Binding and Blocking of Influenza A WT and S31N M2 by Cage Alkyl Amine-Containing Inhibitors

Proton conductance of the wild-type (WT) M2 protein of influenza A is inhibited by cage alkyl amines, which bind in the pore of the channel. Conjugates with a methylene-linked aryl group have been shown to block the channel of the prevalent S31N strain with a different mechanism, but at the same binding site. Here, we investigated the binding and blocking of WT and S31N M2 by a set of these amines using different biophysical methods. The binding behavior of the ligands was explored using magic angle spinning NMR. NMR clearly detected binding for WT but not S31N. Correlation of binding data with ligand cage size suggested a significant structural barrier. Using a liposomal proton flux assay, we confirmed that the compounds identified as binders by NMR also blocked the channel. To assess the binding mode of these small molecules, molecular dynamics simulations were performed. The simulations predicted different binding modes to WT or S31N. No binding to the S31N variant was detected by NMR, nor was the channel effectively blocked in the liposomal proton flux assay or MD simulations. A comparison of M218–60 with ectodomain-truncated M2 (M221–97) suggests that a substantial structural barrier limits entry of bulky cage amines and that the structural barrier is influenced by the presence or absence of cytoplasmic residues.