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 ␣3␤4* nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant ␣3␤4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native ␣3␤4* nAChR, with IC 50 values ranging from 0.4 to 13.0 M. Using cells expressing recombinant ␣3␤4 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 M. 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 M. 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.

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The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between ␣4␤2 AChRs and the presynaptic cell adhesion molecule, neurexin-1␤. In non-neuronal tsA 201 cells, recombinant neurexin-1␤ and mature ␣4␤2 AChRs form complexes. ␣4␤2 AChRs and neurexin-1␤ also coimmunoprecipitate from rat brain lysates. When exogenous ␣4␤2 AChRs and neurexin-1␤ are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1␤. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1␤ lacking the extracellular domain. Additionally, when ␣4␤2 AChRs, ␣7 AChRs, and neurexin-1␤ are coexpressed in the same neuron, only the ␣4␤2 AChR colocalizes with neurexin-1␤ at presynaptic terminals. Collectively, these data suggest that neurexin-1␤ targets ␣4␤2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on ␣4␤2 AChRs, may contribute to the etiology of these neurological disorders.The clustering of ion channels or receptors and precise targeting to pre-and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs) 5 regulate the release of neurotransmitters at presynaptic sites (1) and mediate fast synaptic transmission at postsynaptic sites of neurons (2). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of ␣2-␣10 and ␤2-␤4 subunits (3). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4, 5).The ␣4␤2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for ␣4␤2 AChRs in nicotine addiction. ␣4␤2 AChRs show high affinity for nicotine (6) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7,8). Furthermore, ␤2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9) and show attenuated self-administration of nicotine (10). ␣4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11). Finally, experiments with knock-in mice expressing ␣4␤2 AChRs hypersensitive to nicotine demonstrate that ␣4␤2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sen...

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 ␣3␤4* 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 nicotinemediated functional activation of both native and recombinant ␣3␤4* nAChRs with a wide range of IC 50 values (0.9 -115 M). 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.The physiological roles of neuronal nicotinic acetylcholine receptors (nAChRs) in synaptic release of acetylcholine and their involvement in the modulation of other important neurotransmitters such as norepinephrine, serotonin, GABA, glutamate, and dopamine make nAChRs prime targets for therapeutic interventions. In addition, nAChRs have been linked to pain, epilepsy, and many neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, and psychiatric disorders, such as depression and schizophrenia. The development of new drugs to target these receptors has been slow for several reasons: 1) multiple subtypes of nAChRs are expressed in the central and peripheral nervous systems; 2) few drugs are available that selectively target nAChR subtypes; and 3) information on the physiological roles of specific nAChR subtypes is limited. A key approach to provide a better understanding of physiological processes and pathophysiological conditions involving

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