTANCE: This study presents a detailed analysis of the impact of treating a hepatitis C virus (HCV)-infected animal with synthetic hairpin-shaped RNAs that can degrade the virus's RNA genome. These RNAs can reduce the viral load in these animals by over 99% after 1 to 2 injections. The study results confirm that the viral rebound that often occurred a few weeks after treatment is due to emergence of a virus whose genome is mutated in the sequences targeted by the RNAs. The use of two RNA inhibitors, which is more effective than use of either one by itself, requires that any resistant virus have mutations in the targets sites of both agents, a higher hurdle, if the virus is to retain the ability to replicate efficiently. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNAi mechanism of action.