Anti-influenza Drug Amantadine Research Articles

An effective molecular blocker of ion channel of M2 protein as anti-influenza a drug

Design of a drug compound that can effectively bind to the M2 ion channel and block the diffusion of hydrogen ions (H+) through and inhibit influenza A virus replication is an important task. Known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus. A new class of positively charged, +2 e.u., molecules is proposed here to block diffusion of H+ ion through the M2 channel. Several drug candidates, derivatives of a lead compound (diazabicyclooctane), were proposed and investigated. Molecular dynamics of thermal fluctuations of M2 protein structure and ionization-conformation coupling of all the ionizable residues were simulated at physiological pH. The influence of the most probable mutations of key drug-binding amino acid residues in the M2 ion channel were investigated too. It is shown that the suggested new blocker has high binding affinity for the M2 channel. There are two in-channel binding sites of high affinity, the first one has H-bonds with two of four serine residues Ser-31A (B) or Ser-31C(D), and the second one has H-bonds with two of four histidine residues His-37A (B), or His-37C(D). The main advantage of the new drug molecule is the positive charge, +2 e.u., which creates a positive electrostatic potential barrier (in addition to a steric one) for a transfer of H+ ion through M2 channel and may serve as an effective anti-influenza A virus drug. Communicated by Ramaswamy H. Sarma

Design of an Efficient Inhibitor for the Influenza A Virus M2 Ion Channel

Influenza A virus is capable of rapidly infecting large human populations, warranting the development of novel drugs to efficiently inhibit virus replication. A transmembrane ion channel formed by the M2 protein plays an important role in influenza virus replication. A reasonable approach to designing an effective antivirus drug is constructing a molecule that binds in the M2 transmembrane proton channel, blocks H^(+) proton diffusion through the channel, and thus the influenza A virus cycle. The known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus replication. A new class of positively charged molecules, diazabicyclooctane derivatives with a constant charge of +2, was proposed to block proton diffusion through the M2 ion channel. Molecular dynamics simulations were performed to study the temperature fluctuations in the M2 structure, and ionization states of histidine residues were established at physiological pH values. Two types of diazabicyclooctane derivatives were analyzed for binding with the M2 ion channel. An optimal structure was determined for a blocker to most efficiently bind with the M2 ion channel and block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport through the M2 ion channel in addition to a steric barrier.

Understanding the Evolutionary Relationship of M2 Channel Protein of Influenza A Virus and its Structural Variation and Drug Resistance

Background: M2 channel protein of influenza A virus is one of the specific targets for the anti-influenza drugs amantadine and rimantadine. These drugs have lost their efficacy because of the mutations in their drug interaction sites. Large-scale analysis of these influenza surface proteins may give better elucidation for understanding the evolution of the proteins toward the drug resistant mechanism. Keywords: Amantadine, evolutionary lineage, influenza A virus, M2 channel protein, rimantadine.

Structural Influences: Cholesterol, Drug, and Proton Binding to Full-Length Influenza A M2 Protein

The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue 13C-13C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His37 residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding.

Inhibitors of the influenza A virus M2 proton channel discovered using a high-throughput yeast growth restoration assay.

The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.

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.

Solid-supported membrane technology for the investigation of the influenza A virus M2 channel activity

Influenza A virus encodes an integral membrane protein, A/M2, that forms a pH-gated proton channel that is essential for viral replication. The A/M2 channel is a target for the anti-influenza drug amantadine, although the effectiveness of this drug has been diminished by the appearance of naturally occurring point mutations in the channel pore. Thus, there is a great need to discover novel anti-influenza therapeutics, and, since the A/M2 channel is a proven target, approaches are needed to screen for new classes of inhibitors for the A/M2 channel. Prior in-depth studies of the activity and drug sensitivity of A/M2 channels have employed labor-intensive electrophysiology techniques. In this study, we tested the validity of electrophysiological measurements with solid-supported membranes (SSM) as a less labor-intensive alternative technique for the investigation of A/M2 ion channel properties and for drug screening. By comparing the SSM-based measurements of the activity and drug sensitivity of A/M2 wild-type and mutant channels with measurements made with conventional electrophysiology methods, we show that SSM-based electrophysiology is an efficient and reliable tool for functional studies of the A/M2 channel protein and for screening compounds for inhibitory activity against the channel.

Design and Pharmacological Characterization of Inhibitors of Amantadine-Resistant Mutants of the M2 Ion Channel of Influenza A Virus

The A/M2 proton channel of influenza A virus is a target for the anti-influenza drugs amantadine and rimantadine, whose effectiveness was diminished by the appearance of naturally occurring point mutants in the A/M2 channel pore, among which the most common are S31N, V27A, and L26F. We have synthesized and characterized the properties of a series of compounds, originally derived from the A/M2 inhibitor BL-1743. A lead compound emerging from these investigations, spiro[5.5]undecan-3-amine, is an effective inhibitor of wild-type A/M2 channels and L26F and V27A mutant ion channels in vitro and also inhibits replication of recombinant mutant viruses bearing these mutations in plaque reduction assays. Differences in the inhibition kinetics between BL-1743, known to bind inside the A/M2 channel pore, and amantadine were exploited to demonstrate competition between these compounds, consistent with the conclusion that amantadine binds inside the channel pore. Inhibition by all of these compounds was shown to be voltage-independent, suggesting that their charged groups are within the N-terminal half of the pore, prior to the selectivity filter that defines the region over which the transmembrane potential occurs. These findings not only help to define the location and mechanism of binding of M2 channel-blocking drugs but also demonstrate the feasibility of discovering new inhibitors that target this binding site in a number of amantadine-resistant mutants.

Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel

The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21-61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22-46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pK(a) values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.

Reversibility of Amantadine Inhibition in the M2 Proton Channel of Influenza A Virus

The M2 protein from influenza A virus forms a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drug amantadine. Many observations have shown that amantadine inhibits the channel only when applied extracellulary, probably by residing within the channel pore and disrupting the gating mechanism. The effect of amantadine on AM2 channel activity was found to be slowly reversible in tissue culture cells and oocytes, but the reversibility kinetics of the drug on recombinant AM2 channels has never been directly addressed. In the current work we provide evidence for significant differences in the rate of amantadine reversibility among several heterologous expression systems. We characterized the channel activity, the amantadine sensitivity and reversibility of the AM2 protein expressed in CHO-K1 cells using novel solid supported membrane technology (SURFE2R, IonGate Biosciences GmbH). SURFE2R technology allows time resolved measurements of the electrogenic activity of slowly conducting channels and provides a valuable compliment to electrophysiological studies. We also tested amantadine reversibility in AM2 expressing CHO-K1 cells using pH sensitive green fluorescent protein microscopy and in AM2 expressing oocytes using TEVC technique. We show that amantadine inhibition in AM2-expressing mammalian cells is rapidly reversible. However in oocytes the reversibility of amantadine inhibition is much slower and depends on the channel properties. These findings should be taken into consideration for future investigation of the mechanism of amantadine inhibition and show once again that the properties of recombinant proteins may be significantly influenced by the properties of the expression system.

Structural basis for the function and inhibition of an influenza virus proton channel

The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

Conductance and amantadine binding of a pore formed by a lysine‐flanked transmembrane domain of SARS coronavirus envelope protein

The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM α-helical bundle. We have shown previously that the topology of the α-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM α-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric α-helical bundle model.

Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs.

Influenza virus A/Hong Kong/1774/99, isolated from a young child with mild influenza, was shown to be similar in its antigenic and genetic characteristics to H3N2 viruses circulating in pigs in Europe during the 1990s and in particular to be closely related to viruses isolated from two children in the Netherlands in 1993. Similar viruses had previously not been identified outside Europe. Although there is little evidence as to how the child contracted the infection, it appears likely that pigs in southern China were the source of infection. Characteristics shared with the European swine viruses include resistance to the anti-influenza drugs amantadine and rimantadine. Thus not only does this incident once again highlight the potential of pigs as a source of novel human influenza viruses, but also indicates the potential for emergence of amantadine-resistant human viruses.

Neutron Diffraction Reveals the Site of Amantadine Blockade in the Influenza A M2 Ion Channel

The influenza A M2 protein forms proton channels which are blocked by the anti-influenza drug amantadine. Using the technique of neutron diffraction with both deuterium-labeled amantadine and influenza A M2 peptides, this study has directly located the position of interaction between the drug and the transmembrane domain of M2. Amantadine is found 0.5 nm from the center of the bilayer in an area between Val 27 and Ser 31, a location consistent with the formation of a steric block within the ion channel. Similar experiments with amantadine and an amantadine-resistant mutant peptide showed no such interaction.

Role of the M2 Protein in influenza Virus Membrane Fusion: Effects of Amantadine and Monensin on Fusion Kinetics

We have investigated the effects of the anti-influenza drug amantadine (AMT) and the proton-ionophore monensin on the membrane fusion activity of influenza virus in a liposomal model system, using a kinetic fluorescence lipid mixing assay. Fusion of influenza virus A/turkey/Oregon/71 (H7N3) with liposomes was slowed down in the presence of 2 μ M AMT. The effect of AMT was not observed with an AMT-resistant mutant virus. Fusion inhibition by AMT was reversed by the proton-ionophore monensin. In fact, 1 μ M monensin stimulated fusion of AMT-sensitive or -resistant virus, irrespective of the presence of AMT. The effects of AMT and monensin increased with increasing temperature. They were not observed at 25°, but were very prominent at 45°. Monensin did not influence the fusion rates of reconstituted viral envelopes (virosomes), which lack the nucleocapsid and the M1 protein. These results suggest that intraviral low pH facilitates influenza virus fusion, possibly by weakening interactions of the C-terminus of the viral hemagglutinin with the M1 protein and/or the viral nucleocapsid. The effect of AMT on the fusion capacity of influenza virus may contribute to the anti-influenza action of the drug in the early stages of cellular infection. However, the limited extent of the fusion inhibition suggests that the fusion step is unlikely to be the primary target of AMT.

Influenza virus M2 protein: a molecular modelling study of the ion channel.

The influenza A M2 protein forms cation-selective ion channels which are blocked by the anti-influenza drug amantadine. A molecular model of the M2 channel is presented in which a bundle of four parallel M2 transbilayer helices surrounds a central ion-permeable pore. Analysis of helix amphipathicity was used to aid determination of the orientation of the helices about their long axes. The helices are tilted such that the N-terminal mouth of the pore is wider than the C-terminal mouth. The channel is lined by residues V27, S31 and I42. Residues D24 and D44 are located at opposite mouths of the pore, which is narrowest in the vicinity of I42. Energy profiles for interaction of the channel with Na+, amantadine-H+ and cyclopentylamine-H+ are evaluated. The interaction profile for Na+ exhibits three minima, one at each mouth of the pore, and one in the region of residue S31. The amantadine-H+ profile exhibits a minimum close to S31 and a barrier near residue I42. This provides a molecular model for amantadine-H+ block of M2 channels. The profile for cyclopentylamine-H+ does not exhibit such a barrier. It is predicted that cyclopentylamine-H+ will not act as an M2 channel blocker.

The transmembrane domain of influenza A M2 protein forms amantadine-sensitive proton channels in planar lipid bilayers

In a direct test of the hypothesis that the M2 coat protein of influenza A can function as a proton translocator, we incorporated a synthetic peptide containing its putative transmembrane domain into voltage-clamped planar lipid bilayers. We observed single proton-selective ion channels with a conductance of ∼10 pS at a pH of 2.3, consistent with the association of several monomers around a central water-filled pore. The channels were reversibly blocked by the anti-influenza drug amantadine. These experiments imply a central role for M2 protein in virus replication and assembly and may explain the mechanism of action of amantadine. Analogous proteins may have a similar function in other viruses, and these may be susceptible to similar antiviral agents.