Solid-State NMR Characterization of S31N M2 Transmembrane Domain Bound to Novel Adamantanes with Persistent In Vitro Efficacy

Abstract

The M2 proton channel from Influenza A virus is essential for the viral lifecycle and is an important drug target. Amino acid mutations in the residues lining the pore of the channel have abrogated clinical efficacy of the previously FDA-approved antiviral agents, amantadine and rimantadine. More than 95% of the circulating strains isolated from human clinical cases bear S31N mutation and a subsequent resistance to the licensed small molecule inhibitors. Efforts in rational drug design targeting S31N M2 channel have been impeded by the limited number of experimental techniques with capabilities for structural characterization of the protein-ligand interaction in native-like membrane mimetic environments. Solution and Solid State Nuclear Magnetic Resonance (NMR) investigations of the recently introduced adamantane analogues suggest multiple orientations of the inhibitor molecules bound in the pore. Position of the substituent moiety in the channel varies for different compounds, unlike the fixed orientation in the wild type channel. Here we report a Solid State NMR investigation of the transmembrane domain of S31N M2 (TMD) proton channel bound to the novel inhibitor molecules, while also incorporated into a lipid bilayer environment. Oriented sample solid state NMR experiments of S31N M2 TMD indicate that the channel is sampling two states with helical tilts of 28° and 33° relative to the bilayer normal. The kink in the monomer between two helical fragments, observed in the wild type M2 TMD bound with amantadine, is absent for the S31N M2 TMD bound to all inhibitor molecules tested to date. Stabilizing interactions are further investigated with Rotational Echo Double Resonance Magic Angle Spinning (REDOR MAS) experiments for measuring distances from the inhibitor to the protein.