Discovery of benzamide analogs as negative allosteric modulators of human neuronal nicotinic receptors: pharmacophore modeling and structure-activity relationship studies.

The present study describes our ongoing efforts toward the discovery of drugs that selectively target nAChR subtypes. We exploited knowledge on nAChR ligands and their binding site that were previously identified by our laboratory through virtual screenings and identified benzamide analogs as a novel chemical class of neuronal nicotinic receptor (nAChR) ligands. The lead molecule, compound 1 (4-(allyloxy)-N-(6-methylpyridin-2-yl)benzamide) inhibits nAChR activity with an IC50 value of 6.0 (3.4-10.6) µM on human α4ß2 nAChRs with a ∼5-fold preference against human α3ß4 nAChRs. Twenty-six analogs of compound 1 were also either synthesized or purchased for structure-activity relationship (SAR) studies and provided information relating the chemical/structural properties of the molecules to their ability to inhibit nAChR activity. The discovery of subtype-selective ligands of nAChRs described here should contribute significantly to our understanding of the involvement of specific nAChR subtypes in normal and pathophysiological states.

Alkaloids from Microcos paniculata with cytotoxic and nicotinic receptor antagonistic activities.

Microcos paniculata is a large shrub or small tree that grows in several countries in South and Southeast Asia. In the present study, three new piperidine alkaloids, microgrewiapines A-C (1-3), as well as three known compounds, inclusive of microcosamine A (4), 7'-(3',4'-dihydroxyphenyl)-N-[4-methoxyphenyl)ethyl]propenamide (5), and liriodenine (6), were isolated from cytotoxic fractions of the separate chloroform-soluble extracts of the stem bark, branches, and leaves of M. paniculata. Compounds 1-6 and 1a (microgrewiapine A 3-acetate) showed a range of cytotoxicity values against the HT-29 human colon cancer cell line. When evaluated for their effects on human α3ß4 or α4ß2 nicotinic acetylcholine receptors (nAChRs), several of these compounds were shown to be active as nAChR antagonists. As a result of this study, microgrewiapine A (1) was found to be a selective cytotoxic agent for colon cancer cells over normal colon cells and to exhibit nicotinic receptor antagonistic activity for both the hα3ß4 and hα4ß2 receptor subtypes.

Defining the putative inhibitory site for a selective negative allosteric modulator of human α4ß2 neuronal nicotinic receptors.

Neuronal nicotinic receptors (nAChRs) have been implicated in several diseases and disorders such as autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, and nicotine addiction. To understand the role of nAChRs in these conditions, it would be beneficial to have selective molecules that target specific nAChRs in vitro and in vivo. Our laboratory has previously identified a novel allosteric site on human α4ß2 nAChRs using a series of computational and in vitro approaches. At this site, we have identified negative allosteric modulators that selectively inhibit human α4ß2 nAChRs, a subtype implicated in nicotine addiction. This study characterizes the allosteric site via site-directed mutagenesis. Three amino acids (Phe118, Glu60, and Thr58) on the ß2 subunit were shown to participate in the inhibitory properties of the selective antagonist KAB-18 and provided insights into its antagonism of human α4ß2 nAChRs. SAR studies with KAB-18 analogues and various mutant α4ß2 nAChRs also provided information concerning how different physiochemical features influence the inhibition of nAChRs through this allosteric site. Together, these studies identify the amino acids that contribute to the selective antagonism of human α4ß2 nAChRs at this allosteric site. Finally, these studies define the physiochemical features of ligands that influence interaction with specific amino acids in this allosteric site.

3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate the design of a novel selective antagonist of human α4ß2 neuronal nicotinic acetylcholine receptors.

Subtype selective molecules for α4ß2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4ß2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3ß4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4ß2 nAChRs and human α3ß4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4ß2 nAChRs.

Structure-activity relationship studies of sulfonylpiperazine analogues as novel negative allosteric modulators of human neuronal nicotinic receptors.

Neuronal nicotinic receptors have been implicated in several diseases and disorders such as autism, Alzheimer's disease, Parkinson's disease, epilepsy, and various forms of addiction. To understand the role of nicotinic receptors in these conditions, it would be beneficial to have selective molecules that target specific nicotinic receptors in vitro and in vivo. Our laboratory has previously identified novel negative allosteric modulators of human α4ß2 (Hα4ß2) and human α3ß4 (Hα3ß4) nicotinic receptors. The effects of novel sulfonylpiperazine analogues that act as negative allosteric modulators on both Hα4ß2 nAChRs and Hα3ß4 nAChRs were investigated. This work, through structure-activity relationship (SAR) studies, describes the chemical features of these molecules that are important for both potency and selectivity on Hα4ß2 nAChRs.

Identification of a negative allosteric site on human α4ß2 and α3ß4 neuronal nicotinic acetylcholine receptors.

Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4ß2 and α3ß4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the ß2 subunit were independently mutated to the corresponding residues found on the ß4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4ß2 over α3ß4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 µM. These results demonstrate the selectivity of KAB-18 for human α4ß2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders.

Discovery of Novel α4ß2 Neuronal Nicotinic Receptor Modulators through Structure-Based Virtual Screening.

We performed a hierarchical structure-based virtual screening utilizing a comparative model of the human α4ß2 neuronal nicotinic acetylcholine receptor (nAChR) extracellular domain. Compounds were selected for experimental testing based on structural diversity, binding pocket location, and standard error of the free energy scoring function used in the screening. Four of the eleven in silico hit compounds showed promising activity with low micromolar IC50 values in a calcium accumulation assay. Two of the antagonists were also proven to be selective for human α4ß2 vs human α3ß4 nAChRs. This is the first report of successful discovery of novel nAChR antagonists through the use of structure-based virtual screening with a human nAChR homology model. These compounds may serve as potential novel scaffolds for further development of selective nAChR antagonists.

Evidence for the involvement of adenomatous polyposis coli (APC) protein in maintaining cellular distributions of α3ß4 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 α3ß4 nAChRs was investigated. RT-PCR and Western blotting techniques established the expression of APC protein both in bovine adrenal chromaffin cells, which express native α3ß4* nAChRs, and in a HEK293 cell line expressing recombinant bovine adrenal α3ß4 nAChRs (BMα3ß4 cells). Transfection of BMα3ß4 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 α3ß4 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 α3ß4 nAChRs.

Negative allosteric modulators that target human alpha4beta2 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 alpha4beta2 (Halpha4beta2) and human alpha3beta4 (Halpha3beta4) nAChRs expressed in human embryonic kidney ts201 cells were investigated. During calcium accumulation assays, these NAMs inhibited nAChR activation with IC(50) values ranging from 2.4 microM to more than 100 microM. Several NAMs showed relative selectivity for Halpha4beta2 nAChRs with IC(50) values in the low micromolar range. A lead molecule, KAB-18, was identified that shows relative selectivity for Halpha4beta2 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 Halpha4beta2 nAChR homology model, a binding mode for KAB-18 at the alpha/beta 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.

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 alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. 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 microM. 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.

Pharmacological and immunological identification of native alpha7 nicotinic receptors: evidence for homomeric and heteromeric alpha7 receptors.

Controversy surrounds the expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, alpha7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [(125)I]alpha-bungarotoxin (alphaBGT) binding reaches equilibrium within 4 h and is saturable with a K(d) value of 4.2 nM. Using homologous competition experiments, the K(i) for binding of alphaBGT was 1.9 nM. These data are consistent with the expression of homomeric alpha7 nAChRs. Methyllycaconatine (MLA), which binds alpha7 nAChRs with high affinity, inhibits [(125)I]alphaBGT binding in a concentration-dependent manner with a K(i) of 30.6 nM; this value is approximately 10 fold higher than the reported affinity of MLA for alpha7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate alpha7 nAChRs, as has been previous demonstrated for bovine adrenal alpha3beta4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes alpha7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes alpha3 and alpha5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. Together, these results support the expression of alpha7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric alpha7 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 alpha3beta4* 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 alpha3beta4* nAChRs with a wide range of IC(50) values (0.9-115 microM). 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.

Receptor protection studies comparing recombinant and native nicotinic receptors: Evidence for a subpopulation of mecamylamine-sensitive native alpha3beta4* nicotinic receptors.

Studies involving receptor protection have been used to define the functional involvement of specific receptor subtypes in tissues expressing multiple receptor subtypes. Previous functional studies from our laboratory demonstrate the feasibility of this approach when applied to neuronal tissues expressing multiple nicotinic acetylcholine receptors (nAChRs). In the current studies, the ability of a variety of nAChR agonists and antagonists to protect native and recombinant alpha3beta4 nAChRs from alkylation were investigated using nAChR binding techniques. Alkylation of native alpha3beta4* nAChRs from membrane preparations of bovine adrenal chromaffin cells resulted in a complete loss of specific [(3)H]epibatidine binding. This loss of binding to native nAChRs was preventable by pretreatment with the agonists, carbachol or nicotine. The partial agonist, cytisine, produced partial protection. Several nAChR antagonists were also tested for their ability to protect. Hexamethonium and decamethonium were without protective activity while mecamylamine and tubocurarine were partially effective. Addition protection studies were performed on recombinant alpha3beta4 nAChRs. As with native alpha3beta4* nAChRs, alkylation produced a complete loss of specific [(3)H]epibatidine binding to recombinant alpha3beta4 nAChRs which was preventable by pretreatment with nicotine. However, unlike native alpha3beta4* nAChRs, cytisine and mecamylamine, provide no protection for alkylation. These results highlight the differences between native alpha3beta4* nAChRs and recombinant alpha3beta4 nAChRs and support the use of protection assays to characterize native nAChR subpopulations.

Expression of native alpha3beta4* neuronal nicotinic receptors: binding and functional studies investigating turnover of surface and intracellular receptor populations.

Several pathological conditions involve alterations in expression of neuronal nicotinic acetylcholine receptors (nAChRs). Although some studies have addressed processes involved with muscle nAChR expression, knowledge of the regulation of neuronal nAChRs is particularly sparse. The following studies were designed to investigate cellular mechanisms involved with expression of neuronal alpha3beta4* nAChRs. Catecholamine secretion assays and receptor binding studies coupled with receptor alkylation were used to study the nAChR regulation and turnover. Alkylation of adrenal nAChRs results in a rapid and complete loss of receptor-mediated neurosecretion and surface [(3)H]epibatidine binding sites. After alkylation, both neurosecretory function and nAChR binding slowly (24-48 h) return to prealkylation levels. When cells are treated with the protein synthesis inhibitor puromycin, after alkylation, receptor-mediated neurosecretion does not recover. Long-term treatment (24-48-h) with puromycin, in the absence of alkylation, results in a slow, time-dependent shift to the right, followed by a downward shift, in the nicotine concentration-response curve, documenting a disappearance of surface nAChRs. Puromycin treatment alone also results in a loss to both surface and intracellular [(3)H]epibatidine binding sites. nAChR beta4 subunit levels are significantly decreased after treatment with puromycin. These data support a constitutive turnover of adrenal alpha3beta4* nAChRs, requiring continual de novo synthesis of new receptor protein.

Structure activity studies of ring E analogues of methyllycaconitine. Part 2: Synthesis of antagonists to the alpha3beta4* nicotinic acetylcholine receptors through modifications to the ester.

The development of novel agents for the differentiation of neuronal nicotinic acetylcholine receptors (nAChRs) is important for the treatment of a variety of pathological conditions. We have prepared and evaluated a number of simpler analogues of the norditerpeniod alkaloid methyllycaconitine (MLA) in an effort to understand molecular determinants of nAChR*small molecule interactions. We have previously reported the synthesis and evaluation of a series of ring E analogues of MLA. We report here the optimization of the alpha3beta4* functional activity of this series of compounds through modification of the ester.

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.

Surface and intracellular nicotinic receptors expressed in intact adrenal chromaffin cells: direct measurements using [3H]epibatidine.

The presence and importance of assembled, intracellular neuronal nicotinic acetylcholine receptors (nAChRs) has not been established in native systems. In these studies [3H]epibatidine binding techniques were used to characterize surface and intracellular sites expressed in intact bovine adrenal chromaffin cells in culture. Permeant (300 microM nicotine) and impermeant (5 mM carbachol) cholinergic agents were used to define specific [3H]epibatidine binding to total (surface and intracellular) sites and surface sites, respectively. Intracellular [3H]epibatidine binding sites were characterized after eliminating surface binding sites via alkylation. Equilibrium binding to all sites was reached within 30 min at room temperature. Homologous (epibatidine) competition experiments on total (surface and intracellular) binding sites demonstrated a significant fraction of the high affinity sites were localized to intracellular compartments. Saturation binding assays to surface and intracellular sites revealed K(d) values of 1.9+/-1.1 and 3.6+/-1.9 nM, respectively. These binding studies document the existence of a significant population of high affinity, intracellular binding sites in native neuronal cells and support their characterization as assembled, alpha3beta4* nAChRs. Although the intracellular nAChRs represent approximately 70% of the total, high-affinity nAChRs expressed in cultured chromaffin cells, they do not appear to be involved in functional recovery after nAChR down-regulation.

Pharmacological characterization of recombinant bovine alpha3beta4 neuronal nicotinic receptors stably expressed in HEK 293 cells.

In these studies, [(3)H]epibatidine is used as the radioligand to characterize recombinant bovine alpha3beta4 nicotinic acetylcholine receptors (nAChRs) expressed in HEK 293 cells. Specific binding reaches equilibrium quickly and is saturable with a K(d) value of 0.66 nM. The affinities of the several cholinergic agents were determined, including nicotine (K(i), 0.5 microM), cytisine (K(i), 0.5 microM), carbachol (K(i), 4.1 microM), dihydro-(beta)-erythroidine (K(i), 43.5 microM), d-tubocurarine (K(i), 0.1 microM), 1,1-dimethyl-4-phenylpiperazinium (K(i), 0.5 microM), decamethonium (K(i), 175 microM) and methyllycaconitine (K(i), 0.4 microM). These studies show that the pharmacological characteristics of recombinant bovine alpha3beta4 nAChRs are similar to native bovine alpha3beta4* nAChRs, and indicate that the alpha5 subunit, if present in the native nAChRs, does not affect ligand affinity.

Effects of methyllycaconitine and related analogues on bovine adrenal alpha3beta4* nicotinic acetylcholine receptors.

Adrenal secretion and binding studies were performed using ring E analogues of methyllycaconitine to assess structural determinants affecting activity on bovine adrenal alpha3beta4* nicotinic receptors. The most potent analogues are as potent as many inhibitors of adrenal secretion. Our data support the potential use of methyllycaconitine analogues to generate nicotinic receptor subtype-specific compounds.