M2 Proton Research Articles

Effect of cytosolic pH on inward currents reveals structural characteristics of the proton transport cycle in the influenza A protein M2 in cell-free membrane patches of Xenopus oocytes.

Transport activity through the mutant D44A of the M2 proton channel from influenza virus A was measured in excised inside-out macro-patches of Xenopus laevis oocytes at cytosolic pH values of 5.5, 7.5 and 8.2. The current-voltage relationships reveal some peculiarities: 1. “Transinhibition”, i.e., instead of an increase of unidirectional outward current with increasing cytosolic H+ concentration, a decrease of unidirectional inward current was found. 2. Strong inward rectification. 3. Exponential rise of current with negative potentials. In order to interpret these findings in molecular terms, different kinetic models have been tested. The transinhibition basically results from a strong binding of H+ to a site in the pore, presumably His37. This assumption alone already provides inward rectification and exponential rise of the IV curves. However, it results in poor global fits of the IV curves, i.e., good fits were only obtained for cytosolic pH of 8.2, but not for 7.5. Assuming an additional transport step as e.g. caused by a constriction zone at Val27 resulted in a negligible improvement. In contrast, good global fits for cytosolic pH of 7.5 and 8.2 were immediately obtained with a cyclic model. A “recycling step” implies that the protein undergoes conformational changes (assigned to Trp41 and Val27) during transport which have to be reset before the next proton can be transported. The global fit failed at the low currents at pHcyt = 5.5, as expected from the interference of putative transport of other ions besides H+. Alternatively, a regulatory effect of acidic cytosolic pH may be assumed which strongly modifies the rate constants of the transport cycle.

Viral M2 Ion Channel Protein: A Promising Target for Anti-influenza Drug Discovery

Influenza virus is an important RNA virus causing pandemics (Spanish Flu (1918), Asian Flu (1957), Hong Kong Flu (1968) and Swine Flu (2009)) over the last decades. Due to the spontaneous mutations of these viral proteins, currently available antiviral and anti-influenza drugs quickly develop resistance. To account this, only limited antiinfluenza drugs have been approved for the therapeutic use. These include amantadine and rimantadine (M2 proton channel blockers), zanamivir, oseltamivir and peramivir (neuraminidase inhibitors), favipravir (polymerase inhibitor) and laninamivir. This review provides an outline on the strategies to develop novel, potent chemotherapeutic agents against M2 proton channel. Primarily, the M2 proton channel blockers elicit pharmacological activity through destabilizing the helices by blocking the proton transport across the transmembrane. The biologically important compounds discovered using the scaffolds such as bisnoradmantane, noradamantane, triazine, spiroadamantane, isoxazole, amino alcohol, azaspiro, spirene, pinanamine, etc are reported to exhibit anti-influenza activity against wild or mutant type (S31N and V27A) of M2 proton channel protein. The reported studies explained that the adamantane based compounds (amantadine and rimantadine) strongly interact with His37 (through hydrogen bonding) and Ala30, Ile33 and Gly34 residues (hydrophobic interactions). The adamantane and the non-adamantane scaffolds fit perfectly in the active site pocket present in the wild type and the charged amino groups (ammonium) create positive electrostatic potential, which blocks the transport of protons across the pore. In the mutated proteins, larger or smaller binding pocket are created by small or large mutant residues, which do not allow the molecules fit in the active site. This causes the channel to be unblocked and the protons are allowed to transfer inside the pore. The structural analysis of the M2 proton channel blockers illustrated that the adamantane derivatives have action against both influenza A and B, but have no effect on the mutants.

Discovery of Potential, Non-Toxic Influenza Virus Inhibitor by Computational Techniques.

Influenza infection continues to be a major problem in many parts of the world. Rimantadine is a first-line drug used to treat the influenza infection by targeting M2 proton channel. However, S31N mutation in M2 proton channel diminishes the efficiency of rimantadine and creates resistance. To address this issue, the present study was aimed to screen the effective lead candidate against drug resistance strain of influenza from DrugBank database. Initially, the lead molecules were filtered using Lipinski rule of five and the drug likeliness property. Subsequently, the data reduction was carried out by employing molecular docking study. Finally, molecular dynamics simulations techniques were performed to validate the lead compound. Most importantly, the -p LD50 of the screened lead molecule was calculated using CORAL software to estimate the Rat oral toxicity. Accordingly, memantine may possibly become a promising lead compound of rimantadine-resistant influenza virus strain.

Easily Accessible Polycyclic Amines that Inhibit the Wild-Type and Amantadine-Resistant Mutants of the M2 Channel of Influenza A Virus

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 analogues 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 work, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogues 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.

2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel.

Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel.

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.

Do the omeprazole family compounds exert a protective effect against influenza-like illness?

BackgroundInfections by influenza viruses place a heavy burden on public health and economies worldwide. Although vaccines are the best weapons against influenza, antiviral drugs could offer an opportunity to alleviate the burden of influenza. Since omeprazole family compounds block the “proton pump”, we hypothesized that they could interfere with the mechanism of fusion of the virus envelope and endosomal membrane, thereby hindering the M2 proton pump mechanism of influenza viruses.MethodsA matched case-control study was performed in 2010-2011 in Italy. Cases were subjects aged over 18 years with a diagnosis of Influenza-like Illness (ILI); 254 case-control pairs were recruited. A multivariable conditional logistic regression analysis was used to assess the association between the prevention of ILI and the administration of omeprazole family compounds. The interaction between omeprazole family compounds and influenza vaccination was also examined.ResultsAfter control for potential confounders, subjects treated with omeprazole family compounds displayed a lower risk of catching ILI (ORadj = 0.29, 95% CI: 0.15-0.52). The risk of ILI in unvaccinated non-OFC users was about six times than that in vaccinated OFC users.ConclusionsAlthough confirmation is necessary, these results suggest that omeprazole family compounds could be profitably used in the prevention of ILI.

Aminoadamantanes with persistent in vitro efficacy against H1N1 (2009) influenza A.

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.

Computational investigation of drug-resistant mutant of M2 proton channel (S31N) against rimantadine.

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.

EGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM)

The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500nm in two color channels with time resolution of below 100ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H+ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Expedient Total Synthesis of Small to Medium-Sized Membrane Proteins via Fmoc Chemistry

Total chemical synthesis provides a unique approach for the access to uncontaminated, monodisperse, and more importantly, post-translationally modified membrane proteins. In the present study we report a practical procedure for expedient and cost-effective synthesis of small to medium-sized membrane proteins in multimilligram scale through the use of automated Fmoc chemistry. The key finding of our study is that after the attachment of a removable arginine-tagged backbone modification group, the membrane protein segments behave almost the same as ordinary water-soluble peptides in terms of Fmoc solid-phase synthesis, ligation, purification, and mass spectrometry characterization. The efficiency and practicality of the new method is demonstrated by the successful preparation of Ser64-phosphorylated M2 proton channel from influenza A virus and the membrane-embedded domain of an inward rectifier K(+) channel protein Kir5.1. Functional characterizations of these chemically synthesized membrane proteins indicate that they provide useful and otherwise-difficult-to-access materials for biochemistry and biophysics studies.

The role of a LC3 interacting region motif in influenza M2

BackgroundIn autophagy, cellular components are engulfed in a double membrane and recycled for their nutritional value. This mechanism has been adapted to defend cells against intracellular pathogens. Although viruses must evade autophagocytic destruction, they can also benefit from the resources that autophagy provides. We explored the molecular mechanisms by which influenza manipulates the autophagocytic machinery. MethodsWe used pull-down and fluorescence anisotropy assays to investigate the binding of the influenza A virus M2 proton channel to the essential autophagy protein LC3. The distribution of autophagosomes in infected cells was investigated by confocal microscopy. Construction of LIR-mutant viruses allowed us to investigate the importance of the interaction for subverting autophagy and the consequences for virion morphology and stability. FindingsWe found that binding to LC3 is mediated by a highly conserved LC3 interacting region (LIR) in the cytoplasmic tail of M2, the first such motif discovered in a pathogen. The M2 LIR is required for the redistribution of LC3 to the unusual destination of the plasma membrane in virus-infected cells. Mutations in M2 that abolish binding to LC3 interfere with filamentous budding and reduce virion stability. InterpretationWe conclude that influenza A virus subverts autophagy by mimicking a host short linear protein–protein interaction motif. We suggest that this process occurs to provide adequate supply of membrane for virion production. This efficient strategy might facilitate transmission of infection between organisms by enhancing the stability of viral progeny. FundingAcademy of Medical Sciences.

Solid-State NMR Characterization of S31N M2 Transmembrane Domain Bound to Novel Adamantanes with Persistent In Vitro Efficacy

The M2 proton channel from Influenza A virus is essential for the viral lifecycle and is an important drug target. Amino acid mutations in the residues lining the pore of the channel have abrogated clinical efficacy of the previously FDA-approved antiviral agents, amantadine and rimantadine. More than 95% of the circulating strains isolated from human clinical cases bear S31N mutation and a subsequent resistance to the licensed small molecule inhibitors. Efforts in rational drug design targeting S31N M2 channel have been impeded by the limited number of experimental techniques with capabilities for structural characterization of the protein-ligand interaction in native-like membrane mimetic environments. Solution and Solid State Nuclear Magnetic Resonance (NMR) investigations of the recently introduced adamantane analogues suggest multiple orientations of the inhibitor molecules bound in the pore. Position of the substituent moiety in the channel varies for different compounds, unlike the fixed orientation in the wild type channel. Here we report a Solid State NMR investigation of the transmembrane domain of S31N M2 (TMD) proton channel bound to the novel inhibitor molecules, while also incorporated into a lipid bilayer environment. Oriented sample solid state NMR experiments of S31N M2 TMD indicate that the channel is sampling two states with helical tilts of 28° and 33° relative to the bilayer normal. The kink in the monomer between two helical fragments, observed in the wild type M2 TMD bound with amantadine, is absent for the S31N M2 TMD bound to all inhibitor molecules tested to date. Stabilizing interactions are further investigated with Rotational Echo Double Resonance Magic Angle Spinning (REDOR MAS) experiments for measuring distances from the inhibitor to the protein.

Structure and Inhibition of the M2 Proton Channel from the Influenza a Virus

The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs, amantadine and rimantadine, whose use have been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants of M2, S31N, V27A and L26F were found in more than 99% of the currently circulating viruses. Discovery of inhibitors targeting these M2 mutants has been hampered by the lack of structural information and their limited sizes, polarity, and dynamic nature of their drug binding sites. Nevertheless, using an integrated approach including medicinal chemistry, molecular dynamics simulation, solid/solution-state NMR, X-ray crystallography, and pharmacological characterizations, we have discovered small molecule drugs that inhibit mutant M2 (S31N, V27A and L26F) with potencies greater than amantadine's potency against WT M2. A few compounds exhibiting EC50 around 100 nM are advanced to mice model studies. Structural characterization of S31N drug binding by NMR shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. The S31N inhibitors, like other potent WT M2 inhibitors, contain 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 drug binding hotspots along the channel axis provide a general model of M2 inhibition that can be used to guide the design of other channel blockers.

3-Azatetracyclo[5.2.1.15,8.01,5]undecane Derivatives: From Wild-Type Inhibitors of the M2 Ion Channel of Influenza A Virus to Derivatives with Potent Activity against the V27A Mutant

We have synthesized and characterized a series of compounds containing the 3-azatetracyclo[5.2.1.1(5,8).0(1,5)]undecane scaffold designed as analogues of amantadine, an inhibitor of the M2 proton channel of influenza A virus. Inhibition of the wild-type (WT) M2 channel and the amantadine-resistant A/M2-S31N and A/M2-V27A mutant ion channels were measured in Xenopus oocytes using two-electrode voltage clamp (TEV) assays. Most of the novel compounds inhibited the WT ion channel in the low micromolar range. Of note, several compounds inhibited the A/M2 V27A mutant ion channel, one of them with submicromolar IC50. None of the compounds was found to inhibit the S31N mutant ion channel. The antiviral activity of three novel dual WT and A/M2-V27A channels inhibitors was confirmed by influenza virus yield assays.

Activation and Proton Transport Mechanism in Influenza A M2 Channel

Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His37 tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His37 provide insight into the mechanism of proton transport. The channel is closed at both His37 and Trp41 sites in the singly and doubly protonated states, but it opens at Trp41 upon further protonation. Anions access the charged His37 and by doing so stabilize the protonated states of the channel. The narrow opening at the His37 site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His37 correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val27 remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations.

Asp44 Stabilizes the Trp41 Gate of the M2 Proton Channel of Influenza A Virus

Channel gating and proton conductance of the influenza A virus M2 channel result from complex pH-dependent interactions involving the pore-lining residues His37, Trp41, and Asp44. Protons diffusing from the outside of the virus protonate His37, which opens the Trp41 gate and allows one or more protons to move into the virus interior. The Trp41 gate gives rise to a strong asymmetry in the conductance, favoring rapid proton flux only when the outside is at acid pH. Here, we show that the proton currents recorded for mutants of Asp44, including D44N found in the A/FPV/Rostock/34 strain, lose this asymmetry. Moreover, NMR and MD simulations show that the mutations induce a conformational change similar to that induced by protonation of His37 at low pH, and decrease the structural stability of the hydrophobic seal associated with the Trp41 gate. Thus, Asp44 is able to determine two important properties of the M2 proton channel.

Impact of histidine residues on the transmembrane helices of viroporins

Abstract The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein 'u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.

Hot Spots in a Network of Functional Sites

It is of significant interest to understand how proteins interact, which holds the key phenomenon in biological functions. Using dynamic fluctuations in high frequency modes, we show that the Gaussian Network Model (GNM) predicts hot spot residues with success rates ranging between S 8–58%, C 84–95%, P 5–19% and A 81–92% on unbound structures and S 8–51%, C 97–99%, P 14–50%, A 94–97% on complex structures for sensitivity, specificity, precision and accuracy, respectively. High specificity and accuracy rates with a single property on unbound protein structures suggest that hot spots are predefined in the dynamics of unbound structures and forming the binding core of interfaces, whereas the prediction of other functional residues with similar dynamic behavior explains the lower precision values. The latter is demonstrated with the case studies; ubiquitin, hen egg-white lysozyme and M2 proton channel. The dynamic fluctuations suggest a pseudo network of residues with high frequency fluctuations, which could be plausible for the mechanism of biological interactions and allosteric regulation.

Applications of rare event dynamics on the free energy calculations for membrane protein systems

Techniques of rare event dynamics were reviewed, including string methods, which will be implemented with the biochemical simulation packages. The existing methods were applied to study biological systems with relevance to drug design and drug metabolism. The rare event dynamics simulations were performed to understand the kinetic and thermodynamic free energy information on the drug binding sites in the M2 proton channel, the free energy of insertion and association of membrane proteins and membrane active peptides. Results give a theoretical framework to interpret and reconcile existing and often conflicting opinions.