Recent findings from solid state NMR (ssNMR) studies suggested that the (R)-enantiomer of rimantadine binds to the full M2 protein with higher affinity than the (S)-enantiomer. Intrigued by these findings, we applied functional assays, such as antiviral assay and electrophysiology (EP), to evaluate the binding affinity of rimantadine enantiomers to the M2 protein channel. Unexpectedly, no significant difference was found between the two enantiomers. Our experimental data based on the full M2 protein function were further supported by alchemical free energy calculations and isothermal titration calorimetry (ITC) allowing an evaluation of the binding affinity of rimantadine enantiomers to the M2TM pore. Both enantiomers have similar channel blockage, affinity, and antiviral potency.KEYWORDS: Rimantadine enantiomers, isothermal titration calorimetry, free energy perturbation, Bennett's acceptance ratio, electrophysiology, synthesis, antiviral assay, membrane protein, influenza M2 pore A mantadine (1) and rimantadine (2) (Scheme 1) are channel blockers of proton transit by the influenza virus M2 proton channel 1,2 and long used prophylactics and therapeutics against influenza A viruses. 3 The primary binding site of 1 and 2 is the lumen of the transmembrane domain of a tetrameric M2 protein (M2TM: amino acids 22−46) that forms the proton transit path. 4 Although 1 and 2 have been used as antivirals for decades, it was only after 2008 that high resolution structures from X-ray and ssNMR experiments unveiled the structures of M2TM in complex with 1 or 2. 5−9 According to these findings, the M2TM protein channel is blocked by 1 or 2 via a pore-binding mechanism. 6−10 The adamantane cage in 1 or 2, as well as in other aminoadamantane analogues, 11−13 is tightly contacted on all sides by V27 and A30 side chains, producing a steric occlusion of proton transit 6−9 and thereby preventing the viral replication. The ssNMR results for 2 also demonstrated that the ammonium group of the drug is pointing toward the four H37 residues at the C-terminus. 9 This orientation can be stabilized either through hydrogen bonds between the ammonium group of the aminoadamantane ligand and water molecules in the channel lumen which exist between the imidazoles of H37 and the ligand, 13 and/or with A30 carbonyls in the vicinity, 14 according to experimental 9,14−16 and MD simulations data. 13,17−22 Provided that M2TM is a minimal model for M2 binding, 10 these high resolution structures can be used for the development of new ligands which may bind more effectively to the M2TM pore.The effect of ligand's chirality in its binding with a chiral receptor is of outstanding significance and the characterization of protein−ligand interactions for each enantiomer separately may identify potential stereospecific binding interactions to the receptor. While rimantadine analogues are known antiviral drugs for more than four decades, the relative potency of rimantadine enantiomers has not been studied at the molecular level. The binding affinity...