Differential Binding of Rimantadine Enantiomers to Influenza A M2 Proton Channel

Abstract

Rimantadine hydrochloride (α-methyl-1-adamantane-methalamine hydrochloride) is a chiral compound which exerts antiviral activity against the influenza A virus by inhibiting proton conductance of the M2 ion channel. In complex with M2, rimantadine has always been characterized as a racemic mixture. Here, we report the novel enantioselective synthesis of deuterium-labeled (R)- and (S)-rimantadine and the characterization of their protein-ligand interactions using solid-state NMR. Isotropic chemical shift changes strongly support differential binding of the enantiomers to the proton channel. Position restrained simulations satisfying distance restraints from (13)C-(2)H rotational-echo double-resonance NMR show marked differences in the hydrogen-bonding pattern of the two enantiomers at the binding site. Together these results suggest a complex set of interactions between (R)-rimantadine and the M2 proton channel, leading to a higher stability for this enantiomer of the drug in the channel pore.

Affinity of Rimantadine Enantiomers against Influenza A/M2 Protein Revisited

“…The S31 and G34 resonance frequencies were similar for both 2-R and 2-S. These results provided an experimental evidence that both enantiomers bind strongly and the binding site and orientation of the drug in the pore are similar for the two enantiomers as mentioned in ref 23. However, the authors reported the appearance of an additional resonance of medium intensity for the 2-S enantiomer at 115/ 63 ppm close to the frequencies of the S31 resonance of the unbound M2 state at ∼114/62 ppm and suggested a weaker binding of 2-S.…”

“…The distance between the adamantyl ring and the center of mass between the four A30 (Ad-A30) for 1−3 was measured ∼1 Å, and the distance CH 3 (lig. )-G34Ca for 2-R, 2-S, and 3 was 2.9, 3.2 and 2 Å respectively, close to the REDOR measurements for 2-R, 2-S. 23 The adamantyl ring was embraced by the V27 and A30 side chains, which defined the binding site of the ligands. Compounds 1, 2-R, 2-S, and 3 form hydrogen bonds (average of three hydrogen bonds) through the ammonium group with neighboring water molecules which are positioned between the ligand and H37 residues.…”

“…23 The center of mass between the four V27 and the adamantyl ring of the ligand (V27-Ad) varies between 4.0 and 4.5 Å on average (Table S1). For 2-R and 2-S the average tilt angle was measured ∼14°, in accordance to experimental ssNMR values, 9 and for 3 the average tilt angle The purity of each enantiomer used was 90% for 2-R and 95% for 2-S; the enantiomeric excess (ee) of both 2-R and 2-S is 99% (Mosher's method); the purity of compound 3 used was >99%.…”

“…The binding affinity of each enantiomer results from chiral interactions with the binding area inside the 4-fold symmetric M2 protein. Based on differences in isotropic chemical shift changes measured using ssNMR and MD simulations results, it has been recently suggested that 2-R and 2-S have a strong but differential binding to full length M2, i.e., that 2-R binds more tightly than 2-S. 23 This was the first state of the art ssNMR study of the full M2 protein and analysis of the rimantadine enantiomers binding by ssNMR, but this conclusion appears to be puzzling because: (1) 2-R and 2-S have similar in vivo antiviral activity in protecting mice from lethal influenza; 24 (2) rimantadine was developed prior to the 1992 FDA guidance on the development of stereoisomers. It was approved as commercial drug in the US in 1993 containing both enantiomers.…”

Structural biology of supramolecular assemblies by magic-angle spinning NMR spectroscopy

“…10) and membrane protein architecture by Griffin and co-workers (Andreas et al, 2015). REDOR distance measurements are also used for the studies of ligand-protein interactions, including binding of amantadine and its derivatives in the M2 channel by Cross (Wright et al, 2016) and Hong (Cady et al, 2010) and retinal binding in bacteriorhodopsin (Helmle et al, 2000). REDOR-derived distances can also indicate changes in protein or nucleic acid conformation upon ligand binding, demonstrated for tat peptide-bound TAR RNA of HIV-1 (Olsen et al, 2005).…”