RESUMO
Justicia gendarussa, a medicinal plant collected in Vietnam, was identified as a potent anti-HIV-1 active lead from the evaluation of over 4500 plant extracts. Bioassay-guided separation of the extracts of the stems and roots of this plant led to the isolation of an anti-HIV arylnaphthalene lignan (ANL) glycoside, patentiflorin A (1). Evaluation of the compound against both the M- and T-tropic HIV-1 isolates showed it to possess a significantly higher inhibition effect than the clinically used anti-HIV drug AZT. Patentiflorin A and two congeners were synthesized, de novo, as an efficient strategy for resupply as well as for further structural modification of the anti-HIV ANL glycosides in the search for drug leads. Subsequently, it was determined that the presence of a quinovopyranosyloxy group in the structure is likely essential to retain the high degree of anti-HIV activity of this type of compounds. Patentiflorin A was further investigated against the HIV-1 gene expression of the R/U5 and U5/gag transcripts, and the data showed that the compound acts as a potential inhibitor of HIV-1 reverse transcription. Importantly, the compound displayed potent inhibitory activity against drug-resistant HIV-1 isolates of both the nucleotide analogue (AZT) and non-nucleotide analogue (nevaripine). Thus, the ANL glycosides have the potential to be developed as novel anti-HIV drugs.
Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , HIV-1/efeitos dos fármacos , Justicia/química , Lignanas/isolamento & purificação , Lignanas/farmacologia , Plantas Medicinais/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/química , Glicosídeos/química , HIV-1/genética , Humanos , Lignanas/química , Estrutura Molecular , Raízes de Plantas/química , Caules de Planta/química , Inibidores da Transcriptase Reversa/química , Vietnã , Zidovudina/farmacologiaRESUMO
UNLABELLED: Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients. IMPORTANCE: Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.
Assuntos
Filoviridae/fisiologia , Glicosaminoglicanos/fisiologia , N-Acetilglucosaminiltransferases/fisiologia , Internalização do Vírus , Animais , Linhagem Celular , Ebolavirus/patogenicidade , Ebolavirus/fisiologia , Filoviridae/patogenicidade , Infecções por Filoviridae/etiologia , Infecções por Filoviridae/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Heparina/fisiologia , Heparitina Sulfato/biossíntese , Heparitina Sulfato/deficiência , Interações Hospedeiro-Patógeno , Humanos , Marburgvirus/patogenicidade , Marburgvirus/fisiologia , Camundongos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , Receptores Virais/fisiologia , Proteínas Virais/fisiologia , VirulênciaRESUMO
BACKGROUND: Genome-wide RNAi screening has been widely used to identify host proteins involved in replication and infection of different viruses, and numerous host factors are implicated in the replication cycles of these viruses, demonstrating the power of this approach. However, discrepancies on target identification of the same viruses by different groups suggest that high throughput RNAi screening strategies need to be carefully designed, developed and optimized prior to the large scale screening. METHODS: Two genome-wide RNAi screens were performed in parallel against the entry of pseudotyped Marburg viruses and avian influenza virus H5N1 utilizing an HIV-1 based surrogate system, to identify host factors which are important for virus entry. A comparative analysis approach was employed in data analysis, which alleviated systematic positional effects and reduced the false positive number of virus-specific hits. RESULTS: The parallel nature of the strategy allows us to easily identify the host factors for a specific virus with a greatly reduced number of false positives in the initial screen, which is one of the major problems with high throughput screening. The power of this strategy is illustrated by a genome-wide RNAi screen for identifying the host factors important for Marburg virus and/or avian influenza virus H5N1 as described in this study. CONCLUSIONS: This strategy is particularly useful for highly pathogenic viruses since pseudotyping allows us to perform high throughput screens in the biosafety level 2 (BSL-2) containment instead of the BSL-3 or BSL-4 for the infectious viruses, with alleviated safety concerns. The screening strategy together with the unique comparative analysis approach makes the data more suitable for hit selection and enables us to identify virus-specific hits with a much lower false positive rate.
Assuntos
Interações Hospedeiro-Patógeno , Virus da Influenza A Subtipo H5N1/fisiologia , Marburgvirus/fisiologia , Interferência de RNA , Internalização do Vírus , Testes Genéticos/métodos , HIV-1/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Marburgvirus/genéticaRESUMO
Avian influenza viruses continue to threaten globally with pandemic potential. The first step in a potential pandemic is the ability of the virus to enter human cells which is mediated by the viral surface glycoprotein hemagglutinin (HA). Viral entry of influenza is dependent upon the processing of the HA0 polypeptide precursor protein into HA1 and HA2 which is mediated by host cellular proteases. The sequence of the cleavage site which is recognized by host proteases has been linked with pathogenesis of various influenza viruses. Here we examined the effects of cleavage site sequences between a highly pathogenic H5N1 strain and a low pathogenic H5N2 strain to determine their effects on viral entry. From this analysis we determined that at the level of viral entry, the only observed difference between the low and high pathogenic strains is their ability to be cleaved by host cellular proteases.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A Subtipo H5N2/fisiologia , Peptídeo Hidrolases/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Animais , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A Subtipo H5N2/genética , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
BACKGROUND: Avian influenza virus H5N1 is a major concern as a potential global pandemic. It is thought that multiple key events must take place before efficient human-to-human transmission of the virus occurs. The first step in overcoming host restriction is viral entry which is mediated by HA, responsible for both viral attachment and viral/host membrane fusion. HA binds to glycans-containing receptors with terminal sialic acid (SA). It has been shown that avian influenza viruses preferentially bind to alpha2,3-linked SAs, while human influenza A viruses exhibit a preference for alpha2,6-linked SAs. Thus it is believed the precise linkage of SAs on the target cells dictate host tropism of the viruses. RESULTS: We demonstrate that H5N1 HA/HIV pseudovirus can efficiently transduce several human cell lines including human lung cells. Interestingly, using a lectin binding assay we show that the presence of both alpha2,6-linked and alpha2,3-linked SAs on the target cells does not always correlate with efficient transduction. Further, HA substitutions of the residues implicated in switching SA-binding between avian and human species did not drastically affect HA-mediated transduction of the target cells or target cell binding. CONCLUSION: Our results suggest that a host factor(s), which is yet to be identified, is required for H5N1 entry in the host cells.
Assuntos
Hemaglutininas/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/virologia , Tropismo , Internalização do Vírus , Células 3T3 , Cloreto de Amônio/farmacologia , Animais , Aves , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Análise Mutacional de DNA , Inibidores Enzimáticos/farmacologia , HIV/fisiologia , Células HeLa , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Células Jurkat , Macrolídeos/farmacologia , Camundongos , Neuraminidase/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/metabolismo , Ácidos Siálicos/análise , Células Vero , Internalização do Vírus/efeitos dos fármacosRESUMO
In a search for new anti-HIV active leads from over several thousands of plant extracts, we have identified a potent plant lead. The active plant is determined as Justicia gendarussa (Acanthaceae), a medicinal plant that has been used for the treatment of injury, arthritis and rheumatism in Asia including China. Our bioassay-guided fractionation of the methanol extract of the stems and barks of the plant led to the isolation of two anti-HIV compounds, justiprocumins A and B. The compounds are identified as new arylnaphthalide lignans (ANL) glycosides. We further determined that the ANL glycosides are the chemical constituents that contribute to the anti-HIV activity of this plant. Justiprocumin B displayed potent activity against a broad spectrum of HIV strains with IC50 values in the range of 15-21 nM (AZT, IC50 77-95 nM). The compound also displayed potent inhibitory activity against the NRTI (nucleoside reverse transcriptase inhibitor)-resistant isolate (HIV-11617-1) of the analogue (AZT) as well as the NNRTI (non-nucleoside reverse transcriptase inhibitor)-resistant isolate (HIV-1N119) of the analogue (nevaripine).
Assuntos
Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Benzodioxóis/isolamento & purificação , Benzodioxóis/farmacologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , HIV-1/efeitos dos fármacos , Justicia/química , Lignanas/isolamento & purificação , Lignanas/farmacologia , Fármacos Anti-HIV/química , Benzodioxóis/química , Medicamentos de Ervas Chinesas/química , Glicosídeos/química , Concentração Inibidora 50 , Lignanas/química , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Influenza A viruses have broad host tropism, being able to infect a range of hosts from wild fowl to swine to humans. This broad tropism makes highly pathogenic influenza A strains, such as H5N1, potentially dangerous to humans if they gain the ability to jump from an animal reservoir to humans. How influenza A viruses are able to jump the species barrier is incompletely understood due to the complex genetic nature of the viral surface glycoprotein, hemagglutinin, which mediates entry, combined with the virus's ability to use various receptor linkages. Current therapeutics against influenza A include those that target the uncoating process after entry as well as those that prevent viral budding. While there are therapeutics in development that target entry, currently there are none clinically available. We review here the genetics of influenza A viruses that contribute to entry tropism, how these genetic alterations may contribute to receptor usage and species tropism, as well as how novel therapeutics can be developed that target the major surface glycoprotein, hemagglutinin.
RESUMO
Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre-messenger RNA (mRNA) splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB), is overexpressed in ovarian tumors compared with matched normal controls, and knockdown of PTB expression by short-hairpin RNA impairs ovarian tumor cell growth, colony formation, and invasiveness. Given the complexity of PTB's molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high-throughput screening assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high-throughput assay could be used to identify small-molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors, targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.
Assuntos
Processamento Alternativo , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas , Processamento Alternativo/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Proteínas do Tecido Nervoso/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Precursores de RNA/genética , Reprodutibilidade dos TestesRESUMO
As prophylactic therapies and vaccines against viral infections continue to improve, drug resistant strains are continuing to arise; therefore it is imperative to develop new therapeutics against these diseases. For highly pathogenic viruses, such as Ebola and H5N1 influenza virus, the need for antivirals is even more urgent due to limited therapeutics against these viruses. Furthermore, the high pathogenicity of such viruses often makes it difficult to work with such agents. In this report, we describe a protocol called "One-stone-two-birds" which provides a safe and efficient screening system to identify anti-flu (entry) and anti-HIV (replication) activities. Using plant extracts as an example, we demonstrate the utility of this protocol in antiviral screening.