Stephen P. Arneric

A Novel Positive Allosteric Modulator of the α7 Neuronal Nicotinic Acetylcholine Receptor: In Vitro and In Vivo Characterization

Several lines of evidence suggest a link between the α7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimers disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the α7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human α7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by α4β2, α3β4, and α9α10 nAChRs. PNU-120596 increased the channel mean open time of α7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of α7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances α7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.

Presenilin-1 and 2 are molecular targets for gamma secretase inhibitors

Presenilins are integral membrane protein involved in the production of amyloid β-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimers disease and are thought to alter γ-secretase cleavage of the β-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of Aβ, the 4-kDa β-peptide. Here, we show that radiolabeled γ-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular Aβ formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of γ-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective γ-secretase inhibitors block Aβ formation by binding to presenilin-1 and -2.

Diversity of neuronal nicotinic acetylcholine receptors: lessons from behavior and implications for CNS therapeutics.

Although the molecular biology of neuronal nicotinic acetylcholine receptors (nAChRs) provides evidence for multiple receptor subtypes, few selective pharmacological tools exist to identify these subtypes in vivo. However, the diversity of behavioral effects of available nAChR agonists and antagonists reviewed in this paper suggests that neuronal nAChR subtypes may play distinct roles in a variety of behavioral outcomes. Further characterization of the behavioral effects of the activation of discrete nAChR subtypes may eventually provide information useful in designing selective nAChR ligands targeting a variety of CNS disorders.

Stable expression and pharmacological properties of the human α7 nicotinic acetylcholine receptor

The alpha 7 neuronal nicotinic acetylcholine receptor subtype forms a Ca(2+)-permeable homooligomeric ion channel sensitive to alpha-bungarotoxin in Xenopus oocytes. In this study, we have stably and functionally expressed the human alpha 7 cDNA in a mammalian cell line, HEK-293 and examined its pharmacologic properties. [125I] alpha-Bungarotoxin bound to transfected cells with a Kd value of 0.7 nM and a Bmax value of 973 pmoL/mg protein. No specific binding was detected in untransfected cells. Specific binding could be displaced by unlabeled alpha-bungarotoxin (Ki = 0.5 nM) and an excellent correlation was observed between binding affinities of a series of nicotinic cholinergic ligands in transfected cells and those in the human neuroblastoma IMR-32 cell line. Additionally, cell surface expression of alpha 7 receptors was detected by fluorescein isothiocyanate-conjugated alpha-bungarotoxin in transfected cells. Whole cell currents sensitive to blockade by alpha-bungarotoxin, and with fast kinetics of activation and inactivation, were recorded from transfected cells upon rapid application of (-)-nicotine or acetylcholine with EC50 values of 49 microM and 155 microM respectively. We conclude that the human alpha 7 subunit when expressed alone can form functional ion channels and that the stably transfected HEK-293 cell line serves as a unique system for studying human alpha 7 nicotinic receptor function and regulation, and for examining ligand interactions.

Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).

Human α4β2 Neuronal Nicotinic Acetylcholine Receptor in HEK 293 Cells: A Patch-Clamp Study

The cloning and expression of genes encoding for the human neuronal nicotinic acetylcholine receptors (nAChRs) has opened new possibilities for investigating their physiological and pharmacological properties. Cells (HEK 293) stably transfected with two of the major brain subunits, α4 and β2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC50) of 3 μm and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human α4β2 receptors is (−)-nicotine > ACh > (−)-cytisine > ABT-418. At saturating concentrations, the efficacy of these ligands is ABT-418 ≫ (−)-nicotine > ACh ≫ (−)-cytisine > GTS-21 (previously named DMXB). Coapplication of 1 μm ACh with known nAChR inhibitors such as dihydro-β-erythroidine and methyllycaconitine reversibly reduces the current evoked by the agonist with respective IC50 values of 80 nm and 1.5 μm. The current–voltage relationship of human α4β2 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human α4β2 nAChRs are permeable to sodium and potassium ions. In the “outside-out” configuration, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is observed when the extracellular calcium concentration is increased from 0.2 to 2 mm. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mm.

The Pharmacology of (-)-Nicotine and Novel Cholinergic Channel Modulators

Advances in the understanding of the molecular biology and pharmacology of nAChRs may provide targets for the development of novel and selective modulators of nAChRs in the brain. This contention is supported by the dissimilar behavioral effects observed following systemic administration of currently available nicotinic ligands. The concept of multiple subtypes of nAChRs is not unique, as evidenced by the pharmacology of other ligand-gated ion channels, such as GABA-A receptor, which also exist in multiple subtypes. At present, with respect to the nAChRs, relatively few of the subtypes identified have been cloned from human tissue and pharmacologically evaluated, but several groups are focusing their research efforts in this direction. With a thorough understanding of the pharmacological and functional characteristics of more of the putative human nAChR subtypes, this knowledge will facilitate the discovery of more efficacious and less toxic ChCMs that may provide potential novel therapeutic agents for a variety of CNS conditions.

Antagonism of CRF2 receptors produces anxiolytic behavior in animal models of anxiety

Two pharmacologically distinct CRF receptors are distributed in different brain regions and peripheral tissues. Studies suggest that CRF(1) receptors play an important role in mediating the anxiety provoking effects of CRF. In contrast, far less functional information is available on CRF(2) receptors. Therefore, we conducted dose response studies using antisauvagine-30 (anti-SVG-30, 0-20 microg, 20-min pretreatment, i.c.v.), a potent CRF(2) peptide antagonist, and tested rats in three models of anxiety - the conditioned freezing, the elevated plus maze, and the defensive-withdrawal test. Anti-SVG-30 produced a significant dose-dependent reduction in conditioned freezing. In the elevated plus maze test, administration of anti-SVG-30 effectively increased the number of entries and time spent in the open arms. In the defensive-withdrawal test, anti-SVG-30 treatment facilitated exploratory activity in a large illuminated open field. Thus, in all three animal models, administration of anti-SVG-30 was consistent in producing an anxiolytic-like behavioral effect. In addition, a dose of anti-SVG-30 (10 microg) that produced anxiolytic-like behavior had no significant effects on locomotor activity measured in an automated activity box. This latter finding suggests that antagonism of CRF(2) receptors is not associated with a non-specific increase in behavioral movements. These results provide evidence that, in addition to CRF(1) receptors, CRF(2) receptors may play an important role in the mediation of anxiety behavior.

Nicotinic receptor binding of [3H]cytisine, [3H]nicotine and [3H]methylcarbamylcholine in rat brain.

Three radiolabeled nicotinic receptor agonists were examined for their binding characteristics and for inhibition by cholinergic compounds in order to distinguish possible differential affinities for subtypes of neuronal nicotinic acetylcholine receptors. KD and Bmax values for [3H]cytisine, [3H]methylcarbamylcholine and [3H]nicotine were determined from Scatchard analysis using an enriched whole-brain membrane fraction from male Sprague-Dawley rats. Respective KD values were 0.15, 1.07 and 0.89 nM while Bmax values were 99, 64 and 115 fmol/mg protein respectively. All three ligands fit a one-site model of receptor-ligand interaction. Concentration-inhibition curves were used to determine Ki values for 16 cholinergic compounds. The rank order of potencies for displacement of the three ligands was: (-)-cytisine > (-)-nicotine > (-)-lobeline = methylcarbamylcholine > 1,1-dimethyl-4-phenylpiperazinium, (+)-nicotine, dihydro-beta-erythroidine, (+/-)-nornicotine > carbachol > arecoline >> oxotremorine, tetrahydroaminoacridine, AF102B >> (-)-cotinine > RS86 = heptylphysostigmine. Correlations of the affinities of these compounds determined with the three ligands were very near to unity. In contrast, there was a negative correlation of affinities for [3H]cytisine compared to affinities for the muscarinic receptor agonist, [3H]oxotremorine-M, and the muscarinic receptor antagonist, [3H]quinuclidinyl benzoate. Using membranes from whole rat brain yields data suggesting that all three nicotinic ligands bind to the same nicotinic acetylcholine receptor subtype, and are unable to distinguish subtypes of neuronal nicotinic acetylcholine receptor at the level examined.

A-85380 [3-(2(S)-azetidinylmethoxy) pyridine]: In Vitro pharmacological properties of a novel, high affinity α4β2 nicotinic acetylcholine receptor ligand

Abstract The in vitro pharmacological properties of a novel cholinergic channel ligand, A-85380 [3-(2( S )-azetidinylmethoxy) pyridine], were examined using tissue preparations that express different putative nAChR subtypes. In radioligand binding studies, A-85380 is shown to be a potent and selective ligand for the human α4β2 nAChR subtype ( K i = 0.05 ± 0.01 nM) relative to the human α7 ( K i = 148 ± 13 nM) and the muscle α1β1dg subtype expressed in Torpedo electroplax ( K i = 314 ± 12 nM). The R -enantiomer of A-85380, A-159470, displays little enantioselectivity towards the α4β2 and α1β1δγ subtypes but does display 12-fold enantioselectivity towards the α7 subtype ( K i = 1275 ± 199 nM). (+)- and (−)-Epibatidine display similar potencies at the human human α4β2 ( K i = 0.04 ± 0.02 nM and 0.07 ± 0.02 nM, respectively), human α7 ( K i = 16 ± 2 nM and 22 ± 3 nM, respectively) and muscle α1β1δg ( K i = 2.5 ± 0.9 nM and 5.7 ± 1.0 nM, respectively) nAChRs. Functionally, A-85380 is a potent activator of cation efflux through the human α4β2 (EC 50 = 0.7 ± 0.1 μ M) and ganglionic (EC 50 = 0.8 ± 0.09 μ M) subtypes, effects that are attenuated by pretreatment with mecamylamine (10 μM). Further, A-85380 can activate (EC 50 = 8.9 ± 1.9 μ M) currents through channels formed by injection of the human α7 subunit into Xenopus oocytes, effects that are attenuated by pretreatment with the α7 nAChR antagonist, methyllycaconitine (10 nM). In all cases, A-85380 is more potent than (−)-nicotine but less potent than (±)-epibatidine. In neurotransmitter release studies, A-85380 stimulates the release of dopamine with an EC 50 value of 0.003 ± 0.001 μ M which is equipotent to (±)-epibatidine, and 20-fold more potent that (−)-nicotine (EC 50 = 0.04 ± 0.009 μ M). Thus, A-85380 displays a profile of robust activation of a number of nAChR subtypes with substantially less affinity for [ 125 I]α-BgT sites than [ 3 H](-)-cytisine sites, suggesting that it may serve as a more selective pharmacologic probe for the αx4β2 subtype relative to the α7 and α1β1δg nAChRs than (±)-epibatidine.