Differential Binding of Rimantadine Enantiomers to Influenza A M2 Proton Channel.
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ABSTRACT:
SUBMITTER: Wright AK
PROVIDER: S-EPMC9328162 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recent findings from solid state NMR (ssNMR) studies suggested that the (R)-enantiomer of rimantadine binds to the full M2 protein with higher affinity than the (S)-enantiomer. Intrigued by these findings, we applied functional assays, such as antiviral assay and electrophysiology (EP), to evaluate the binding affinity of rimantadine enantiomers to the M2 protein channel. Unexpectedly, no significant difference was found between the two enantiomers. Our experimental data based on the full M2 protein function were further supported by alchemical free energy calculations and isothermal titration calorimetry (ITC) allowing an evaluation of the binding affinity of rimantadine enantiomers to the M2TM pore. Both enantiomers have similar channel blockage, affinity, and antiviral potency.
| S-EPMC5312807 | biostudies-literature
Project description:Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.
| S-EPMC7418971 | biostudies-literature
Project description:Recently, the binding kinetics of a ligand-target interaction, such as the residence time of a small molecule on its protein target, are seen as increasingly important for drug efficacy. Here, we investigate these concepts to explain binding and proton blockage of rimantadine variants bearing progressively larger alkyl groups to influenza A virus M2 wild type (WT) and M2 S31N protein proton channel. We showed that resistance of M2 S31N to rimantadine analogues compared to M2 WT resulted from their higher koff rates compared to the kon rates according to electrophysiology (EP) measurements. This is due to the fact that, in M2 S31N, the loss of the V27 pocket for the adamantyl cage resulted in low residence time inside the M2 pore. Both rimantadine enantiomers have similar channel blockage and binding kon and koff against M2 WT. To compare the potency between the rimantadine variants against M2, we applied approaches using different mimicry of M2, i.e., isothermal titration calorimetry and molecular dynamics simulation, EP, and antiviral assays. It was also shown that a small change in an amino acid at site 28 of M2 WT, which does not line the pore, seriously affects M2 WT blockage kinetics.
| S-EPMC5846047 | biostudies-literature
Project description:Structures of truncated versions of the influenza A virus M2 proton channel have been determined recently by x-ray crystallography in the open conformation of the channel, and by NMR in the closed state. The structures differ in the position of the bound inhibitors. The x-ray structure shows a single amantadine molecule in the middle of the channel, whereas in the NMR structure four drug molecules bind at the channel's outer surface. To study this controversy we applied computational solvent mapping, a technique developed for the identification of the most druggable binding hot spots of proteins. The method moves molecular probes--small organic molecules containing various functional groups--around the protein surface, finds favorable positions using empirical free energy functions, clusters the conformations, and ranks the clusters on the basis of the average free energy. The results of the mapping show that in both structures the primary hot spot is an internal cavity overlapping the amantadine binding site seen in the x-ray structure. However, both structures also have weaker hot spots at the exterior locations that bind rimantadine in the NMR structure, although these sites are partially due to the favorable interactions with the interfacial region of the lipid bilayer. As confirmed by docking calculations, the open channel binds amantadine at the more favorable internal site, in good agreement with the x-ray structure. In contrast, the NMR structure is based on a peptide/micelle construct that is able to accommodate the small molecular probes used for the mapping, but has a too narrow pore for the rimantadine to access the internal hot spot, and hence the drug can bind only at the exterior sites.
| S-EPMC2776279 | biostudies-literature
Project description:Prevalent resistance to inhibitors that target the influenza A M2 proton channel has necessitated a continued drug design effort, supported by a sustained study of the mechanism of channel function and inhibition. Recent high-resolution X-ray crystal structures present the first opportunity to see how the adamantyl amine class of inhibitors bind to M2 and disrupt and interact with the channel's water network, providing insight into the critical properties that enable their effective inhibition in wild-type M2. In this work, we examine the hypothesis that these drugs act primarily as mechanism-based inhibitors by comparing hydrated excess proton stabilization during proton transport in M2 with the interactions revealed in the crystal structures, using the Multiscale Reactive Molecular Dynamics (MS-RMD) methodology. MS-RMD, unlike classical molecular dynamics, models the hydrated proton (hydronium-like cation) as a dynamic excess charge defect and allows bonds to break and form, capturing the intricate interactions between the hydrated excess proton, protein atoms, and water. Through this, we show that the ammonium group of the inhibitors is effectively positioned to take advantage of the channel's natural ability to stabilize an excess protonic charge and act as a hydronium mimic. Additionally, we show that the channel is especially stable in the drug binding region, highlighting the importance of this property for binding the adamantane group. Finally, we characterize an additional hinge point near Val27, which dynamically responds to charge and inhibitor binding. Altogether, this work further illuminates a dynamic understanding of the mechanism of drug inhibition in M2, grounded in the fundamental properties that enable the channel to transport and stabilize excess protons, with critical implications for future drug design efforts.
| S-EPMC7564090 | biostudies-literature
Project description:The M2 protein of influenza A viruses forms a tetrameric pH-activated proton-selective channel that is targeted by the amantadine class of antiviral drugs. Its ion channel function has been extensively studied by electrophysiology and mutagenesis; however, the molecular mechanism of proton transport is still elusive, and the mechanism of inhibition by amantadine is controversial. We review the functional data on proton channel activity, molecular dynamics simulations of the proton conduction mechanism, and high-resolution structural and dynamical information of this membrane protein in lipid bilayers and lipid-mimetic detergents. These studies indicate that elucidation of the structural basis of M2 channel activity and inhibition requires thorough examination of the complex dynamics and conformational plasticity of the protein in different lipid bilayers and lipid-mimetic environments.
| S-EPMC2879269 | biostudies-literature
Project description:The homotetrameric influenza A M2 channel (AM2) is an acid-activated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.
| S-EPMC5111692 | biostudies-literature
Project description:Molecular dynamics trajectories 2 ?s 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 His(37) tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His(37) provide insight into the mechanism of proton transport. The channel is closed at both His(37) and Trp(41) sites in the singly and doubly protonated states, but it opens at Trp(41) upon further protonation. Anions access the charged His(37) and by doing so stabilize the protonated states of the channel. The narrow opening at the His(37) site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His(37) 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 Val(27) 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.
| S-EPMC3824548 | biostudies-literature
Project description:The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K(+) or Na(+) efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K(+) to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH(in)) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH(in) first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH(in) is achieved.
| S-EPMC2932607 | biostudies-literature
Project description:M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591-595) may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus.Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors.1) The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2) The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3) The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a "barrel hoop", holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4) The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.
| S-EPMC2826421 | biostudies-literature