RESUMO
Despite the global prevalence of gastric disease, there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/ß-catenin signalling in mouse embryos led to conversion of fundic to antral epithelium, and that ß-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signalling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology, and also represent a new platform for drug discovery.
Assuntos
Fundo Gástrico/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Descoberta de Drogas/métodos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Fundo Gástrico/citologia , Fundo Gástrico/embriologia , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Organoides/citologia , Organoides/embriologia , Organoides/metabolismo , Células Parietais Gástricas/citologia , Células Parietais Gástricas/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Transativadores/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/agonistasRESUMO
The Ebola virus is a deadly human pathogen responsible for several outbreaks in Africa. Its genome encodes the 'large' L protein, an essential enzyme that has polymerase, capping and methyltransferase activities. The methyltransferase activity leads to RNA co-transcriptional modifications at the N7 position of the cap structure and at the 2'-O position of the first transcribed nucleotide. Unlike other Mononegavirales viruses, the Ebola virus methyltransferase also catalyses 2'-O-methylation of adenosines located within the RNA sequences. Herein, we report the crystal structure at 1.8 Å resolution of the Ebola virus methyltransferase domain bound to a fragment of a camelid single-chain antibody. We identified structural determinants and key amino acids specifically involved in the internal adenosine-2'-O-methylation from cap-related methylations. These results provide the first high resolution structure of an ebolavirus L protein domain, and the framework to investigate the effects of epitranscriptomic modifications and to design possible antiviral drugs against the Filoviridae family.
Assuntos
Ebolavirus/enzimologia , Metiltransferases/química , Proteínas Virais/química , Domínio Catalítico , Cristalografia por Raios X , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica em alfa-Hélice , Anticorpos de Domínio Único/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The large (L) protein of Ebola virus is a key protein for virus replication. Its N-terminal region harbors the RNA-dependent RNA polymerase activity, and its C terminus contains a cap assembling line composed of a capping domain and a methyltransferase domain (MTase) followed by a C-terminal domain (CTD) of unknown function. The L protein MTase catalyzes methylation at the 2'-O and N-7 positions of the cap structures. In addition, the MTase of Ebola virus can induce cap-independent internal adenosine 2'-O-methylation. In this work, we investigated the CTD role in the regulation of the cap-dependent and cap-independent MTase activities of the L protein. We found that the CTD, which is enriched in basic amino acids, plays a key role in RNA binding and in turn regulates the different MTase activities. We demonstrated that the mutation of CTD residues modulates specifically the different MTase activities. Altogether, our results highlight the pivotal role of the L protein CTD in the control of viral RNA methylation, which is critical for Ebola virus replication and escape from the innate response in infected cells.IMPORTANCE Ebola virus infects human and nonhuman primates, causing severe infections that are often fatal. The epidemics, in West and Central Africa, emphasize the urgent need to develop antiviral therapies. The Ebola virus large protein (L), which is the central protein for viral RNA replication/transcription, harbors a methyltransferase domain followed by a C-terminal domain of unknown function. We show that the C-terminal domain regulates the L protein methyltransferase activities and consequently participates in viral replication and escape of the host innate immunity.
Assuntos
Ebolavirus/genética , Metiltransferases/genética , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Ebolavirus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Metilação , Metiltransferases/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
The health emergency caused by the recent Covid-19 pandemic highlights the need to identify effective treatments against the virus causing this disease (SARS-CoV-2). The first clinical trials have been testing repurposed drugs that show promising anti-SARS-CoV-2 effects in cultured cells. Although more than 2400 clinical trials are already under way, the actual number of tested compounds is still limited to approximately 20, alone or in combination. In addition, knowledge on their mode of action (MoA) is currently insufficient. Their first results reveal some inconsistencies and contradictory results and suggest that cohort size and quality of the control arm are two key issues for obtaining rigorous and conclusive results. Moreover, the observed discrepancies might also result from differences in the clinical inclusion criteria, including the possibility of early treatment that may be essential for therapy efficacy in patients with Covid-19. Importantly, efforts should also be made to test new compounds with a documented MoA against SARS-CoV-2 in clinical trials. Successful treatment will probably be based on multitherapies with antiviral compounds that target different steps of the virus life cycle. Moreover, a multidisciplinary approach that combines artificial intelligence, compound docking, and robust in vitro and in vivo assays will accelerate the development of new antiviral molecules. Finally, large retrospective studies on hospitalized patients are needed to evaluate the different treatments with robust statistical tools and to identify the best treatment for each Covid-19 stage. This review describes different candidate antiviral strategies for Covid-19, by focusing on their mechanism of action.
Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Terapia Combinada , Gerenciamento Clínico , Suscetibilidade a Doenças , Desenvolvimento de Medicamentos , Reposicionamento de Medicamentos , Interações Hospedeiro-Patógeno , Humanos , Resultado do TratamentoRESUMO
PURPOSE: The aim of the study was to evaluate organogel nanoparticles as a lipophilic vehicle to increase the oral bioavailability of poorly soluble compounds. Efavirenz (EFV), a Biopharmaceutical Classification System (BCS) Class II, was used as drug model. METHODS: Organogel nanoparticles loaded with EFV were formulated with sunflower oil, 12-hydroxystearic acid (HSA) and polyvinyl alcohol (PVA). Various parameters have been investigated in the current study such as (i) the release profile of organogel assessed by USP 4 cell flow dialysis, (ii) the impact of organogel on intestinal absorption, using Caco-2 cells as in vitro model and jejunum segments as ex vivo assay and (iii) the bioavailability of organogel following oral pharmacokinetic study. RESULTS: 250-300 nm spherical particles with a final concentration of 4.75 mg/mL drug loading were obtained, corresponding to a thousand fold increase in EFV solubility, combined to a very high encapsulation efficiency (>99.8%). Due to rapid diffusion, drug was immediately released from the nanoparticles. The biopharmaceutical evaluation on ex vivo jejunum segments demonstrated an increased absorption of EFV from organogel nanoparticles compare to a native EFV suspension. In vitro assays combining Caco-2 cell cultures with TEM and confocal microscopy demonstrated passive diffusion, while paracellular integrity and endocytosis activity remain expelled. Oral pharmacokinetics of EFV organogel nanoparticles improve oral bioavailability (Fr: 249%) and quick absorption compared to EFV suspension. CONCLUSION: Organogel nanoparticles increase the bioavailability of BCS Class II drugs. The main phenomena is simply oil transfer from the gelled particles through the cell membrane.
Assuntos
Benzoxazinas/química , Portadores de Fármacos/química , Géis/química , Nanocápsulas/química , Álcool de Polivinil/química , Ácidos Esteáricos/química , Óleo de Girassol/química , Alcinos , Animais , Benzoxazinas/administração & dosagem , Benzoxazinas/farmacocinética , Disponibilidade Biológica , Células CACO-2 , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclopropanos , Difusão , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Excipientes/química , Humanos , Absorção Intestinal , Masculino , Solubilidade , Suspensões/química , Distribuição TecidualRESUMO
Flaviviruses, such as Dengue (DENV) and Zika (ZIKV) viruses, represent a severe health burden. There are currently no FDA-approved treatments, and vaccines against most flaviviruses are still lacking. We have developed several flexible analogues ("fleximers") of the FDA-approved nucleoside Acyclovir that exhibit activity against various RNA viruses, demonstrating their broad-spectrum potential. The current study reports activity against DENV and Yellow Fever Virus (YFV), particularly for compound 1. Studies to elucidate the mechanism of action suggest the flex-analogue triphosphates, especially 1-TP, inhibit DENV and ZIKV methyltransferases, and a secondary, albeit weak, effect on the DENV RNA-dependent RNA polymerase was observed at high concentrations. The results of these studies are reported herein.
Assuntos
Antivirais/farmacologia , Flavivirus/efeitos dos fármacos , Nucleosídeos/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2'O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2'O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2'O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting internal adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2'O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA internal methylations in the viral replication.
Assuntos
Adenosina/metabolismo , Ebolavirus/genética , Regulação Viral da Expressão Gênica , Metiltransferases/genética , RNA Viral/genética , Proteínas não Estruturais Virais/genética , Adenosina/genética , Motivos de Aminoácidos , Clonagem Molecular , Ebolavirus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanosina/genética , Guanosina/metabolismo , Metilação , Metiltransferases/metabolismo , Domínios Proteicos , Capuzes de RNA , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genéticaRESUMO
The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2'-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2'-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor (S-adenosyl-l-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the αX α-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs.IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns, and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune mechanisms. We demonstrate that the ZIKV methyltransferase methylates the mRNA cap and adenosines located in RNA sequences. The structure of ZIKV methyltransferase shows high structural similarities to the dengue virus methyltransferase, but conformational specificities highlight the role of a conserved Ser/Arg motif, which participates in RNA and SAM recognition during the reaction turnover. In addition, the SAM binding site accommodates a sulfate and a glycerol, offering structural information to initiate structure-based drug design. Altogether, these results contribute to a better understanding of the Flavivirus methyltransferases, which are central players in the virus replication.
Assuntos
Antivirais/química , Metiltransferases/química , Proteínas não Estruturais Virais/química , Zika virus/enzimologia , Sítio Alostérico , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Escherichia coli , Ligação de Hidrogênio , Metiltransferases/biossíntese , Modelos Moleculares , Ligação Proteica , Proteínas não Estruturais Virais/biossínteseRESUMO
The purpose of the study was to evaluate organogel nanoparticles as a drug delivery system by investigating their stability, according to the formulation strategy, and their release profile. The gelled nanoparticles were prepared by hot emulsification (above the gelation temperature) of an organogel in water, and cooling at room temperature. In the first step, we used DLS and DSC to select the most suitable formulations by optimizing the proportion of ingredients (HSA, PVA, castor oil) to obtain particles of the smallest size and greatest stability. Then, two lipophilic drug models, indomethacin and ketoconazole were entrapped in the nanoparticles made of castor oil gelled by 12-hydroxystearic acid. Thermal studies (DSC) confirmed that there was no significant alteration of gelling due to the entrapped drugs, even at 3% w/w. Very stable dispersions were obtained (>3 months), with gelled oil nanoparticles presenting a mean diameter between 250 and 300 nm. High encapsulation efficiency (>98%) was measured for indomethacin and ketoconazole. The release profile determined by in vitro dialysis showed an immediate release of the drug from the organogel nanoparticles, due to rapid diffusion. The study demonstrates the interest of these gelled oil nanoparticles for the encapsulation and the delivery of lipophilic active compounds.
Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Liberação Controlada de Fármacos , Géis , Indometacina/administração & dosagem , Cetoconazol/administração & dosagem , Tamanho da Partícula , TemperaturaRESUMO
Toll-like receptor 3 (TLR3) agonists such as polyinosinic:polycytidylic acid (poly(I:C)) have immunostimulatory effects that can be taken advantage of to induce anticancer immune responses in preclinical models. In addition, poly(I:C) has been introduced into clinical trials to demonstrate its efficacy as an adjuvant and to enhance the immunogenicity of locally injected tumors, thus reverting resistance to PD-L1 blockade in melanoma patients. Here, we report the pharmacokinetic, pharmacodynamic, mechanistic and toxicological profile of a novel TLR3 agonist, TL-532, a chemically synthesized double-stranded RNA that is composed by blocks of poly(I:C) and poly(A:U) (polyadenylic - polyuridylic acid). In preclinical models, we show that TL-532 is bioavailable after parenteral injection, has an acceptable toxicological profile, and stimulates the production of multiple chemokines and interleukins that constitute pharmacodynamic markers of its immunostimulatory action. When given at a high dose, TL-532 monotherapy reduced the growth of bladder cancers growing on mice. In addition, in immunodeficient mice lacking formylpeptide receptor-1 (FPR1), TL-532 was able to restore the response of orthotopic subcutaneous fibrosarcoma to immunogenic chemotherapy. Altogether, these findings may encourage further development of TL-532 as an immunotherapeutic anticancer agent.
Assuntos
Melanoma , Receptor 3 Toll-Like , Animais , Camundongos , Adjuvantes Imunológicos , Melanoma/tratamento farmacológico , Poli I-C/farmacologiaRESUMO
Polymer composites of molecular spin crossover complexes have emerged as promising mechanical actuator materials, but their effective thermomechanical properties remain elusive. In this work, we investigated a series of iron(ii)-triazole@P(VDF-TrFE) particulate composites using a tensile testing stage with temperature control. From these measurements, we assessed the temperature dependence of the Young's modulus as well as the free deformation and blocking stress, associated with the thermally-induced spin transition. The results denote that the expansion of the particles at the spin transition is effectively transferred to the macroscopic composite material, providing ca. 1-3% axial strain for 25% particle load. This strain is in excess of the 'neat' particle strain, which we attribute to particle-matrix mechanical coupling. On the other hand, the blocking stress (â¼1 MPa) appears reduced by the softening of the composite around the spin transition temperature.
RESUMO
BACKGROUND: This translational study explores multi-tracer PET imaging for the non-invasive detection of the IDH1 mutation which is a positive prognostic factor in glioma. METHODS: U87 human high-grade glioma (HGG) isogenic cell lines with or without the IDH1 mutation (CRISP/Cas9 method) were stereotactically grafted into rat brains, and examined, in vitro, in vivo and ex vivo. PET imaging sessions, with radiotracers specific for glycolytic metabolism ([18F]FDG), amino acid metabolism ([18F]FDopa), and inflammation ([18F]DPA-714), were performed sequentially during 3-4 days. The in vitro radiotracer uptake was expressed as percent per million cells. For each radiotracer examined in vivo, static analyses included the maximal and mean tumor-to-background ratio (TBRmax and TBRmean) and metabolic tumor volume (MTV). Dynamic analyses included the distribution volume ratio (DVR) and the relative residence time (RRT) extracted from a reference Logan model. Ex vivo analyses consisted of immunological analyses. RESULTS: In vitro, IDH1+ cells (i.e. cells expressing the IDH1 mutation) showed lower levels of [18F]DPA-714 uptake compared to IDH1- cells (p < 0.01). These results were confirmed in vivo with lower [18F]DPA-714 uptake in IDH+ tumors (3.90 versus 5.52 for TBRmax, p = 0.03). Different values of [18F]DPA-714 and [18F] FDopa RRT (respectively 11.07 versus 22.33 and 2.69 versus - 1.81 for IDH+ and IDH- tumors, p < 0.02) were also observed between the two types of tumors. RRT [18F]DPA-714 provided the best diagnostic performance to discriminate between the two cell lines (AUC of 100%, p < 0.01). Immuno-histological analyses revealed lower expression of Iba-1 and TSPO antibodies in IDH1+ tumors. CONCLUSIONS: [18F]DPA-714 and [18F] FDopa both correlate with the presence of the IDH1 mutation in HGG. These radiotracers are therefore good candidates for translational studies investigating their clinical applications in patients.
Assuntos
Glioma , Animais , Fluordesoxiglucose F18 , Glioma/diagnóstico por imagem , Glioma/genética , Glioma/metabolismo , Humanos , Mutação , Tomografia por Emissão de Pósitrons/métodos , Ratos , Receptores de GABA/genéticaRESUMO
The Crimean-Congo hemorrhagic fever virus (CCHFV) is the etiological agent of a severe hemorrhagic fever affecting Africa, Asia and southern Europe. Climate changes of recent decades have recently led to a rise in the distribution of this virus. Still few scientific data are available on the biology of its vector, the tick, or its own biology, but the proven presence of human infections observed in Spain and animals with positive serology in Corsica should focus our attention on this pathogen. This review takes stock of the epidemiologic evolution of CCHF in Europe, notably in France.
TITLE: La fièvre hémorragique de Crimée-Congo, une future problématique de santé en France ? ABSTRACT: Le virus de la fièvre hémorragique de Crimée-Congo (CCHFV) est l'agent étiologique d'une fièvre hémorragique grave affectant l'Afrique, l'Asie et le sud de l'Europe. Les modifications climatiques de ces dernières décennies induisent depuis peu une remontée de l'aire de distribution de ce virus. Encore peu de données scientifiques sont disponibles sur les interactions avec son vecteur, la tique, ou sur sa biologie propre. Cependant, la présence avérée d'infections humaines en Espagne et des sérologies positives dans le cheptel corse pourraient bien concentrer l'attention sur ce pathogène. Cette revue fait le point sur l'évolution des connaissances éco-épidémiologiques de ce virus, notamment en Europe et plus particulièrement en France.
Assuntos
Febre Hemorrágica da Crimeia/epidemiologia , Animais , Europa (Continente)/epidemiologia , França/epidemiologia , Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Febre Hemorrágica da Crimeia/virologia , Humanos , Estudos SoroepidemiológicosRESUMO
In the recent years, Ebola virus has been responsible for several major epidemics. Research efforts have allowed the development and evaluation in the field of several vaccine candidates. At present, two of them are already approved and used in the fight against the virus in the Democratic Republic of Congo. This review aims to describe the different candidates, the clinical trials that have been conducted as well as the first results in the field.
TITLE: Ebola, des premiers vaccins disponibles. ABSTRACT: Ces dernières années, le virus Ebola a été responsable d'épidémies de grande ampleur. Les efforts de recherche ont permis la mise au point et l'évaluation sur le terrain de plusieurs candidats vaccins. À l'heure actuelle, deux sont déjà homologués et utilisés dans la lutte contre le virus en République démocratique du Congo. Cette revue se propose de faire le point sur les différents candidats vaccins, les essais cliniques qui ont été menés et les premiers résultats de terrain.
Assuntos
Vacinas contra Ebola/uso terapêutico , Ebolavirus/imunologia , Doença pelo Vírus Ebola/terapia , Surtos de Doenças , Epidemias , Necessidades e Demandas de Serviços de Saúde , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/imunologia , Humanos , Vacinação em Massa/organização & administração , Vacinação em Massa/estatística & dados numéricos , Fatores SocioeconômicosRESUMO
Ebola virus is an important pathogen that emerged in Central Africa where it was responsible of numerous outbreaks of haemorrhagic fevers associated with a extremely high mortality rate (up to 90%). The filovirus pathogenicity is related to an inappropriate antiviral response. Indeed, this family of viruses has developed evasion strategies from early innate immunity mechanisms. As a result, a massive viral replication induces an unsuitable immune response causing an acute inflammatory reaction associated with the haemorrhagic syndrome. In this review, we describe the mechanisms adopted by filoviruses like Ebola virus to escape innate immunity response.
Assuntos
Infecções por Filoviridae/imunologia , Infecções por Filoviridae/virologia , Filoviridae/imunologia , Filoviridae/fisiologia , Evasão da Resposta Imune/fisiologia , Animais , Filoviridae/patogenicidade , Humanos , Imunidade Inata/fisiologiaRESUMO
There are no approved medications for the treatment of Marburg or Ebola virus infection. In two previous articles (Martin et al., 2016, Martin et al., 2017), we reviewed surface glycoprotein and replication proteins structure/function relationship to decipher the molecular mechanisms of filovirus life cycle and identify antiviral strategies. In the present article, we recapitulate knowledge about the viral proteins involved in filovirus assembly and budding. First we describe the structural data available for viral proteins associated with virus assembly and virion egress and then, we integrate the structural features of these proteins in the functional context of the viral replication cycle. Finally, we summarize recent advances in the development of innovative antiviral strategies to target filovirus assembly and egress. The development of such prophylactic or post-exposure treatments could help controlling future filovirus outbreaks.
Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Filoviridae/efeitos dos fármacos , Filoviridae/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Montagem de Vírus/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacos , Antivirais/química , Descoberta de Drogas/métodos , Filoviridae/classificação , Genoma Viral , Genômica/métodos , Humanos , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidoresRESUMO
Filoviruses are important pathogens that cause severe and often fatal hemorrhagic fever in humans, for which no approved vaccines and antiviral treatments are yet available. In an earlier article (Martin et al., Antiviral Research, 2016), we reviewed the role of the filovirus surface glycoprotein in replication and as a target for drugs and vaccines. In this review, we focus on recent findings on the filovirus replication machinery and how they could be used for the identification of new therapeutic targets and the development of new antiviral compounds. First, we summarize the recent structural and functional advances on the molecules involved in filovirus replication/transcription cycle, particularly the NP, VP30, VP35 proteins, and the "large" protein L, which harbors the RNA-dependent RNA polymerase (RdRp) and mRNA capping activities. These proteins are essential for viral mRNA synthesis and genome replication, and consequently they constitute attractive targets for drug design. We then describe how these insights into filovirus replication mechanisms and the structure/function characterization of the involved proteins have led to the development of new and innovative antiviral strategies that may help reduce the filovirus disease case fatality rate through post-exposure or prophylactic treatments.
Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Filoviridae/efeitos dos fármacos , Filoviridae/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Desenho de Fármacos , Ebolavirus/química , Ebolavirus/efeitos dos fármacos , Ebolavirus/metabolismo , Infecções por Filoviridae/tratamento farmacológico , Marburgvirus/química , Marburgvirus/efeitos dos fármacos , Marburgvirus/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Two highly pathogenic human coronaviruses associated with severe respiratory syndromes emerged since the beginning of the century. The severe acute respiratory syndrome SARS-coronavirus (CoV) spread first in southern China in 2003 with about 8000 infected cases in few months. Then in 2012, the Middle East respiratory syndrome (MERS-CoV) emerged from the Arabian Peninsula giving a still on-going epidemic associated to a high fatality rate. CoVs are thus considered a major health threat. This is especially true as no vaccine nor specific therapeutic are available against either SARS- or MERS-CoV. Therefore, new drugs need to be identified in order to develop antiviral treatments limiting CoV replication. In this study, we focus on the nsp14 protein, which plays a key role in virus replication as it methylates the RNA cap structure at the N7 position of the guanine. We developed a high-throughput N7-MTase assay based on Homogenous Time Resolved Fluorescence (HTRF®) and screened chemical libraries (2000 compounds) on the SARS-CoV nsp14. 20 compounds inhibiting the SARS-CoV nsp14 were further evaluated by IC50 determination and their specificity was assessed toward flavivirus- and human cap N7-MTases. Our results reveal three classes of compounds: 1) molecules inhibiting several MTases as well as the dengue virus polymerase activity unspecifically, 2) pan MTases inhibitors targeting both viral and cellular MTases, and 3) inhibitors targeting one viral MTase more specifically showing however activity against the human cap N7-MTase. These compounds provide a first basis towards the development of more specific inhibitors of viral methyltransferases.
Assuntos
Antivirais/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Fluorometria , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade MicrobianaRESUMO
This review focuses on the recent progress in our understanding of filovirus protein structure/function and its impact on antiviral research. Here we focus on the surface glycoprotein GP1,2 and its different roles in filovirus entry. We first describe the latest advances on the characterization of GP gene-overlapping proteins sGP, ssGP and Δ-peptide. Then, we compare filovirus surface GP1,2 proteins in terms of structure, synthesis and function. As they bear potential in drug-design, the discovery of small organic compounds inhibiting filovirus entry is a currently very active field. Although it is at an early stage, the development of antiviral drugs against Ebola and Marburg virus entry might prove essential to reduce outbreak-associated fatality rates through post-exposure treatment of both suspected and confirmed cases.