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Neuronal nicotinic acetylcholine receptors (nAChRs) have an established role in pain pathways and devastating neurodegenerative diseases; however, few drugs have been successfully developed to target them. The most abundant nAChR in the brain, the α4β2 nAChR, is assembled from five subunits in a 3α:2β stoichiometry-(α4) 3 (β2) 2 . This receptor contains a unique agonist-binding site at the α4α4 interface in addition to two classical agonist-binding sites at α4β2 interfaces. Most known agonists target both α4α4 and α4β2 sites, however, a few compounds with selectivity for the α4α4 site have been identified. These α4α4 selective compounds have a modulator-like effect akin to benzodiazepines in the γ-aminobutyric acid type A receptor, which is desirable from a drug development perspective. The two most well characterised α4α4 selective compounds are CMPI and NS9283. Both are structurally very different from classical agonists, and it is puzzling how they occupy the same binding site. In the search for a common pharmacophore, we conducted extensive molecular dynamics simulations with both classical agonists and site-selective non-classical compounds. Analyses of the simulations revealed that the α4α4 binding site contains a unique pocket not found in the α4β2 binding site. CMPI and NS9283 were observed to bind in this pocket, thereby explaining why they are selective for the α4α4 binding site. The proposed binding mode featured a closed-loop C conformation, which is strongly correlated with agonism in nAChRs and explained key site-directed mutagenesis data for both compounds. Based on this binding mode, we proposed a pharmacophore for drugs targeting the α4α4 binding site. The proposed pharmacophore captures the essence of the original model, that is, nicotinic agonists act as a bridge between protein subunits. The pharmacophore model we propose is unique to the α4α4 binding site and provides a template for developing new site-selective therapeutic agents., (© 2025 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

In contrast to G protein-coupled receptors or receptor tyrosine kinases, the mechanism underlying ERK activation through nicotine acetylcholine receptors (nAChRs), members of the ligand-gated ion channel family, remains poorly elucidated. This study aimed to delineate the signaling pathway responsible for ERK activation by the α4β2 nAChR subtype, which is implicated in nicotine addiction and various mental disorders. Loss-of-function strategies and mutants of arrestin2/PKCβII with distinct functional characteristics were employed to identify the cellular components and processes involved in ERK activation. ERK activation via α4β2 nAChR was observed within the nucleus and necessitated the nuclear translocation of arrestin2 and PKCβII, which exhibited mutual augmentation. Activation of PKCβII by α4β2 nAChR stimulation facilitated the nuclear translocation of arrestin2 by enhancing its interaction with importin β1. Apart from scaffolding ERK activation in the nucleus, arrestin2, in cooperation with GRK2, facilitated the activation of the Src/Syk/PKCβII signaling cascade, leading to the nuclear entry of PKCβII in a Gβγ-dependent manner. Upon nuclear localization, PKCβII underwent ubiquitination by Mdm2 and interacted with MEK1, resulting in ERK activation. In summary, α4β2 nAChR-mediated ERK activation in the nucleus involves the nuclear translocation of arrestin2 and PKCβII, which is reciprocally facilitated via positive feedback augmentation. As α4β2 nAChRs play a pivotal role in various cellular processes including drug addiction and mental disorders, our findings will offer insights into understanding the pathogenesis of α4β2 nAChR-related disorders and may facilitate the development of targeted therapeutic interventions. [Display omitted] [ABSTRACT FROM AUTHOR]

The β3 subunit of nicotinic acetylcholine receptors (nAChRs) participates in heteropentameric assemblies with some α and other β neuronal subunits forming a plethora of various subtypes, differing in their electrophysiological and pharmacological properties. While β3 has for several years been considered an accessory subunit without direct participation in the formation of functional binding sites, recent electrophysiology data have disputed this notion and indicated the presence of a functional (+) side on the extracellular domain (ECD) of β3. In this study, we present the 2.4 Å resolution crystal structure of the monomeric β3 ECD, which revealed rather distinctive loop C features as compared to those of α nAChR subunits, leading to intramolecular stereochemical hindrance of the binding site cavity. Vigorous molecular dynamics simulations in the context of full length pentameric β3-containing nAChRs, while not excluding the possibility of a β3 (+) binding site, demonstrate that this site cannot efficiently accommodate the agonist nicotine. From the structural perspective, our results endorse the accessory rather than functional role of the β3 nAChR subunit, in accordance with earlier functional studies on β3-containing nAChRs. [ABSTRACT FROM AUTHOR]

Background and Purpose: The α4β2 nicotinic ACh receptor (nAChR), a subtype of the ligand‐gated ion channel, is abundantly expressed in the brain and is implicated in several neurological disorders. The endocytosis of nAChRs plays important roles in the pathogenesis of neurological diseases, but the underlying molecular mechanisms remain poorly understood. Experimental Approach Loss‐of‐function approaches and mutants of α4β2 nAChRs that display different endocytic properties were used to identify the cellular components and processes responsible for endocytosis. The signalling cascade that leads to endocytosis was deduced via protein interactions in predicted cellular components. The endocytosis of α4β2 nAChRs was determined and crosschecked using an ELISA and radioligand assay. Key Results: Endocytosis of α4β2 nAChRs occurred through clathrin‐mediated endocytosis in a dynamin‐dependent manner. 14‐3‐3η‐dependent Src‐mediated phosphorylation of the nAChR α4 subunit at Y575 was required for nAChR endocytosis, and this occurred with the assistance of β‐arrestin1 and GPCR kinase 2 (GRK2) without the need for kinase activity. Endocytosis triggered the mouse double minute 2 homologue‐mediated ubiquitination and subsequent down‐regulation of α4β2 nAChRs. Conclusions and Implications: α4β2 nAChR, an ionophore receptor, employs the metabotropic signalling pathway required for endocytosis, which leads to ubiquitination and down‐regulation. Further, GRK2 and β‐arrestin1, usually associated with GPCR signalling, are involved in the endocytosis of α4β2 nAChRs via different mechanisms. Considering the functional and pathological implications of nAChR endocytosis, results obtained in this study are crucial for the progression of basic research and clinical investigations. [ABSTRACT FROM AUTHOR]

The α4β2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson’s and Alzheimer’s diseases. Designing α4β2 nAChR selective inhibitors could help define the role of the α4β2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4β2 nAChR through competitive inhibition of the α4(+)β2(−) or α4(+)α4(−) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3β2, α4β2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4β2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4β2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)3(β2)2 nAChR was deduced using the information from the cryo-electron structure of (α4)3(β2)2 nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(−) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)3(β2)2 nAChR.

Neuronal α4β2 nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels (LGIC) that have been implicated in nicotine addiction, reward, cognition, pain disorders, anxiety, and depression. Nicotine has been widely used as a template for the synthesis of ligands that prefer α4β2 nAChRs subtypes. The most important therapeutic use for α4β2 nAChRs is as replacement therapy for smoking cessation and withdrawal and the most successful therapeutic ligands are partial agonists. In this case, we use the N-methylpyrrolidine moiety of nicotine to design and synthesize new α4β2 nicotinic derivatives, coupling the pyrrolidine moiety to an aromatic group by introducing an ether-bonded functionality. Meta-substituted phenolic derivatives were used for these goals. Radioligand binding assays were performed on clonal cell lines of hα4β2 nAChR and two electrode voltage-clamp experiments were used for functional assays. Molecular docking was performed in the open state of the nAChR in order to rationalize the agonist activity shown by our compounds.

Each of the four aromatic –CH= of ( S,R )-2-pyrrolidinyl-1,4-benzodioxane [( S,R )- 6 ] and of its epimer at the dioxane stereocenter ( S,S )- 6 , previously reported as α4β2 nAChR ligands, was replaced with nitrogen. The resulting four diastereoisomeric pairs of pyrrolidinyl-pyridodioxanes were studied for the nicotinic affinity and activity at α4β2, α3β4 and α7 nAChR subtypes and compared to their common carbaisostere. It turned out that such isosteric substitutions are highly detrimental, but with the important exception of the S,R stereoisomer of the pyrrolidinyl-pyridodioxane with the pyridine nitrogen adjacent to the dioxane and seven atoms distant from the pyrrolidine nitrogen. Indeed, this stereo/regioisomer not only maintained the α4β2 affinity of [( S,R )- 6 ], but also greatly improved in selectivity over the α3β4 and α7 subtypes and, most importantly, exhibited a highly selective α4β2 partial agonism. The finding that [( S,R )- 6 ] is, instead, an unselective α4β2 antagonist indicates that the benzodioxane substructure confers affinity for the α4β2 nAChR binding site, but activation of this receptor subtype needs benzodioxane functionalization under strict steric requirements, such as the previously reported 7-OH substitution or the present isosteric modification. [ABSTRACT FROM AUTHOR]

Aim Neuropathic pain is often refractory to conventional analgesics including opioids and non-steroidal anti-inflammatory drugs. Evidence suggests nicotinic acetylcholine receptor ligands regulate pain transmission. Effects of α4 β2 nicotinic acetylcholine receptor activation on pain behaviours after nerve injury were studied. Methods Mice were subjected to partial sciatic nerve ligation ( PSL). Nicotinic acetylcholine receptor α4 and β2 subunits localization in injured nerves were evaluated by immunohistochemistry. Neuropathic pain, assessed by tactile allodynia and thermal hyperalgesia, was examined by von Frey test and Hargreaves test respectively. Results Nicotinic acetylcholine receptor α4 and β2 subunits were up-regulated in injured nerves and were expressed on F4/80-positive macrophages. When nicotine was perineurally administered daily for 4 days (day 7-10; maintenance phase) after nerve injury, pain behaviours were significantly alleviated. The inhibitory effects of nicotine were reversed by co-administration of mecamylamine (non-selective nicotinic acetylcholine receptor antagonist) and dihydro- β-erythroidine (selective α4 β2 nicotinic acetylcholine receptor antagonist). Likewise, when α4 β2 nicotinic acetylcholine receptor agonists ( TC2559 or ABT418) were administered daily for 4 days (day 7-10) after nerve injury, pain behaviours were significantly attenuated. On the other hand, nicotine administered daily for 4 days (day 0-3; initiation phase) after nerve injury alleviated pain behaviours, which were antagonized by co-administration of dihydro- β-erythroidine. TC2559 administered daily for 4 days (day 0-3) also attenuated nerve injury-induced pain behaviours. Conclusion The activation of α4 β2 nicotinic acetylcholine receptor expressed on infiltrating macrophages in injured nerves may participate in the relief of PSL-induced neuropathic pain during maintenance and initiation phases. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors (nAChR) are abundant in the brain and are essential in cognitive function, learning and memory. Previous efforts on α4β2 nAChR had been focused on functional and pharmacological characterization, where high expression yield is not essential. For structural studies though, large amounts of pure protein is important but heterologous overexpression of membrane proteins can be a burdensome task, especially if high amounts are required. In the current study, a truncated mutant of the human α4β2 nAChR was designed in order to improve expression and solubility and to obtain material suitable for high resolution structural studies. We showed that the wild type α4β2 nAChR presented low expression and solubilization yield both of which were improved with the truncated construct. The truncated nAChR showed similar binding profile to the wild type, was purified by a two-step chromatography and isolated in high purity and adequate quantity. [ABSTRACT FROM AUTHOR]

Neural stem/progenitor cells (NSPCs) of the subgranular zone have been implicated in cognitive processes, which represent a potentially important source of regenerative medicine for the treatment of neurodegenerative diseases such as Alzheimer's disease (AD). In our previous studies, ZY-1, a novel nicotinic analog, improved cognitive function in transgenic mice model of AD. However, the effect of ZY-1 on the NSPCs remains unclear. Here, we show that ZY-1 significantly increased proliferation and migration of NSPCs, but failed to affect NSPCs differentiation in vitro. Furthermore, during the proliferative period, ZY-1 enhanced intracellular reactive oxygen species (ROS) levels. Meanwhile, ZY-1 also inhibited the levels of Aβ-induced ROS. Our data indicate that ZY-1 regulates adult hippocampal neurogenesis in vitro, at least partly due to modulating intracellular ROS levels. These results, taken together with those of our previous studies, suggest that ZY-1 might have a potential therapeutic effect for the treatment of AD. [ABSTRACT FROM AUTHOR]

Abstract: We have carried out computational studies on interactions of diazabicyclic amide analogs with α4β2 nAChR using homology modeling, docking and pharmacophore elucidation techniques. We have found alternative ligand binding modes in most cases. All these diverse poses exhibit the quintessential hydrogen-bonding interaction between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the highly conserved Trp-149. This hydrogen bond was always found to be shorter than the one contracted by the ligand carbonyl group and a second hydrogen-bond made by the cationic center with Tyr-93 of the principal face of the protein. In most of the poses observed, cation–π interactions involved three aromatic residues located in the principal face of the protein: Trp-149, Tyr-190 and Tyr-197. The latter amino acid residue appears to often donate a hydrogen-bond to the ligand carbonyl oxygen atom. We also describe two rings of alternative receptor-based hydrogen-bond donor features equidistantly separated from the carbonyl oxygen of the highly conserved Trp-149 approximately by 5 and 8Å, respectively. These findings could be exploited to design diverse and selective novel chemical libraries for the treatment of diseases and conditions where the α4β2 nAChR is disrupted, such as Alzheimer disease, Parkinson’s disease and l-dopa-induced dyskinesia (LID). [Copyright &y& Elsevier]

Abstract: The α4β2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the α4β2 nAChR at clinically relevant concentrations, but their binding sites in α4β2 remain unclear. The recently determined NMR structures of the α4β2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the α4β2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the β2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., [32]). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of α4 and β2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the α4β2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions. [Copyright &y& Elsevier]

Abstract: Neuronal cholinergic transmission is a prerequisite for proper CNS function. Consequently, disturbance of this system is associated with a number of pathophysiological conditions such as Parkinson''s disease, Alzheimer''s disease, schizophrenia and ADHD. Consequently, drug discovery efforts have spurred considerable research endeavours into identifying specific compounds for this system. Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels involved in cholinergic transmission. nAChRs are homo- or heteromeric pentamers with α4β2 receptors being the most abundant heteromer. The stoichiometry of α4β2 receptors can be either (α4)3(β2)2 or (α4)2(β2)3 representing channels with low (LS) or high (HS) sensitivity, respectively, to endogenous ligands. In the present study we applied the partial nAChR α4β2 LS and HS agonist NS3956 and the LS selective positive allosteric modulator NS9283 to investigate the role of α4β2 in Parkinson and pain models. In 6-OHDA lesioned rats, NS3956 increased rotational behaviour when rats were co-treated with nomifensine. This effect was absent in the presence of mecamylamine. In contrast, co-treatment with NS3956 and NS9283 reduced rotational behaviour in the animals. In a rat formalin pain model NS3956 induced an analgesic response that was strongly potentiated by NS9283. Finally in vitro experiments were applied to determine dopamine release from striatal minces. NS3956 induced a concentration dependent release while NS9283 was unable to potentiate agonist induced release. Together these results emphasize involvement of α4β2 nAChR in rotational and analgesic responses and confirm striatal α4β2 receptors to be of the HS form. [Copyright &y& Elsevier]

Abstract: Brain nicotinic acetylcholine receptors are involved in several neuropsychiatric disorders, e.g. Alzheimer''s and Parkinson''s diseases, Tourette''s syndrome, schizophrenia, depression, autism, attention deficit hyperactivity disorder, and anxiety. Currently, approaches selectively targeting the activation of specific nicotinic acetylcholine receptors are in clinical development for treatment of memory impairment of Alzheimer''s disease patients. These are α4β2 and α7 nicotinic acetylcholine receptor agonists which are believed to enhance cholinergic and glutamatergic neurotransmission, respectively. In order to gain a better insight into the mechanistic role of these two nicotinic acetylcholine receptors in learning and memory, we investigated the effects of the α4β2 nicotinic acetylcholine receptor agonist TC-1827 and the α7 nicotinic acetylcholine receptor partial agonist SSR180711 on hippocampal long-term potentiation (LTP), a widely accepted cellular experimental model of memory formation. Generally, LTP is distinguished in an early and a late form, the former being protein-synthesis independent and the latter being protein-synthesis dependent. TC-1827 was found to increase early LTP in a bell-shaped dose dependent manner, but did not affect late LTP. In contrast, the α7 nicotinic acetylcholine receptor partial agonist SSR180711 showed enhancing effects on both early and late LTP in a bell-shaped manner. Furthermore, SSR180711 not only increased early LTP, but also transformed it into late LTP, which was not observed with the α4β2 nicotinic acetylcholine receptor agonist. Therefore, based on these findings α7 nicotinic acetylcholine receptor (partial) agonists appear to exhibit stronger efficacy on memory improvement than α4β2 nicotinic acetylcholine receptor agonists. [Copyright &y& Elsevier]

Abstract: Nicotinic acetylcholine receptors (nAChRs) function as ligand-gated ion channels activated by the neurotransmitter acetylcholine. Gene knockout and antisense studies coupled with pharmacological studies with nAChR agonists have documented a role of α4β2 nAChR activation in analgesia. ABT-594, for the first time, provided clinical validation to the nAChR agonist pharmacology as a novel mechanism for treatment of pain. However, ABT-594 was poorly tolerated at the efficacious doses, particularly with respect to the side effects of nausea and emesis, which is thought to be mediated by activation of the ganglionic-type (α3-containing) nAChRs. An alternate approach is to selectively modulate the α4β2 nAChR via positive allosteric modulation. Positive allosteric modulators (PAMs) are compounds that do not interact with the agonist binding sites or possess intrinsic activity at the receptor per se, but potentiate the effects of the agonist. NS9283 (also known as A-969933), the first oxadiazole analog, was found to selectively enhance the potency of a range of nAChR agonists at α4β2, but not α3β4, nAChRs. Studies reported here, along with the accompanying manuscript collectively point to the conclusion, based on preclinical models, that the analgesic efficacy of clinically well-tolerated doses of ABT-594 in humans can be significantly enhanced by co-administration with the α4β2 PAM. Additionally, studies in ferrets demonstrate no exaggeration of emetic effect when ABT-594 is co-dosed with NS9283. Cardiovascular studies in anesthetized dogs achieve supra-therapeutic plasma concentrations of ABT-594 (>20-fold) without hemodynamic or electrophysiological effects using the co-administration paradigm. [Copyright &y& Elsevier]

Abstract: We have reported that systemic application of nicotinic agonists expresses a long-term potentiation (LTP)-like facilitation, a model of synaptic plasticity, in vivo in the mouse hippocampus. The present study conducted to clarify the involvement of synaptotagmin1 in synaptic plasticity by investigating the time-dependent change of the mRNA and protein levels of synaptotagmin1 during LTP-like facilitation in the mouse hippocampus. The mRNA expression of synaptotagmin1 increased during 2- to 8-h period by intraperitoneal application of nicotine (3mg/kg), returning to the basal level in 12-h. Also, the protein level of synaptotagmin1, but not synaptophysin, in a total fraction from hippocampus increased during 4- to 12-h period by the same treatment, returning to the basal level in 24-h. The protein level of synaptotagmin1 in a membrane fraction from hippocampus also increased during 4- to 8-h period by nicotine, returning to the basal level in 12-h. This nicotine-enhanced synaptotagmin1 protein in a membrane fraction was inhibited by pretreatment of mecamylamine (0.3mg/kg, i.p.), a nonselective nicotinic acetylcholine receptors (nAChRs) antagonist. Furthermore, choline (30mg/kg, i.p.), a selective α7 nAChR agonist, or ABT-418 (10mg/kg, i.p.), a selective α4β2 nAChR agonist, enhanced the level of synaptotagmin1 in a membrane fraction. Our findings demonstrate that synaptotagmin1 protein following mRNA which is enhanced without increasing the number of synapse gathers around pre-synaptic membrane during hippocampal LTP-like facilitation through activation of α7 and/or α4β2 nAChRs in the brain. These results suggest that new-synthesized synaptotagmin1 following synaptic plasticity may contribute to long-lasting synaptic plasticity via positive, feedfoward mechanisms. [ABSTRACT FROM AUTHOR]

[Display omitted] • Benzofurans bearing N -methyl-2-pyrrolidinyl at furan are α4β2 nAChR ligands. • rac -2-(N -Methyl-2-pyrrolidinyl)-6-hydroxybenzofuran (12) shows the best affinity. • 12 and three benzodioxane based α4β2 agonists were tested at LS and HS α4β2 isoform. • (2R,2′S)-2-(N -Methyl-2′-pyrrolidinyl)-7-hydroxybenzodioxane is HS selective agonist. • (2S,2′S)-2-(N -Methyl-2′-pyrrolidinyl)-5-aminobenzodioxane is LS selective agonist. A series of racemic benzofurans bearing N -methyl-2-pyrrolidinyl residue at C(2) or C(3) has been synthesized and tested for affinity at the α4β2 and α3β4 nicotine acetylcholine receptors (nAChRs). As previously reported for the benzodioxane based analogues, hydroxylation at proper position of benzene ring results in high α4β2 nAChR affinity and α4β2 vs. α3β4 nAChR selectivity. 7-Hydroxy- N -methyl-2-pyrrolidinyl-1,4-benzodioxane (2) and its 7- and 5-amino benzodioxane analogues 3 and 4 , which are all α4β2 nAChR partial agonists, and 2-(N -methyl-2-pyrrolidinyl)-6-hydroxybenzofuran (12) were selected for functional characterization at the two α4β2 stoichiometries, the high sensitivity (α4) 2 (β2) 3 and the low sensitivity (α4) 3 (β2) 2. The benzene pattern substitution, which had previously been found to control α4β2 partial agonist activity and α4β2 vs. α3β4 selectivity, proved to be also involved in stoichiometry-selectivity. The 7-hydroxybenzodioxane derivative 2 selectively activates (α4) 2 (β2) 3 nAChR, which cannot be activated by its 5-amino analogue 4. A marginal structural modification, not altering the base pyrrolidinyl benzodioxane scaffold, resulted in opposite activity profiles at the two α4β2 nAChR isoforms providing an interesting novel case study. [ABSTRACT FROM AUTHOR]

Abstract: Compounds based on the 3-(dimethylamino)butyl dimethylcarbamate (DMABC) scaffold were synthesized and pharmacologically characterized at the α4β2, α3β4, α4β4 and α7 neuronal nicotinic acetylcholine receptors (nAChRs). The carbamate functionality and a small hydrophobic substituent in the C-3 position were found to be vital for the binding affinity to the nAChRs, whereas the carbamate nitrogen substituents were important for nAChR subtype selectivity. Finally, the compounds were found to be agonists at the α3β4 nAChR. [Copyright &y& Elsevier]

Abstract: The preclinical pharmacology of the α4β2 nicotinic acetylcholine receptor (nAChR) partial agonist varenicline, a novel smoking cessation agent is described. Varenicline binds with subnanomolar affinity only to α4β2 nAChRs and in vitro functional patch clamp studies in HEK cells expressing nAChRs show that varenicline is a partial agonist with 45% of nicotine''s maximal efficacy at α4β2 nAChRs. In neurochemical models varenicline has significantly lower (40–60%) efficacy than nicotine in stimulating [3H]-dopamine release from rat brain slices in vitro and in increasing dopamine release from rat nucleus accumbens in vivo, while it is more potent than nicotine. In addition, when combined with nicotine, varenicline effectively attenuates the nicotine-induced dopamine release to the level of the effect of varenicline alone, consistent with partial agonism. Finally, varenicline reduces nicotine self-administration in rats and supports lower self-administration break points than nicotine. These data suggest that varenicline can reproduce to some extent the subjective effects of smoking by partially activating α4β2 nAChRs, while preventing full activation of these receptors by nicotine. Based on these findings, varenicline was advanced into clinical development and recently shown to be an effective and safe aid for smoking cessation treatment. [Copyright &y& Elsevier]

The α4β2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson's and Alzheimer's diseases. Designing α4β2 nAChR selective inhibitors could help define the role of the α4β2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4β2 nAChR through competitive inhibition of the α4(+)β2(−) or α4(+)α4(−) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3β2, α4β2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4β2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4β2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)3(β2)2 nAChR was deduced using the information from the cryo-electron structure of (α4)3(β2)2 nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(−) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)3(β2)2 nAChR. [ABSTRACT FROM AUTHOR]

Nicotine has been known to enhance recognition memory in various species. However, the brain region where nicotine acts and exerts its effect remains unclear. Since the medial prefrontal cortex (mPFC) is associated with memory, we examined the role of the mPFC in nicotine-induced enhancement of recognition memory using the novel object recognition test in male C57BL/6J mice. Systemic nicotine administration 10 min before training session significantly enhanced object recognition memory in test session that was performed 24 h after the training. Intra-mPFC infusion of mecamylamine, a non-selective nicotinic acetylcholine receptor (nAChR) antagonist, 5 min before nicotine administration blocked the effect of nicotine. Additionally, intra-mPFC infusion of dihydro-β-erythroidine, a selective α4β2 nAChR antagonist, or methyllycaconitine, a selective α7 nAChR antagonist, significantly suppressed the nicotine-induced object recognition memory enhancement. Finally, intra-mPFC infusion of nicotine 1 min before the training session augmented object recognition memory in a dose-dependent manner. These findings suggest that mPFC α4β2 and α7 nAChRs mediate the nicotine-induced object recognition memory enhancement.

Neuronal α4β2 nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels (LGIC) that have been implicated in nicotine addiction, reward, cognition, pain disorders, anxiety, and depression. Nicotine has been widely used as a template for the synthesis of ligands that prefer α4β2 nAChRs subtypes. The most important therapeutic use for α4β2 nAChRs is as replacement therapy for smoking cessation and withdrawal and the most successful therapeutic ligands are partial agonists. In this case, we use the N-methylpyrrolidine moiety of nicotine to design and synthesize new α4β2 nicotinic derivatives, coupling the pyrrolidine moiety to an aromatic group by introducing an ether-bonded functionality. Meta-substituted phenolic derivatives were used for these goals. Radioligand binding assays were performed on clonal cell lines of hα4β2 nAChR and two electrode voltage-clamp experiments were used for functional assays. Molecular docking was performed in the open state of the nAChR in order to rationalize the agonist activity shown by our compounds. [ABSTRACT FROM AUTHOR]