RESUMEN
Viruses hijack and modify host cell functions to maximize viral proliferation. Hepatitis C virus (HCV) reorganizes host cell metabolism to produce specialized membrane structures and to modify organelles such as double-membrane vesicles and enlarged lipid droplets (LDs), thereby enabling virus replication and assembly. However, the molecular bases of these host-HCV interactions are largely unknown. Here, using a chemical screen, we demonstrate that the benzamide derivative flutamide reduces the host capacity to produce infectious HCV. Flutamide disrupted the formation of enlarged LDs in HCV-infected cells, thereby abolishing HCV assembly. We also report that aryl hydrocarbon receptor (AhR), a known flutamide target, plays a key role in mediating LD accumulation and HCV production. This AhR function in lipid production was also observed in HCV-uninfected Huh-7 cells and primary human hepatocytes, suggesting that AhR signaling regulates lipid accumulation independently of HCV infection. We further observed that a downstream activity, that of cytochrome P450 1A1 (CYP1A1), was the primary regulator of AhR-mediated lipid production. Specifically, blockade of AhR-induced CYP1A1 up-regulation counteracted LD overproduction, and overproduction of CYP1A1, but not of CYP1B1, in AhR-inactivated cells restored lipid accumulation. Of note, HCV infection up-regulated the AhR-CYP1A1 pathway, resulting in the accumulation of enlarged LDs. In conclusion, we demonstrate that the AhR-CYP1A1 pathway has a significant role in lipid accumulation, a hallmark of HCV infection that maximizes progeny virus production. Our chemical-genetic analysis reveals a new strategy and lead compounds to control hepatic lipid accumulation as well as HCV infection.
Asunto(s)
Citocromo P-450 CYP1A1/metabolismo , Hepacivirus/fisiología , Metabolismo de los Lípidos , Receptores de Hidrocarburo de Aril/metabolismo , Ensamble de Virus , Línea Celular , Flutamida/farmacología , Hepacivirus/efectos de los fármacos , Humanos , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Unión Proteica , Ensamble de Virus/efectos de los fármacosRESUMEN
With the introduction of direct-acting antivirals (DAAs), treatment against hepatitis C virus (HCV) has significantly improved. To manage and control this worldwide infectious disease better, the "best" multidrug treatment is demanded based on scientific evidence. However, there is no method available that systematically quantifies and compares the antiviral efficacy and drug-resistance profiles of drug combinations. Based on experimental anti-HCV profiles in a cell culture system, we quantified the instantaneous inhibitory potential (IIP), which is the logarithm of the reduction in viral replication events, for both single drugs and multiple-drug combinations. From the calculated IIP of 15 anti-HCV drugs from different classes [telaprevir, danoprevir, asunaprevir, simeprevir, sofosbuvir (SOF), VX-222, dasabuvir, nesbuvir, tegobuvir, daclatasvir, ledipasvir, IFN-α, IFN-λ1, cyclosporin A, and SCY-635], we found that the nucleoside polymerase inhibitor SOF had one of the largest potentials to inhibit viral replication events. We also compared intrinsic antiviral activities of a panel of drug combinations. Our quantification analysis clearly indicated an advantage of triple-DAA treatments over double-DAA treatments, with triple-DAA treatments showing enhanced antiviral activity and a significantly lower probability for drug resistance to emerge at clinically relevant drug concentrations. Our framework provides quantitative information to consider in designing multidrug strategies before costly clinical trials.
Asunto(s)
Antivirales/farmacología , Hepacivirus/fisiología , Hepatitis C/tratamiento farmacológico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Antivirales/uso terapéutico , Células Cultivadas , Evaluación Preclínica de Medicamentos , Farmacorresistencia Viral/efectos de los fármacos , Quimioterapia Combinada/mortalidad , Hepacivirus/efectos de los fármacos , Hepatitis C/virología , HumanosRESUMEN
UNLABELLED: Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery. IMPORTANCE: Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.
Asunto(s)
Alcaloides/metabolismo , Antivirales/metabolismo , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Hongos/química , Hepacivirus/efectos de los fármacos , Receptores X del Hígado/antagonistas & inhibidores , Piperazinas/metabolismo , Alcaloides/aislamiento & purificación , Antivirales/aislamiento & purificación , Técnicas de Cultivo de Célula , Línea Celular , Virus del Dengue/efectos de los fármacos , Virus del Dengue/fisiología , Sinergismo Farmacológico , Hepacivirus/fisiología , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Piperazinas/aislamiento & purificación , Poliovirus/efectos de los fármacos , Poliovirus/fisiología , Unión Proteica , Replicación Viral/efectos de los fármacosRESUMEN
New diazabicyclo[2.2.2]octane derivatives, peniciherquamides A-C (1-3), and a novel herqueinone derivative, neoherqueinone (5), were isolated from a fungal culture broth of Penicillium herquei. The structures of these novel compounds were determined by interpretation of spectroscopic data (1D/2D NMR, MS, and IR). Four known compounds, preparaherquamide (4), peniciherqueinone (6), and herqueinone/isoherqueinone (7/7a), were also obtained. The isolated compounds were tested for anti-hepatitis C virus (HCV) activity, and peniciherquamide C (3) was found to display an IC50 value of 5.1 µM. To our knowledge, this is the first report of a diazabicyclo[2.2.2]octane derivative with anti-HCV activity.
Asunto(s)
Antivirales/aislamiento & purificación , Antivirales/farmacología , Compuestos Aza/aislamiento & purificación , Compuestos Aza/farmacología , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Ciclooctanos/aislamiento & purificación , Ciclooctanos/farmacología , Hepacivirus/efectos de los fármacos , Penicillium/química , Antivirales/química , Compuestos Aza/química , Productos Biológicos/química , Ciclooctanos/química , Estructura MolecularRESUMEN
BACKGROUND & AIMS: Cyclophilin inhibitors are being developed for treatment of hepatitis C virus (HCV) infection. They are believed to inhibit the HCV replication complex. We investigated whether cyclophilin inhibitors interact with interferon (IFN) signaling in cultured cells infected with HCV. METHODS: We used immunoblot assays to compare expression of IFN-stimulated genes (ISGs) and of components of IFN signaling in HCV-infected and uninfected cells. RESULTS: Incubation with IFN alfa induced expression of ISGs in noninfected cells and, to a lesser extent, in HCV-infected cells; addition of the cyclophilin inhibitor SCY-635 restored expression of ISG products in HCV-infected cells. SCY-635 reduced phosphorylation of double-strand RNA-dependent protein kinase (PKR) and its downstream factor eIF2α; the phosphorylated forms of these proteins are negative regulators of ISG translation. Cyclophilin A interacted physically with PKR; this interaction was disrupted by SCY-635. SCY-635 also suppressed PKR-mediated formation of stress granules. Cyclophilin inhibitors were found to inhibit PKR phosphorylation and stress granule formation in HCV-infected and uninfected cells. CONCLUSIONS: In cultured cells, cyclophilin inhibitors reverse the attenuation of the IFN response by HCV, in addition to their effects on HCV replication complex. Cyclophilin A regulation of PKR has been proposed as a mechanism for observed effects of cyclophilin inhibitors on IFN signaling. We found that cyclophilin inhibitors reduce phosphorylation of PKR and eIF2α during HCV infection to allow for translation of ISG products. Proteins in this pathway might be developed as targets for treatment of HCV infection.
Asunto(s)
Antivirales/farmacología , Ciclofilina A/antagonistas & inhibidores , Ciclosporinas/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Factores Reguladores del Interferón/metabolismo , Hígado/efectos de los fármacos , eIF-2 Quinasa/metabolismo , Línea Celular Tumoral , Ciclofilina A/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Hepacivirus/metabolismo , Hepatitis C/enzimología , Hepatitis C/virología , Humanos , Factores Reguladores del Interferón/genética , Interferón-alfa/metabolismo , Hígado/enzimología , Hígado/virología , Fosforilación , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
UNLABELLED: Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti-HBV agents, emergence of drug-resistant viruses highlights the need for new anti-HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti-HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection-susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti-HBV potency, having a median inhibitory concentration <0.2 µM. CONCLUSION: This study provides a proof of concept for the novel strategy to identify anti-HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform.
Asunto(s)
Antivirales/farmacología , Ciclosporina/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Hepatocitos/virología , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Simportadores/antagonistas & inhibidores , Internalización del Virus/efectos de los fármacos , Células Cultivadas , Virus de la Hepatitis B/fisiología , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
The first total synthesis of MA026 and the identification of its candidate target protein for anti-hepatitis C virus activity are presented. MA026, a novel lipocyclodepsipeptide isolated from the fermentation broth of Pseudomonas sp. RtIB026, consists of a cyclodepsipeptide, a chain peptide, and an N-terminal (R)-3-hydroxydecanoic acid. The first subunit, side chain 2, was prepared by coupling fatty acid moiety 4 with tripeptide 5. The key macrocyclization of the decadepsipeptide at L-Leu(10)-D-Gln(11) provided the second subunit, cyclodepsipeptide 3. Late-stage condensation of the two key subunits and final deprotection afforded MA026. This convergent, flexible, solution-phase synthesis will be invaluable in generating MA026 derivatives for future structure-activity relationship studies. An infectious hepatitis C virus (HCV) cell culture assay revealed that MA026 suppresses HCV infection into host hepatocytes by inhibiting the entry process in a dose-dependent manner. Phage display screening followed by surface plasmon resonance (SPR) binding analyses identified claudin-1, an HCV entry receptor, as a candidate target protein of MA026.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Depsipéptidos/síntesis química , Depsipéptidos/farmacología , Hepacivirus/efectos de los fármacos , FermentaciónRESUMEN
Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. Although various classes of anti-HCV agents have been under clinical development, most of these agents target RNA replication in the HCV life cycle. To achieve a more effective multidrug treatment, the development of new, less expensive anti-HCV agents that target a different step in the HCV life cycle is needed. We prepared an in-house natural product library consisting of compounds derived from fungal strains isolated from seaweeds, mosses, and other plants. A cell-based functional screening of the library identified sulochrin as a compound that decreased HCV infectivity in a multi-round HCV infection assay. Sulochrin inhibited HCV infection in a dose-dependent manner without any apparent cytotoxicity up to 50 µM. HCV pseudoparticle and trans-complemented particle assays suggested that this compound inhibited the entry step in the HCV life cycle. Sulochrin showed anti-HCV activities to multiple HCV genotypes 1a, 1b, and 2a. Co-treatment of sulochrin with interferon or a protease inhibitor telaprevir synergistically augmented their anti-HCV effects. Derivative analysis revealed anti-HCV compounds with higher potencies (IC50<5 µM). This is the first report showing an antiviral activity of methoxybenzoate derivatives. Thus, sulochrin derivatives are anti-HCV lead compounds with a new mode of action.