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The development of new drugs is costly and time-consuming, with estimates of over $US1 billion and 15 years for a product to reach the market. As understanding of the molecular basis of disease improves, various approaches have been used to target specific molecular interactions in the search for effective drugs. These include high-throughput screening (HTS) for novel drug identification and computer-aided drug design (CADD) to assess the properties of putative drugs before experimental work begins. We have applied conventional HTS and CADD approaches to the problem of identifying antiviral compounds to limit infection by Venezuelan equine encephalitis virus (VEEV). Nuclear targeting of the VEEV capsid (CP) protein through interaction with the host nuclear import machinery has been shown to be essential for viral pathogenicity, with viruses incapable of this interaction being greatly attenuated. Our previous conventional HTS and in silico structure-based drug design (SBDD) screens were successful in identifying novel inhibitors of CP interaction with the host nuclear import machinery, thus providing a unique opportunity to assess the relative value of the two screening approaches directly. This focused review compares and contrasts the two screening approaches, together with the properties of the inhibitors identified, as a case study for parallel use of the two approaches to identify antivirals. The utility of SBDD screens, especially when used in parallel with traditional HTS, in identifying agents of interest to target the host-pathogen interface is highlighted.
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Dengue virus (DENV) threatens almost 70% of the world's population, with no therapeutic currently available. The severe, potentially lethal forms of DENV disease (dengue hemorrhagic fever/dengue shock syndrome) are associated with the production of high level of cytokines, elicited as part of the host antiviral response, although the molecular mechanisms have not been fully elucidated. We previously showed that infection by DENV serotype 2 (DENV2) disrupts promyelocytic leukemia (PML) gene product nuclear bodies (PML-NBs) after viral protein translation in infected cells. Apart from playing a key role as the nucleating agent in forming PML-NBs, PML has antiviral activity against various viruses, including DENV. The present study builds on this work, showing for the first time that all four DENV serotypes elicit PML-NB breakdown. Importantly, we show for the first time that of the nuclear localizing proteins of DENV, DENV non-structural protein (NS) 5 polymerase alone is sufficient to elicit PML-NB disassembly, in part through complexing with PML isoforms III and IV, but not other PML isoforms or other PML-NB components. The results raise the possibility that PML-NB disruption by nuclear localized NS5 contributes to DENV's suppression of the host antiviral response.
Assuntos
Núcleo Celular/metabolismo , Vírus da Dengue/fisiologia , Dengue/metabolismo , Dengue/virologia , Interações Hospedeiro-Patógeno , Proteína da Leucemia Promielocítica/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Vírus da Dengue/classificação , Expressão Gênica , Humanos , Ligação Proteica , Isoformas de Proteínas , Transporte Proteico , Sorogrupo , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
There are currently no therapeutics to treat infection with the alphavirus Venezuelan equine encephalitis virus (VEEV), which causes flu-like symptoms leading to neurological symptoms in up to 14% of cases. Large outbreaks of VEEV can result in 10,000 s of human cases and mass equine death. We previously showed that mifepristone (RU486) has anti-VEEV activity (EC50 = 20 µM) and only limited cytotoxicity (CC50 > 100 µM), but a limitation in its use is its abortifacient activity resulting from its ability to antagonize the progesterone receptor (PR). Here we generate a suite of new mifepristone analogues with enhanced antiviral properties, succeeding in achieving >11-fold improvement in anti-VEEV activity with no detectable increase in toxicity. Importantly, we were able to derive a lead compound with an EC50 of 7.2 µM and no detectable PR antagonism activity. Finally, based on our SAR analysis we propose avenues for the further development of these analogues as safe and effective anti-VEEV agents.
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Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Mifepristona/análogos & derivados , Mifepristona/farmacologia , Receptores de Progesterona/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células HeLa , Humanos , Mifepristona/síntese química , Mifepristona/química , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Relação Estrutura-AtividadeRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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Although the alphavirus Venezuelan equine encephalitis virus (VEEV) has been the cause of multiple outbreaks resulting in extensive human and equine mortality and morbidity, there are currently no anti-VEEV therapeutics available. VEEV pathogenicity is largely dependent on targeting of the viral capsid protein (CP) to the host cell nucleus through the nuclear transporting importin (Imp) α/ß1 heterodimer. Here we perform a high-throughput screen, combined with nested counterscreens to identify small molecules able to inhibit the Impα/ß1:CP interaction for the first time. Several compounds were able to significantly reduce viral replication in infected cells. Compound G281-1564 in particular could inhibit VEEV replication at low µM concentration, while showing minimal toxicity, with steady state and dynamic quantitative microscopic measurements confirming its ability to inhibit CP nuclear import. This study establishes the principle that inhibitors of CP nucleocytoplasmic trafficking can have potent antiviral activity against VEEV, and represents a platform for future development of safe anti-VEEV compounds with high efficacy and specificity.
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Antivirais/farmacologia , Proteínas do Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Encefalomielite Equina Venezuelana/virologia , Carioferinas/antagonistas & inibidores , Carioferinas/metabolismo , Replicação Viral/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Antivirais/química , Sobrevivência Celular , Chlorocebus aethiops , Encefalomielite Equina Venezuelana/metabolismo , Ensaios de Triagem em Larga Escala , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Concentração Inibidora 50 , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Células VeroRESUMO
Therapeutics are currently unavailable for Venezuelan equine encephalitis virus (VEEV), which elicits flu-like symptoms and encephalitis in humans, with an estimated 14% of cases resulting in neurological disease. Here we identify anti-VEEV agents using in silico structure-based-drug-design (SBDD) for the first time, characterising inhibitors that block recognition of VEEV capsid protein (C) by the host importin (IMP) α/ß1 nuclear transport proteins. From an initial screen of 1.5 million compounds, followed by in silico refinement and screening for biological activity in vitro, we identified 21 hit compounds which inhibited IMPα/ß1:C binding with IC50s as low as 5 µM. Four compounds were found to inhibit nuclear import of C in transfected cells, with one able to reduce VEEV replication at µM concentration, concomitant with reduced C nuclear accumulation in infected cells. Further, this compound was inactive against a mutant VEEV that lacks high affinity IMPα/ß1:C interaction, supporting the mode of its antiviral action to be through inhibiting C nuclear localization. This successful application of SBDD paves the way for lead optimization for VEEV antivirals, and is an exciting prospect to identify inhibitors for the many other viral pathogens of significance that require IMPα/ß1 in their infectious cycle.
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Proteínas do Capsídeo/efeitos dos fármacos , Descoberta de Drogas/métodos , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Antivirais/farmacologia , Capsídeo , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Simulação por Computador , Desenho de Fármacos , Vírus da Encefalite Equina Venezuelana/patogenicidade , Humanos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/metabolismoRESUMO
The ß-dystroglycan (ß-DG) protein has the ability to target to multiple sites in eukaryotic cells, being a member of diverse protein assemblies including the transmembranal dystrophin-associated complex, and a nuclear envelope-localised complex that contains emerin and lamins A/C and B1. We noted that the importin α2/ß1-recognised nuclear localization signal (NLS) of ß-DG is also a binding site for the cytoskeletal-interacting protein ezrin, and set out to determine whether ezrin binding might modulate ß-DG nuclear translocation for the first time. Unexpectedly, we found that ezrin enhances rather than inhibits ß-DG nuclear translocation in C2C12 myoblasts. Both overexpression of a phosphomimetic activated ezrin variant (Ez-T567D) and activation of endogenous ezrin through stimulation of the Rho pathway resulted in both formation of actin-rich surface protrusions and significantly increased nuclear translocation of ß-DG as shown by quantitative microscopy and subcellular fractionation/Western analysis. In contrast, overexpression of a nonphosphorylatable inactive ezrin variant (Ez-T567A) or inhibition of Rho signaling, decreased nuclear translocation of ß-DG concomitant with a lack of cell surface protrusions. Further, a role for the actin cytoskeleton in ezrin enhancement of ß-DG nuclear translocation was implicated by the observation that an ezrin variant lacking its actin-binding domain failed to enhance nuclear translocation of ß-DG, while disruption of the actin cytoskeleton led to a reduction in ß-DG nuclear localization. Finally, we show that ezrin-mediated cytoskeletal reorganization enhances nuclear translocation of the cytoplasmic but not the transmembranal fraction of ß-DG. This is the first study showing that cytoskeleton reorganization can modulate nuclear translocation of ß-DG, with the implication that ß-DG can respond to cytoskeleton-driven changes in cell morphology by translocating from the cytoplasm to the nucleus to orchestrate nuclear processes in response to the functional requirements of the cell.
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Citoesqueleto de Actina/metabolismo , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/fisiologia , Distroglicanas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Biotinilação , Linhagem Celular , Camundongos , Mioblastos Esqueléticos/metabolismo , Transdução de Sinais , beta Carioferinas/metabolismo , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
Targeting host responses to invading viruses has been the focus of recent antiviral research. Venezuelan Equine Encephalitis Virus (VEEV) is able to modulate host transcription and block nuclear trafficking at least partially due to its capsid protein forming a complex with the host proteins importin α/ß1 and CRM1. We hypothesized that disrupting the interaction of capsid with importin α/ß1 or the interaction of capsid with CRM1 would alter capsid localization, thereby lowering viral titers in vitro. siRNA mediated knockdown of importin α, importin ß1, and CRM1 altered capsid localization, confirming their role in modulating capsid trafficking. Mifepristone and ivermectin, inhibitors of importin α/ß-mediated import, were able to reduce nuclear-associated capsid, while leptomycin B, a potent CRM1 inhibitor, confined capsid to the nucleus. In addition to altering the level and distribution of capsid, the three inhibitors were able to reduce viral titers in a relevant mammalian cell line with varying degrees of efficacy. The inhibitors were also able to reduce the cytopathic effects associated with VEEV infection, hinting that nuclear import inhibitors may be protecting cells from apoptosis in addition to disrupting the function of an essential viral protein. Our results confirm that VEEV uses host importins and exportins during part of its life cycle. Further, it suggests that temporarily targeting host proteins that are hijacked for use by viruses is a viable antiviral therapy.
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Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Ivermectina/farmacologia , Mifepristona/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Astrocitoma/patologia , Compartimento Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Efeito Citopatogênico Viral , Vírus da Encefalite Equina Venezuelana/fisiologia , Ácidos Graxos Insaturados/farmacologia , Humanos , Carioferinas/antagonistas & inibidores , Substâncias Macromoleculares , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Células Vero , Cultura de Vírus , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/genética , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genética , Proteína Exportina 1RESUMO
Beta-dystroglycan (beta-DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of beta-DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of beta-DG, characterizing a functional nuclear localization signal (NLS) in the beta-DG cytoplasmic domain, within amino acids 776-782. The NLS either alone or in the context of the whole beta-DG protein was able to target the heterologous GFP protein to the nucleus, with site-directed mutagenesis indicating that amino acids R(779) and K(780) are critical for NLS functionality. The nuclear transport molecules Importin (Imp)alpha and Impbeta bound with high affinity to the NLS of beta-DG and were found to be essential for NLS-dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of beta-DG may result in cytoplasmic retention, with Y(892) playing a key role. beta-DG thus follows a conventional Impalpha/beta-dependent nuclear import pathway, with important implications for its potential function in the nucleus.