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Flaviviruses , including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans -membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics simulations. Models contained a predicted lumenal rimantadine-binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted therapies., Competing Interests: EB, GS, DL, RS, AM, RT, DM, HB, MB, CD, AK, AM, NR, RF, CM, AK, SG No competing interests declared, (© 2024, Brown et al.)

Repurposing of approved drugs allows strong savings in time and investment. Rimantadine is an FDA-approved drug for prevention and treatment of influenza A infection. Patent US2021330605 describes the use of rimantadine, an adamantane derivative, for the treatment of melanoma, breast cancer and head and neck squamous cell carcinoma. Rimantadine inhibited proliferation of cell lines of melanoma, breast cancer, and head and neck squamous cell carcinoma, increased the survival of mice injected with cancer cell lines and restores the expression of MHC class I. Rimantadine has the potential to be used successfully in the treatment of head and neck squamous cell carcinoma.

Hepatitis A virus (HAV) infection is a major cause of acute viral hepatitis worldwide. Furthermore, HAV causes acute liver failure or acute-on-chronic liver failure. However, no potent anti-HAV drugs are currently available in the clinical situations. There have been some reports that amantadine, a broad-spectrum antiviral, suppresses HAV replication in vitro . Therefore, we examined the effects of amantadine and rimantadine, derivates of adamantane, on HAV replication, and investigated the mechanisms of these drugs. In the present study, we evaluated the effects of amantadine and rimantadine on HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in cell culture infection systems. Amantadine and rimantadine significantly inhibited HAV replication at the post-entry stage in Huh7 cells. HAV infection inhibited autophagy by suppressing the autophagy marker light chain 3 and reducing number of lysosomes. Proteomic analysis on HAV-infected Huh7 cells treated by amantadine and rimantadine revealed the changes of the expression levels in 42 of 373 immune response-related proteins. Amantadine and rimantadine inhibited HAV replication, partially through the enhancement of autophagy. Taken together, our results suggest a novel mechanism by which HAV replicates along with the inhibition of autophagy and that amantadine and rimantadine inhibit HAV replication by enhancing autophagy. IMPORTANCE Amantadine, a nonspecific antiviral medication, also effectively inhibits HAV replication. Autophagy is an important cellular mechanism in several virus-host cell interactions. The results of this study provide evidence indicating that autophagy is involved in HAV replication and plays a role in the HAV life cycle. In addition, amantadine and its derivative rimantadine suppress HAV replication partly by enhancing autophagy at the post-entry phase of HAV infection in human hepatocytes. Amantadine may be useful for the control of acute HAV infection by inhibiting cellular autophagy pathways during HAV infection processes.

Most of the patients infected with Chikungunya virus (CHIKV) develop chronic manifestations characterized by pain and deformity in joints, impacting their quality of life. The aminoadamantanes, in their turn, have been exploited due to their biological activities, with amantadine and memantine recently described with anti-CHIKV activities. Here we evaluated the antiviral activity of rimantadine hydrochloride (rtdH), a well-known antiviral agent against influenza A, its platinum complex (Pt-rtd), and the precursor cis-[PtCl 2 (dmso) 2 ], against CHIKV infection in vitro. The rtdH demonstrated significant antiviral activity in all stages of CHIKV replication (29% in pre-treatment; 57% in early stages of infection; 60% in post-entry stages). The Pt-rtd complex protected the cells against infection in 92%, inhibited 100% of viral entry, mainly by a virucidal effect, and impaired 60% of post-entry stages. Alternatively, cis-[PtCl 2 (dmso) 2 ] impaired viral entry in 100% and post-entry steps in 60%, but had no effect in protecting cells when administered prior to CHIKV infection. Collectively, the obtained data demonstrated that rtdH and Pt-rtd significantly interfered in the early stages of CHIKV life cycle, with the strongest effect observed to Pt-rtd complex, which reduced up to 100% of CHIKV infection. Moreover, molecular docking analysis and infrared spectroscopy data (ATR-FTIR) suggest an interaction of Pt-rtd with CHIKV glycoproteins, potentially related to the mechanism of inhibition of viral entry by Pt-rtd. Through a migration retardation assay, it was also shown that Pt-rtd and cis-[PtCl 2 (dmso) 2 ] interacted with the dsRNA in 87% and 100%, respectively. The obtained results highlight the repurposing potential of rtdH as an anti-CHIKV drug, as well as the synthesis of promising platinum(II) metallodrugs with potential application for the treatment of CHIKV infections. Importance Chikungunya fever is a disease that can result in persistent symptoms due to the chronic infection process. Infected patients can develop physical disability, resulting and high costs to the health system and significant impacts on the quality of life of affected individuals. Additionally, there are no licensed vaccines or antivirals against the Chikungunya virus (CHIKV) and the virus is easily transmitted due to the abundance of viable vectors in epidemic regions. In this context, our study highlights the repurposing potential of the commercial drug rimantadine hydrochloride (rtdH) as an antiviral agent for the treatment of CHIKV infections. Moreover, our data demonstrated that a platinum(II)-rimantadine metallodrug (Pt-rtd) poses as a potent anti-CHIKV molecule with potential application for the treatment of Chikungunya fever. Altogether, rtdH and Pt-rtd significantly interfered in the early stages of CHIKV life cycle, reducing up to 100% of CHIKV infection in vitro., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment. We therefore propose amantadine as a novel, cheap, readily available and effective way to treat COVID-19., (© 2021. The Author(s).)

We report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.

The p7 viroporin of the hepatitis C virus (HCV) forms an intracellular proton-conducting transmembrane channel in virus-infected cells, shunting the pH of intracellular compartments and thus helping virus assembly and release. This activity is essential for virus infectivity, making viroporins an attractive target for drug development. The protein sequence and drug sensitivity of p7 vary between the seven major genotypes of the hepatitis C virus, but the essential channel activity is preserved. Here, we investigated the effect of several inhibitors on recombinant HCV p7 channels corresponding to genotypes 1a-b, 2a-b, 3a and 4a using patch-clamp electrophysiology and cell-based assays. We established a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assay for recombinant p7 expressed in HEK293 cells to assess channel activity and its sensitivity to inhibitors. The results from the cell viability assay were consistent with control measurements using established assays of haemadsorption and intracellular pH, and agreed with data from patch-clamp electrophysiology. Hexamethylene amiloride (HMA) was the most potent inhibitor of p7 activity, but possessed cytotoxic activity at higher concentrations. Rimantadine was active against p7 of all genotypes, while amantadine activity was genotype-dependent. The alkyl-chain iminosugars N B-DNJ, N N-DNJ and N N-DGJ were tested and their activity was found to be genotype-specific. In the current study, we introduce cell viability assays as a rapid and cost-efficient technique to assess viroporin activity and identify channel inhibitors as potential novel antiviral drugs.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Dr. Paul Targett-Adams is a former employee of Pfizer Ltd. He was involved collaboratively with this project during inception and early stages and acted as a contact and advocate during the application for the Pfizer pump-priming award. He is presently an employee of Sygnature Discovery, who are not involved with this work. C. Scott is a previous employee of ReViral Ltd. and is now an employee of Covance. However, neither body supplied funding to, nor influenced this manuscript.

The recent outbreak of coronavirus disease-19 (COVID-19) continues to drastically affect healthcare throughout the world. To date, no approved treatment regimen or vaccine is available to effectively attenuate or prevent the infection. Therefore, collective and multidisciplinary efforts are needed to identify new therapeutics or to explore effectiveness of existing drugs and drug-like small molecules against SARS-CoV-2 for lead identification and repurposing prospects. This study addresses the identification of small molecules that specifically bind to any of the three essential proteins (RdRp, 3CL-protease and helicase) of SARS-CoV-2. By applying computational approaches we screened a library of 4574 compounds also containing FDA-approved drugs against these viral proteins. Shortlisted hits from initial screening were subjected to iterative docking with the respective proteins. Ranking score on the basis of binding energy, clustering score, shape complementarity and functional significance of the binding pocket was applied to identify the binding compounds. Finally, to minimize chances of false positives, we performed docking of the identified molecules with 100 irrelevant proteins of diverse classes thereby ruling out the non-specific binding. Three FDA-approved drugs showed binding to 3CL-protease either at the catalytic pocket or at an allosteric site related to functionally important dimer formation. A drug-like molecule showed binding to RdRp in its catalytic pocket blocking the key catalytic residues. Two other drug-like molecules showed specific interactions with helicase at a key domain involved in catalysis. This study provides lead drugs or drug-like molecules for further in vitro and clinical investigation for drug repurposing and new drug development prospects., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have used the light-inducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis- regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti-PD-L1 immunotherapy significantly enhanced local control, CD8 + memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti-PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti-PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade. SIGNIFICANCE: This study identifies a novel mechanism of resistance to anti-PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/732/F1.large.jpg., (©2019 American Association for Cancer Research.)

Introduction: Adamantanthane-type drugs such as rimantadine and amantadine have long been used to treat diseases caused by influenza A virus. However, as a result of the mutations, influenza viruses have become resistant to aminoadamantans. The target for these drugs was the protein channel M2. Influenza A virus M2 viroporin in the protein shell forms fairly specific ion channels with a diameter of about 11 Å, specializing in transporting protons inside the viral particle (virion). Restoration of the antiviral properties of adamantanthane-type drugs consists in the selection of advanced functional groups bound by the carbocycle to find new sites of binding to the protein target M2. The purpose of the study is to identify the antiviral properties of new adamantanum derivatives to the pandemic strain of influenza A virus in vitro., Material and Methods: Compounds of aminoadamantans with amino acids and other organic molecules were obtained by classical peptide synthesis methods. The structure of the compound was tested by means of physical and chemical methods. Antiviral properties of synthetic compounds were studied in vitro on monolayer MDCK cells infected with pandemic strain of influenza A/California/07/2009 virus in two schemes of administration of investigated compounds and virus., Results: The reference strain of the influenza virus A/California/07/2009(H1N1) was sensitive to the compounds under test in varying degrees. The antiviral activity of the compounds was expressed in a 50% inhibitory concentration (IС 50 ) ranging from 0.5 to 2.5 мкM, which is generally a good indicator for the Rimantadine/Amantadine resistant strain., Discussion: The values of the IС 50 for compounds introduced two hours before contact with the virus were slightly higher than those for single-moment introduction of the substance and virus. The effect of increasing the inhibitory concentration in the prophylactic scheme of compounds was valid for all compounds of the experiment., Conclusion: The presented synthetic compounds are active against the variant of influenza A virus resistant to Rimantadine and Amantadine preparations. The obtained compounds can be used as model structures for creation of a new drug of direct action against advanced strains of influenza A virus., Competing Interests: The authors declare no conflict of interest.

HCV p7 protein is a cation-selective ion channel, playing an essential role during the life cycle of HCV viruses. To understand the cation-selective mechanism, we constructed a hexameric model in lipid bilayers of HCV p7 protein for HCB JFH-1 strain, genotype 2a. In this structural model, His9 and Val6 were key factors for the HCV cation-selective ion channel. The histidine residues at position 9 in the hexameric model formed a first gate for HCV p7 channel, acting as a selectivity filter for cations. The valines mentioned above formed a second gate for HCV p7 channel, serving as a hydrophobic filter for the dehydrated cations. The binding pocket for the channel blockers, e.g., amantadine and rimantadine, was composed of residues 20-26 in H2 helix and 52-60 in H3 helix in i + 2 monomer. However, the molecular volumes for both amantadine and rimantadine were too small for the binding pocket of HCV p7 channel. Thus, designing a compound similar with rimantadine and having much larger volume would be an effective strategy for discovering inhibitors against HCV p7 channel. To achieve this point, we used rimantadine as a structural template to search ChEMBL database for the candidates employing favorable binding affinities to HCV p7 channel. As a result, six candidates were identified to have potential to be novel inhibitors against HCV p7 channel.

Despite the availability of prophylactic vaccines the burden of human papillomavirus (HPV) associated malignancy remains high and there is a need to develop additional therapeutic strategies to complement vaccination. We have previously shown that the poorly characterised E5 oncoprotein forms a virus-coded ion channel or viroporin that was sensitive to the amantadine derivative rimantadine. We now demonstrate that alkylated imino sugars, which have antiviral activity against a number of viruses, inhibit E5 channel activity in vitro. Using molecular modelling we predict that imino sugars intercalate between E5 protomers to prevent channel oligomerisation. We explored the ability of these viroporin inhibitors to block E5-mediated activation of mitogenic signalling in keratinocytes. Treatment with either rimantadine or imino sugars prevented ERK-MAPK phosphorylation and reduced cyclin B1 expression in cells expressing E5 from a number of high-risk HPV types. Moreover, viroporin inhibitors also reduced ERK-MAPK activation and cyclin B1 expression in differentiating primary human keratinocytes containing high-risk HPV18. These observations provide evidence of a key role for E5 viroporin function during the HPV life cycle. Viroporin inhibitors could be utilised for stratified treatment of HPV associated tumours prior to virus integration, or as true antiviral therapies to eliminate virus prior to malignant transformation., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Adamantane-based compounds have been identified to interfere with the ion-channel activity of viroporins and thereby inhibit viral infection. To better understand the difference in the inhibition mechanism of viroporins, we synthesized symmetric dimeric adamantane analogs of various alkyl-spacer lengths., Methods: Symmetric dimeric adamantane derivatives were synthesized where two amantadine or rimantadine molecules were linked by various alkyl-spacers. The inhibitory activity of the compounds was studied on two viroporins: the influenza virus M2 protein, expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique, and the hepatitis C virus (HCV) p7 channels for five different genotypes (1a, 1b, 2a, 3a, and 4a) expressed in HEK293 cells using whole-cell patch-clamp recording techniques., Results: Upon testing on M2 protein, dimeric compounds showed significantly lower inhibitory activity relative to the monomeric amantadine. The lack of channel blockage of the dimeric amantadine and rimantadine analogs against M2 wild type and M2-S31N mutant was consistent with previously proposed drug-binding mechanisms and further confirmed that the pore-binding model is the pharmacologically relevant drug-binding model. On the other hand, these dimers showed similar potency to their respective monomeric analogs when tested on p7 protein in HCV genotypes 1a, 1b, and 4a while being 700-fold and 150-fold more potent than amantadine in genotypes 2a and 3a, respectively. An amino group appears to be important for inhibiting the ion-channel activity of p7 protein in genotype 2a, while its importance was minimal in all other genotypes., Conclusion: Symmetric dimeric adamantanes can be considered a prospective class of p7 inhibitors that are able to address the differences in adamantane sensitivity among the various genotypes of HCV., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

A series of (R)- and (S)-isomers of new adamantane-substituted heterocycles (1,3-oxazinan-2-one, piperidine-2,4-dione, piperidine-2-one and piperidine) with potent activity against rimantadine-resistant strains of influenza A virus were synthesized through the transformation of adamantyl-substituted N-Boc-homoallylamines 8 into piperidine-2,4-diones 11 through the cyclic bromourethanes 9 and key intermediate enol esters 10. Biological assays of the prepared compounds were performed on the rimantadine-resistant S31N mutated strains of influenza A - A/California/7/2009(H1N1)pdm09 and modern pandemic strain A/IIV-Orenburg/29-L/2016(H1N1)pdm09. The most potent compounds were both enantiomers of the enol ester 10 displaying IC 50 = 7.7 μM with the 2016 Orenburg strain.

Hepatitis C is a major worldwide disease and health hazard, affecting ∼3% of the world population. The p7 protein of hepatitis C virus (HCV) is an intracellular ion channel and pH regulator that is involved in the viral replication cycle. It is targeted by various classical ion channel blockers. Here, we generated p7 constructs corresponding to HCV genotypes 1a, 2a, 3a, and 4a for recombinant expression in HEK293 cells, and studied p7 channels using patch-clamp recording techniques. The pH50 values for recombinant p7 channels were between 6.0 and 6.5, as expected for proton-activated channels, and current-voltage dependence did not show any differences between genotypes. Inhibition of p7-mediated currents by amantadine, however, exhibited significant, genotype-specific variation. The IC50 values of p7-1a and p7-4a were 0.7 ± 0.1 nM and 3.2 ± 1.2 nM, whereas p7-2a and p7-3a had 50- to 1000-fold lower sensitivity, with IC50 values of 2402 ± 334 nM and 344 ± 64 nM, respectively. The IC50 values for rimantadine were low across all genotypes, ranging from 0.7 ± 0.1 nM, 1.6 ± 0.6 nM, and 3.0 ± 0.8 nM for p7-1a, p7-3a, and p7-4a, respectively, to 24 ± 4 nM for p7-2a. Results from patch-clamp recordings agreed well with cellular assays of p7 activity, namely, measurements of intracellular pH and hemadsorption assays, which confirmed the much reduced amantadine sensitivity of genotypes 2a and 3a. Thus, our results establish patch-clamp studies of recombinant viroporins as a valid analytical tool that can provide quantitative information about viroporin channel properties, complementing established techniques., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

New amino acid derivatives with carbocycles of adamantine and quinaldic acid were synthesized and their in vitro antiviral activity against influenza A/H5N1 virus was evaluated. Experiments on cultured embryonic porcine kidney epithelial cells showed that amino acid derivatives suppressed viral replication. Tret-butyloxycarbonyl-DL-methionylsulfonyl-1-adamantayl ethylamine and benzyloxycarbonyl-L-trypthophanyl-1-adamantayl ethylamine compounds demonstrated high activity in all in vitro experiments. Moreover, some compounds showed virucidal activity against influenza A/H5N1 virus.

The nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome, a multiprotein complex, is an essential intracellular mediator of antiviral immunity. In murine dendritic cells, this complex responds to a wide array of signals, including ion efflux and influenza A virus infection, to activate caspase-1-mediated proteolysis of IL-1β and IL-18 into biologically active cytokines. However, the presence and function of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells, in response to various triggers, including viral infection, has not been defined clearly. Here, we delineate the contribution of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome to the secretion of IL-1β, IL-18, and IL-1α by human dendritic cells (monocyte-derived and primary conventional dendritic cells). Activation of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells by various synthetic activators resulted in the secretion of bioactive IL-1β, IL-18, and IL-1α and induction of pyroptotic cell death. Cellular IL-1β release depended on potassium efflux and the activity of proteins nucleotide-binding oligomerization domain-like receptor protein 3 and caspase-1. Likewise, influenza A virus infection of dendritic cells resulted in priming and activation of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome and secretion of IL-1β and IL-18 in an M2- and nucleotide-binding oligomerization domain-like receptor protein 3-dependent manner. The magnitude of priming by influenza A virus varied among different strains and inversely corresponded to type I IFN production. To our knowledge, this is the first report describing the existence and function of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in human dendritic cells and the ability of influenza A virus to prime and activate this pathway in human dendritic cells, with important implications for antiviral immunity and pathogenesis., (© Society for Leukocyte Biology.)

Influenza virus has two major structural modules, an external lipid envelope and an internal ribonucleocapsid containing the genomic RNA in the form of the ribonucleoprotein (RNP) complex, both of which are interlinked by the matrix protein M1. Here we studied M1-RNP cohesion within virus exposed to acidic pH in vitro. The effect of acidification was dependent on the cleavage of the surface glycoprotein HA. Acidic pH caused a loss of intravirion RNP-M1 cohesion and activated RNP polymerase activity in virus with cleaved HA (HA1/2) but not in the uncleaved (HA0) virus. The in vitro acidified HA1/2 virus rapidly lost infectivity whereas the HA0 one retained infectivity, following activation by trypsin, suggesting that premature activation and release of the RNP is detrimental to viral infectivity. Rimantadine, an inhibitor of the M2 ion channel, was found to protect the HA1/2 virus interior against acidic disintegration, confirming that M2-dependent proton translocation is essential for the intravirion RNP release and suggesting that the M2 ion channel is only active in virions with cleaved HA. Acidic treatment of both HA0 and HA1/2 influenza viruses induces formation of spikeless bleb-like protrusion of ~ 25 nm in diameter on the surface of the virion, though only the HA1/2 virus was permeable to protons and permitted RNP release. It is likely that this bleb corresponds to the M2-enriched and M1-depleted focus arising from pinching off of the virus during the completion of budding. Cooperatively, the data suggest that the influenza virus has an asymmetric structure where the M1-mediated organization of the RNP inside the virion is a prerequisite for infectious entry into target cell., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The purpose of the experiment was to evaluate the local anesthetic effect of rimantadine in spinal anesthesia. Rimantadine in a dose-dependent fashion was constructed after intrathecally injecting the rats with four different doses. The potency and duration of rimantadine were compared with that of the local anesthetic lidocaine at producing spinal motor, nociceptive, and proprioceptive blockades. We demonstrated that intrathecal rimantadine dose-dependently produced spinal motor, nociceptive, and proprioceptive blockades. On the 50% effective dose (ED50) basis, the ranks of potencies at inducing spinal motor, nociceptive, and proprioceptive blockades was lidocaine>rimantadine (P<0.01). Rimantadine exhibited more nociceptive block (ED50) than motor block (P<0.05). At equi-anesthetic doses (ED25, ED50, and ED75), the spinal block duration produced by rimantadine was longer than that produced by lidocaine (P<0.01). Furthermore, rimantadine (26.52μmol/kg) prolonged the nociceptive nerve block more than the motor block (P<0.001). Our preclinical data showed that rimantadine, with a more sensory-selective action over motor block, was less potent than lidocaine. Rimantadine produced longer duration in spinal anesthesia when compared with lidocaine., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The purpose of this work was to study the mechanism of drug resistance of M2 channel proteins by analyzing the interactions between the drugs amantadine and rimantadine and M2 channel proteins (including the wild type and the three mutants V27A, S31N, and G34A) and the drug binding pathways, by use of a computational approach. Our results showed that multiple drug-binding sites were present in the M2 channel, and the trajectory of the drugs through the M2 channel was determined. A novel method was developed to investigate of free energy profiles of the ligand-protein complexes. Our work provides a new explanation of the large amount of experimental data on drug efficacy.

The annual flu season causes thousands of deaths and millions of hospitalizations, which pose a great burden to global health and economy. Moreover, a flu pandemic arising from reassortment viruses, such as H5N1 and H1N1, raises even greater concern due to the lack of effective vaccines at the initial stage of flu outbreak. The influenza virus is the causative agent of flu infection. Currently there are four drugs in use to combat influenza infection. Amantadine and rimantadine are M2 proton channel blockers that inhibit virus uncoating; oseltamivir and zanamivir are neuraminidase (NA) inhibitors that inhibit virus release. However, recent years have witnessed a drastic increase in instances of drug resistance, and flu strains that are resistant to both classes of drugs have been reported. Thus, there is a pressing need to develop the next generation of anti-influenza drugs. Among a handful of anti-influenza drug targets, the viral fusion protein hemagglutinin (HA) is one of the most advanced. This review discusses the biological roles of HA during viral replication and highlights peptide- and small molecule-based HA inhibitors, including recent computationally designed HA binders. The text is organized into four sections based on the maturation stages of HA: inhibitors targeting the glycosylation of HA, the proteolytic activation of HA, the attachment of HA to host cell receptors, and peptide- and small molecule-based inhibitors targeting HA-mediated membrane fusion. Of particular interest are advances in the areas of developing dual inhibitors targeting both HA and NA and broad-spectrum HA inhibitors targeting both groups of HAs.

M2 proton channel is the target for treating the patients who ere suffering from influenza A infection, which facilitates the spread of virions. Amantadine and rimantadine are adamantadine-based drugs, which target M2 proton channel and inhibit the viral replication. Preferably, rimantadine drug is used more than amantadine because of its fewer side effects. However, S31N mutation in the M2 proton channel was highly resistant to the rimantadine drug. Therefore, in the present study, we focused to understand the drug-resistance mechanism of S31N mutation with the aid of molecular docking and dynamics approach. The docking analysis undoubtedly indicates that affinity for rimantadine with mutant-type M2 proton channel is significantly lesser than the native-type M2 proton channel. In addition, RMSD, RMSF, and principal component analysis suggested that the mutation shows increased flexibility. Furthermore, the intermolecular hydrogen bonds analysis showed that there is a complete loss of hydrogen bonds in the mutant complex. On the whole, we conclude that the intermolecular contact was maintained by D-44, a key residue for stable binding of rimantadine. These findings are certainly helpful for better understanding of drug-resistance mechanism and also helpful for designing new drugs for treating influenza infection against drug-resistance target.

Pentameric ligand-gated ion channels (pLGIC) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules, including the Alzheimer's drug memantine, block ion conduction at vertebrate pLGICs by plugging the channel pore. We show that memantine has similar potency in ELIC, a prokaryotic pLGIC, when it contains an F16'S pore mutation. X-ray crystal structures, using both memantine and its derivative, Br-memantine, reveal that the ligand is localized at the extracellular entryway of the channel pore, and the pore is in a more closed conformation than wild-type ELIC in both the presence and absence of memantine. However, using voltage clamp fluorometry we observe fluorescence changes in opposite directions during channel activation and pore block, revealing an additional conformational transition not apparent from the crystal structures. These results have important implications for drugs such as memantine, which block channel pores., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

HIV-1 Vpu is a small, single-span membrane protein with two attributed functions that increase the virus' pathogenicity: degradation of CD4 and inactivation of BST-2. Vpu has also been shown to possess ion channel activity, yet no correlation has been found between this attribute and Vpu's role in viral release. In order to gain further insight into the channel activity of Vpu we devised two bacteria-based assays that can examine this function in detail. In the first assay Vpu was over-expressed, such that it was deleterious to bacterial growth due to membrane permeabilization. In the second and more sensitive assay, the channel was expressed at low levels in K(+) transport deficient bacteria. Consequently, Vpu expression enabled the bacteria to grow at otherwise non permissive low K(+) concentrations. Hence, Vpu had the opposite impact on bacterial growth in the two assays: detrimental in the former and beneficial in the latter. Furthermore, we show that channel blockers also behave reciprocally in the two assays, promoting growth in the first assay and hindering it in the second assay. Taken together, we investigated Vpu's channel activity in a rapid and quantitative approach that is amenable to high-throughput screening, in search of novel blockers.

A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

We studied the capacity amino acid derivatives of adamantane to inhibit replication of highly virulent avian influenza A/duck/Novosibirsk/56/05 (H5N1) virus in cultures of swine embryonic kidney cells. Amino acid derivatives of adamantane H-His-Rem and Ad(CH2-Ser-OMe)2 were characterized by lower toxicity than remantadine previously used in the treatment of influenza. Histidine-containing adamantane derivative (H-His-Rem) was the most effective and low-toxic inhibitor of influenza А/H5N1 virus replication and can be recommended for clinical trials to produce a preparation for the treatment and prevention of influenza.

The aim of this study was to investigate infiltrative cutaneous anesthesia of 2-adamantanamine and rimantadine. After subcutaneous injections of drugs in rats, the blockade of cutaneous trunci muscle reflex by 2-adamantanamine and rimantadine was evaluated. Lidocaine, a common local anesthetic, was used as control. We showed that rimantadine and 2-adamantanamine as well as the local anesthetic lidocaine produced infiltrative anesthesia of skin in a dose-dependent fashion. Saline (vehicle) group displayed no cutaneous anesthesia. The relative potency of these drugs was rimantadine [23.8 (21.1-26.8)] = lidocaine [26.4 (22.7-30.6)] > 2-adamantanamine [64.6 (55.0-75.9)] (P < 0.01). On an equianesthetic basis [25% effective dose (ED25 ), ED50 , and ED75 ], rimantadine and 2-adamantanamine had longer duration of action than lidocaine (P < 0.05). Neither local injection of saline nor intraperitoneal administration of a large dose of drugs elicited cutaneous anesthesia (data not shown). These data demonstrated for the first time that rimantadine had a similar potent and longer duration of skin infiltrative anesthesia than did lidocaine, whereas 2-adamantanamine had a less potency but longer duration of cutaneous anesthesia than did lidocaine., (© 2012 The Authors Fundamental and Clinical Pharmacology © 2012 Société Française de Pharmacologie et de Thérapeutique.)

Inhibitors targeting the hepatitis C virus (HCV) encoded viroporin, p7 prevent virus release in vitro. HCV can transmit by cell-free particle infection of new target cells and via cell-to-cell dependent contact with limited exposure to the extracellular environment. The role of assembly inhibitors in preventing HCV transmission via these pathways has not been studied. We compared the efficacy of three published p7 inhibitors to inhibit cell-free and cell-to-cell transmission of two chimeric HCV strains encoding genotype 2 (GT2) or 5 (GT5) p7 using a recently developed single cycle co-culture assay. The inhibitors reduced the infectivity of extracellular GT2 and GT5 virus by 80-90% and GT2 virus cell-to-cell transmission by 50%. However, all of the p7 inhibitors had minimal effect on GT5 cell contact dependent transmission. Screening a wider panel of diverse viral genotypes demonstrated that p7 viroporin inhibitors were significantly more effective at blocking cell-free virus than cell-to-cell transmission. These results suggest an altered assembly or trafficking of cell-to-cell transmitted compared to secreted virus. These observations have important implications for the validation, therapeutic design and testing of HCV assembly inhibitors., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

By attaching multiple copies of the influenza M2 ion channel inhibitors amantadine (1) and rimantadine (2) to polymeric chains, we endeavored to recover their potency in inhibiting drug-resistant influenza viruses. Depending on loading densities, as well as the nature of the drug, the polymer, and the spacer arm, polymer-conjugated drugs were up to 30-fold more potent inhibitors of drug-resistant strains than their monomeric parents. In particular, a 20% loading density and a short linker group on the negatively charged poly-l-glutamate resulted in one of the most potent inhibitors for 2's conjugates against drug-resistant influenza strains. Although full recovery of the inhibitory action against drug-resistant strains was not achieved, this study may be a step toward salvaging anti-influenza drugs that are no longer effective., (Copyright © 2013 Wiley Periodicals, Inc.)

The hepatitis C virus (HCV) has developed a small membrane protein, p7, which remarkably can self-assemble into a large channel complex that selectively conducts cations. We wanted to examine the structural solution that the viroporin adopts in order to achieve selective cation conduction, because p7 has no homology with any of the known prokaryotic or eukaryotic channel proteins. The activity of p7 can be inhibited by amantadine and rimantadine, which are potent blockers of the influenza M2 channel and licensed drugs against influenza infections. The adamantane derivatives have been used in HCV clinical trials, but large variation in drug efficacy among the various HCV genotypes has been difficult to explain without detailed molecular structures. Here we determine the structures of this HCV viroporin as well as its drug-binding site using the latest nuclear magnetic resonance (NMR) technologies. The structure exhibits an unusual mode of hexameric assembly, where the individual p7 monomers, i, not only interact with their immediate neighbours, but also reach farther to associate with the i+2 and i+3 monomers, forming a sophisticated, funnel-like architecture. The structure also points to a mechanism of cation selection: an asparagine/histidine ring that constricts the narrow end of the funnel serves as a broad cation selectivity filter, whereas an arginine/lysine ring that defines the wide end of the funnel may selectively allow cation diffusion into the channel. Our functional investigation using whole-cell channel recording shows that these residues are critical for channel activity. NMR measurements of the channel-drug complex revealed six equivalent hydrophobic pockets between the peripheral and pore-forming helices to which amantadine or rimantadine binds, and compound binding specifically to this position may allosterically inhibit cation conduction by preventing the channel from opening. Our data provide a molecular explanation for p7-mediated cation conductance and its inhibition by adamantane derivatives.

We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M2(18-60) from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the (15)N-labeled ammonium of rimantadine was observed near A30 (13)Cβ and G34 (13)Cα, suggesting a possible hydrogen bond to A30 carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of ~10 at 400 MHz using only 4-6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 Å.

BM2 channel plays an important role in the replication of influenza virus B. However, few studies attempt to investigate the mechanism of the proton conductance in BM2 channel, as well as the drug resistance of the BM2 channel. The first experimental structure of the BM2 protein channel has recently been solved, enabling us to theoretically study BM2 systems with different protonation states of histidine. By performing molecular dynamics simulations on the BM2 systems with different protonation states of four His19 residues, we provided our understanding of the structure, dynamics, and drug resistance of the BM2 channel. In general, the results of our study and other investigations both have demonstrated that whether the BM2 channel adopts an open or a closed form depends on the protonation state of His19. Meanwhile, we discovered that a drug (amantadine) was unable to enter into the center of the BM2 channel even at a low pH condition probably due to the number of hydrophilic residues of the BM2 channel. Finally, potentials of mean force (PMF) calculations were performed for the drug binding BM2 channel, energetically explaining why the BM2 channel exhibited drug resistance to two inhibitors of the AM2 channel, amantadine and rimantadine.

The combined effect of rimantadine and oseltamivir in a prophylactic context (therapy beginning 4 h pre-virus infection) and therapeutic context (therapy started at 24 h post-viral inoculation) course on influenza H3N2 virus infection in mice was studied. In the prophylactic course 5 and 10 mg/kg/day rimantadine with 0.2 and 0.4 mg/kg/day (25:1 dose ratio) oseltamivir showed a protection index (PI) of 79.6% and 75%, respectively and a mean survival time (MST) of 13.1 and 12.9 days. The individual effects of the same doses ranged from 0% to 33.3% PI and 8.2 to 10.3 days MST, respectively. Lung virus titers were decreased 630-fold in the combination-treated groups as compared to monotherapy and placebo groups. The reduction of surface lung pathology in combination-treated groups demonstrated a protective effect for the combination of both antivirals. In the therapeutic course 5 and 10 mg/kg rimantadine combined with 0.2 and 0.4 mg/kg oseltamivir showed no beneficial effect. At higher dosage (0.8, 1.6, 3.2 mg/kg oseltamivir and 20, 40, 80 mg/kg rimantadine) preserving the 25:1 ratio, the resultant PI ranged from 57.6% to 80.5% and the MST was 12.8-13.4 days. Used alone at the same doses the compounds' protection varied between 10.7% and 71.8% PI, MST 9.8-12.8 days (8.7 days in PBS control). Compared to vehicle and individual treatment, a decrease in infectious viral titers of up to 1000-fold and other viral pneumonia parameters were also recorded. The therapeutic effect of the drugs' optimal effective doses combinations was characterized as synergistic. Survival of animals was 81.2-100% and MST was extended by 5-7 days compared to placebos. Monotherapy protection was from 9.1% to a maximum of 56.5%, MST being prolonged only by 1.3-4.2 days compared to 7.5 days in the PBS control group. Lung viral titers were decreased 1445-fold for the most efficacious combination groups and a significant reduction in lung parameters was observed. These data emphasize that prophylactic and therapeutic courses using a combination of oseltamivir and rimantadine have a significant protective effect in mice experimentally infected with drug-sensitive influenza virus A (H3N2)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

New adamantane derivatives with amino acid residues and other bifunctional compounds were synthesized and their antiviral activity towards influenza A(H1N1)pdm and A(H3N2) viruses was studied. Some of these adamantane derivatives completely suppressed replication of remantadine-resistant influenza A virus strains.

We report chemical shift assignments of the drug-resistant S31N mutant of M2(18-60) determined using 3D magic-angle-spinning (MAS) NMR spectra acquired with a (15)N-(13)C ZF-TEDOR transfer followed by (13)C-(13)C mixing by RFDR. The MAS spectra reveal two sets of resonances, indicating that the tetramer assembles as a dimer of dimers, similar to the wild-type channel. Helicies from the two sets of chemical shifts are shown to be in close proximity at residue H37, and the assignments reveal a difference in the helix torsion angles, as predicted by TALOS+, for the key resistance residue N31. In contrast to wild-type M2(18-60), chemical shift changes are minimal upon addition of the inhibitor rimantadine, suggesting that the drug does not bind to S31N M2., (© 2012 American Chemical Society)

A novel gold(I) complex with rimantadine (RTD) was obtained and structurally characterized by a set of chemical and spectroscopic analysis. 1H, 13C and 15N nuclear magnetic resonance (NMR) and infrared (IR) spectroscopic measurements suggest coordination of the ligand to Au(I) through the N atom of the ethanamine group. Theoretical (DFT) calculations confirmed the IR assignments and permit proposing an optimized geometry for the complex. The gold(I)-rimantadine complex (Au-RTD) is soluble in methanol, ethanol, dimethylsulfoxide, acetone and acetonitrile. The preliminary kinetic studies based on UV-vis spectroscopic measurements indicate the stability of the compound in solution. Antibacterial activities of the complex were evaluated by an antibiogram assay. The Au-RTD complex showed an effective in vitro antibacterial activity against the Pseudomonas aeruginosa, Escherichia coli (Gram-negative), and Staphylococcus aureus (Gram-positive) bacterial strains., (Copyright © 2011 Elsevier B.V. All rights reserved.)

M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m) of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

The Gloeobacter ligand-gated ion channel (GLIC) is a bacterial homolog of vertebrate Cys-loop ligand-gated ion channels. Its pore-lining region in particular has a high sequence homology to these related proteins. Here we use electrophysiology to examine a range of compounds that block the channels of Cys-loop receptors to probe their pharmacological similarity with GLIC. The data reveal that a number of these compounds also block GLIC, although the pharmacological profile is distinct from these other proteins. The most potent compound was lindane, a GABA(A) receptor antagonist, with an IC₅₀ of 0.2 μM. Docking studies indicated two potential binding sites for this ligand in the pore, at the 9' or between the 0' and 2' residues. Similar experiments with picrotoxinin (IC₅₀ = 2.6 μM) and rimantadine (IC₅₀ = 2.6 μM) reveal interactions with 2'Thr residues in the GLIC pore. These locations are strongly supported by mutagenesis data for picrotoxinin and lindane, which are less potent in a T2'S version of GLIC. Overall, our data show that the inhibitory profile of the GLIC pore has considerable overlap with those of Cys-loop receptors, but the GLIC pore has a unique pharmacology., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In order to understand how rimantadine (RMT) inhibits the proton conductance in the influenza A M2 channel via the recently proposed "allosteric mechanism", molecular dynamics simulations were applied to the M2-tetrameric protein with four RMTs bound outside the channel at the three protonation states: the 0H-closed, 1H-intermediate and 3H-open situations. In the 0H-closed state, a narrow channel with the RMT-Asp44-Trp41 H-bond network was formed, therefore the water penetration through the channel was completely blocked. The Trp41-Asp44 interaction was absent in the 1H-intermediate state, whilst the binding of RMT to Asp44 remained, which resulted in a weakened helix-helix packing, therefore the channel was partially prevented. In the 3H-open state it was found that the electrostatic repulsion from the three charged His37 residues allowed the Trp41 gate to open, permitting water to penetrate through the channel. This agreed well with the potential of the means force which is in the following order: 0H > 1H > 3H.

The amino acid and peptide derivatives of 1-adamantane carboxylic acid and rimantadine (18 compounds) have been first synthesized and investigated for their activity against influenza A virus (H1N1, H1N1v). In a series of obtained adamantine derivatives, some compounds have been found to be able to inhibit rimantadine-resistant influenza A virus strains. Thus, the antiviral properties of rimantadine can be restored.

The recent outbreaks of avian flu in Southeast Asia and swine flu in Mexico City painfully exemplify the ability of the influenza virus to rapidly mutate and develop resistance to modern medicines. This review seeks to detail the molecular mechanism by which the influenza virus has obtained resistance to amino-adamantyls, one of only two classes of drugs that combat the flu. Amino-adamantyls target the viral M2 H(+) channel and have become largely ineffective due to mutations in the transmembrane domain of the protein. Herein we describe these resistance rendering mutations and the compounded effects they have upon the protein's function and resulting virus viability., (2010 Elsevier B.V. All rights reserved.)

The influenza M2 H(+) channel enables the concomitant acidification of the viral lumen upon endosomic internalization. This process is critical to the viral infectivity cycle, demonstrated by the fact that M2 is one of only two targets for anti-flu agents. However, aminoadamantyls that block the M2 channel are of limited therapeutic use due to the emergence of resistance mutations in the protein. Herein, using an assay that involves expression of the protein in Escherichia coli with resultant growth retardation, we present quantitative measurements of channel blocker interactions. Comparison of detailed K(s) measurements of different drugs for several influenza channels, shows that the swine flu M2 exhibits the highest resistance to aminoadamantyls of any channel known to date. From the perspective of the blocker, we show that rimantadine is consistently a better blocker of M2 than amantadine. Taken together, such detailed and quantitative analyses provide insight into the mechanism of this important and pharmaceutically relevant channel blocker system., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The paper describes the specific features of the 2009-2010 epidemic season in Russia and the world, which are due to the wide spread of a new pandemic strain of influenza A(H1N1)v virus. There is an unusual early upsurge in the incidence of influenza and acute respiratory viral infection (ARVI) (in October-November 2009) with its peak at weeks 45 to 48 of the year with a succeeding reduction to the seasonal values by its end. The circulation of influenza B virus strains was recorded in February-April 2010, which was responsible for the higher epidemic thresholds of morbidity in a number of Russia's regions. A study of the antigenic properties of the strains defined their relationship to the reference strains A/California/07/2009 (H1N1)v and B/Brisbene/60/2008. There were strains with amino acid substitutions at position 222 of hemagglutinin in the population of pandemic influenza A(H1N1)v virus. The strains of the new pandemic influenza A(H1N1)v virus were resistant to remantadine and susceptible to oseltamivir, zanamivir, and arbidol. The influenza B virus strains were susceptible to oseltamivir, zanamivir, and arbidol. The proportion of pathogens of some ARVIs was as follows: parainfluenza viruses, 9.8%; adenoviruses, 5.5%; respiratory syncytial virus, 4.8%; and Mycoplasma pneumonia, 0.6%. There is evidence that there is a need for further monitoring of influenza viruses in Russia.

The influence of antivirals, such as rimantadine, ribavirine and triazavirine on influenza virus replication in human cell cultures was evaluated. All the antivirals inhibited viral nucleoprotein NP synthesis. The strongest effect was shown for ribavirine in lung carcinoma A-549 cells and endothelial ECV-304 cells. Hoechst-33258 staining revealed induction of apoptosis in all the cell lines. Rimantadine and ribavirine inhibited virus-induced apoptosis while ribavirine enhanced it. The effect was registered in monolayer cell cultures as well as in suspension cell cultures. The influence of the antiviral drugs on the virus-induced cell proliferation in the suspension cell cultures is also described.

The data on cytotoxicity and antiviral activity of commercial antivirals, such as Remantadine, Oseltamivir, Arbidol and Ribavirin in the MDCK cell culture infected with highly pathogenic (H5N1) and pandemic 2009 (H1N1) influenza A viruses are presented. The study of the antiviral activity of antivirals in the MDCK cells culture demonstrated that Arbidol, Rimantadine and Ribavirin efficiently inhibited reproduction of the highly pathogenic H5N1 influenza viruses isolated from sick birds. Arbidol and Oseltamivir carboxylate selectively inhibited reproduction of the pandemic 2009 H1N1 influenza A viruses with changed specificity to the cell receptors, causing severe influenza in men, while remantadine had no effect on their reproduction.

Efficacy of combination of the intramuscularly administered neuraminidase (NA) inhibitor, peramivir, and the orally administered M2 ion channel blocker, rimantadine was evaluated in mouse influenza A/Victoria/3/75 (H3N2) model. Mice were challenged with a sub-lethal virus dose (0-40% mortality in placebo group) and changes in body weights were analyzed by three-dimensional effect analysis to assess mode of drug interactions. Compounds were administered in a 5-day treatment course starting 1h before viral inoculation. The peramivir and rimantadine doses ranged from 0.3-3 mg/kg/d and 5-30 mg/kg/d, respectively. The maximum mean weight loss of 5.19 g was observed in the vehicle-infected group on day 10. In the 1 and 3 mg/kg/d peramivir monotherapy groups, the weight losses were 4.3 and 3.55 g, respectively. In the rimantadine monotherapy group, the weight losses were 3.43, 2.1, and 1.64 g for the 5, 10, and 30 mg/kg/d groups, respectively. Combination of 1mg/kg/d peramivir with 5 and 10 mg/kg/d rimantadine produced weight losses of 1.69 and 0.69 (p<0.05 vs. vehicle and individual agent), respectively, whereas the combination of 3.0 mg/kg/d peramivir with 10 and 30 mg/kg/d rimantadine did not show any weight loss (p<0.05 vs. vehicle and individual agent). The three-dimensional analysis of the weight loss for the majority of the drug combinations of peramivir and rimantadine tested demonstrated synergistic antiviral effects., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Introduction: In December 2008, new interim guidelines on the use of influenza antiviral agents were released in response to a high prevalence of circulating oseltamivir-resistant seasonal influenza A(H1N1) and adamantane-resistant influenza A(H3N2) viruses. Zanamivir, oseltamivir +/- an adamantane, or oseltamivir was recommended, depending on virus type, subtype, and local surveillance data., Materials and Methods: Information about antiviral prescribing practices among IDSA Emerging Infections Network (EIN) members was obtained using two web-based questionnaires; one in January 2009 regarding the prior 2007-08 influenza season and one in April 2009 (prepandemic), regarding the concurrent 2008-09 season., Results: In the 2007-08 survey, 646 (52%) of 1249 EIN members responded and in the 2008-09 season survey, 350 (27%) of 1281 responded. In 2008-09 vs. 2007-08: 59% vs. 69% prescribed or recommended antivirals for treatment (p<.0001); 48% vs. 80% prescribed oseltamivir alone and 39% vs. 10% prescribed zanamivir alone (p<.0001 for both). During 2008-09 28% reported treating fewer patients compared with 2007-08; 42% felt antivirals were less effective due to resistance and 40% felt patients had less severe illness. During 2008-09, 42% of respondents reported difficulty providing zanamivir to patients vs. 5% for oseltamivir (p<.0001). Only 11% of respondents could test for influenza A subtype. During both seasons, ~55% used local surveillance data to make treatment decisions., Discussion: A mild winter influenza season, difficulty obtaining recommended agents, and lack of access to subtype diagnosis and surveillance data may have contributed to reduced antiviral use during 2008-09., (Copyright © 2010 Elsevier B.V. All rights reserved.)