Dennis B. McKay

Cloning and expression of zebrafish neuronal nicotinic acetylcholine receptors

We propose to use the zebrafish (Danio rerio) as a vertebrate model to study the role of neuronal nicotinic acetylcholine receptors (nAChR) in development. As a first step toward using zebrafish as a model, we cloned three zebrafish cDNAs with a high degree of sequence similarity to nAChR beta3, alpha2 and alpha7 subunits expressed in other species. RT-PCR was used to show that the beta3 and alpha2 subunit RNAs were present in zebrafish embryos only 2-5hours post-fertilization (hpf) while alpha7 subunit RNA was not detected until 8hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish beta3, alpha2, and alpha7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity [3H]-epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC(50) values of 28.6pM and 29.7nM and in 5dpf embryos with IC(50) values of 28.4pM and 8.9nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.

Ring E analogs of methyllycaconitine (MLA) as novel nicotinic antagonists.

We have prepared ring E analogs of the diterpenoid alkaloid methyllycaconitine. These compounds have been assayed for nicotinic activity and were found to act as functional antagonists on adrenal nicotinic receptors.

Structure–activity studies with ring E analogues of methyllycaconitine on bovine adrenal α3β4* nicotinic receptors

The development of new agents that selectively interact with subtypes of neuronal nicotinic receptors (nAChRs) is of primary importance for the study of physiological processes and pathophysiological conditions involving these receptors. Our laboratory has evidence that simple ring E analogues of methyllycaconitine (MLA) act as antagonists to bovine adrenal alpha3beta4* nAChRs. The following studies were designed to characterize the concentration-response effects of several ring E analogues of MLA in order to assess structural requirements involved with their inhibitory activity on bovine adrenal alpha3beta4* nAChRs. Ring E analogues with various substitutions on the ring E nitrogen were tested for their ability to inhibit nicotinic stimulated adrenal catecholamine release and [3H]epibatidine binding to a bovine adrenal membrane preparation. Several N-alkyl derivatives inhibited secretion with IC50 values in the low micromolar range. The N-phenpropyl analogue was the most potent of the analogues tested (IC50, 11 microM) on adrenal secretion. Competition binding studies suggest a noncompetitive interaction of the analogues with bovine adrenal nAChRs. These studies identify several structural features of ring E analogues of MLA which significantly affect their inhibitory activity on bovine adrenal alpha3beta4* nAChRs.

Negative Allosteric Modulators that Target Human α4β2 Neuronal Nicotinic Receptors

Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. We have previously reported on the pharmacological activity of several compounds that act as negative allosteric modulators (NAMs) of nAChRs. In the following studies, the effects of 30 NAMs from our small chemical library on both human α4β2 (Hα4β2) and human α3β4 (Hα3β4) nAChRs expressed in human embryonic kidney ts201 cells were investigated. During calcium accumulation assays, these NAMs inhibited nAChR activation with IC50 values ranging from 2.4 μM to more than 100 μM. Several NAMs showed relative selectivity for Hα4β2 nAChRs with IC50 values in the low micromolar range. A lead molecule, KAB-18, was identified that shows relative selectivity for Hα4β2 nAChRs. This molecule contains three phenyl rings, one piperidine ring, and one ester bond linkage. Structure–activity relationship (SAR) analyses of our data revealed three regions of KAB-18 that contribute to its relative selectivity. Predictive three-dimensional quantitative SAR (comparative molecular field analysis and comparative molecular similarity indices analysis) models were generated from these data, and a pharmacophore model was constructed to determine the chemical features that are important for biological activity. Using docking approaches and molecular dynamics on a Hα4β2 nAChR homology model, a binding mode for KAB-18 at the α/β subunit interface that corresponds to the predicted pharmacophore is described. This binding mode was supported by mutagenesis studies. In summary, these studies highlight the importance of SAR, computational, and molecular biology approaches for the design and synthesis of potent and selective antagonists targeting specific nAChR subtypes.

Analogs of Methyllycaconitine as Novel Noncompetitive Inhibitors of Nicotinic Receptors: Pharmacological Characterization, Computational Modeling, and Pharmacophore Development

As a novel approach to drug discovery involving neuronal nicotinic acetylcholine receptors (nAChRs), our laboratory targeted nonagonist binding sites (i.e., noncompetitive binding sites, negative allosteric binding sites) located on nAChRs. Cultured bovine adrenal cells were used as neuronal models to investigate interactions of 67 analogs of methyllycaconitine (MLA) on native α3β4* nAChRs. The availability of large numbers of structurally related molecules presents a unique opportunity for the development of pharmacophore models for noncompetitive binding sites. Our MLA analogs inhibited nicotine-mediated functional activation of both native and recombinant α3β4* nAChRs with a wide range of IC50 values (0.9–115 μM). These analogs had little or no inhibitory effects on agonist binding to native or recombinant nAChRs, supporting noncompetitive inhibitory activity. Based on these data, two highly predictive 3D quantitative structure-activity relationship (comparative molecular field analysis and comparative molecular similarity index analysis) models were generated. These computational models were successfully validated and provided insights into the molecular interactions of MLA analogs with nAChRs. In addition, a pharmacophore model was constructed to analyze and visualize the binding requirements to the analog binding site. The pharmacophore model was subsequently applied to search structurally diverse molecular databases to prospectively identify novel inhibitors. The rapid identification of eight molecules from database mining and our successful demonstration of in vitro inhibitory activity support the utility of these computational models as novel tools for the efficient retrieval of inhibitors. These results demonstrate the effectiveness of computational modeling and pharmacophore development, which may lead to the identification of new therapeutic drugs that target novel sites on nAChRs.

Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN‐62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K‐252a, H‐7, and H‐8) and reportedly selective protein kinase inhibitors (KN‐62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and 45Ca2+ uptake. Treatment of chromaffin cells with 10 µM K‐252a, 50 µM H‐7, or 50 µM H‐8 induced changes in cell morphology within 3 h; these compounds also induced a time‐dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca2+/phospholipid‐dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN‐62, a selective inhibitor of Ca2+/calmodulin‐dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC50 value of 0.32 µM), but had no effect on cell morphology. The reduction of secretory function induced by 1 µM KN‐62 was significant within 5 min and rapidly reversible. Unlike H‐7, H‐8, and staurosporine, KN‐62 significantly inhibited stimulated 45Ca2+ uptake. KN‐04, a structural analogue of KN‐62 that does not inhibit CaMK II, inhibited stimulated 45Ca2+ uptake and catecholamine release like KN‐62. These studies indicate that KN‐62 inhibits secretory function via the direct blockade of activated Ca2+ influx. The nonselective inhibitors, K‐252a, H‐7, H‐8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.

[3H]Epibatidine binding to bovine adrenal medulla: evidence for α3β4* nicotinic receptors

Abstract In these studies, [ 3 H]epibatidine is used as the radioligand to characterize nicotinic acetylcholine receptors (nAChRs) from bovine adrenal medulla. Specific binding reaches equilibrium within 30 min, and is saturable with a K d value of 0.5 nM. The affinities of several cholinergic agents were determined, including nicotine ( K i , 0.2 μM), cytisine ( K i , 0.4 μM), carbachol ( K i , 4.7 μM), dihydro-β-erythrodine ( K i , 33.6 μM), d -tubocurarine ( K i , 0.4 μM), 1,1-dimethyl-4-phenyl-piperazinium ( K i , 0.8 μM), decamethonium ( K i , 234 μM) and methyllycaconitine ( K i , 1.3 μM). These values are similar to reported values for recombinant α3β4 nAChRs in transfected cell lines. These studies demonstrate [ 3 H]epibatidine binding to an easily obtainable adrenal membrane preparation and support the characterization of adrenal nAChRs as α3β4* nAChRs.

Receptor protection studies to characterize neuronal nicotinic receptors: tubocurarine prevents alkylation of adrenal nicotinic receptors

Our laboratory has evidence that multiple nicotinic acetylcholine receptor subtypes regulate bovine adrenal catecholamine release. In the following studies, receptor protection assays were used to differentiate adrenal nicotinic receptor subpopulations. Under alkylating conditions, bromoacetylcholine (30 microM) reduced nicotinic receptor-stimulated adrenal catecholamine secretion by approximately 80%. When 100 microM tubocurarine was present during alkylation, nicotine-stimulated secretion was reduced by less than 30%. Hexamethonium (500 microM), decamethonium (500 microM), mecamylamine (50 microM), pentolinium (50 microM), adiphenine (50 microM), methyllycaconitine (1 microM) and alpha-bungarotoxin (1 microM) afforded no protection when present during alkylation. When the pharmacology of residual, tubocurarine-protected receptors was investigated, the EC50 value for nicotines stimulatory effects on secretion significantly increased from 4.0 (2.5-6.5) microM in control cells to 9.1 (7.2-11.4) microM in tubocurarine-protected cells. In addition, the IC50 value for tubocurarines inhibitory effects on release significantly decreased from 0.7 (0.5-0.9) microM in control cells to 0.3 (0.2-0.4) microM in tubocurarine-protected cells. These studies support the use of protection assays to characterize nicotinic receptor subpopulations.

Effect of Novel Negative Allosteric Modulators of Neuronal Nicotinic Receptors on Cells Expressing Native and Recombinant Nicotinic Receptors: Implications for Drug Discovery

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of α3β4* nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant α3β4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native α3β4* nAChR, with IC50 values ranging from 0.4 to 13.0 μM. Using cells expressing recombinant α3β4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC50 values ranging from 0.7 to 38.2 μM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 μM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.

Effect of Noncompetitive Nicotinic Receptor Blockers on Catecholamine Release from Cultured Adrenal Chromaffin Cells

Nicotinic acetylcholine receptors are found on both muscle tissue and neuronal tissue, including adrenal chromaffin cells. Certain drugs, such as the noncompetitive ion-channel blockers and the vinca alkaloids, have been demonstrated to interact with muscle-type nicotinic receptor-associated ion channels. The objectives of these studies were: (1) to determine the effects of the noncompetitive blockers of the nicotinic receptor-associated ion-channel drugs on adrenal chromaffin cells, and (2) to establish whether the vinca alkaloids and the ion-channel drugs have similar actions on adrenal nicotinic receptors. The ion-channel drugs, adiphenine, tetracaine, quinacrine, amantadine, lidocaine and procaine, inhibited receptor-stimulated catecholamine release from cultured adrenal chromaffin cells in a concentration-dependent manner (IC50s: 2, 4, 6, 41, 55 and 87 microM, respectively). The inhibitory effects of these drugs appeared to be noncompetitive. These drugs had little or no effects on catecholamine release stimulated by depolarizing concentrations of K+ or on basal release. Our results demonstrate that the ion-channel blockers interact with adrenal nicotinic receptors in a manner similar to their interaction with muscle-type nicotinic receptors and they interfere with adrenal receptor function in a manner similar to the vinca alkaloids.