Application of Solid State Nuclear Magnetic Resonance in Drug Discovery

The M2 proton channel from Influenza A is an established drug target, with multiple functions during the viral lifecycle. Amino acid mutations, in the residues lining the channel pore, have rendered M2 resistant to previously licensed inhibitors. Given the propensity for genetic reassortment of Influenza A and history of pandemics due to emergence of novel human strains, M2 has been subject of numerous structural characterization efforts. Attempts at rational drug design targeting M2 proton channel have been impeded by the limited number of experimental techniques having capabilities for elucidating atomic level interactions of the protein-ligand complexes in the native-like membrane mimetic environment. Solid state Nuclear Magnetic Resonance (ssNMR) is a technique that has all of these capabilities for structural characterization of membrane protein drug targets in lipid bilayers. Coupling ssNMR with Computer Aided Drug Discovery (CADD) is the precise approach needed to decrease time and resources required to generate novel therapeutics. Here, we present a first structural characterization of the full length wild type M2 channel in complex with inhibitor, and of the S31N mutant in the apo and drug-bound state. Through the use of diverse ssNMR experiments we tested stereoselectivity of drug binding in the channel pore, structural changes due to mutation, and characterized novel inhibitor interactions. Molecular dynamic simulations were performed by our collaborators and were in good agreement with our experimental findings. Together these results deepen our understanding of the atomic level interactions stabilizing wild type inhibitors in the channel pore, and structural changes in the mutant leading to loss of compound efficacy. Most importantly, specific interaction described herein are essential for successful outcomes from structure based CADD and be used in future computational efforts.