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Concept: M2 protein


The influenza A virus M2 channel (AM2) is crucial in the viral life cycle. Despite many previous experimental and computational studies, the mechanism of the activating process in which proton permeation acidifies the virion to release the viral RNA and core proteins is not well understood. Herein the AM2 proton permeation process has been systematically characterized using multiscale computer simulations, including quantum, classical, and reactive molecular dynamics methods. We report, to our knowledge, the first complete free-energy profiles for proton transport through the entire AM2 transmembrane domain at various pH values, including explicit treatment of excess proton charge delocalization and shuttling through the His37 tetrad. The free-energy profiles reveal that the excess proton must overcome a large free-energy barrier to diffuse to the His37 tetrad, where it is stabilized in a deep minimum reflecting the delocalization of the excess charge among the histidines and the cost of shuttling the proton past them. At lower pH values the His37 tetrad has a larger total charge that increases the channel width, hydration, and solvent dynamics, in agreement with recent 2D-IR spectroscopic studies. The proton transport barrier becomes smaller, despite the increased charge repulsion, due to backbone expansion and the more dynamic pore water molecules. The calculated conductances are in quantitative agreement with recent experimental measurements. In addition, the free-energy profiles and conductances for proton transport in several mutants provide insights for explaining our findings and those of previous experimental mutagenesis studies.

Concepts: Influenzavirus A, M2 protein, PH, Electron, Virus, Chemistry, Computer simulation, Protein


The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine, whose use has been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants, S31N is found in more than 95% of the currently circulating viruses and shows greatly decreased inhibition by amantadine. The discovery of inhibitors of S31N has been hampered by the limited size, polarity, and dynamic nature of its amantadine-binding site. Nevertheless, we have discovered small-molecule drugs that inhibit S31N with potencies greater than amantadine’s potency against WT M2. Drug binding locks the protein into a well-defined conformation, and the NMR structure of the complex shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. Unrestrained molecular dynamics simulations predicted the same binding site. This S31N inhibitor, like other potent M2 inhibitors, contains a charged ammonium group. The ammonium binds as a hydrate to one of three sites aligned along the central cavity that appear to be hotspots for inhibition. These sites might stabilize hydronium-like species formed as protons diffuse through the outer channel to the proton-shuttling residue His37 near the cytoplasmic end of the channel.

Concepts: Pharmacology, Inhibitor, M2 protein, Enzyme inhibitor, Virus, Influenza, Antiviral drug, Protein


The influenza A virus matrix protein 2 (M2 protein) is a pH-regulated proton channel embedded in the viral membrane. Inhibition of the M2 proton channel has been used to treat influenza infections for decades due to the crucial role of this protein in viral infection and replication. However, the widely-used M2 inhibitors, amantadine and rimantadine, have gradually lost their efficiencies because of naturally-occurring drug resistant mutations. Therefore, investigation of the structure and function of the M2 proton channel will not only increase our understanding of this important biological system, but also lead to the design of novel and effective anti-influenza drugs. Despite the simplicity of the M2 molecular structure, the M2 channel is highly flexible and there have been controversies and arguments regarding the channel inhibition mechanism and the proton conduction mechanism. In this book chapter, we will first carefully review the experimental and computational studies of the two possible drug binding sites on the M2 protein and explain the mechanisms regarding how inhibitors prevent proton conduction. Then, we will summarize our recent molecular dynamics simulations of the drug-resistant mutant channels and propose mechanisms for drug resistance. Finally, we will discuss two existing proton conduction mechanisms and talk about the remaining questions regarding the proton-relay process through the channel. The studies reviewed here demonstrate how molecular modeling and simulations have complemented experimental work and helped us understand the M2 channel structure and function.

Concepts: Pharmacology, Rimantadine, Computational chemistry, Microbiology, Influenza, Antibiotic resistance, Virus, M2 protein


The M2 proton channel of influenza A virus is an integral membrane protein involved in the acidification of the viral interior, a step necessary for the release of the viral genetic material and replication of new virions. The aim of this study is to explore the mechanism of drug (un)binding to the M2 channel in order to gain insight into the structural and energetic features relevant for the development of novel inhibitors. To this end, we have investigated the binding of amantadine (Amt) to the wild type (wt) M2 channel and its V27A variant using multiple independent molecular dynamics simulations, exploratory conventional metadynamics, and multiple-walkers well-tempered metadynamics calculations. The results allow us to propose a sequential mechanism for the (un)binding of Amt to the wt M2 channel, which involves the adoption of a transiently populated intermediate (up state) leading to the thermodynamically favored down binding mode in the channel pore. Furthermore, they suggest that chloride anions play a relevant role in stabilizing the down binding mode of Amt to the wt channel, giving rise to a kinetic trapping that explains the experimentally observed pseudoirreversible inhibition of the wt channel by Amt. We propose that this trapping mechanism underlies the inhibitory activity of potent M2 channel blockers, as supported by the experimental confirmation of the irreversible binding of a pyrrolidine analogue from electrophysiological current assays. Finally, the results reveal that the thermodynamics and kinetics of Amt (un)binding is very sensitive to the V27A mutation, providing a quantitative rationale to the drastic decrease in inhibitory potency against the V27A variant. Overall, these findings pave the way to explore the inhibitory activity of Amt-related analogues in mutated M2 channel variants, providing guidelines for the design of novel inhibitors against resistant virus strains.

Concepts: Genome, DNA, Integral membrane protein, Influenza, Gene, M2 protein, Virus, Protein


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.

Concepts: Influenzavirus A, Inhibitor, Rimantadine, Action potential, Amantadine, Influenza, Antiviral drug, M2 protein


Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogs has revolved around the synthesis of novel adamantane derivatives, we have recently found that other polycyclic scaffolds effectively block the M2 proton channel, including amantadine-resistant mutant channels. In this paper, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogs of amantadine. Inhibition of the wild-type M2 channel and the A/M2-S31N, A/M2-V27A and A/M2-L26F mutant forms of the channel were measured in Xenopus oocytes using two-electrode voltage clamp assays. Most of the novel compounds inhibited the wild type ion channel in the low micromolar range. Of note, two of the compounds inhibited the amantadine-resistant A/M2-V27A and A/M2-L26F mutant ion channels with submicromolar and low micromolar IC50, respectively. None of the compounds was found to inhibit the S31N mutant ion channel.

Concepts: Patch clamp, Amantadine, Action potential, Electrophysiology, Protein, Gene, Influenza, M2 protein


M2 protein of influenza A virus is a proton channel spanning the viral envelope. This proton channel activity is required for uncoating of viral particles and equilibrating the pH across the trans-Golgi apparatus to prevent a conformational change of hemagglutinin. Amantadine is an anti-influenza A virus drug which inhibits M2 proton channel activity by binding to the channel pore, however, the majority of currently circulating influenza A viruses are amantadine-resistant. The most prevalent resistant mutation is a substitution from Ser31 to Asn31 in M2. To provide new approaches for the design of novel M2 channel blockers, further atomistic analysis of ligand-M2 complexes is needed. Here we examined the free energy profiles of the binding kinetics of M2 channel blockers by well-tempered metadynamics simulations. We found that amantadine first binds to Asp24 of S31 M2 and forms a metastable conformation. In contrast, the free energy profiles of adamantyl bromothiophene dual inhibitor with either S31 M2 or N31 M2 were a broad funnel-shaped curve, suggesting that adamantyl bromothiophene does not form metastable complexes with M2. The trajectory of well-tempered metadynamics simulations revealed that the steric hindrance between adamantyl bromothiophene and S31 M2 interrupts the formation of metastable conformation at Asp24, and a halogen bond between the bromine atom and N31 is responsible for pulling down the ligand to the channel pore of N31 M2 without metastable state. Based on these results, we propose binding pathways of M2 channel blockers to M2 for providing new approaches to design further M2 channel blockers.

Concepts: Rimantadine, Amantadine, Orthomyxoviridae, Microbiology, Influenza A virus, Virus, M2 protein, Influenza


Lasting activations of toll-like receptors (TLRs), MAPK and NF-κB pathways can support influenza A virus (IAV) infection and promote pneumonia. In this study, we have investigated the effect and mechanism of action of emodin on IAV infection using qRT-PCR, western blotting, ELISA, Nrf2 luciferase reporter, siRNA and plaque inhibition assays. The results showed that emodin could significantly inhibit IAV (ST169, H1N1) replication, reduce IAV-induced expressions of TLR2/¾/7, MyD88 and TRAF6, decrease IAV-induced phosphorylations of p38/JNK MAPK and nuclear translocation of NF-κB p65. Emodin also activated the Nrf2 pathway, decreased ROS levels, increased GSH levelss and GSH/GSSG ratio, and upregulated the activities of SOD, GR, CAT and GSH-Px after IAV infection. Suppression of Nrf2 via siRNA markedly blocked the inhibitory effects of emodin on IAV-induced activations of TLR4, p38/JNK, and NF-κB pathways and on IAV-induced production of IL-1β, IL-6 and expression of IAV M2 protein. Emodin also dramatically increased the survival rate of mice, reduced lung edema, pulmonary viral titer and inflammatory cytokines, and improved lung histopathological changes. In conclusion, emodin can inhibit IAV replication and influenza viral pneumonia, at least in part, by activating Nrf2 signaling and inhibiting IAV-induced activations of the TLR4, p38/JNK MAPK and NF-κB pathways.

Concepts: M2 protein, Toll-like receptor, Inhibitor, Gene expression, Inflammation, Influenza vaccine, Pneumonia, Influenza


The influenza A and B viruses are the primary cause of seasonal flu epidemics. Common to both viruses is the M2 protein, a homo-tetrameric transmembrane ™ proton channel that acidifies the virion after endocytosis. Although influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HxxxW motif, which is responsible for proton selectivity and channel gating. Importantly, BM2 contains a second titratable histidine, His27, in the TM domain, which forms a reverse WxxxH motif with the gating tryptophan. To understand how His27 affects the proton conduction property of BM2, we have used solid-state NMR (SSNMR) to characterize the pH-dependent structure and dynamics of His27. In cholesterol-containing lipid membranes mimicking the virus envelope, 15N NMR spectra show that the His27 tetrad protonates with higher pKa’s than His19, indicating that the solvent-accessible His27 facilitates proton conduction of the channel by increasing the proton dissociation rates of His19. AM2 is inhibited by the amantadine class of antiviral drugs while BM2 has no known inhibitors. We measured the N-terminal interhelical separation of the BM2 channel using fluorinated Phe5. The interhelical (19)F-(19)F distances show a bimodal distribution of a short distance of 7 Å and a long distance of 15-20 Å, indicating that the phenylene rings do not block small-molecule entry into the channel pore. These results give insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels.

Concepts: Orthomyxoviridae, Cell membrane, Pandemic, M2 protein, Antiviral drug, Protein, Virus, Influenza


The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in “wires” inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.

Concepts: Hydrogen, Orthomyxoviridae, Atom, Electron, Water, M2 protein, Electric charge, Influenza