RESUMO
To face the COVID-19 pandemic, prophylactic vaccines have been developed in record time, but vaccine coverage is still limited, accessibility is not equitable worldwide, and the vaccines are not fully effective against emerging variants. Therefore, therapeutic treatments are urgently needed to control the pandemic and treat vulnerable populations, but despite all efforts made, options remain scarce. However, the knowledge gained during 2020 constitutes an invaluable platform from which to build future therapies. In this review, we highlight the main drug repurposing strategies and achievements made over the first 18 months of the pandemic, but also discuss the antivirals, immunomodulators and drug combinations that could be used in the near future to cure COVID-19.
Assuntos
COVID-19 , Vacinas , Reposicionamento de Medicamentos , Humanos , Pandemias , SARS-CoV-2RESUMO
Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.
Assuntos
Carcinoma Hepatocelular/virologia , Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Neoplasias Hepáticas/virologia , Proteínas Repressoras/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas de Ciclo Celular/genética , Vírus da Hepatite B/genética , Hepatócitos/virologia , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteômica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Fatores de Processamento de Serina-Arginina/genética , Proteínas do Core Viral/genética , Replicação ViralRESUMO
BACKGROUND: A link between HBV and PLK1 was clearly evidenced in HBV-driven carcinogenesis, and we have also recently shown that PLK1 is a proviral factor in the early phases of HBV infection. Moreover, we have shown that BI-2536, a small molecule PLK1 inhibitor, was very efficient at inhibiting HBV DNA neosynthesis, notably by affecting nucleocapsid assembly as a result of the modulation of HBc phosphorylation. Yet, as small molecule kinase inhibitors often feature poor selectivity, a more specific and safer strategy to target PLK1 would be needed for a potential development against chronic HBV infections. METHODS: Here, we analysed using both freshly isolated primary human hepatocytes and differentiated HepaRG, the anti-HBV properties of an LNP-encapsulated PLK1-targeting siRNA. Standard assays were used to monitor the effect of LNP siPLK1, or controls (LNP siHBV and LNP siNon-targeting), on HBV replication and cell viability. RESULTS: A dose as low as 100 ng/ml of LNP-siPLK1 resulted in a >75% decrease in secreted HBV DNA (viral particles), which was comparable to that obtained with LNP siHBV or 10 µM of tenofovir (TFV), without affecting cell viability. Interestingly, and in contrast to that obtained with TFV, a strong inhibition of viral RNA and HBe/HBsAg secretions was also observed under LNP siPLK1 treatment. This correlated with a significant intracellular decrease of vRNA accumulation, which was independent of any change in cccDNA levels, thus suggesting a transcriptional or post-transcriptional modulation. Such an effect was not obtained with a biochemical approach of PLK1 inhibition, suggesting an enzymatic-independent role of PLK1. CONCLUSIONS: This study emphasizes that a specific PLK1 inhibition could help in achieving an improved HBsAg loss in CHB patients, likely in combination with other HBsAg-targeting strategies.
Assuntos
Antivirais/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Hepatite B/tratamento farmacológico , Hepatócitos/virologia , Lipossomos , Nanopartículas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , RNA Interferente Pequeno/uso terapêutico , Antivirais/administração & dosagem , Proteínas de Ciclo Celular/genética , Células Cultivadas , Hepatite B/virologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Replicação Viral/efeitos dos fármacos , Quinase 1 Polo-LikeRESUMO
Chronic infection by hepatitis B virus (HBV) is a major public health problem with more than 250 millions of people chronically infected worldwide who have a high risk to develop cirrhosis and hepatocellular carcinoma. Available treatments reduce viremia but do not eradicate the virus from hepatocytes. Therefore, there is an urgent need to develop new classes of antiviral molecules and the viral capsid protein, Core, constitutes a new favored target. Core protein Allosteric Modulators (CAMs) targeting its assembly functions are in clinical development. In addition, investigation of Core regulatory functions may lead to the development of compounds targeting cellular factors (HTA) that could be used in combined therapies aiming to achieve a better control of HBV replication.
Assuntos
Pesquisa Biomédica/tendências , Descoberta de Drogas , Vírus da Hepatite B/química , Hepatite B/tratamento farmacológico , Proteínas do Core Viral/fisiologia , Antivirais/isolamento & purificação , Antivirais/uso terapêutico , Descoberta de Drogas/normas , Descoberta de Drogas/tendências , Humanos , Terapia de Alvo Molecular/normas , Terapia de Alvo Molecular/tendênciasRESUMO
The assembly of hepatitis B virus (HBV) core protein (HBc) into capsids represents a critical step of viral replication. HBc has multiple functions during the HBV life cycle, which makes it an attractive target for antiviral therapies. Capsid assembly modulators (CAMs) induce the formation of empty capsid or aberrant capsid devoid of pregenomic RNA (pgRNA) and finally block relaxed circular DNA neosynthesis and virion progeny. In this study, the novel CAMs JNJ-827 and JNJ-890 were found to be potent inhibitors of HBV replication with respective half-maximal effective concentrations of 4.7 and 66 nM, respectively, in HepG2.117 cells. Antiviral profiling in differentiated HepaRG (dHepaRG) cells and primary human hepatocytes revealed that these compounds efficiently inhibited HBV replication, as well as de novo establishment of covalently closed circular DNA (cccDNA). In addition to these two known effects of CAMs, we observed for the first time that a CAM, here JNJ-827, when added postinfection for a short-term period, significantly reduced hepatitis B e antigen (HBeAg) secretion without affecting the levels of cccDNA amount, transcription, and hepatitis B surface antigen (HBsAg) secretion. This inhibitory activity resulted from a direct effect of JNJ-827 on HBeAg biogenesis. In a long-term treatment condition using persistently infected dHepaRG cells, JNJ-827 and JNJ-890 reduced HBsAg concomitantly with a decrease in viral total RNA and pgRNA levels. Altogether, these data demonstrate that some CAMs could interfere with multiple functions of HBc in the viral life cycle.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Antivirais/farmacologia , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , DNA Circular/genética , DNA Circular/metabolismo , Células Hep G2 , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos E da Hepatite B , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/virologia , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
In the liver, HBV and HCV infections, exposure to toxics, genetic and metabolic disorders may induce endoplasmic reticulum (ER) stress and the unfolding protein response (UPR). The UPR allows cells to reach ER homeostasis after lumen overload, but also fosters survival of damaged cells and therefore HCC onset. Dependence receptors such as UNC5A trigger apoptosis when unbound to their ligands. We have previously shown that the main dependence receptor ligand, netrin-1, could protect cells against UPR-induced apoptosis through sustained translation. In this study, we show that UNC5A is cumulatively downregulated by the UPR at the transcriptional level in vitro and at the translational level both in vitro and in vivo. We have found that the 5'-untranslated region of the UNC5A mRNA shares a certain homology degree with that of netrin-1, suggesting linked translational regulatory mechanisms, at least during the initial stages of the UPR. RNAi and forced expression studies identified UNC5A as a modulator of cell death in the context of the UPR. UNC5A decrease of association with polysomes and expression oriented cells towards UPR-associated hepatocytic survival. Such data indicate that cooperation between the UPR and UNC5A depletion as previously observed by ourselves in HCC patients samples may foster liver cancer development and growth.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Netrina-1/genética , Receptores de Superfície Celular/genética , Resposta a Proteínas não Dobradas/genética , Apoptose/genética , Carcinogênese , Linhagem Celular Tumoral , Repressão Epigenética/genética , Humanos , Receptores de NetrinaRESUMO
Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in drugs with potentially enhanced therapeutic properties. In the present work, the anti-HCV chimeric inhibitor RNA HH363-10, which has a hammerhead catalytic domain and an aptamer RNA domain, was subjected to an in vitro selection strategy to isolate ten different optimised chimeric inhibitor RNAs. The catalytic domain was preserved while the aptamer RNA domain was evolved to contain two binding sites, one mapping to the highly conserved IIIf domain of the HCV genome's internal ribosome entry site (IRES), and the other either to IRES domain IV (which contains the translation start codon) or the essential linker region between domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC50 values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved RNA molecules with potential clinical applications.
Assuntos
Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Genoma Viral/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Catalítico/farmacologia , Antivirais/química , Aptâmeros de Nucleotídeos/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Genes Reporter , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Sítios Internos de Entrada Ribossomal/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , Replicação Viral/efeitos dos fármacosRESUMO
Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for chronic hepatitis B and HCC are suboptimal. Herein, we identified cellular serine/threonine Polo-like-kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthesis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of primary human hepatocytes (PHHs) and differentiated HepaRG cells in conjunction with pharmacologic PLK1 inhibitors, small interfering RNA (siRNA)-mediated knockdown, and overexpression of constitutively active PLK1 (PLK1CA ). In addition, a humanized liver Fah-/- /Rag2-/- /Il2rg-/- (FRG) mouse model was used to determine the antiviral effect of PLK1 inhibitor BI-2536 on HBV infection in vivo. Finally, in vitro PLK1 kinase assays and site-directed mutagenesis were employed to demonstrate that HBV core protein (HBc) is a PLK1 substrate. We demonstrated that HBV infection activated cellular PLK1 in PHHs and differentiated HepaRG cells. PLK1 inhibition by BI-2536 or siRNA-mediated knockdown suppressed HBV DNA biosynthesis, whereas overexpression of PLK1CA increased it, suggesting that the PLK1 effects on viral biosynthesis are specific and that PLK1 is a proviral cellular factor. Significantly, BI-2536 administration to HBV-infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as an antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI-2536 leads to its decreased intracellular formation/accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein. CONCLUSION: PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitumoral strategies against HBV infection and HBV-mediated carcinogenesis. (Hepatology 2017;66:1750-1765).
Assuntos
Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/uso terapêutico , Proteínas do Core Viral/metabolismo , Replicação Viral , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Hepatócitos/enzimologia , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Fosforilação , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/farmacologia , Quinase 1 Polo-LikeAssuntos
Carcinoma Hepatocelular/etiologia , Receptores ErbB/fisiologia , Hepatite C/complicações , Cirrose Hepática/complicações , Neoplasias Hepáticas/etiologia , Fatores de Crescimento Neural/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Suscetibilidade a Doenças , Amplificação de Genes , Hepacivirus/fisiologia , Hepatite C/patologia , Hepatite C/virologia , Humanos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Fatores de Crescimento Neural/genética , Netrina-1 , Fatores de Risco , Proteínas Supressoras de Tumor/genética , Replicação ViralRESUMO
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.
Assuntos
Receptores ErbB/metabolismo , Hepatite C/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Autoantígenos/metabolismo , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Transformação Celular Neoplásica , Hepatite C/complicações , Hepatite C/virologia , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/virologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Netrina-1 , Ribonucleoproteínas/metabolismo , Regulação para Cima , Proteínas não Estruturais Virais/metabolismo , Internalização do Vírus , Antígeno SS-BRESUMO
BACKGROUND & AIMS: Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. METHODS: Isolation of translational complexes, determination of RNA secondary structures by selective 2'-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro-grown hepatocytic cells, wild-type, and netrin-1 transgenic mice. RESULTS: HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA-like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region-driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. CONCLUSIONS: UPR-resistant, internal ribosome entry site-driven netrin-1 translation leads to the inhibition of uncoordinated phenotype-5/death-associated protein kinase 1-mediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease.
RESUMO
BACKGROUND: The endothelium lines blood and lymph vessels and protects underlying tissues against external agents such as viruses, bacteria and parasites. Yet, microbes and particularly viruses have developed sophisticated ways to bypass the endothelium in order to gain access to inner organs. De novo infection of the liver parenchyma by many viruses and notably hepatitis viruses, is thought to occur through recruitment of virions on the sinusoidal endothelial surface and subsequent transfer to the epithelium. Furthermore, the liver endothelium undergoes profound changes with age and in inflammation or infection. However, primary human liver sinusoidal endothelial cells (LSECs) are difficult to obtain due to scarcity of liver resections. Relevant derived cell lines are needed in order to analyze in a standardized fashion the transfer of pathogens across the liver endothelium. By lentiviral transduction with hTERT only, we have immortalized human LSECs isolated from a hereditary hemorrhagic telangiectasia (HHT) patient and established the non-transformed cell line TRP3. TRP3 express mesenchymal, endothelial and liver sinusoidal markers. Functional assessment of TRP3 cells demonstrated a high capacity of endocytosis, tube formation and reactivity to immune stimulation. However, TRP3 displayed few fenestrae and expressed C-type lectins intracellularly. All these findings were confirmed in the original primary LSECs from which TRP3 were derived suggesting that these features were already present in the liver donor. We consider TRP3 as a model to investigate the functionality of the liver endothelium in hepatic inflammation in infection.