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.

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.

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.

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.

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.

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.