Auxiliary protein and chaperone regulation of neuronal nicotinic receptor subtype expression and function.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of pentameric, ligand-gated ion channels that are located on the surface of neurons and non-neuronal cells and have multiple physiological and pathophysiological functions. In order to reach the cell surface, many nAChR subtypes require the help of chaperone and/or auxiliary/accessory proteins for their assembly, trafficking, pharmacological modulation, and normal functioning in vivo. The use of powerful genome-wide cDNA screening has led to the identification and characterisation of the molecules and mechanisms that participate in the assembly and trafficking of receptor subtypes, including chaperone and auxiliary or accessory proteins. The aim of this review is to describe the latest findings concerning nAChR chaperones and auxiliary proteins and pharmacological chaperones, and how some of them control receptor biogenesis or regulate channel activation and pharmacology. Some auxiliary proteins are subtype selective, some regulate various subtypes, and some not only modulate nAChRs but also target other receptors and signalling pathways. We also discuss how changes in auxiliary proteins may be involved in nAChR dysfunctions.

Evidence of a dual mechanism of action underlying the anti-proliferative and cytotoxic effects of ammonium-alkyloxy-stilbene-based α7- and α9-nicotinic ligands on glioblastoma cells.

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

Cytisine and cytisine derivatives. More than smoking cessation aids.

Cytisine, a natural bioactive compound that is mainly isolated from plants of the Leguminosae family (especially the seeds of Laburnum anagyroides), has been marketed in central and eastern Europe as an aid in the clinical management of smoking cessation for more than 50 years. Its main targets are neuronal nicotinic acetylcholine receptors (nAChRs), and pre-clinical studies have shown that its interactions with various nAChR subtypes located in different areas of the central and peripheral nervous systems are neuroprotective, have a wide range of biological effects on nicotine and alcohol addiction, regulate mood, food intake and motor activity, and influence the autonomic and cardiovascular systems. Its relatively rigid conformation makes it an attractive template for research of new derivatives. Recent studies of structurally modified cytisine have led to the development of new compounds and for some of them the biological activities are mediated by still unidentified targets other than nAChRs, whose mechanisms of action are still being investigated. The aim of this review is to describe and discuss: 1) the most recent pre-clinical results obtained with cytisine in the fields of neurological and non-neurological diseases; 2) the effects and possible mechanisms of action of the most recent cytisine derivatives; and 3) the main areas warranting further research.

Choline and nicotine increase glioblastoma cell proliferation by binding and activating α7- and α9- containing nicotinic receptors.

Glioblastomas (GBMs), the most frequent and aggressive human primary brain tumours, have altered cell metabolism, and one of the strongest indicators of malignancy is an increase in choline compounds. Choline is also a selective agonist of some neuronal nicotinic acetylcholine receptor (nAChR) subtypes. As little is known concerning the expression of nAChR in glioblastoma cells, we analysed in U87MG human grade-IV astrocytoma cell line and GBM5 temozolomide-resistant glioblastoma cells selected from a cancer stem cell-enriched culture, molecularly, pharmacologically and functionally which nAChR subtypes are expressed and,whether choline and nicotine can affect GBM cell proliferation. We found that U87MG and GBM5 cells express similar nAChR subtypes, and choline and nicotine increase their proliferation rate and activate the anti-apoptotic AKT and pro-proliferative ERK pathways. These effects are blocked by the presence of non-cell-permeable peptide antagonists selective for α7- and α9-containing nicotinic receptors. siRNA-mediated silencing of α7 or α9 subunit expression also selectively prevents the effects of nicotine and choline on GBM cell proliferation. Our findings indicate that nicotine and choline activate the signalling pathways involved in the proliferation of GBM cells, and that these effects are mediated by α7 and α9-containing nAChRs. This suggests that these nicotinic receptors may contribute to the aggressive behaviour of this tumor and may indicate new therapeutic strategies against high-grade human brain tumours.

Persistent cognitive and affective alterations at late withdrawal stages after long-term intermittent exposure to tobacco smoke or electronic cigarette vapour: Behavioural changes and their neurochemical correlates.

Smoking cessation induces a withdrawal syndrome associated with anxiety, depression, and impaired neurocognitive functions, but much less is known about the withdrawal of e-cigarettes (e-CIG). We investigated in Balb/c mice the behavioural and neurochemical effects of withdrawal for up to 90 days after seven weeks' intermittent exposure to e-CIG vapour or cigarette smoke (CIG). The withdrawal of e-CIG and CIG induced early behavioural alterations such as spatial memory deficits (spatial object recognition task), increased anxiety (elevated plus maze test) and compulsive-like behaviour (marble burying test) that persisted for 60-90 days. Notably, attention-related (virtual object recognition task) and depression-like behaviours (tail suspension and sucrose preference tests) appeared only 15-30 days after withdrawal and persisted for as long as up to 90 days. At hippocampal level, the withdrawal-induced changes in the levels of AMPA receptor GluA1 and GluA2/3 subunits, PSD 95 protein, corticotropin-releasing factor (Crf) and Crf receptor 1 (CrfR1) mRNA were biphasic: AMPA receptor subunit and PSD95 protein levels initially remained unchanged and decreased after 60-90 days, whereas Crf/CrfR1 mRNA levels initially increased and then markedly decreased after 60 days. These late reductions correlated with the behavioural impairments, particularly the appearance of depression-like behaviours. Our findings show that major behavioural and neurochemical alterations persist or even first appear late after the withdrawal of chronic CIG smoke or e-CIG vapour exposure, and underline importance of conducting similar studies of humans, including e-CIG vapers.

A Genetic Algorithm Procedure for the Automatic Updating of FEM Based on Ambient Vibration Tests.

The dynamic identification of the modal parameters of a structure, in order to gain control of its functionality under operating conditions, is currently under discussion from a scientific and technical point of views. The experimental observations obtained through structural health monitoring (SHM) are a useful calibration reference of numerical models (NMs). In this paper, the procedures for the identification of modal parameters in historical bell towers using a stochastic subspace identification (SSI) algorithm are presented. Then, NMs are manually calibrated on the identification's results. Finally, the applicability of a genetic algorithm for the automatic calibration of the elastic parameters is considered with the aim of searching for the properties of the autochthonous material, in order to reduce modelling error following the model assurance criterion (MAC). In this regard, several material values on the same model are examined to see how to approach the evolution and the distribution of these features, comparing the characterization proposed by the genetic algorithm with the results considered by the manual iterative procedure.

Nicotine-modulated subunit stoichiometry affects stability and trafficking of α3ß4 nicotinic receptor.

Heteromeric nAChRs are pentameric cation channels, composed of combinations of two or three α and three or two ß subunits, which play key physiological roles in the central and peripheral nervous systems. The prototypical agonist nicotine acts intracellularly to upregulate many nAChR subtypes, a phenomenon that is thought to contribute to the nicotine dependence of cigarette smokers. The α3ß4 subtype has recently been genetically linked to nicotine dependence and lung cancer; however, the mode of action of nicotine on this receptor subtype has been incompletely investigated. Here, using transfected mammalian cells as model system, we characterized the response of the human α3ß4 receptor subtype to nicotine and the mechanism of action of the drug. Nicotine, when present at 1 mm concentration, elicited a ∼5-fold increase of cell surface α3ß4 and showed a more modest upregulatory effect also at concentrations as low as 10 µM. Upregulation was obtained if nicotine was present during, but not after, pentamer assembly and was caused by increased stability and trafficking of receptors assembled in the presence of the drug. Experimental determinations as well as computational studies of subunit stoichiometry showed that nicotine favors assembly of pentamers with (α3)2(ß4)3 stoichiometry; these are less prone than (α3)3(ß4)2 receptors to proteasomal degradation and, because of the presence in the ß subunit of an endoplasmic reticulum export motif, more efficiently transported to the plasma membrane. Our findings uncover a novel mechanism of nicotine-induced α3ß4 nAChR upregulation that may be relevant also for other nAChR subtypes.

Adolescent nicotine exposure transiently increases high-affinity nicotinic receptors and modulates inhibitory synaptic transmission in rat medial prefrontal cortex.

Adolescence is a critical developmental period during which most adult smokers initiate their habit. Adolescents are more vulnerable than adults to nicotine's long-term effects on addictive and cognitive behavior. We investigated whether adolescent nicotine exposure in rats modifies expression of nicotinic acetylcholine receptors (nAChRs) in medial prefrontal cortex (mPFC) in the short and/or long term, and whether this has functional consequences. Using receptor binding studies followed by immunoprecipitation of nAChR subunits, we showed that adolescent nicotine exposure, as compared with saline, caused an increase in mPFC nAChRs containing α4 or ß2 subunits (24 and 18%, respectively) 24 h after the last injection. Nicotine exposure in adulthood had no such effect. This increase was transient and was not observed 5 wk following either adolescent or adult nicotine exposure. In line with increased nAChRs expression 1 d after adolescent nicotine exposure, we observed a 34% increase in amplitude of nicotine-induced spontaneous inhibitory postsynaptic currents in layer II/III mPFC pyramidal neurons. These effects were transient and specific, and observed only acutely after adolescent nicotine exposure, but not after 5 wk, and no changes were observed in adult-exposed animals. The acute nicotine-induced increase in α4ß2-containing receptors in adolescents interferes with the normal developmental decrease (37%) of these receptors from early adolescence (postnatal day 34) to adulthood (postnatal day 104) in the mPFC. Together, this suggests that these receptors play a role in mediating the acute rewarding effects of nicotine and may underlie the increased sensitivity of adolescents to nicotine.

Subnanomolar Affinity and Selective Antagonism at α7 Nicotinic Receptor by Combined Modifications of 2-Triethylammonium Ethyl Ether of 4-Stilbenol (MG624).

Modifications of the cationic head and the ethylene linker of 2-(triethylammonium)ethyl ether of 4-stilbenol (MG624) have been proved to produce selective α9*-nAChR antagonism devoid of any effect on the α7-subtype. Here, single structural changes at the styryl portion of MG624 lead to prevailing α7-nAChR antagonism without abolishing α9*-nAChR antagonism. Nevertheless, rigidification of the styryl into an aromatic bicycle, better if including a H-bond donor NH, such as 5-indolyl (31), resulted in higher and more selective α7-nAChR affinity. Hybridization of this modification with the constraint of the 2-triethylammoniumethyloxy portion into (R)-N,N-dimethyl-3-pyrrolidiniumoxy substructure, previously reported as the best modification for the α7-nAChR affinity of MG624 (2), was a winning strategy. The resulting hybrid 33 had a subnanomolar α7-nAChR affinity and was a potent and selective α7-nAChR antagonist, producing at the α7-, but not at the α9*-nAChR, a profound loss of subsequent ACh function.

Engineering of α-conotoxin MII-derived peptides with increased selectivity for native α6ß2* nicotinic acetylcholine receptors.

α6ß2* Nicotinic acetylcholine receptors are expressed in selected central nervous system areas, where they are involved in striatal dopamine (DA) release and its behavioral consequences, and other still uncharacterized brain activities. α6ß2* receptors are selectively blocked by the α-conotoxins MII and PIA, which bear a characteristic N-terminal amino acid tail [arginine (R), aspartic acid (D), and proline (P)]. We synthesized a group of PIA-related peptides in which R1 was mutated or the RDP motif gradually removed. Binding and striatal DA release assays of native rat α6ß2* receptors showed that the RDP sequence, and particularly residue R1, is essential for the activity of PIA. On the basis of molecular modeling analyses, we synthesized a hybrid peptide (RDP-MII) that had increased potency (7-fold) and affinity (13-fold) for α6ß2* receptors but not for the very similar α3ß2* subtype. As docking studies also suggested that E11 of MII might be a key residue engendering α6ß2* vs. α3ß2* selectivity, we prepared MII[E11R] and RDP-MII[E11R] peptides. Their affinity and potency for native α6ß2* receptors were similar to those of their parent analogues, whereas, for the oocyte expressed rat α3ß2* subtype, they showed a 31- and 14-fold lower affinity and 21- and 3.5-fold lower potency. Thus, MII[E11R] and RDP-MII[E11R] are potent antagonists showing a degree of α6ß2* vs. α3ß2* selectivity in vivo.

Synthesis and binding affinity at α4ß2 and α7 nicotinic acetylcholine receptors of new analogs of epibatidine and epiboxidine containing the 7-azabicyclo[2.2.1]hept-2-ene ring system.

A group of novel racemic nicotinic ligands structurally related to epibatidine or epiboxidine [(±)-10-(±)-17] was synthesized through a palladium-catalyzed cross-coupling between the appropriate vinyl triflate and a range of organometallic heterocycles. The target compounds were evaluated for binding affinity at the α4ß2 and α7 neuronal nicotinic receptors (nAChRs). The set of 3-pyridinyl derivatives (±)-10, (±)-11 and (±)-12 exhibited an affinity for the α4ß2 nAChR subtype in the subnanomolar range (K(i) values of 0.20, 0.40 and 0.50nM, respectively) and behaved as α4ß2 versus α7 subtype selective ligands. Interestingly, the epiboxidine-related dimethylammonium iodide (±)-17, which retained a good affinity for the α4ß2 nAChR (K(i)=13.30nM), tightly bound also to the α7 subtype (K(i)=1.60nM), thus displaying a reversal of the affinity trend among the reference and new nicotinic ligands under investigation.

The enantiomers of epiboxidine and of two related analogs: synthesis and estimation of their binding affinity at α4ß2 and α7 neuronal nicotinic acetylcholine receptors.

Epiboxidine hydrochlorides (+)-2 and (-)-2, which are the structural analogs of the antipodes of epibatidine (±)-1, as well as the enantiomeric pairs (+)-3/(-)-3 and (+)-4/(-)-4 were synthesized and tested for binding affinity at α4ß2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes.

Nicotinic acetylcholine receptors in the mesolimbic pathway: primary role of ventral tegmental area alpha6beta2* receptors in mediating systemic nicotine effects on dopamine release, locomotion, and reinforcement.

alpha6* nicotinic acetylcholine receptors (nAChRs) are highly and selectively expressed by mesostriatal dopamine (DA) neurons. These neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Yet the functional role of alpha6* nAChRs in midbrain DA neurons is mostly unknown. The aim of this study was to determine the composition and in vivo functional role of alpha6* nAChR in mesolimbic DA neurons of male rats. Immunoprecipitation and immunopurification techniques coupled with cell-specific lesions showed that the composition of alpha6* nAChR in the mesostriatal system is heterogeneous, with (non-alpha4)alpha6beta2* being predominant in the mesolimbic pathway and alpha4alpha6beta2* in the nigrostriatal pathway. We verified whether alpha6* receptors mediate the systemic effects of nicotine on the mesolimbic DA pathway by perfusing the selective antagonists alpha-conotoxin MII (CntxMII) (alpha3/alpha6beta2* selective) or alpha-conotoxin PIA (CntxPIA) (alpha6beta2* selective) into ventral tegmental area (VTA). The intra-VTA perfusion of CntxMII or CntxPIA markedly decreased systemic nicotine-elicited DA release in the nucleus accumbens and habituated locomotion; the intra-VTA perfusion of CntxMII also decreased the rate of nicotine infusion in the maintenance phase of nicotine, but not of food, self-administration. Overall, the results of these experiments show that the alpha6beta2* nAChRs expressed in the VTA are necessary for the effects of systemic nicotine on DA neuron activity and DA-dependent behaviors such as locomotion and reinforcement, and suggest that alpha6beta2*-selective compounds capable of crossing the blood-brain barrier may affect the addictive properties of nicotine and therefore be useful in the treatment of tobacco dependence.

α9-Containing Nicotinic Receptors in Cancer.

Neuronal nicotinic acetylcholine receptors containing the α9 or the α9 and α10 subunits are expressed in various extra-neuronal tissues. Moreover, most cancer cells and tissues highly express α9-containing receptors, and a number of studies have shown that they are powerful regulators of responses that stimulate cancer processes such as proliferation, inhibition of apoptosis, and metastasis. It has also emerged that their modulation is a promising target for drug development. The aim of this review is to summarize recent data showing the involvement of these receptors in controlling the downstream signaling cascades involved in the promotion of cancer.

Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the alpha3beta4* and alpha3beta3beta4* subtypes mediate acetylcholine release.

Recent studies suggest that the neuronal nicotinic receptors (nAChRs) present in the habenulo-interpeduncular (Hb-IPn) system can modulate the reinforcing effect of addictive drugs and the anxiolytic effect of nicotine. Hb and IPn neurons express mRNAs for most nAChR subunits, thus making it difficult to establish the subunit composition of functional receptors. We used immunoprecipitation and immunopurification studies performed in rat and wild-type (+/+) and beta2 knock-out (-/-) mice to establish that the Hb and IPn contain significant beta2* and beta4* populations of nAChR receptors (each of which is heterogeneous). The beta4* nAChR are more highly expressed in the IPn. We also identified novel native subtypes (alpha2beta2*, alpha4beta3beta2*, alpha3beta3beta4*, alpha6beta3beta4*). Our studies on IPn synaptosomes obtained from +/+ and alpha2, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4(-/-) mice show that only the alpha3beta4 and alpha3beta3beta4 subtypes facilitate acetylcholine (ACh) release. Ligand binding, immunoprecipitation, and Western blotting studies in beta3(-/-) mice showed that, in the IPn of these mice, there is a concomitant reduction of ACh release and alpha3beta4* receptors, whereas the receptor number remains the same in the Hb. We suggest that, in habenular cholinergic neurons, the beta3 subunit may be important for transporting the alpha3beta4* subtype from the medial habenula to the IPn. Overall, these studies highlight the presence of a wealth of uncommon nAChR subtypes in the Hb-IPn system and identify alpha3beta4 and alpha3beta3beta4, transported from the Hb and highly enriched in the IPn, as the subtypes modulating ACh release in the IPn.

A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes.

Long-term nicotine exposure changes neuronal acetylcholine nicotinic receptor (nAChR) subtype expression in the brains of smokers and experimental animals. The aim of this study was to investigate nicotine-induced changes in nAChR expression in two models commonly used to describe the effects of nicotine in animals: operant (two-lever presses) intravenous self-administration (SA) and passive subcutaneous nicotine administration via an osmotic minipump (MP). In the MP group, alpha4beta2 nAChRs were up-regulated in all brain regions, alpha6beta2* nAChRs were down-regulated in the nucleus accumbens (NAc) and caudate-putamen, and alpha7 nAChRs were up-regulated in the caudal cerebral cortex (CCx); the up-regulation of alpha4beta2alpha5 nAChRs in the CCx was also suggested. In the SA group, alpha4beta2 up-regulation was lower and limited to the CCx and NAc; there were no detectable changes in alpha6beta2* or alpha7 nACRs. In the CCx of the MP rats, there was a close correlation between the increase in alpha4beta2 binding and alpha4 and beta2 subunit levels measured by means of Western blotting, demonstrating that the up-regulation was due to an increase in alpha4beta2 proteins. Western blotting also showed that the increase in the beta2 subunit exceeded that of the alpha4 subunit, suggesting that a change in alpha4beta2 stoichiometry may occur in vivo as has been shown in vitro. These results show that nicotine has an area-specific effect on receptor subtypes, regardless of its administration route, but the effect is quantitatively greater in the case of MP administration.

Novel tricyclic Delta(2)-isoxazoline and 3-oxo-2-methyl-isoxazolidine derivatives: synthesis and binding affinity at neuronal nicotinic acetylcholine receptor subtypes.

A group of novel tricyclic Delta(2)-isoxazolines (4b, 5b, 7a-b, and 8a-b) and 3-oxo-isoxazolidines (6a-b and 9a-b), structurally related to cytisine or norferruginine, was prepared through 1,3-dipolar cycloadditions involving suitable olefins and bromonitrile oxide. The target compounds were assayed at alpha4beta2 and alpha7 neuronal acetylcholine receptors (nAChRs). The results of competition binding experiments indicated for the new derivatives a reduction of the affinity at the alpha4beta2 subtype in comparison with the reference molecules, coupled with an overall negligible affinity at the alpha7 subtype. The binding mode of the bromo-Delta(2)-isoxazolines 4b and 7b, which were the highest affinity ligands in the series (K(i)=0.92 and 0.75 microM, respectively), was analyzed by applying a recently developed model of the alpha4beta2 nAChRs.

5-(2-Pyrrolidinyl)oxazolidinones and 2-(2-pyrrolidinyl)benzodioxanes: synthesis of all the stereoisomers and alpha4beta2 nicotinic affinity.

The four stereoisomers of 2-oxazolidinone 5-substituted with 1-methyl-2-pyrrolidinyl (1), of 1,4-benzodioxane 2-substituted with the same residue (2) and of the nor-methyl analogue of this latter (2a) were synthesized as candidate nicotinoids. Of the 12 compounds, two N-methylated pyrrolidinyl-benzodioxane stereoisomers, namely those with the same relative configuration at the pyrrolidine stereocentre as (S)-nicotine, bind at alpha4beta2 nicotinic acetylcholine receptor with submicromolar affinity. Consistently with the biological data, docking analysis enlightens significant differences in binding site interactions not only between 1 and 2, but also between 2 and 2a and between the stereoisomers of 2 accounting for the critical role played, in the case of the pyrrolidinyl-benzodioxanes, by the chirality of both the stereolabile and stereostable stereogenic atoms, namely the protonated tertiary nitrogen and the two asymmetric carbons.

New analogues of epiboxidine incorporating the 4,5-dihydroisoxazole nucleus: synthesis, binding affinity at neuronal nicotinic acetylcholine receptors, and molecular modeling investigations.

A group of novel 4,5-dihydro-3-methylisoxazolyl derivatives, structurally related to epiboxidine (=(1R,4S,6S)-6-(3-methylisoxazol-5-yl)-7-azabicyclo[2.2.1]heptane), was prepared via 1,3-dipolar cycloaddition of acetonitrile oxide to different olefins. Target compounds 1a and 1b, 2a and 2b, 3, 4, and 5 were tested for affinity at neuronal nicotinic heteromeric (alpha4beta2) and homomeric (alpha7) acetylcholine receptors. Notably, diastereoisomers 1a and 1b were characterized by a massive drop of the affinity at the alpha4beta2 subtypes (K(i) values spanning the range 4.3-126 microM), when compared with that of epiboxidine (K(i)=0.6 nM). Therefore, the replacement of the 3-methylisoxazole ring of epiboxidine with the 4,5-dihydro-3-methylisoxazole nucleus is detrimental for the affinity at alpha4beta2 receptors. A comparable lack of affinity/selectivity for the two nAChR subtypes under study was evidenced for the remaining epiboxidine-related dihydroisoxazole derivatives 2a and 2b, and 3-5. Diastereoisomers 1a and 1b, and spirocyclic derivative 3 were docked into molecular models of the receptor subtypes under study, and their binding mode was compared with that of reference ligands endowed with high binding affinity.