μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level.

A pharmacophore model for butyrylcholinesterase (BChE) inhibitors was applied to a human cannabinoid subtype 2 receptor (hCB2 R) agonist and verified it as a first-generation lead for respective dual-acting compounds. The design, synthesis, and pharmacological evaluation of various derivatives led to the identification of aminobenzimidazoles as second-generation leads with micro- or sub-micromolar activities at both targets and excellent selectivity over hCB1 and AChE, respectively. Computational studies of the first- and second-generation lead structures by applying molecular dynamics (MD) on the active hCB2 R model, along with docking and MD on hBChE, has enabled an explanation of their binding profiles at the protein levels and opened the way for further optimization. Dual-acting compounds with "balanced" affinities and excellent selectivities could be obtained that represent leads for treatment of both cognitive and pathophysiological impairment occurring in neurodegenerative disorders.

Adamantane derivatives, such as amantadine and rimantadine, have been reported to block the transmembrane domain (TM) of the M2 protein of influenza A virus (A/M2) but their clinical use has been discontinued due to evolved resistance in humans. Although experiments and simulations have provided adequate information about the binding interaction of amantadine or rimantadine to the M2 protein, methods for predicting binding affinities of whole series of M2 inhibitors have so far been scarcely applied. Such methods could assist in the development of novel potent inhibitors that overcome A/M2 resistance. Here we show that alchemical free energy calculations of ligand binding using the Bennett acceptance ratio (BAR) method are valuable for determining the relative binding potency of A/M2 inhibitors of the aminoadamantane type covering a binding affinity range of only ∼2 kcal mol(-1). Their binding affinities measured by isothermal titration calorimetry (ITC) against the A/M2TM tetramer from the Udorn strain in its closed form at pH 8 were used as experimental probes. The binding constants of rimantadine enantiomers against M2TMUdorn were measured for the first time and found to be equal. Two series of alchemical free energy calculations were performed using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids to mimic the membrane environment. A fair correlation was found for DPPC that was significantly improved using DMPC, which resembles more closely the DPC lipids used in the ITC experiments. This demonstrates that binding free energy calculations by the BAR approach can be used to predict relative binding affinities of aminoadamantane derivatives toward M2TM with good accuracy.

Recently, the binding kinetics of a ligand-target interaction, such as the residence time of a small molecule on its protein target, are seen as increasingly important for drug efficacy. Here, we investigate these concepts to explain binding and proton blockage of rimantadine variants bearing progressively larger alkyl groups to influenza A virus M2 wild type (WT) and M2 S31N protein proton channel. We showed that resistance of M2 S31N to rimantadine analogues compared to M2 WT resulted from their higher rates compared to the rates according to electrophysiology (EP) measurements. This is due to the fact that, in M2 S31N, the loss of the V27 pocket for the adamantyl cage resulted in low residence time inside the M2 pore. Both rimantadine enantiomers have similar channel blockage and binding and against M2 WT. To compare the potency between the rimantadine variants against M2, we applied approaches using different mimicry of M2, i.e., isothermal titration calorimetry and molecular dynamics simulation, EP, and antiviral assays. It was also shown that a small change in an amino acid at site 28 of M2 WT, which does not line the pore, seriously affects M2 WT blockage kinetics.

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...