title: 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.
authors.affiliation: Department of Nutrition, Food Science and Gastronomy, Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine (IBUB), University of Barcelona , Avgda. Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain.
authors.forename: Salomé
authors.initials: S
authors.lastname: Llabrés
authors.affiliation: Department of Nutrition, Food Science and Gastronomy, Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine (IBUB), University of Barcelona , Avgda. Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Juárez-Jiménez
authors.affiliation: Department of Pharmacy and Biotecnology (FaBit), Alma Mater Studiorum, University of Bologna , via Belmeloro 6, 40126 Bologna, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Masetti
authors.affiliation: Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia i Ciències de l'Alimentació, and Institute of Biomedicine (IBUB), Universitat de Barcelona , Av. Joan XXIII 27-31, 08028 Barcelona, Spain.
authors.forename: Rosana
authors.initials: R
authors.lastname: Leiva
authors.affiliation: Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia i Ciències de l'Alimentació, and Institute of Biomedicine (IBUB), Universitat de Barcelona , Av. Joan XXIII 27-31, 08028 Barcelona, Spain.
authors.forename: Santiago
authors.initials: S
authors.lastname: Vázquez
authors.affiliation: Department of Biosciences and National Research Council (CNR) Biophysics Institute (IBF), University of Milan , Via Celoria 26, 20133 Milan, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Gazzarrini
authors.affiliation: Department of Biosciences and National Research Council (CNR) Biophysics Institute (IBF), University of Milan , Via Celoria 26, 20133 Milan, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Moroni
authors.affiliation: Department of Pharmacy and Biotecnology (FaBit), Alma Mater Studiorum, University of Bologna , via Belmeloro 6, 40126 Bologna, Italy.|CompuNet, Istituto Italiano di Tecnologia (IIT) , via Morego 30, 16163 Genova, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cavalli
authors.affiliation: Department of Nutrition, Food Science and Gastronomy, Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine (IBUB), University of Barcelona , Avgda. Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain.
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Luque