Antiviral Drug Amantadine Research Articles

Determining the Functional Core for Proton Transport, Ion Selectivity and Amantadine Sensitivity of the A/M2 Protein from Influenza A Virus

Influenza continues to be an epidemic and pandemic disease. The M2 protein from influenza A virus is a pH-activated proton channel. Its function is essential for efficient replication of the virus. Moreover, the M2 protein is the target of the antiviral drug amantadine, which is one of few available antiviral drugs that inhibit influenza A replication. Although M2 protein is only a 97-amino acid protein, it possesses multiple roles by its different domains in different stages of virus life cycles. In spite of the importance of the ion channel function of the M2 protein, the part of the protein that possesses its central role-proton channel-has not been defined clearly. Moreover, recent structural studies used truncated constructs that have not yet been evaluated for proton channel function. Here we report findings from experiments designed to investigate the functional core for proton transport, ion selectivity and amantadine sensitivity of M2 ion channel protein. We constructed a series of truncation mutants, measured low-pH activated, amantadine sensitive current in oocytes of Xenopus laevis and also determined the relative M2 surface expression on the Xenopus laevis oocyte membrane. We found that a construct of residues 21-61 (“shortie”), which includes the TM domain and 18-residues of the cytoplasmic tail, has the ion channel activity indistinguishable from that of the full length M2 protein. Functional reconstitution vesicle assay also showed that this construct was sufficient for proton channel function. Further truncated peptides (residues 22-46 and residues 22-50) showed amantadine-sensitive proton fluxes similar to “shortie” M2 (residues 19-62), however these peptides displayed a lower proton-selectivity and some potassium ion flux.

Structure of Amantadine-Bound M2 Transmembrane Peptide of Influenza A in Lipid Bilayers from Magic-Angle-Spinning Solid-State NMR: The Role of Ser31 in Amantadine Binding

The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the side-chain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting structural findings from non-bilayer samples.

Identification of the Pore-lining Residues of the BM2 Ion Channel Protein of Influenza B Virus

The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleo-protein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. It was found that mutation of transmembrane domain residues Ser(9), Ser(12), Phe(13), Ser(16), His(19), and Trp(23) to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser(9), Ser(12), Ser(16), or Trp(23). Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.

A Secondary Gate As a Mechanism for Inhibition of the M2 Proton Channel by Amantadine

The mechanism of inhibition of the influenza A virus M2 proton channel by the antiviral drug amantadine has been under intense investigation. The importance of a mechanistic understanding is heightened by the prevalence of amantadine-resistant mutations. To gain mechanistic insight at the molecular level, we carried out extensive molecular dynamics simulations of the tetrameric M2 proton channel in both apo and amantadine-bound forms in a lipid bilayer. The simulation of the apo form revealed that Val27 from the four M2 subunits can form a secondary gate near the channel entrance and break the water wire in the channel pore. This gate arises from physical occlusion and the elimination of hydrogen-bonding partners for water molecules. In the presence of amantadine, the secondary gate formed by Val27 and the drug molecule lying just below form an extended blockage, which breaks the water wire throughout the simulation. The location and orientation of amantadine inside of the channel pore as found in our simulation are supported by a host of experimental observations. Our study suggests a novel role for Val27 in the inhibition of the M2 proton channel by amantadine.

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Influenza A virus and influenza B virus particles both contain small integral membrane proteins (A/M2 and BM2, respectively) that function as a pH-sensitive proton channel and are essential for virus replication. The mechanism of action of the M2 channels is a subject of scientific interest particularly as A/M2 channel was shown to be a target for the action of the antiviral drug amantadine. Unfortunately, an inhibitor of the BM2 channel activity is not known. Thus, knowledge of the structural and functional properties of the BM2 channel is essential for the development of potent antiviral drugs. The characterization of the oligomeric state of the BM2 channel is an essential first step in the understanding of channel function. Here we describe determination of the stoichiometry of the BM2 proton channel by utilizing three different approaches. 1) We demonstrated that BM2 monomers can be chemically cross-linked to yield species consistent with dimers, trimers, and tetramers. 2) We studied electrophysiological and biochemical properties of mixed oligomers consisting of wild-type and mutated BM2 subunits and related these data to predicted binomial distribution models. 3) We used fluorescence resonance energy transfer (FRET) in combination with biochemical measurements to estimate the relationships between BM2 channel subunits expressed in the plasma membrane. Our experimental data are consistent with a tetrameric structure of the BM2 channel. Finally, we demonstrated that BM2 transmembrane domain is responsible for the channel oligomerization.

Solid-State NMR and MD Simulations of the Antiviral Drug Amantadine Solubilized in DMPC Bilayers

The interactions of 15N-labeled amantadine, an antiinfluenza A drug, with DMPC bilayers were investigated by solid-state NMR and by a 12.6-ns molecular dynamics (MD) simulation. The drug was found to assume a single preferred orientation and location when incorporated in these bilayers. The experimental and MD computational results demonstrate that the long axis of amantadine is on average parallel to the bilayer normal, and the amine group is oriented toward the headgroups of the lipid bilayers. The localization of amantadine was determined by paramagnetic relaxation and by the MD simulation showing that amantadine is within the interfacial region and that the amine interacts with the lipid headgroup and glycerol backbone, while the hydrocarbon portion of amantadine interacts with the glycerol backbone and much of the fatty acyl chain as it wraps underneath the drug. The lipid headgroup orientation changes on drug binding as characterized by the anisotropy of 31P chemical shielding and 14N quadrupolar interactions and by the MD simulation.

Structure and mechanism of the M2 proton channel of influenza A virus

The integral membrane protein M2 of influenza virus forms pH-gated proton channels in the viral lipid envelope. The low pH of an endosome activates the M2 channel before haemagglutinin-mediated fusion. Conductance of protons acidifies the viral interior and thereby facilitates dissociation of the matrix protein from the viral nucleoproteins--a required process for unpacking of the viral genome. In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm. Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication. Here we present the structure of the tetrameric M2 channel in complex with rimantadine, determined by NMR. In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a 'tryptophan gate' is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix-helix packing, thus facilitating channel opening.

Side-Chain Conformation of the M2 Transmembrane Peptide Proton Channel of Influenza A Virus from 19F Solid-State NMR

The M2 transmembrane peptide (M2TMP) of the influenza A virus forms a tetrameric helical bundle that acts as a proton-selective channel important in the viral life cycle. The side-chain conformation of the peptide is largely unknown and is important for elucidating the proton-conducting mechanism and the channel stability. Using a 19F spin diffusion NMR technique called CODEX, we have measured the oligomeric states and interhelical side chain-side chain 19F-19F distances at several residues using singly fluorinated M2TMP bound to DMPC bilayers. 19F CODEX data at a key residue of the proton channel, Trp41, confirm the tetrameric state of the peptide and yield a nearest-neighbor interhelical distance of approximately 11 A under both neutral and acidic pH. Since the helix orientation is precisely known from previous 15N NMR experiments and the backbone channel diameter has a narrow allowed range, this 19F distance constrains the Trp41 side-chain conformation to t90 (chi1 approximately 180 degrees , chi2 approximately 90 degrees ). This Trp41 rotamer, combined with a previously measured 15N-13C distance between His37 and Trp411, suggests that the His37 rotamer is t-160. The implication of the proposed (His37, Trp41) rotamers to the gating mechanism of the M2 proton channel is discussed. Binding of the antiviral drug amantadine to the peptide does not affect the F-F distance at Trp41. Interhelical 19F-19F distances are also measured at residues 27 and 38, each mutated to 4-19F-Phe. For V27F-M2TMP, the 19F-19F distances suggest a mixture of dimers and tetramers, whereas the L38F-M2TMP data indicate two tetramers of different sizes, suggesting side chain conformational heterogeneity at this lipid-facing residue. This work shows that 19F spin diffusion NMR is a valuable tool for determining long-range intermolecular distances that shed light on the mechanism of action and conformational heterogeneity of membrane protein oligomers.

The Chemical and Dynamical Influence of the Anti-Viral Drug Amantadine on the M 2 Proton Channel Transmembrane Domain

The M 2 proton channel plays a vital role in the life cycle of the influenza A virus. His 37, the key residue in the M 2 transmembrane domain (M 2-TMD), plays a central role in the proton conductance mechanism. The anti-influenza drug, amantadine, inhibits the channel activity through binding to the pore of the M 2 channel. The nuclear spin relaxation data and polarization inversion spin exchange at the magic angle spectra show that both the polypeptide backbone and His 37 side chain are more constrained in the presence of amantadine. Using 15N cross polarization magic-angle spinning NMR spectroscopy, the protonation of His 37 of M 2-TMD in lipid bilayers was monitored in the absence and presence of amantadine as a function of pH. Binding amantadine lowers the His 37 pK a values by approximately three orders of magnitude compared with the first pK a of histidine in amantadine-free M 2-TMD. Amantadine’s influence on the His 37 chemical properties suggests a novel mechanism by which amantadine may inhibit proton conductance.

Influenza virus proton channels

The M2 ion channel proteins of influenza A and B viruses are essential to viral replication. The two ion channels share a common motif, HXXXW, that is responsible for proton selectivity and activation. The ion channel for the influenza A virus, but not influenza B virus, is inhibited by the antiviral drug amantadine and amantadine-resistant escape mutants form in treated influenza A patients. The studies reviewed suggest that an antiviral compound directed against the conserved motif would be more useful than amantadine in inhibiting viral replication.

インフルエンザパンデミック

In most cases, influenza is not fatal, even without treatment. Moreover, vaccination and antivirals have reduced influenza-related mortality in recent years. However, the direct transmission of avian influenza viruses to humans with lethal outcomes in Hong Kong in 1997 was a potent reminder of the devastating potential of the disease. Currently, H5N1 avian influenza viruses are circulating in many Asian countries, and the human death toll continues to rise as the virus spreads to European countries as well. Since the beginning of the outbreak in Asia, more than 120 cases have been confirmed and the mortality rate has been no less than 50%. Current vaccines for H3N2 and H1N1 viruses, of course, have no effect on infection by H5N1 viruses. In addition, H5N1 viruses that are resistant to the antiviral drugs amantadine and oseltamivir have emerged. Fortunately, a virus that is capable of efficient transmission among humans has not emerged. However, it is not a matter of if, but when, such a virus will appear. Here, we review the current situation of avian influenza and pandemic preparedness.

A randomised, open label, controlled trial on the effect of interferon plus amantadine compared with interferon alone for treatment of chronic hepatitis C

Background: Combination of the antiviral drug amantadine with interferon (IFN) may be more effective than IFN monotherapy for treatment of chronic hepatitis C. Methods: We randomised 93 patients with chronic hepatitis C to IFNIFN 6 MU 3 times/week for 24 weeks, followed by IFN 3 MU 3 times/week for further 24 weeks given in combination with amantadine 100 mg t.i.d. (regimen A, n=48) or as monotherapy (regimen B, n=45). Control liver biopsies were obtained 6 months post treatment. Results: At the end of the trial a similar proportion of patients had normal serum ALT levels (35% for regimen A, and 44% for regimen B) as measured by intention to treat criteria. Sustained biochemical response at 6 months post treatment was 15 and 20%, and sustained virological response was 10 and 20% for regimen A and B, respectively. The effect on liver histology was also similar: the inflammatory components of the Knodell score decreased by 1.3±0.6 points for regimen A and by 1.6±0.6 for regimen B, and score for fibrosis remained unchanged with both regimens. Conclusions: Combination of IFN therapy with amantadine does not enhance the effect of IFN as shown by biochemical, virological and histological criteria.

Conditional ablation of T-cell development by a novel viral ion channel transgene.

A novel conditional-lethal transgene system is defined in which a mutated influenza A virus ion-channel protein, which is permeable to monovalent cations, is lethal to cells on heterotypic expression and whose activity can be blocked by an antiviral drug (amantadine), is used to reversibly disrupt T-cell development. In vivo expression of the M2 ion channel, as a transgene under control of the T-cell specific p56(Lck) proximal promoter, resulted in total ablation of T-cell development with the accumulation of three distinct populations of early progenitor cells (CD44(+) CD25(-); CD44(+) CD25(+); CD44(+) CD25(hi)) in the thymic rudiment. In vitro development of transgenic fetal thymic progenitors to single-positive T cells could be rescued by antiviral drug treatment. Moreover, there was a radical reduction in B-cell lymphopoiesis, evident at the pre-B-cell stage, with a twofold increase of lymphoid cells 'in cycle' in transgenic bone marrow, indicative of major changes in haematopoietic homeostasis. This system may provide a generic protocol for conditional, lineage-specific cell ablation with available tissue-specific promoters for any eukaryotic developmental system, and provide a window on early T-cell development.

Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture.

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656-5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del(29-31) virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del(29-31) virus. We found that the M2-del(29-31) virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M2-del(29-31) virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.

Amantadine in Parkinson's disease: lymphocyte subsets and IL-2 secreting T cell precursor frequencies

The antiviral drug amantadine, that is effective in idiopathic Parkinson's disease (PD), may affect the composition and function of peripheral blood lymphocytes. In an explorative study, we therefore compared lymphocyte subpopulations and IL-2 secreting T cell precursors frequencies (HTLp-frequencies) in 15 PD patients without amantadine and six patients on long-term treatment. Five patients were investigated before and three months after the start of treatment. Group comparisons for long-term amantadine treatment showed no differences in subpopulations of B-, T-, and NK cells, and HTLp-frequencies. However, three months after initiation of treatment we noted in all five patients an increase of CD3+CD4+ and decrease of CD3+CD8+ cells, associated with an increase of the CD3+CD4+/CD3+CD8+ ratio. These changes had no effect on the HTLp-frequencies. Thus, at least for a short period of time, amantadine improves the T cell mediated immune system in PD patients.

Influenza: vaccination and treatment.

Few conditions exert such an enormous toll of absenteeism, suffering, medical consultations, hospitalization, death and economic loss as influenza. Patients at high risk of complications and mortality include the elderly and those with pre-existing cardiopulmonary disease. The outbreak in 1997 in Hong Kong, of avian H5N1 influenza in man, which resulted in six deaths among 18 hospitalized cases, and the recent isolation of H9N2 viruses from two children in Hong Kong, are reminders that preparation must be made for the next pandemic. Since the 1970s, efforts to control influenza have mostly focussed on the split product and surface antigen vaccines. These vaccines are of proven efficacy in healthy adults and are effective in elderly people with and without medical conditions putting them at high risk of complications and death following influenza infection. However, vaccine coverage is patchy and often low, and outbreaks of influenza are not uncommon in well-immunized residents of nursing homes. New vaccines and methods of vaccine delivery are being developed in attempts to overcome the limitations of existing vaccines. The antiviral drugs amantadine and rimantadine were developed in the 1960s, but have not been used widely due to their spectrum of activity, rapid emergence of resistance, and adverse effects associated with amantadine. The site of enzyme activity of the influenza neuraminidase is highly conserved between types, subtypes and strains of influenza and has emerged as the target of an exciting new class of antiviral agents that are effective both prophylactically and as therapy.

Pre-resonance Raman and surface-enhanced Raman spectroscopy study of the complex of the antiviral and antiparkinsonian drug amantadine with histidine

UV-pre-resonance Raman spectroscopy (PRS) and surface-enhanced Raman spectroscopy (SERS) were applied to study the interaction of the antiviral and antiparkinsonian agent amantadine with histidine. The binding sites of amantadine and histidine molecules were identified in the complex. In this model, (i) the amino group of amantadine and the N1H group of histidine are included in the complex formation, (ii) the creation of the complex is mediated via formation of an H-bond between the nitrogen of the amantadine amino group and the hydrogen of the histidine N1H group. This model of the amantadine–histidine complex formation is discussed in the relation to a possible mode of interaction of the M2 protein transmembrane region with amantadine.

Developments in Influenza Prevention and Treatment

Until recently, control of influenza has focused on increasing the use of the inactivated influenza vaccine and, in some countries, encouraging the appropriate use of the antiviral drugs amantadine and rimantadine. Now that a new class of antiviral drugs, the neuraminidase inhibitors, has been approved for use in many countries, and a live attenuated vaccine is on the horizon, novel opportunities have arisen for the prevention and treatment of influenza. The clinical effectiveness of these innovations has not been fully determined in many patient populations, particularly in managed-care environments; clinical and economic impacts are similarly considered in making decisions regarding access to new technologies in most healthcare delivery systems. However, it is possible to use available information to speculate on how these technologies might be used, while the relative clinical and cost consequences of these innovations are further clarified.

Exploring Models of the Influenza A M2 Channel: MD Simulations in a Phospholipid Bilayer

The M2 protein of influenza A virus forms homotetrameric helix bundles, which function as proton-selective channels. The native form of the protein is 97 residues long, although peptides representing the transmembrane section display ion channel activity, which (like the native channel) is blocked by the antiviral drug amantadine. As a small ion channel, M2 may provide useful insights into more complex channel systems. Models of tetrameric bundles of helices containing either 18 or 22 residues have been simulated while embedded in a fully hydrated 1-palmitoyl-2-oleoyl- sn-glycerol-3-phosphatidylcholine bilayer. Several different starting models have been used. These suggest that the simulation results, at least on a nanosecond time scale, are sensitive to the exact starting structure. Electrostatics calculations carried out on a ring of four ionizable aspartate residues at the N-terminal mouth of the channel suggest that at any one time, only one will be in a charged state. Helix bundle models were mostly stable over the duration of the simulation, and their helices remained tilted relative to the bilayer normal. The M2 helix bundles form closed channels that undergo breathing motions, alternating between a tetramer and a dimer-of-dimers structure. Under these conditions either the channel forms a pocket of trapped waters or it contains a column of waters broken predominantly at the C-terminal mouth of the pore. These waters exhibit restricted motion in the pore and are effectively “frozen” in a way similar to those seen in previous simulations of a proton channel formed by a four-helix bundle of a synthetic leucine-serine peptide (Randa et al., 1999, Biophys. J. 77:2400–2410).

FT-Raman, FTIR and surface-enhanced Raman spectroscopy of the antiviral and antiparkinsonian drug amantadine

FT-Raman, FTIR and surface-enhanced Raman spectroscopy (SERS) are applied to the vibrational characterization of the antiviral and antiparkinsonian drug amantadine. SERS spectroscopy is employed for the first time for characterizing the interfacial behavior of this molecule and to study its interaction with colloidal silver. The comparison of SERS spectrum with the Raman spectra of amantadine in solid state and in aqueous solution reveals remarkable changes attributed to the interaction of the drug with the metal through the unprotonated amino group and the formation of a self-assembled amantadine layer on the metal surface. A tentative assignment of the obtained vibrational spectra is carried out on the basis of the vibrational spectra of the structurally related molecules adamantane and tert-buthylamine and the ab initio calculations accomplished for amantadine.