Dihydromyricetin is a new inhibitor of influenza polymerase PB2 subunit and influenza-induced inflammation

Development of new and effective anti-influenza drugs is critical for the treatment of influenza virus infection. The polymerase basic 2 (PB2) subunit as a core subunit of influenza A virus RNA polymerase complex is considered to be an attractive drug target for anti-influenza drug discovery. Dihydromyricetin, as a natural flavonoid, has a wide range of biological activities, but its anti-influenza A virus activity is ambiguous. Here, we found dihydromyricetin could inhibit the replication of a variety of influenza A virus strains. Mechanism studies demonstrated that dihydromyricetin reduced viral polymerase activity via selective inhibition of viral PB2 subunit, and decreased relative amounts of viral mRNA and genomic RNA during influenza A virus infection. The binding affinity and molecular docking analyses revealed that dihydromyricetin interacted with the PB2 cap-binding pocket, functioned as a cap-binding competitor. Interestingly, dihydromyricetin also reduced cellular immune injury by inhibiting TLR3 signaling pathway. Additionally, combination treatment of dihydromyricetin with zanamivir exerted a synergistic anti-influenza effect. Altogether, our experiments reveal the antiviral and anti-inflammatory activities of dihydromyricetin in vitro against influenza virus infection, which provides a new insight into the development of novel anti-influenza drugs.     

A Drosophila model for genetic analysis of influenza viral/host interactions

Influenza viruses impose a constant threat to vertebrates susceptible to this family of viruses. We have developed a new tool to study virus-host interactions that play key roles in viral replication and to help identify novel anti-influenza drug targets. Via the UAS/Gal4 system we ectopically expressed the influenza virus M2 gene in Drosophila melanogaster and generated dose-sensitive phenotypes in the eye and wing. We have confirmed that the M2 proton channel is properly targeted to cell membranes in Drosophila tissues and functions as a proton channel by altering intracellular pH. As part of the efficacy for potential anti-influenza drug screens, we have also demonstrated that the anti-influenza drug amantadine, which targets the M2 proton channel, suppressed the UAS-M2 mutant phenotype when fed to larvae. In a candidate gene screen we identified mutations in components of the vacuolar V1V0 ATPase that modify the UAS-M2 phenotype. Importantly, in this study we demonstrate that Drosophila genetic interactions translate directly to physiological requirements of the influenza A virus for these components in mammalian cells. Overexpressing specific V1 subunits altered the replication capacity of influenza virus in cell culture and suggests that drugs targeting the enzyme complex via these subunits may be useful in anti-influenza drug therapies. Moreover, this study adds credence to the idea of using the M2 "flu fly" to identify new and previously unconsidered cellular genes as potential drug targets and to provide insight into basic mechanisms of influenza virus biology.     

Gene Expression Signature-Based Screening Identifies New Broadly Effective Influenza A Antivirals

Classical antiviral therapies target viral proteins and are consequently subject to resistance. To counteract this limitation, alternative strategies have been developed that target cellular factors. We hypothesized that such an approach could also be useful to identify broad-spectrum antivirals. The influenza A virus was used as a model for its viral diversity and because of the need to develop therapies against unpredictable viruses as recently underlined by the H1N1 pandemic. We proposed to identify a gene-expression signature associated with infection by different influenza A virus subtypes which would allow the identification of potential antiviral drugs with a broad anti-influenza spectrum of activity. We analyzed the cellular gene expression response to infection with five different human and avian influenza A virus strains and identified 300 genes as differentially expressed between infected and non-infected samples. The most 20 dysregulated genes were used to screen the connectivity map, a database of drug-associated gene expression profiles. Candidate antivirals were then identified by their inverse correlation to the query signature. We hypothesized that such molecules would induce an unfavorable cellular environment for influenza virus replication. Eight potential antivirals including ribavirin were identified and their effects were tested in vitro on five influenza A strains. Six of the molecules inhibited influenza viral growth. The new pandemic H1N1 virus, which was not used to define the gene expression signature of infection, was inhibited by five out of the eight identified molecules, demonstrating that this strategy could contribute to identifying new broad anti-influenza agents acting on cellular gene expression. The identified infection signature genes, the expression of which are modified upon infection, could encode cellular proteins involved in the viral life cycle. This is the first study showing that gene expression-based screening can be used to identify antivirals. Such an approach could accelerate drug discovery and be extended to other pathogens.     

甲型流感病毒RNA聚合酶——抗病毒药物靶点

甲型流感病毒的RNA聚合酶由PB1、PB2和PA 三个亚基组成,在病毒的生命周期中负责行使病毒基因组的转录与复制等多方面功能. 甲型流感病毒由于频繁变异,导致其对传统抗病毒药物的敏感性降低,因此开发疗效好、针对性强、毒性低的新型抗病毒药物已成为当前亟待解决的问题.由于RNA聚合酶是甲型流感病毒生命周期重要的调控蛋白,并且编码聚合酶各亚基的基因序列具有高度保守性,故成为当前抗病毒药物的重要靶点.     

Pterostilbene effectively inhibits influenza A virus infection by promoting the type I interferon production

With the prevalence of novel strains and drug-resistant influenza viruses, there is an urgent need to develop effective and low-toxicity anti-influenza therapeutics. Regulation of the type I interferon antiviral response is considered an attractive therapeutic strategy for viral infection. Pterostilbene, a 3,5-dimethoxy analog of resveratrol, is known for its remarkable pharmacological activity. Here, we found that pterostilbene effectively inhibited influenza A virus infection and mainly affected the late stages of viral replication. A mechanistic study showed that the antiviral activity of pterostilbene might promote the induction of antiviral type I interferon and expression of its downstream interferon-stimulated genes during viral infection. The same effect of pterostilbene was also observed in the condition of polyinosinic-polycytidylic acid (poly I:C) transfection. Further study showed that pterostilbene interacted with influenza non-structural 1 (NS1) protein, inhibited ubiquitination mediated degradation of RIG-I and activated the downstream antiviral pathway, orchestrating an antiviral state against influenza virus in the cell. Taken together, pterostilbene could be a promising anti-influenza agent for future antiviral drug exploitation and compounds with similar structures may provide new options for the development of novel inhibitors against influenza A virus (IAV).     

Pomegranate (Punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir

Influenza epidemics cause numerous deaths and millions of hospitalizations each year. Because of the alarming emergence of resistance to anti-influenza drugs, there is a need to identify new naturally occurring antiviral molecules. We tested the hypothesis that pomegranate polyphenol extract (PPE) has anti-influenza properties. Using real time PCR, plaque assay, and TCID 50% hemagglutination assay, we have shown that PPE suppresses replication of influenza A virus in MDCK cells. PPE inhibits agglutination of chicken red blood cells (cRBC) by influenza virus and is virucidal. The single-cycle growth conditions indicated that independent of the virucidal effect PPE also inhibits viral RNA replication. PPE did not alter virus ribonucleoprotein (RNP) entry into nucleus or translocation of virus RNP from nucleus to cytoplasm in MDCK cells. We evaluated four major Polyphenols in PPE (ellagic acid, caffeic acid, luteolin, and punicalagin) and demonstrated that punicalagin is the effective, anti-influenza component of PPE. Punicalagin blocked replication of the virus RNA, inhibited agglutination of chicken RBC's by the virus and had virucidal effects. Furthermore, the combination of PPE and oseltamivir synergistically increased the anti-influenza effect of oseltamivir. In conclusion, PPE inhibited the replication of human influenza A/Hong Kong (H3N2) in vitro. Pomegranate extracts should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics.     

The NF‐κB inhibitor SC75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance

Ongoing human infections with highly pathogenic avian H5N1 viruses and the emergence of the pandemic swine‐origin influenza viruses (IV) highlight the permanent threat elicited by these pathogens. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti‐influenza drugs. The recently identified virus‐supportive function of the cellular IKK/NF‐κB signalling pathway suggests this signalling module as a potential target for antiviral intervention. We characterized the NF‐κB inhibitor SC75741 as a broad and efficient blocker of IV replication in non‐toxic concentrations. The underlying molecular mechanism of SC75741 action involves impaired DNA binding of the NF‐κB subunit p65, resulting in reduced expression of cytokines, chemokines, and pro‐apoptotic factors, subsequent inhibition of caspase activation and block of caspase‐mediated nuclear export of viralribonucleoproteins. SC75741 reduces viral replication and H5N1‐induced IL‐6 and IP‐10 expression in the lung of infected mice. Besides its virustatic effect the drug suppresses virus‐induced overproduction of cytokines and chemokines, suggesting that it might prevent hypercytokinemia that is discussed to be an important pathogenicity determinant of highly pathogenic IV. Importantly the drug exhibits a high barrier for development of resistant virus variants. Thus, SC75741‐derived drugs may serve as broadly non‐toxic anti‐influenza agents.     

Synthesis and biological evaluation of dihydrofuran-fused perhydrophenanthrenes as a new anti-influenza agent having novel structural characteristic

Dihydrofuran-fused perhydrophenanthrenes were synthesized by means of o-quinodimethane chemistry with high generality and stereoselectivity, and found to exhibit potent anti-influenza activity. These compounds exerted an inhibitory effect on various strains of influenza virus growth, including influenza A and B, with a concentration dependent manner, and direct cytotoxicity was low. Several biological experiments suggested that these new drugs affected a virus replication process before mRNA synthesis stage. Novel rigid cage-type of structural characteristic of the compounds has not been found in hitherto anti-influenza drugs, and will provide new basis and motif for exploring promising and unprecedented anti-influenza agents.     

Avian influenza virus, a very sticky situation

The appearance of the highly pathogenic avian influenza virus H5N1 highlighted the potential impact of influenza virus on humanity. The emergence of this high profile virus stimulated much research towards a better understanding of the key determinants for successful human-to-human transmission and as such has provided new directions for therapeutic intervention strategies. For example, a phylogenetic-based grouping of influenza virus sialidases into either Group 1 or 2 has been proposed. This has provided new opportunity for the development of Group 1-specific anti-influenza drugs. Furthermore, a number of next generation sialidase inhibitors as anti-influenza drugs have also been developed.     

ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases

Background

Influenza viruses are a major cause of morbidity and mortality around the world. More recently, a swine-origin influenza A (H1N1) virus that is spreading via human-to-human transmission has become a serious public concern. Although vaccination is the primary strategy for preventing infections, influenza antiviral drugs play an important role in a comprehensive approach to controlling illness and transmission. In addition, a search for influenza-inhibiting drugs is particularly important in the face of high rate of emergence of influenza strains resistant to several existing influenza antivirals.

Methods

We searched for novel anti-influenza inhibitors using a cell-based neutralization (inhibition of virus-induced cytopathic effect) assay. After screening 20,800 randomly selected compounds from a library from ChemDiv, Inc., we found that BPR1P0034 has sub-micromolar antiviral activity. The compound was resynthesized in five steps by conventional chemical techniques. Lead optimization and a structure-activity analysis were used to improve potency. Time-of-addition assay was performed to target an event in the virus life cycle.

Results

The 50% effective inhibitory concentration (IC₅₀) of BPR1P0034 was 0.42 ± 0.11 μM, when measured with a plaque reduction assay. Viral protein and RNA synthesis of A/WSN/33 (H1N1) was inhibited by BPR1P0034 and the virus-induced cytopathic effects were thus significantly reduced. BPR1P0034 exhibited broad inhibition spectrum for influenza viruses but showed no antiviral effect for enteroviruses and echovirus 9. In a time-of-addition assay, in which the compound was added at different stages along the viral replication cycle (such as at adsorption or after adsorption), its antiviral activity was more efficient in cells treated with the test compound between 0 and 2 h, right after viral infection, implying that an early step of viral replication might be the target of the compound. These results suggest that BPR1P0034 targets the virus during viral uncoating or viral RNA importation into the nucleus.

Conclusions

To the best of our knowledge, BPR1P0034 is the first pyrazole-based anti-influenza compound ever identified and characterized from high throughput screening to show potent (sub-μM) antiviral activity. We conclude that BPR1P0034 has potential antiviral activity, which offers an opportunity for the development of a new anti-influenza virus agent.     

抗流感病毒小RNAs研究进展

流行性感冒是一类由流感病毒引起的呼吸道传染病, 通过季节性流行或全球性爆发严重威胁着人类健康。目前防治流感的主要方法是疫苗和药物, 但存在神经毒性、肠胃副作用、易耐药等诸多限制因素。新的技术特别是小RNAs介导的RNA干扰(RNAi)技术, 因其具有高效、特异、快速等特点, 已成为抗病毒治疗的候选方法之一。随着近年来流感病毒的流行, 应用小RNAs抗流感病毒的报导越来越多, 其中靶向PA、NP和M2的PA-2087, NP-1496和M-950是目前报道的抑制流感病毒效果最好的siRNA。靶向不同流感病毒基因保守区域的siRNA具有更广泛的病毒毒株抑制效果, 靶向不同基因的siRNAs联合使用可取得更好的病毒抑制效果。文章就目前siRNAs和miRNAs在抗流感病毒方面的研究进展及RNAi治疗的前景和问题进行了综述。     

抗流感病毒小RNAs研究进展

流行性感冒是一类由流感病毒引起的呼吸道传染病,通过季节性流行或全球性爆发严重威胁着人类健康。目前防治流感的主要方法是疫苗和药物,但存在神经毒性、肠胃副作用、易耐药等诸多限制因素。新的技术特别是小RNAs介导的RNA干扰(RNAi)技术,因其具有高效、特异、快速等特点,已成为抗病毒治疗的候选方法之一。随着近年来流感病毒的流行,应用小RNAs抗流感病毒的报导越来越多,其中靶向PA、NP和M2的PA-2087,NP-1496和M-950是目前报道的抑制流感病毒效果最好的siRNA。靶向不同流感病毒基因保守区域的siRNA具有更广泛的病毒毒株抑制效果,靶向不同基因的siRNAs联合使用可取得更好的病毒抑制效果。文章就目前siRNAs和miRNAs在抗流感病毒方面的研究进展及RNAi治疗的前景和问题进行了综述。     

Pyrazole compound BPR1P0034 with potent and selective anti-influenza virus activity

Background

Influenza viruses are a major cause of morbidity and mortality around the world. More recently, a swine-origin influenza A (H1N1) virus that is spreading via human-to-human transmission has become a serious public concern. Although vaccination is the primary strategy for preventing infections, influenza antiviral drugs play an important role in a comprehensive approach to controlling illness and transmission. In addition, a search for influenza-inhibiting drugs is particularly important in the face of high rate of emergence of influenza strains resistant to several existing influenza antivirals.

Methods

We searched for novel anti-influenza inhibitors using a cell-based neutralization (inhibition of virus-induced cytopathic effect) assay. After screening 20,800 randomly selected compounds from a library from ChemDiv, Inc., we found that BPR1P0034 has sub-micromolar antiviral activity. The compound was resynthesized in five steps by conventional chemical techniques. Lead optimization and a structure-activity analysis were used to improve potency. Time-of-addition assay was performed to target an event in the virus life cycle.

Results

The 50% effective inhibitory concentration (IC₅₀) of BPR1P0034 was 0.42 ± 0.11 μM, when measured with a plaque reduction assay. Viral protein and RNA synthesis of A/WSN/33 (H1N1) was inhibited by BPR1P0034 and the virus-induced cytopathic effects were thus significantly reduced. BPR1P0034 exhibited broad inhibition spectrum for influenza viruses but showed no antiviral effect for enteroviruses and echovirus 9. In a time-of-addition assay, in which the compound was added at different stages along the viral replication cycle (such as at adsorption or after adsorption), its antiviral activity was more efficient in cells treated with the test compound between 0 and 2 h, right after viral infection, implying that an early step of viral replication might be the target of the compound. These results suggest that BPR1P0034 targets the virus during viral uncoating or viral RNA importation into the nucleus.

Conclusions

To the best of our knowledge, BPR1P0034 is the first pyrazole-based anti-influenza compound ever identified and characterized from high throughput screening to show potent (sub-μM) antiviral activity. We conclude that BPR1P0034 has potential antiviral activity, which offers an opportunity for the development of a new anti-influenza virus agent.     

Inhibition of Influenza Virus Replication by Targeting Broad Host Cell Pathways

Antivirals that are currently used to treat influenza virus infections target components of the virus which can mutate rapidly. Consequently, there has been an increase in the number of resistant strains to one or many antivirals in recent years. Here we compared the antiviral effects of lysosomotropic alkalinizing agents (LAAs) and calcium modulators (CMs), which interfere with crucial events in the influenza virus replication cycle, against avian, swine, and human viruses of different subtypes in MDCK cells. We observed that treatment with LAAs, CMs, or a combination of both, significantly inhibited viral replication. Moreover, the drugs were effective even when they were administered 8 h after infection. Finally, analysis of the expression of viral acidic polymerase (PA) revealed that both drugs classes interfered with early events in the viral replication cycle. This study demonstrates that targeting broad host cellular pathways can be an efficient strategy to inhibit influenza replication. Furthermore, it provides an interesting avenue for drug development where resistance by the virus might be reduced since the virus is not targeted directly.     

Influenza virus partially counteracts restriction imposed by tetherin/BST-2

Influenza virus infections lead to a burst of type I interferon (IFN) in the human respiratory tract, which most probably accounts for a rapid control of the virus. Although in mice, IFN-induced Mx1 factor mediates a major part of this response, the situation is less clear in humans. Interestingly, a recently identified IFN-induced cellular protein, tetherin (also known as CD317, BST-2, or HM1.24), exerts potent antiviral activity against a broad range of retroviruses, as well as several other enveloped viruses, by impeding the release of newly generated viral particles from the cell surface. Here we show that influenza virus belongs to the targets of this potent antiviral factor. Ectopic expression of tetherin strongly inhibited fully replicative influenza virus. In addition, depleting endogenous tetherin increased viral production of influenza virions, both in cells constitutively expressing tetherin and upon its induction by IFN. We further demonstrate, by biochemical and morphological means, that tetherin exerts its antiviral action by tethering newly budded viral particles, a mechanism similar to the one that operates against HIV-1. In addition, we determined that the magnitude of tetherin antiviral activity is comparable with or higher than the one of several previously identified anti-influenza cellular factors, such as MxA, ADAR1, ISG15, and viperin. Finally, we demonstrate that influenza virus reduces the impact of tetherin-mediated restriction on its replication by several mechanisms. First, the influenza virus NS1 protein impedes IFN-mediated tetherin induction. Second, influenza infection leads to a decrease of tetherin steady state levels, and the neuraminidase surface protein partly counteracts its activity. Overall, our study helps to delineate the intricate molecular battle taking place between influenza virus and its host cells.     

Evaluation of cytotoxicity and antiviral activity of Rhazya stricta Decne leaves extract against influenza A/PR/8/34 (H1N1)

Influenza viruses have developed resistance to the current classes of drugs, which means they could eventually become more virulent and cause more mortality and hospitalization. Our study aims to investigate the antiviral activity of Rhazya stricta Decne leaves extract in vitro and search for new promising drugs from R. stricta identified compounds in silico. The study was performed in vitro by utilizing Madin-Darby Canine Kidney cell line (MDCK) as a substrate for the influenza virus and estimating the inhibition performance of the plant leaves extract. Additionally, in silico screening was conducted to explore the antiviral activity of R. stricta phytochemicals. We investigated the cytotoxicity of R. stricta leaves extract and its antiviral activity against influenza virus (A/Puerto Rico/8/34 (H1N1)) using the MTT assay. The mode of action of the plant leaves extract during the viral life cycle was tested using time-of-addition assay. In silico analyses were performed, including molecular docking, drug-likeness analysis, and toxicity risk assessment, to state the leading compounds to be developed into an anti-influenza virus drug. The 50% cytotoxicity concentration of the leaves extract was CC50: 184.6 µg/mL, and the 50% inhibition concentration was CI50: 19.71 µg\mL. The time of addition assay revealed that R. stricta leaves extract exerted its activity in the late step of the influenza virus replication cycle. In comparison to Oseltamivir, the leading compounds showed better binding affinity and can be developed into oral drugs with low toxicity risk. Isolation and purification of the leading compounds and testing their antiviral activity in vitro and in vivo are required.