Rimantadine Binds to and Inhibits the Influenza A M2 Proton Channel without Enantiomeric Specificity

Abstract

The influenza A M2 wild-type (WT) proton channel is the target of the anti-influenza drug rimantadine. Rimantadine has two enantiomers, though most investigations into drug binding and inhibition have used a racemic mixture. Solid-state NMR experiments using the full length-M2 WT have shown significant spectral differences that were interpreted to indicate tighter binding for (R)- vs (S)-rimantadine. However, it was unclear if this correlates with a functional difference in drug binding and inhibition. Using X-ray crystallography, we have determined that both (R)- and (S)-rimantadine bind to the M2 WT pore with slight differences in the hydration of each enantiomer. However, this does not result in a difference in potency or binding kinetics, as shown by similar values for k on, k off, and K d in electrophysiological assays and for EC50 values in cellular assays. We concluded that the slight differences in hydration for the (R)- and (S)-rimantadine enantiomers are not relevant to drug binding or channel inhibition. To further explore the effect of the hydration of the M2 pore on binding affinity, the water structure was evaluated by grand canonical ensemble molecular dynamics simulations as a function of the chemical potential of the water. Initially, the two layers of ordered water molecules between the bound drug and the channel’s gating His37 residues mask the drug’s chirality. As the chemical potential becomes more unfavorable, the drug translocates down to the lower water layer, and the interaction becomes more sensitive to chirality. These studies suggest the feasibility of displacing the upper water layer and specifically recognizing the lower water layers in novel drugs.

Enhanced Grand Canonical Sampling of Occluded Water Sites Using Nonequilibrium Candidate Monte Carlo

“…As a result, they have been applied in a wide range of contexts such as investigating the binding of hydrogen to metal–organic frameworks and simulating the movement of ions through channels in membranes. − Sampling the grand canonical ensemble requires the chemical potential (μ), volume ( V ), and temperature ( T ) to be held constant. − Simulating at a constant chemical potential allows the number of particles in the system to fluctuate, which can be used to bypass kinetic barriers to the sampling of buried water molecules through randomly attempting their insertion and deletion within a user-defined region of interest such as a binding site. ,,,− These attempted moves are accepted and rejected based on rigorous probabilities derived using the Metropolis-Hastings algorithm. The use of GCMC sampling has been found to significantly improve the accuracy of ligand binding free energy calculations, where displaced waters that are not expelled sufficiently quickly from the binding site can have a serious impact on the free energy results, when using conventional sampling methods. ,,, However, the acceptance rates for unbiased and instantaneous particle insertions and deletions in condensed phases are typically very low, with around 1 in every 10,000 moves attempting to insert/delete water molecules to/from a bulk water system being accepted . A number of enhanced sampling techniques have been developed to improve the acceptance rates of GCMC, including cavity biasing, continuous fractional component Monte Carlo, configurational biasing, and molecular exchange approaches .…”

A proof-of-concept study of the secondary structure of influenza A, B M2 and MERS- and SARS-CoV E transmembrane peptides using folding molecular dynamics simulations in a membrane mimetic solvent

“…In the X-ray structure of AM2TM (PDB ID 35 ) or its ssNMR in the membrane (PBD ID 33 ), the structure of the peptide in the tetrameric bundle is α-helical and there is a kink at G13 (G34 in the AM2TM numbering scheme) in the middle of the TM domain, which allows the peptide to change conformations and adopt distinct orientations between the C- and N-terminal regions. 97 This G13 kink has been observed also in the ssNMR (PDB ID , 33 43 ) or X-ray (PDB ID , 44 98 ) drug-bound AM2 structures. This pattern almost exactly matches that in the middle of the membrane.…”

From Acid Activation Mechanisms of Proton Conduction to Design of Inhibitors of the M2 Proton Channel of Influenza A Virus

“…Finally, the stability of the protonated states of the His37 tetrad may be affected by the presence of chloride anions in the interior of the pore. Inspection of the X-ray crystallographic data available for the M2 proton channel in the Protein Data Bank (Berman et al, 2000;Burley et al, 2021) reveals the occurrence of a subset of structures where chloride anions are found in the interior of the pore (this subset is collected in Table 2) (Thomaston and DeGrado, 2016;Thomaston et al, 2018;Thomaston et al, 2020;Thomaston et al, 2021). In four out of the five cases, the chloride anion occupies a well-defined position close to the plane formed by the Trp41 residues.…”