RESUMO
Pain is the most common reason for patients seeking medical care resulting in an estimated world market for analgesics of more than USD 50 billion. Pain is a highly complex, heterogeneous and dynamic process characterized by specific patterns of phenotypic sensory neuronal change. Current treatment options for pain include opioids and non-opioid analgesics, acetaminophen and non-steroidal anti-inflammatory drugs and other drug classes such as antidepressants and anticonvulsants and a combination thereof. Novel approaches are focusing on the optimization of side-effect profiles of opioid based analgesics, the improvement of selectivity for specific opioid receptors, or by addressing molecular gateways implicated in pain. Promising candidates in development target various types of voltage-gated ion channels and receptors for capsaicin and analogs. Currently, after decades of pain research it has to be stated that the assessment, prevention and treatment of pain in industrialized countries as well as in low-income and middle-income countries are neither adequate nor equitable. Further research is needed so that specifically chronic pain control can be improved and individualized.
Assuntos
Analgésicos/uso terapêutico , Anti-Inflamatórios não Esteroides/uso terapêutico , Dor/tratamento farmacológico , Analgésicos/química , Anti-Inflamatórios não Esteroides/química , Antidepressivos/química , Antidepressivos/uso terapêutico , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canabinoides/química , Canabinoides/uso terapêutico , Humanos , Medição da Dor , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/uso terapêutico , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Influenza virus uses a unique cap-snatching mechanism characterized by hijacking and cleavage of host capped pre-mRNAs, resulting in short capped RNAs, which are used as primers for viral mRNA synthesis. The PA subunit of influenza polymerase carries the endonuclease activity that catalyzes the host mRNA cleavage reaction. Here, we show that PA is a sequence selective endonuclease with distinct preference to cleave at the 3' end of a guanine (G) base in RNA. The G specificity is exhibited by the native influenza polymerase complex associated with viral ribonucleoprotein particles and is conferred by an intrinsic G specificity of the isolated PA endonuclease domain PA-Nter. In addition, RNA cleavage site choice by the full polymerase is also guided by cap binding to the PB2 subunit, from which RNA cleavage preferentially occurs at the 12th nt downstream of the cap. However, if a G residue is present in the region of 10-13 nucleotides from the cap, cleavage preferentially occurs at G. This is the first biochemical evidence of influenza polymerase PA showing intrinsic sequence selective endonuclease activity.
Assuntos
Endorribonucleases/metabolismo , Vírus da Influenza A/enzimologia , Clivagem do RNA , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Bases , Endorribonucleases/química , Estrutura Terciária de Proteína , RNA/química , Capuzes de RNA/metabolismo , RNA Polimerase Dependente de RNA/química , Ribonucleoproteínas/metabolismo , Especificidade por Substrato , Proteínas Virais/químicaRESUMO
It is generally recognised that novel antiviral drugs, less prone to resistance, would be a desirable alternative to current drug options in order to be able to treat potentially serious influenza infections. The viral polymerase, which performs transcription and replication of the RNA genome, is an attractive target for antiviral drugs since potent polymerase inhibitors could directly stop viral replication at an early stage. Recent structural studies on functional domains of the heterotrimeric polymerase, which comprises subunits PA, PB1 and PB2, open the way to a structure based approach to optimise inhibitors of viral replication. In particular, the unique cap-snatching mechanism of viral transcription can be inhibited by targeting either the PB2 cap-binding or PA endonuclease domains. Here we describe high resolution X-ray co-crystal structures of the 2009 pandemic H1N1 (pH1N1) PA endonuclease domain with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme. Comparison of the binding mode of the different compounds and that of a mononucleotide phosphate highlights, firstly, how different substituent groups on the basic metal binding scaffold can be orientated to bind in distinct sub-pockets within the active site cavity, and secondly, the plasticity of certain structural elements of the active site cavity, which result in induced fit binding. These results will be important in optimising the design of more potent inhibitors targeting the cap-snatching endonuclease activity of influenza virus polymerase.
Assuntos
Antivirais/química , Quelantes/química , Endorribonucleases , Vírus da Influenza A Subtipo H1N1/enzimologia , Manganês/química , RNA Polimerase Dependente de RNA , Proteínas Virais , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Cães , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/química , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/químicaRESUMO
The influenza virus infection remains a significant threat to public health and the increase of antiviral resistance to available drugs generates an urgent need for new antiviral compounds. Starting from the natural, antivirally active compound glycyrrhizin, spacer-bridged derivatives were generated with improved antiviral activity against the influenza A virus infection. Simplified analogues of the triterpene saponin glycyrrhizin containing 1-thio-ß-D-glucuronic acid residues have been prepared in good yields by alkylation of 3-amino and 3-thio derivatives of glycyrrhetinic acid with a 2-iodoethyl 1-thio-ß-D-glucopyranosiduronate derivative. The spacer-connected 3-amino derivatives were further transformed into N-acetylated and N-succinylated derivatives. The deprotected compounds containing these carboxylic acid appendices mimic the glycon part of glycyrrhizin as well as the hemisuccinate derivative of glycyrrhetinic acid, carbenoxolone. Antiviral activities of the compounds were determined in a biological test based on influenza A virus-infected cells, wherein the 3-(2-thioethyl)-N-acetylamino- and 3-(2-thioethyl)-thio-linked glucuronide derivatives were effective inhibitors with IC(50) values as low as 54 µM.
RESUMO
The triterpene glycoside glycyrrhizin is the main active compound in liquorice. It is used as a herbal medicine owing to its anticancer, antiviral and anti-inflammatory properties. Its mode of action, however, remains widely unknown. In the present study, we aimed to elucidate the molecular mechanism of glycyrrhizin in attenuating inflammatory responses in macrophages. Using microarray analysis, we found that glycyrrhizin caused a broad block in the induction of pro-inflammatory mediators induced by the TLR (Toll-like receptor) 9 agonist CpG-DNA in RAW 264.7 cells. Furthermore, we found that glycyrrhizin also strongly attenuated inflammatory responses induced by TLR3 and TLR4 ligands. The inhibition was accompanied by decreased activation not only of the NF-kappaB (nuclear factor kappaB) pathway but also of the parallel MAPK (mitogen-activated protein kinase) signalling cascade upon stimulation with TLR9 and TLR4 agonists. Further analysis of upstream events revealed that glycyrrhizin treatment decreased cellular attachment and/or uptake of CpG-DNA and strongly impaired TLR4 internalization. Moreover, we found that the anti-inflammatory effects were specific for membrane-dependent receptor-mediated stimuli, as glycyrrhizin was ineffective in blocking Tnfa (tumour necrosis factor alpha gene) induction upon stimulation with PMA, a receptor- and membrane-independent stimulus. These observations suggest that the broad anti-inflammatory activity of glycyrrhizin is mediated by the interaction with the lipid bilayer, thereby attenuating receptor-mediated signalling.
Assuntos
Anti-Inflamatórios/farmacologia , Glycyrrhiza/química , Ácido Glicirrízico/farmacologia , Receptores de Superfície Celular/imunologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anti-Inflamatórios/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Glycyrrhiza/imunologia , Ácido Glicirrízico/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
Novel antiviral drugs, which are less prone to resistance development, are desirable alternatives to the currently approved drugs for the treatment of potentially serious influenza virus infections. The viral polymerase is highly conserved and serves as an attractive target for antiviral drugs since potent inhibitors would directly stop viral replication at an early stage. Recent structural studies on the functional domains of the heterotrimeric influenza polymerase, which comprises subunits PA, PB1, and PB2, opened the way to a structure-based approach for optimizing inhibitors of viral replication. These strategies, however, are limited by the use of isolated protein fragments instead of employing the entire ribonucleoprotein complex (RNP), which represents the functional form of the influenza polymerase in infected cells. In this study, we have established a screening assay for efficient and reliable analysis of potential influenza polymerase inhibitors of various molecular targets such as monoselective polymerase inhibitors targeting the endonuclease site, the cap-binding domain, and the polymerase active site, respectively. By utilizing whole viral RNPs and a radioactivity-free endpoint detection with the capability for efficient compound screening while offering high-content information on potential inhibitors to drive medicinal chemistry program in a reliable manner, this biochemical assay provides significant advantages over the currently available conventional assays. We propose that this assay can eventually be adapted for coinstantaneous analysis and subsequent optimization of two or more different chemical scaffold classes targeting multiple active sites within the polymerase complex, thus enabling the evaluation of drug combinations and characterization of molecules with dual functionality.
Assuntos
Antivirais/análise , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/análise , Vírus da Influenza A/enzimologia , Ribonucleoproteínas/análise , Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Ribonucleoproteínas/genética , Ribonucleoproteínas/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
The heterotrimeric influenza virus polymerase performs replication and transcription of viral RNA in the nucleus of infected cells. Transcription by "cap-snatching" requires that host-cell pre-mRNAs are bound via their 5' cap to the PB2 subunit. Thus, the PB2 cap-binding site is potentially a good target for new antiviral drugs that will directly inhibit viral replication. Docking studies using the structure of the PB2 cap-binding domain suggested that 7-alkylguanine derivatives substituted at position N-9 and N-2 could be good candidates. Four series of 7,9-di- and 2,7,9-trialkyl guanine derivatives were synthesized and evaluated by an AlphaScreen assay in competition with a biotinylated cap analogue. Three synthesized compounds display potent in vitro activity with IC50 values lower than 10 µM. High-resolution X-ray structures of three inhibitors in complex with the H5N1 PB2 cap-binding domain confirmed the binding mode and provide detailed information for further compound optimization.
Assuntos
Guanina/análogos & derivados , Vírus da Influenza A/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Guanina/síntese química , Guanina/química , Guanina/farmacologia , Modelos Moleculares , Estrutura Molecular , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/metabolismoRESUMO
We investigated the mechanism by which glycyrrhizin (GL), the main active component of licorice roots, protects cells from infection with influenza A virus (IAV). We found that GL treatment leads to a clear reduction in the number of IAV-infected human lung cells as well as a reduction in the CCID50 titer by 90%. The antiviral effect, however, was limited to one or two virus replication cycles. Analysis of different GL treatment protocols suggested that the antiviral effect of GL was limited to an early step in the virus replication cycle. A direct inhibitory action of GL on IAV particles could be excluded and GL did not interact with virus receptor binding either. The antiviral effect of GL was abolished by treatment 1h after virus infection, whereas pre-treatment and treatment during and after virus adsorption led to a reduction in the cytopathic effect, reduced viral RNA within the cells and in the cell supernatants, and reduced viral hemagglutination titers. Detailed virus uptake analyses unambiguously demonstrated reduced virus uptake in various GL-treated cells. These observations lead to the conclusion, that the antiviral activity of GL is mediated by an interaction with the cell membrane which most likely results in reduced endocytotic activity and hence reduced virus uptake. These insights might help in the design of structurally related compounds leading to potent anti-influenza therapeutics.
Assuntos
Antivirais/farmacologia , Ácido Glicirrízico/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/virologia , Glycyrrhiza/química , Humanos , Vírus da Influenza A/fisiologiaRESUMO
In this study, several influenza NS1 mutants were examined for their growth ability in interferon (IFN)-deficient Vero cells treated with human interferon alpha (IFN-alpha). Mutants with an intact RNA binding domain showed similar growth properties as the wild-type virus, whereas viruses carrying an impaired RNA binding domain were dramatically attenuated. Relying on the ability of the first half of the NS1 protein to antagonize the IFN action, we established a rescue system for the NS gene based on the transfection of one plasmid expressing recombinant NS vRNA and subsequent coinfection with an IFN sensitive helper virus followed by adding of human IFN-alpha as a selection drug. Using this method, a recombinant influenza A virus expressing green fluorescence protein (GFP) from the NS1 reading frame was rescued. To ensure the posttranslational cleavage of GFP from the N-terminal 125 amino acids (aa) of NS1 protein, a peptide sequence comprising a caspase recognition site (CRS) was inserted upstream the GFP protein. Although a rather long sequence of 275 aa was inserted into the NS1 reading frame, the rescued recombinant vector appeared to be genetically stable while passaging in Vero cells and was able to replicate in PKR knockout mice.