Vladimir P. Grinevich

bis-azaaromatic quaternary ammonium analogues: Ligands for α4β2* and α7* subtypes of neuronal nicotinic receptors

Abstract A series of bis-nicotinium, bis-pyridinium, bis-picolinium, bis-quinolinium and bis-isoquinolinium compounds was evaluated for their binding affinity at nicotinic acetylcholine receptors (nAChRs) using rat brain membranes. N , N ′-Decane-1,12-diyl-bis-nicotinium diiodide (bNDI) exhibited the highest affinity for [ 3 H]nicotine binding sites ( K i =330 nM), but did not inhibit [ 3 H]methyllycaconitine binding ( K i >100 μM), indicative of an interaction with α4β2*, but not α7* receptor subtypes, respectively. Also, bNDI inhibited (IC 50 =3.76 μM) nicotine-evoked 86 Rb + efflux from rat thalamic synaptosomes, indicating antagonist activity at α4β2* nAChRs. N , N ′-Dodecane-1,12-diyl-bis-quinolinium dibromide (bQDDB) exhibited highest affinity for [ 3 H]methyllycaconitine binding sites ( K i =1.61 μM), but did not inhibit [ 3 H]nicotine binding ( K i >100 μM), demonstrating an interaction with α7*, but not α4β2* nAChRs. Thus, variation of N - n -alkyl chain length together with structural modification of the azaaromatic quaternary ammonium moiety afforded selective antagonists for the α4β2* nAChR subtype, as well as ligands with selectivity at α7* nAChRs.

Comparison of acetylcholine receptor interactions of the marine toxins, 13-desmethylspirolide C and gymnodimine

The interaction of 13-desmethylspirolide C (SPX-desMe-C) and gymnodimine with several nicotinic and muscarinic acetylcholine receptors was investigated. Interaction at the muscarinic receptors was minimal. At nicotinic receptors, both SPX-desMe-C and gymnodimine displayed greatest affinity for the α7 receptor. The rank order for binding affinity (Ki) for SPX-desMe-C was α7 > α6β3β4α5 >> rat α3β4, α1βγδ > α4β4, human α3β4 > human α4β2 > rat α4β2 and for gymnodimine was α7, α6β3β4α5 > rat α3β4 > human α3β4, α4β4 > rat α4β2, human α4β2 > α1βγδ. Both molecules antagonized agonist-induced nicotinic responses. The antagonism rank order of potency (IC(50)) for SPX-desMe-C was α7 > low sensitivity (LS) α4β2 > human α3β4 > high sensitivity (HS) α4β2, α1βγδ > α4β4 > rat α3β4 and for gymnodimine was LS α4β2 > human α3β4 > α7 > HS α4β2 > α4β4 > rat α3β4 > α1βγδ. Neither gymnodimine nor SPX-desMe-C antagonism could be surmounted by increasing concentrations of nicotine. To elucidate the nature of this insurmountable blockade, we carried out homology modelling and molecular docking studies of both ligands with α7 nAChR. Their very high binding affinity results from very tight hydrophobic enclosures, in addition to previously reported hydrogen-bond and cation-π interactions. Also, the higher the hydrophilic surface area of the binding site of nAChRs, the weaker the binding affinity of both ligands. Together these results show the targets of action are nicotinic and define these marine toxins as additional tools to advance our understanding regarding interactions between antagonists and the nAChR ligand binding domain.

Heterologous Expression of Human α6β4β3α5 Nicotinic Acetylcholine Receptors: Binding Properties Consistent with Their Natural Expression Require Quaternary Subunit Assembly Including the α5 Subunit

Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing α6 subunits (α6* nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human α6β2, α6β4, α6β2β3, α6β4β3, or α6β4β3α5 nAChR. Cells expressing subunits needed to form α6β4β3α5 nAChR exhibited saturable [3H]epibatidine binding (Kd = 95.9 ± 8.3 pM and Bmax = 84.5 ± 1.6 fmol/mg of protein). The rank order of binding competition potency (Ki) for prototypical nicotinic compounds was α-conotoxin MII (6 nM) > nicotine (156 nM) ∼ methyllycaconitine (200 nM) > α-bungarotoxin (>10 μM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that β3 and α5 subunits are likely partners of the α6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that α6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human α5 subunit makes a critical contribution to α6β4β3α5 nAChR assembly into a ligand-binding form with native α6*-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.

Atypical antipsychotics as noncompetitive inhibitors of α4β2 and α7 neuronal nicotinic receptors

It has been suggested that the interaction of antipsychotic medications with neuronal nicotinic receptors may increase the cognitive dysfunction associated with schizophrenia and may explain why current therapies only partially address this core feature of the illness. In the present studies we compared the effects of the atypical antipsychotics quetiapine, clozapine and N-desmethylclozapine to those of the typical antipsychotics haloperidol and chlorpromazine on the alpha4beta2 and alpha7 nicotinic receptor subtypes. The binding of [(3)H]-nicotine to rat cortical alpha4beta2 receptors and [(3)H]-methyllycaconitine to rat hippocampal alpha7 receptors was not affected by any of the compounds tested. However, Rb(+) efflux evoked either by nicotine or the selective alpha4beta2 agonist TC-1827 from alpha4beta2 receptors expressed in SH-EP1 cells and nicotine-evoked [(3)H]-dopamine release from rat striatal synaptosomes were non-competitively inhibited by all of the antipsychotics. Similarly, alpha-bungarotoxin-sensitive epibatidine-evoked [(3)H]-norepinephrine release from rat hippocampal slices and acetylcholine-activated currents of alpha7 nicotinic receptors expressed in oocytes were inhibited by haloperidol, chlorpromazine, clozapine and N-desmethylclozapine. The inhibitory effects on nicotinic receptor function produced by the antipsychotics tested occurred at concentrations similar to plasma levels achieved in schizophrenia patients, suggesting that they may lead to clinically relevant effects on cognition.

Potential therapeutic uses of mecamylamine and its stereoisomers.

Mecamylamine (3-methylaminoisocamphane hydrochloride) is a nicotinic parasympathetic ganglionic blocker, originally utilized as a therapeutic agent to treat hypertension. Mecamylamine administration produces several deleterious side effects at therapeutically relevant doses. As such, mecamylamines use as an antihypertensive agent was phased out, except in severe hypertension. Mecamylamine easily traverses the blood-brain barrier to reach the central nervous system (CNS), where it acts as a nicotinic acetylcholine receptor (nAChR) antagonist, inhibiting all known nAChR subtypes. Since nAChRs play a major role in numerous physiological and pathological processes, it is not surprising that mecamylamine has been evaluated for its potential therapeutic effects in a wide variety of CNS disorders, including addiction. Importantly, mecamylamine produces its therapeutic effects on the CNS at doses 3-fold lower than those used to treat hypertension, which diminishes the probability of peripheral side effects. This review focuses on the pharmacological properties of mecamylamine, the differential effects of its stereoisomers, S(+)- and R(-)-mecamylamine, and the potential for effectiveness in treating CNS disorders, including nicotine and alcohol addiction, mood disorders, cognitive impairment and attention deficit hyperactivity disorder.

Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters.

Vesicular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for psychostimulant abuse. Lobeline (1) is a potent antagonist at alpha4beta2 * nicotinic acetylcholine receptors, has moderate affinity (K(i)=5.46microM) for VMAT2, and is being investigated currently as a clinical candidate for treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2-20 of lobeline were synthesized and evaluated for interaction with alpha4beta2 * and alpha7 * neuronal nicotinic acetylcholine receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2. Both carboxylic acid and sulfonic acid esters had low affinity at alpha7 * nAChRs. Similar to lobeline (K(i)=4nM), sulfonic acid esters had high affinity at alpha4beta2 * (K(i)=5-17nM). Aromatic carboxylic acid ester analogs of lobeline (2-4) were 100-1000-fold less potent than lobeline at alpha4beta2 * nAChRs, whereas aliphatic carboxylic acid ester analogs were 10-100-fold less potent than lobeline at alpha4beta2 *. Two representative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the (36)Rb(+) efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC(50) values of 0.85microM and 1.60microM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies (equipotent to 13-45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respectively, and like lobeline, had moderate affinity (K(i)=1.98-10.8microM) for VMAT2. One of the more interesting analogs, p-methoxybenzoic acid ester 4, had low affinity at alpha4beta2 * nAChRs (K(i)=19.3microM) and was equipotent with lobeline, at VMAT2 (K(i)=2.98microM), exhibiting a 6.5-fold selectivity for VMAT2 over alpha4beta2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for the development of lobeline analogs with improved selectivity at VMAT2.

Pharmacological properties and predicted binding mode of arylmethylene quinuclidine-like derivatives at the α3β4 nicotinic acetylcholine receptor (nAChR).

We have carried out a pharmacological evaluation of arylmethylene quinuclidine derivatives interactions with human α3β4 nAChRs subtype, using cell-based receptor binding, calcium-influx, electrophysiological patch-clamp assays and molecular modeling techniques. We have found that the compounds bind competitively to the α3β4 receptor with micromolar affinities and some of the compounds behave as non-competitive antagonists (compounds 1, 2 and 3), displaying submicromolar IC(50) values. These evidences suggest a mixed mode of action for these compounds, having interactions at the orthosteric site and more pronounced interactions at an allosteric site to block agonist effects. One of the compounds, 1-benzyl-3-(diphenylmethylene)-1-azoniabicyclo[2.2.2]octane chloride (compound 3), exhibited poorly reversible use-dependent block of α3β4 channels. We also found that removal of a phenyl group from compound 1 confers a partial agonism to the derived analog (compound 6). Introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative (compound 7) increases agonism potency at the α3β4 receptor subtype. Docking into the orthosteric binding site of a α3β4 protein structure derived by comparative modeling accurately predicted the experimentally-observed trend in binding affinity. Results supported the notion that binding requires a hydrogen bond formation between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the conserved Trp-149.

QM-polarized ligand docking accurately predicts the trend in binding affinity of a series of arylmethylene quinuclidine-like derivatives at the α4β2 and α3β4 nicotinic acetylcholine receptors (nAChRs)

Compounds containing a quinuclidine scaffold are promising drug candidates for pharmacological management of the central nervous system (CNS) pathologies implicating nAChRs. We have carried out binding affinity and in-silico docking studies of arylmethylene quinuclidine-like derivatives at the α4β2 receptor using in-vitro receptor binding assay and comparative modeling, respectively. We found that introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative resulted in a 266-fold increase in binding affinity and confers agonism properties. By contrast, addition of a phenyl group to 3-benzylidene quinuclidine derivative only results in an 18-fold increase in binding affinity, without conferring agonism. We also found that docking into the orthosteric binding site of the α4β2 nAChR is consistent with the fact that the basic nitrogen atom donates a hydrogen-bond to the carbonyl group of the highly conserved Trp-149, as initially observed by Dougherty and co-workers.(1) The experimentally-observed trend in binding affinity at both α4β2 and α3β4 nAChRs was accurately and independently confirmed by quantum mechanics (QM)-polarized docking. The reduction in binding affinity to the α3β4 subtype primarily results from a dampening of both coulombic and cation-π interactions.