RESUMO
Filoviridae, including Ebola (EBOV) and Marburg (MARV) viruses, are emerging pathogens that pose a serious threat to public health. No agents have been approved to treat filovirus infections, representing a major unmet medical need. The selective estrogen receptor modulator (SERM) toremifene was previously identified from a screen of FDA-approved drugs as a potent EBOV viral entry inhibitor, via binding to EBOV glycoprotein (GP). A focused screen of ER ligands identified ridaifen-B as a potent dual inhibitor of EBOV and MARV. Optimization and reverse-engineering to remove ER activity led to a novel compound 30 (XL-147) showing potent inhibition against infectious EBOV Zaire (0.09 µM) and MARV (0.64 µM). Mutagenesis studies confirmed that inhibition of EBOV viral entry is mediated by the direct interaction with GP. Importantly, compound 30 displayed a broad-spectrum antifilovirus activity against Bundibugyo, Tai Forest, Reston, and Menglà viruses and is the first submicromolar antiviral agent reported for some of these strains, therefore warranting further development as a pan-filovirus inhibitor.
Assuntos
Antivirais/farmacologia , Filoviridae/efeitos dos fármacos , Receptores de Estrogênio/efeitos dos fármacos , Antivirais/química , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Filoviridae/fisiologia , Humanos , Ligantes , Fusão de Membrana/efeitos dos fármacos , Modelos Biológicos , Relação Estrutura-AtividadeRESUMO
Filoviruses, which include Ebola virus (EBOV) and Marburg virus, are negative-sense RNA viruses associated with sporadic outbreaks of severe viral hemorrhagic fever characterized by uncontrolled virus replication. The extreme virulence and emerging nature of these zoonotic pathogens make them a significant threat to human health. Replication of the filovirus genome and production of viral RNAs require the function of a complex of four viral proteins, the nucleoprotein (NP), viral protein 35 (VP35), viral protein 30 (VP30) and large protein (L). The latter performs the enzymatic activities required for production of viral RNAs and capping of viral mRNAs. Although it has been recognized that interactions between the virus-encoded components of the EBOV RNA polymerase complex are required for viral RNA synthesis reactions, specific molecular details have, until recently, been lacking. New efforts have combined structural biology and molecular virology to reveal in great detail the molecular basis for critical protein-protein interactions (PPIs) necessary for viral RNA synthesis. These efforts include recent studies that have identified a range of interacting host factors and in some instances demonstrated unique mechanisms by which they act. For a select number of these interactions, combined use of mutagenesis, over-expressing of peptides corresponding to PPI interfaces and identification of small molecules that disrupt PPIs have demonstrated the functional significance of virus-virus and virus-host PPIs and suggest several as potential targets for therapeutic intervention.
Assuntos
Filoviridae/fisiologia , Interações entre Hospedeiro e Microrganismos , RNA Viral/biossíntese , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Ebolavirus/efeitos dos fármacos , Ebolavirus/fisiologia , Filoviridae/efeitos dos fármacos , Humanos , Marburgvirus/efeitos dos fármacos , Marburgvirus/fisiologia , Ligação Proteica , Proteínas Virais/antagonistas & inibidores , Viroses/tratamento farmacológicoRESUMO
Ebola and Marburg are filoviruses and biosafety level 4 pathogens responsible for causing severe hemorrhagic fevers in humans with mortality rates up to 90%. The most recent outbreak in West Africa resulted in approximately 11,310 deaths in 28,616 reported cases. Currently there are no FDA-approved vaccines or therapeutics to treat infections of these deadly viruses. Recently we screened an FDA-approved drug library and identified numerous G protein-coupled receptor (GPCR) antagonists including antihistamines possessing anti-filovirus properties. Antihistamines are attractive targets for drug repurposing because of their low cost and ease of access due to wide use. In this report we identify common over the counter antihistamines, such as diphenhydramine (Benadryl) and chlorcyclizine (Ahist) as potential candidates for repurposing as anti-filovirus agents. Furthermore, we demonstrate that this potential is wide-spread through the 1st generation of H1-specific antihistamines but is not present in newer drugs or drugs targeting H2, H3 and H4 receptors. We showed that the filovirus entry inhibition is not dependent on the classical antagonism of cell surface histamine or muscarinic acetylcholine receptors but occurs in the endosome, like the cathepsin inhibitor CA-074. Finally, using extensive docking studies we showed the potential for these drugs to bind directly to the EBOV-GP at the same site as toremifene. These findings suggest that the 1st generation antihistamines are excellent candidates for repurposing as anti-filovirus therapeutics and can be further optimized for removal of unwanted histamine or muscarinic receptor interactions without loss of anti-filovirus efficacy.
Assuntos
Antivirais/farmacologia , Reposicionamento de Medicamentos , Filoviridae/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos/farmacologia , Células A549 , Difenidramina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Piperazinas/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP-VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.
Assuntos
Antivirais/farmacologia , Filoviridae/fisiologia , Nucleoproteínas/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Sequência Conservada , Avaliação Pré-Clínica de Medicamentos , Filoviridae/efeitos dos fármacos , Filoviridae/genética , Polarização de Fluorescência , Ensaios de Triagem em Larga Escala , Técnicas In Vitro , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Novel therapeutic options are urgently needed to improve global treatment of virus infections. Herbal products with confirmed clinical safety features are attractive starting material for the identification of new antiviral activities. Here we demonstrate that Cistus incanus (Ci) herbal products inhibit human immunodeficiency virus (HIV) infections in vitro. Ci extract inhibited clinical HIV-1 and HIV-2 isolates, and, importantly, a virus isolate with multiple drug resistances, confirming broad anti-HIV activity. Antiviral activity was highly selective for virus particles, preventing primary attachment of the virus to the cell surface and viral envelope proteins from binding to heparin. Bioassay-guided fractionation indicated that Ci extract contains numerous antiviral compounds and therefore has favorably low propensity to induce virus resistance. Indeed, no resistant viruses emerged during 24 weeks of continuous propagation of the virus in the presence of Ci extracts. Finally, Ci extracts also inhibited infection by virus particles pseudotyped with Ebola and Marburg virus envelope proteins, indicating that antiviral activity of Ci extract extends to emerging viral pathogens. These results demonstrate that Ci extracts show potent and broad in vitro antiviral activity against viruses that cause life-threatening diseases in humans and are promising sources of agents that target virus particles.
Assuntos
Antivirais/farmacologia , Cistus/química , Filoviridae/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proteínas do Envelope Viral/antagonistas & inibidores , Antivirais/química , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Farmacorresistência Viral , Humanos , Testes de Sensibilidade Microbiana , Extratos Vegetais/química , Polifenóis/química , Polifenóis/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
We previously described the generation of a novel Ebola virus (EBOV) vaccine based on inactivated rabies virus (RABV) containing EBOV glycoprotein (GP) incorporated in the RABV virion. Our results demonstrated safety, immunogenicity, and protective efficacy in mice and nonhuman primates (NHPs). Protection against viral challenge depended largely on the quality of the humoral immune response against EBOV GP.Here we present the extension and improvement of this vaccine by increasing the amount of GP incorporation into virions via GP codon-optimization as well as the addition of Sudan virus (SUDV) and Marburg virus (MARV) GP containing virions. Immunogenicity studies in mice indicate similar immune responses for both SUDV GP and MARV GP compared to EBOV GP. Immunizing mice with multiple antigens resulted in immune responses similar to immunization with a single antigen. Moreover, immunization of NHP with the new inactivated RABV EBOV vaccine resulted in high titer neutralizing antibody levels and 100% protection against lethal EBOV challenge when applied with adjuvant.Our results indicate that an inactivated polyvalent vaccine against RABV filoviruses is achievable. Finally, the novel vaccines are produced on approved VERO cells and a clinical grade RABV/EBOV vaccine for human trials has been produced.
Assuntos
Filoviridae/imunologia , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Raiva/imunologia , Vacinas de Produtos Inativados/imunologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos/métodos , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Macaca fascicularis , Marburgvirus/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Raiva/virologia , Sudão , Vacinação/métodos , Células VeroRESUMO
Cynomolgus macaques were vaccinated by intramuscular electroporation with DNA plasmids expressing codon-optimized glycoprotein (GP) genes of Ebola virus (EBOV) or Marburg virus (MARV) or a combination of codon-optimized GP DNA vaccines for EBOV, MARV, Sudan virus and Ravn virus. When measured by ELISA, the individual vaccines elicited slightly higher IgG responses to EBOV or MARV than did the combination vaccines. No significant differences in immune responses of macaques given the individual or combination vaccines were measured by pseudovirion neutralization or IFN-γ ELISpot assays. Both the MARV and mixed vaccines were able to protect macaques from lethal MARV challenge (5/6 vs. 6/6). In contrast, a greater proportion of macaques vaccinated with the EBOV vaccine survived lethal EBOV challenge in comparison to those that received the mixed vaccine (5/6 vs. 1/6). EBOV challenge survivors had significantly higher pre-challenge neutralizing antibody titers than those that succumbed.
Assuntos
Eletroporação , Filoviridae/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença do Vírus de Marburg/prevenção & controle , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Códon , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Filoviridae/genética , Glicoproteínas/genética , Glicoproteínas/imunologia , Imunoglobulina G/sangue , Injeções Intramusculares , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Macaca fascicularis , Masculino , Testes de Neutralização , Plasmídeos , Análise de Sobrevida , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
Filoviruses cause severe hemorrhagic fevers with case fatality rates of up to 90%, for which no antivirals are currently available. Their categorization as biosafety level 4 agents restricts work with infectious viruses to a few maximum containment laboratories worldwide, which constitutes a significant obstacle for the development of countermeasures. Reverse genetics facilitates the generation of recombinant filoviruses, including reporter-expressing viruses, which have been increasingly used for drug screening and development in recent years. Further, reverse-genetics based lifecycle modeling systems allow modeling of the filovirus lifecycle without the need for a maximum containment laboratory and have recently been optimized for use in high-throughput assays. The availability of these reverse genetics-based tools will significantly improve our ability to find novel antivirals against filoviruses.
Assuntos
Antivirais/farmacologia , Infecções por Filoviridae/tratamento farmacológico , Filoviridae/genética , Modelos Teóricos , Genética Reversa/métodos , Antivirais/uso terapêutico , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Ebolavirus/efeitos dos fármacos , Ebolavirus/genética , Ebolavirus/fisiologia , Filoviridae/efeitos dos fármacos , Filoviridae/fisiologia , Genoma Viral , Humanos , Testes de Sensibilidade Microbiana , Replicação ViralRESUMO
INTRODUCTION: Ebolaviruses and marburgviruses cause severe and often lethal human hemorrhagic fevers. As no FDA-approved therapeutics are available for these infections, efforts to discover new therapeutics are important, especially because these pathogens are considered biothreats and emerging infectious diseases. All methods for discovering new therapeutics should be considered, including compound library screening in vitro against virus and in silico structure-based drug design, where possible, if sufficient biochemical and structural information is available. AREAS COVERED: This review covers the structure and function of filovirus proteins, as they have been reported to date, as well as some of the current antiviral screening approaches. The authors discuss key studies mapping small-molecule modulators that were found through library and in silico screens to potential sites on viral proteins or host proteins involved in virus trafficking and pathogenesis. A description of ebolavirus and marburgvirus diseases and available animal models is also presented. EXPERT OPINION: To discover novel therapeutics with potent efficacy using sophisticated computational methods, more high-resolution crystal structures of filovirus proteins and more details about the protein functions and host interaction will be required. Current compound screening efforts are finding active antiviral compounds, but an emphasis on discovery research to investigate protein structures and functions enabling in silico drug design would provide another avenue for finding antiviral molecules. Additionally, targeting of protein-protein interactions may be a future avenue for drug discovery since disrupting catalytic sites may not be possible for all proteins.
Assuntos
Infecções por Filoviridae/tratamento farmacológico , Proteínas Virais/fisiologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Simulação por Computador , Desenho de Fármacos , Filoviridae , Infecções por Filoviridae/fisiopatologia , Humanos , Proteínas Virais/químicaRESUMO
Filamentous particles were detected by negative contrast electron microscopy of extracts from the leafhopper Psammotettix alienus (Dahlbom) reared on healthy Festuca gigantea plants. The particles were straight, slightly curved or flexuous, sometimes with the one end curled into a ring with an outer diameter of about 200 nm. Length distribution of 280 particles showed a minor and a major group with median length of about 600 and 1,100 nm, respectively. Projections, 8-10 nm long and about 10 nm apart, were evenly distributed on the surface. The diameter of particles, including projections, was 55-70 nm. Partly disintegrated revealed an internal structure about 30 nm in diameter and with cross-striation with a periodicity of 5-5.5 nm. In some particles, a central canal, 5-10 nm in diameter, could be seen at one end. Ultramicrotomy of leafhopper heads showed that some cells contained intracytoplasmic clusters of particles together with filamentous structures. The particles described in this paper resemble virions in the virus family Filoviridae, but can be distinguished by having a smaller particle diameter. The name Taastrup virus is suggested for the putative virus from Psammotettix alienus, according to the place it was first detected.