Susan B. McKay

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.

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.

[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.

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.

The synthesis of 5-substituted ring E analogs of methyllycaconitine via the Suzuki-Miyaura cross-coupling reaction.

Novel 3,5-disubstituted ring E analogs of methyllycaconitine were prepared and evaluated in nicotinic acetylcholine receptor binding assays. The desired analogs were prepared through the Suzuki-Miyaura cross-coupling reaction of methyl 5-bromo-nicotinate. The Suzuki-Miyaura cross-coupling reactions of pyridines with electron withdrawing substituents have not been extensively described previously.

Comparison of the effects of antimitotic drugs on α-tubulin mRNA, microtubules and nicotinic receptor-mediated catecholamine secretion in adrenal chromaffin cells in culture

1. Four hour treatments of adrenal chromaffin cells with colchicine (10, 100 microM and 1 mM), tubulozole (10 microM) or podophyllotoxin (100 microM) decreases alpha-tubulin mRNA content. Vinblastine (10 microM) and taxol (10 microM), however, do not decrease alpha-tubulin mRNA content. 2. Immunocytochemical techniques demonstrate that 4 hr treatments with all of the antimitotic drugs (colchicine, podophyllotoxin, taxol, tubulozole and vinblastine) produce abnormal microtubule arrays. 3. The effects of 4 hr treatments with the antimitotic drugs on adrenal catecholamine secretion are all qualitatively the same; each drug selectively inhibits adrenal nicotinic acetylcholine receptor-stimulated catecholamine release, while having no inhibitory actions on release stimulated through noncholinergic mechanisms. 4. These studies demonstrate that autoregulation of tubulin synthesis occurs in cultured adrenal chromaffin cells. 5. All of the antimitotic drugs selectively inhibit nicotinic receptor-mediated adrenal catecholamine release under treatment conditions that affect adrenal microtubules. These results support the possibility that the actions of the antimitotic drugs on adrenal nicotinic receptors may involve microtubules.

Evidence for the involvement of adenomatous polyposis coli (APC) protein in maintaining cellular distributions of α3β4 nicotinic receptors

Evidence exists supporting the involvement of adenomatous polyposis coli (APC) protein in the assembly of neuronal nicotinic acetylcholine receptors (nAChRs) in the postsynaptic complex. In the following studies, the effects of APC protein on cellular distribution of recombinant α3β4 nAChRs was investigated. RT-PCR and Western blotting techniques established the expression of APC protein both in bovine adrenal chromaffin cells, which express native α3β4* nAChRs, and in a HEK293 cell line expressing recombinant bovine adrenal α3β4 nAChRs (BMα3β4 cells). Transfection of BMα3β4 cells with siRNA to APC, reduced APC protein levels to 52.4% and 61.9% of control values at 24 and 48 h after transfection. To investigate the effects of APC on the cellular distribution of α3β4 nAChRs, [(3)H]epibatidine binding approaches, coupled with APC siRNA treatment, were used. Twenty-four and 48 h after APC siRNA transfection, intracellular nAChRs were significantly reduced to 71% and 68% of control, respectively, while the total population of nAChRs were not significantly changed. Given that total cellular nAChRs represent the sum of surface and intracellular nAChRs, these studies support a re-distribution of nAChRs to the plasma membrane with APC siRNA treatment. Treatment of the cells with the protein synthesis inhibitor, puromycin, also caused a significant reduction (55%) in APC protein levels, and produced a similar re-distribution of cellular nAChRs. These studies support the involvement of APC protein in the maintenance of normal cellular distribution of α3β4 nAChRs.