Mechanism of the Pseudoirreversible Binding of Amantadine to the M2 Proton Channel

Abstract

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.

Analysis by metadynamics simulation of binding pathway of influenza virus M2 channel blockers

“…The binding trajectories of the M2 channel blockers in the channel pore of S31 M2 showed that amantadine first binds to two of the four Asp24 residues via salt bridges to yield a metastable conformation separated by a high free energy barrier. Previous conventional and multiple‐walkers well‐tempered metadynamics simulations of amantadine unbinding pathway from S31 M2 have shown that Asp24 interacts with amantadine and this binding mediates the amantadine flipping at the entrance of channel pore . Therefore, it is possible that these data support the hypothesis that the metastable binding of amantadine to Asp24 is important in forming a thermodynamically favored binding of amantadine to the channel pore of S31 M2.…”

“…Conventional MD and stirred MD simulation studies have shown that, although amantadine specifically binds to S31 M2 through amino acid residues such as Val27, Ala30 and His37, the amino group of amantadine oppositely faces up toward the N‐terminus and does not have specific binding residues in the channel pore of N31 M2; thus, water molecules occupy space in the channel pore and proton conduction is not impaired . Conventional and multiple‐walkers well‐tempered metadynamics simulations have revealed that amantadine transiently binds to Asp24 during the unbinding pathway of amantadine from the channel pore of S31 M2 , suggesting that Asp24 is one of the metastable binding sites located at the entrances of channel pores. However, the details of the kinetics of binding of amantadine to M2 remain unclear.…”

Development of a multisite model for Ni(II) ion in solution from thermodynamic and kinetic data

“…The Well‐Tempered variant of Metadynamics (WTmetaD) was performed in the NVT ensemble for a total simulation time of about 150 ns. The software NAMD‐2.10 patched with PLUMED‐2.1 was used to execute the enhanced sampling calculations. Two collective variables (CVs) were chosen to efficiently sample the different Ni(II)‐complexed configurations.…”

Spotting Differences in Molecular Dynamic Simulations of Influenza A M2 Protein-Ligand Complexes by Varying M2 construct, Lipid Bilayer and Force Field

“…This is in contrast with the C open M2TM structure [27][28][29] and Amt-bound C open M2TM structure 28 at pH 5 in which the MD simulation of M2TM-Amt complex reveals multiple anions simultaneously inside the pore in the Q3 or Q4 state, 28 or in the MD simulation of M2AH-Amt complex, with M2AH in the Q2 state in which the presence of more than one chloride anion was suggested as possible. 30 (A) Recently, the potential of mean force (PMF) for Trp41 dihedral χ 2 was calculated and concluded that t-105 rotamer corresponds to a metastable state, while t-90 rotamer is energetically preferred. 26 The ssNMR structure of M2AH (PDB ID 2L0J) has the rotamers for all Trp41 with χ 2 = 105º.…”