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

ABSTRACT M2 protein of influenza A virus is a proton channel spanning the viral envelope. Activity of this proton channel is required for uncoating of viral particles and equilibrating the pH across the trans Golgi apparatus, which prevents conformational change in hemagglutinin. Amantadine, an anti‐influenza A virus drug, inhibits M2 proton channel activity by binding to the channel pore; however, most currently circulating influenza A viruses are amantadine‐resistant. The most prevalent resistant mutation is a substitution from Ser31 to Asn31 in M2. Further atomistic analysis of ligand‐M2 complexes is needed to provide new approaches for the design of novel M2 channel blockers. Here, the free energy profiles of the binding kinetics of M2 channel blockers were examined by well‐tempered metadynamics simulations and it was found that amantadine first binds to Asp24 of S31 M2 and forms a metastable conformation. In contrast, the free energy profiles of adamantyl bromothiophene dual inhibitor with either S31 M2 or N31 M2 are broad funnel‐shaped curves, suggesting that adamantyl bromothiophene does not form metastable complexes with M2. The trajectory of well‐tempered metadynamics simulations revealed that steric hindrance between adamantyl bromothiophene and S31 M2 interrupts formation of a metastable conformation at Asp24 and that a halogen bond between the bromine atom and N31 is responsible for pulling down the ligand to the channel pore of N31 M2 in the absence of a metastable state. Binding pathways of M2 channel blockers to M2 are here proposed on the basis of these findings; they may provide new approaches to designing further M2 channel blockers.