Murali Gopalakrishnan

Distinct Profiles of α7 nAChR Positive Allosteric Modulation Revealed by Structurally Diverse Chemotypes

Selective modulation of α7 nicotinic acetylcholine receptors (nAChRs) is thought to regulate processes impaired in schizophrenia, Alzheimers disease, and other dementias. One approach to target α7 nAChRs is by positive allosteric modulation. Structurally diverse compounds, including PNU-120596, 4-naphthalene-1-yl-3a,4,5,9b-tetrahydro-3-H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), and 5-hydroxyindole (5-HI) have been identified as positive allosteric modulators (PAMs), but their receptor interactions and pharmacological profiles remain to be fully elucidated. In this study, we investigated interactions of these compounds at human α7 nAChRs, expressed in Xenopus laevis oocytes, along with genistein, a tyrosine kinase inhibitor. Genistein was found to function as a PAM. Two types of PAM profiles were observed. 5-HI and genistein predominantly affected the apparent peak current (type I) whereas PNU-120596 and TQS increased the apparent peak current and evoked a distinct weakly decaying current (type II). Concentration-responses to agonists [ACh, 3-[(3E)-3-[(2,4-dimethoxyphenyl)methylidene]-5,6-dihydro-4H-pyridin-2-yl]pyridine dihydrochloride (GTS-21), and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)] were potentiated by both types, although type II PAMs had greater effects. When applied after α7 nAChRs were desensitized, type II, but not type I, PAMs could reactivate α7 currents. Both types of PAMs also increased the ACh-evoked α7 window currents, with type II PAMs generally showing larger potentiation. None of the PAMs tested increased nicotine-evoked Ca2+ transients in human embryonic kidney 293 cells expressing human α4β2 or α3β4 nAChRs, although some inhibition was noted for 5-HI, genistein, and TQS. In summary, our studies reveal two distinct α7 PAM profiles, which could offer unique opportunities for modulating α7 nAChRs in vivo and in the development of novel therapeutics for central nervous system indications.

An Allosteric Modulator of the α7 Nicotinic Acetylcholine Receptor Possessing Cognition-Enhancing Properties in Vivo

Augmentation of nicotinic α7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimers disease and schizophrenia. In the present report, a novel positive allosteric modulator of the α7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through α7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of α7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (–)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (–)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that α7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

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.

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.

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.

Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation.

The effects of 1-ethyl-2-benzimidazolinone (1-EBIO) and riluzole on human prostate cancer cells, LNCaP and PC-3, were evaluated using rubidium (86Rb(+)) efflux and proliferation assays. 1-EBIO and riluzole evoked concentration-dependent increases in 86Rb(+) efflux from LNCaP and PC-3 cells that were sensitive to inhibition by intermediate-conductance Ca(2+)-activated K(+) channel (IK(Ca)) blockers clotrimazole and charybdotoxin. Blockers of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, iberiotoxin, or small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel, apamin or scyllatoxin, had no effect. Concurrently, both 1-EBIO and riluzole evoked concentration-dependent increases in proliferation from human prostate cancer cell lines (LNCaP and PC-3 cells). Clotrimazole and charybdotoxin, but not iberiotoxin, apamin or scyllatoxin, inhibited 1-EBIO- and riluzole-evoked increases in proliferation from LNCaP and PC-3 cells. N-(3-(trifluoromethyl)phenyl)-N-(2-hydroxy-5-chlorophenyl)urea (NS-1608) and 2-amino-5-(2-fluorophenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BK(Ca) channel openers had no effect on LNCaP and PC-3 proliferation. These results demonstrate that IK(Ca) channels play an important role in the regulation of human prostate cancer cell proliferation.

Spontaneous phasic activity of the pig urinary bladder smooth muscle: characteristics and sensitivity to potassium channel modulators.

A hallmark for unstable bladder contractions is hyperexcitability and changes in the nature of spontaneous phasic activity of the bladder smooth muscle. In this study, we have characterized the spontaneous activity of the urinary bladder smooth muscle from the pig, a widely used model for studying human bladder function. Our studies demonstrate that phasic activity of the pig detrusor is myogenic and is influenced by the presence of urothelium. Denuded strips exhibit robust spontaneous activity measured as mean area under the contraction curve (AUC=188.9±15.63u2003mNs) compared to intact strips (AUC=7.3±1.94u2003mNs). Spontaneous phasic activity, particularly the amplitude, is dependent on both calcium entry through voltage‐dependent calcium channels and release from ryanodine receptors as shown by inhibition of spontaneous activity by nifedipine and ryanodine respectively. Inhibition of BKCa channels by iberiotoxin (100u2003nM) resulted in an increase in contraction amplitude (89.1±20.4%) and frequency (92.5±31.0%). The SKCa channel blocker apamin (100u2003nM) also increased contraction amplitude (69.1±24.3%) and frequency (53.5±13.6%) demonstrating that these mechanisms are critical to the regulation of phasic spontaneous activity. Inhibition of KATP channels by glyburide (10u2003μM) did not significantly alter myogenic contractions (AUC=18.5±12.3%). However, KATP channel openers (KCOs) showed an exquisite sensitivity for suppression of spontaneous myogenic activity. KCOs were generally 15 fold more potent in suppressing spontaneous activity compared to contractions evoked by electrical field‐stimulation. These studies suggest that potassium channel modulation, particularly KATP channels, may offer a unique mechanism for controlling spontaneous myogenic activity especially those associated with the hyperexcitability occurring in unstable bladders.

Up-regulation of human α7 nicotinic receptors by chronic treatment with activator and antagonist ligands

This study examined the binding and functional properties of human alpha7 neuronal nicotinic acetylcholine receptors stably expressed in human embryonic kidney (HEK) 293 cells following chronic treatment with nicotinic receptor ligands. Treatment of cells with (-)-nicotine (100 microM) for 120 h increased the Bmax values of [125I]alpha-bungarotoxin binding 2.5-fold over untreated cells. This effect was concentration-dependent (EC50) = 970 microM) and a 6-fold upregulation was observed with the maximal concentration of (-)-nicotine tested. Also, treatment of cells with ligands of varying intrinsic activities including (+/-)-epibatidine, (2,4)-dimethoxybenzylidene anabaseine (GTS-21) and 1,1-dimethyl-4-phenyl piperazinium iodide (DMPP) also upregulated [125I]alpha-bungarotoxin binding. A concentration-dependent upregulation of binding sites was also observed following treatment with the alpha7 nicotinic receptor antagonist, methyllycaconitine (EC50 = 92 microM) with a maximal upregulation of about 7-fold. Functionally, the peak amplitude of the whole-cell currents recorded by fast application of (-)-nicotine after chronic treatment of cells with concentrations of (-)-nicotine (1000 microM) or methyllycaconitine (10 microM) that elicited similar increases in binding levels (3.5-fold) resulted in increases of 2-fold (505 +/- 21 pA) and 6-fold (1820 +/- 137 pA) respectively in whole cell current amplitude compared to untreated cells (267 +/- 24 pA). These studies clearly demonstrate that long-term exposure to both activator and antagonist ligands can increase the density of alpha7 nicotinic receptors and can differentially enhance nicotinic receptor function.

Validation of FLIPR Membrane Potential Dye for High Throughput Screening of Potassium Channel Modulators

A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive Ki channel; KATP) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference KATP channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.

High content screen microscopy analysis of Aβ1–42-induced neurite outgrowth reduction in rat primary cortical neurons: Neuroprotective effects of α7 neuronal nicotinic acetylcholine receptor ligands

beta-Amyloid peptide 1-42 (A beta(1-42)) is generated from amyloid precursor protein (APP) and associated with neurodegeneration in Alzheimers disease (AD). A beta(1-42) has been shown to be cytotoxic when incubated with cultured neurons. However, APP transgenic mice over-expressing A beta(1-42) do not show substantial loss of neurons, despite deficits in learning and memory. It is thus emerging that A beta(1-42)-induced memory deficits may involve subtler neuronal alternations leading to synaptic deficits, prior to frank neurodegeneration in AD brains. In this study, high content screen (HCS) microscopy, an advanced high-throughput cellular image processing and analysis technique, was utilized in establishing an in vitro model of A beta(1-42)-induced neurotoxicity utilizing rat neonatal primary cortical cells. Neurite outgrowth was found to be significantly reduced by A beta(1-42) (300 nM to 30 microM), but not by the scrambled control peptide control, in a time- and concentration-dependent manner. In contrast, no reduction in the total number of neurons was observed. The A beta(1-42)-induced reduction of neurite outgrowth was attenuated by the NMDA receptor antagonist memantine and the alpha 7 nicotinic acetylcholine receptor (nAChR) selective agonist PNU-282987. Interestingly, the alpha 7 nAChR antagonist methyllycaconitine also significantly prevented reduction in A beta(1-42)-induced neurite outgrowth. The observed neuroprotective effects could arise either from interference of A beta(1-42) interactions with alpha 7 nAChRs or by modification of receptor-mediated signaling pathways. Our studies demonstrate that reduction of neurite outgrowth may serve as a model representing A beta(1-42)-mediated neuritic and synaptic toxicity, which, in combination of HCS, provides a high-throughput cell-based assay that can be used to evaluate compounds with neuroprotective properties in neurons.