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1.  AT-1001: A High Affinity and Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist Blocks Nicotine Self-Administration in Rats 
Neuropsychopharmacology  2012;37(6):1367-1376.
Genomic and pharmacologic data have suggested the involvement of the α3β4 subtype of nicotinic acetylcholine receptors (nAChRs) in drug seeking to nicotine and other drugs of abuse. In order to better examine this receptor subtype, we have identified and characterized the first high affinity and selective α3β4 nAChR antagonist, AT-1001, both in vitro and in vivo. This is the first reported compound with a Ki below 10 nM at α3β4 nAChR and >90-fold selectivity over the other major subtypes, the α4β2 and α7 nAChR. AT-1001 competes with epibatidine, allowing for [3H]epibatidine binding to be used for structure-activity studies, however, both receptor binding and ligand-induced Ca2+ flux are not strictly competitive because increasing ligand concentration produces an apparent decrease in receptor number and maximal Ca2+ fluorescence. AT-1001 also potently and reversibly blocks epibatidine-induced inward currents in HEK cells transfected with α3β4 nAChR. Importantly, AT-1001 potently and dose-dependently blocks nicotine self-administration in rats, without affecting food responding. When tested in a nucleus accumbens (NAcs) synaptosomal preparation, AT-1001 inhibits nicotine-induced [3H]dopamine release poorly and at significantly higher concentrations compared with mecamylamine and conotoxin MII. These results suggest that its inhibition of nicotine self-administration in rats is not directly due to a decrease in dopamine release from the NAc, and most likely involves an indirect pathway requiring α3β4 nAChR. In conclusion, our studies provide further evidence for the involvement of α3β4 nAChR in nicotine self-administration. These findings suggest the utility of this receptor as a target for smoking cessation medications, and highlight the potential of AT-1001 and congeners as clinically useful compounds.
PMCID: PMC3327842  PMID: 22278092
nicotine; self-administration; α3β4 nicotinic acetylcholine receptor antagonist; AT-1001; addiction and substance abuse; neuropharmacology; animal models; acetylcholine; nicotine; self-administration; nicotinic receptor; AT-1001
2.  Re-Evaluation of Nicotinic Acetylcholine Receptors in Rat Brain by a Tissue-Segment Binding Assay 
Nicotinic acetylcholine receptors (nAChRs) of the cerebral cortex and cerebellum of rats were evaluated by a radioligand binding assay, employing tissue segments, or homogenates as materials. [3H]-epibatidine specifically bound to nAChRs in rat cortex or cerebellum, but the dissociation constants for [3H]-epibatidine differed between segments and homogenates (187 pM for segments and 42 pM for homogenates in the cortex and 160 pM for segments and 84 pM for homogenates in the cerebellum). The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum). Most of the [3H]-epibatidine binding sites in the cortex segments (approximately 70% of the population) showed high affinity for nicotine (pKi = 7.9), dihydro-β-erythroidine, and cytisine, but the binding sites in the cerebellum segments had slightly lower affinity for nicotine (pKi = 7.1). An upregulation of nAChRs by chronic administration of nicotine was observed in the cortex segments but not in the cerebellum segments with [3H]-epibatidine as a ligand. The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4β2). The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.
PMCID: PMC3198036  PMID: 22025914
nicotinic receptor; tissue-segment binding; upregulation
3.  John Daly’s Compound, Epibatidine, Facilitates Identification of Nicotinic Receptor Subtypes 
The diversity of nicotinic acetylcholine receptor (nAChR) subtypes was explored by measuring the effects of gene deletion and pharmacological diversity of epibatidine binding sites in mouse brain. All epibatidine binding sites require expression of either the α7, β2, or β4 subunit. In agreement with general belief, the α4β2*-nAChR and α7-nAChR subtypes are major components of the epibatidine binding sites. α4β2*-nAChR sites account for approximately 70% of total high- and low-affinity epibatidine binding sites, while α7-nAChR accounts for 16% of the total sites all of which have lower affinity for epibatidine. The other subtypes are structurally diverse. Although these minor subtypes account for only 14% of total binding in whole brain, they are expressed at relatively high concentrations in specific brain areas indicating unique functional roles.
PMCID: PMC3042357  PMID: 19672723
Neuronal nicotinic acetylcholine receptors; Epibatidine; Null mutant mice; α-Bungarotoxin; Cytisine; α-Conotoxin MII
4.  Epibatidine Binds to Four Sites on the Torpedo Nicotinic Acetylcholine Receptor 
The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabelled ligand binding techniques, we show that the binding of [3H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 µM. These classes of sites exist in approximately equal numbers and all [3H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.
PMCID: PMC2536623  PMID: 18083118
acetylcholine; nicotinic; epibatidine; α-bungarotoxin; suberyldicholine
5.  An Autoradiographic Survey of Mouse Brain Nicotinic Acetylcholine Receptors Defined by Null Mutants 
Biochemical pharmacology  2011;82(8):828-841.
Nine nicotinic receptor subunits are expressed in the central nervous system indicating that a variety of nicotinic acetylcholine receptors (nAChR) may be assembled. A useful method with which to identify putative nAChR is radioligand binding. In the current study the binding of [125I]α-bungarotoxin, [125I]α-conotoxinMII, 5[125I]-3-((2S)-azetidinylmethoxy)pyridine (A-85380), and [125I]epibatidine has been measured autoradiographically to provide data on many nAChR binding sites. Each binding sites was evaluated semiquantitatively for samples prepared from wild-type and α2, α4, α6, α7, β2, β4, α5 and β3 null mutant mice. Deletion of the α7 subunit completely and selectively eliminated [125I]α-bungarotoxin binding. The binding of [125I]αConotoxinMII was eliminated in most brain regions by deletion of either the α6 or β2 subunit and is reduced by deletion of either the α4 or β3 subunit. The binding of 5[125I]A-85380 was completely eliminated by deletion of the β2 subunit and significantly reduced by deletion of the α4 subunit. Most, but not all, α4-independent sites require expression of the α6 subunit. The effect of gene deletion on total [125I]epibatidine binding was very similar to that on [125I]A-85380 binding. [125I]Epibatidine also labels β4* nAChR, which was readily apparent for incubations conducted in the presence of 100 nM cytisine. The effects of α3 gene deletion could not be evaluated, but persistence of residual sites implies the expression of α3* nAChR. Taken together these results confirm and extend previously published evaluations of the effect of nAChR gene deletion and help to define the nAChR subtypes measurable by ligand binding.
PMCID: PMC3162045  PMID: 21575611
nicotinic acetylcholine receptor; null mutant mice; epibatidine; A-85380; α-conotoxin MII; α-bungarotoxin
6.  Pharmacological and Immunological Identification of Native α7 Nicotinic Receptors: Evidence for Homomeric and Heteromeric α7 Receptors 
Life sciences  2007;81(16):1317-1322.
Controversy surrounds the expression of α7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, α7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [125I]α-bungarotoxin (αBGT) binding reaches equilibrium within 4 hours and is saturable with a Kd value of 4.2 nM. Using homologous competition experiments, the Ki for binding of αBGT was 1.9 nM. These data are consistent with the expression of homomeric α7 nAChRs. Methyllycaconatine (MLA), which binds α7 nAChRs with high affinity, inhibits [125I]αBGT binding in a concentration-dependent manner with a Ki of 30.6 nM; this value is ∼10 fold higher than the reported affinity of MLA for α7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate α7 nAChRs, as has been previous demonstrated for bovine adrenal α3β4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb 319, an antibody which recognizes α7 nAChR protein, and then probed with mAb 319 using Western blot analysis, a single band of ∼53 kD is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes α3 and α5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of ∼53 kD is identified. Together, these results support the expression of α7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric α7 nAChRs.
PMCID: PMC2083560  PMID: 17928008
Nicotinic acetylcholine receptor; binding; adrenal medulla; methyllycaconitine; α-bungarotoxin; α7
7.  Quantitative analysis of the heteromeric neuronal nicotinic receptors in the rat hippocampus 
Journal of neurochemistry  2010;115(3):625-634.
The objective of this study was to identify and quantify the heteromeric neuronal nicotinic receptors (nAChRs) in the rat hippocampus. The density of nAChR subtypes was assessed by labeling them with [3H]epibatidine followed by immunoprecipitation with subunit-selective antibodies. Sequential immunoprecipitation assays were used to establish associations between two different subunits, which then allowed the full subunit composition of the receptors to be deduced. Our results show that most of the hippocampal heteromeric nAChRs contain α4 and β2 subunits. In fact, we identified two populations containing these two predominant subunits, the α4β2 and α4β2α5 subtypes which account for ~40% and ~35%, respectively, of the total [3H]epibatidine-labeled receptors. An additional heteromeric subtype with the subunit composition of α4β2α3 represented ~10% of the total nAChRs, and another 10% of the immunoprecipitated receptors contained α4 and β4 subunits, with or without the α3 subunit. To determine if α4β2 and α4β2α5 nAChR subtypes differ in their ligand binding affinities, the α3- and β4-containing receptors were first removed by immunoprecipitation and then, competition studies with acetylcholine, nicotine, cytisine and sazetidine-A against [3H]epibatidine were carried out on the remaining α4β2 and α4β2α5 subtypes. Results suggested these subtypes have comparable binding affinities for the nicotinic ligands used here.
PMCID: PMC2976603  PMID: 20796176
Neuronal nicotinic receptor; heteromeric subtypes; α5 subunit; hippocampus; immunoprecipitation; binding affinity
8.  Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines 
Analytical chemistry  2008;80(22):8673-8680.
Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [3H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric α7 nicotinic acetylcholine receptors (α7 nAChR) and heteromeric nicotinic acetylcholine receptors (αxβy nAChRs), which was confirmed by the addition of subtype-specific inhibitors, κ-bungarotoxin (α7 nAChR) and K-bungarotoxin (αxβy nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), γ-aminobutyric acid (GABAA) and N-methyl-d-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABAA receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.
PMCID: PMC2583165  PMID: 18847217
9.  Synthesis, Nicotinic Acetylcholine Receptor Binding, and Antinociceptive Properties of 3′-(Substituted Phenyl)epibatidine Analogues. Nicotinic Partial Agonists⊥ 
Journal of natural products  2010;73(3):306-312.
In 1992, John Daly et al. reported the isolation and structure determination of epibatidine. Epibatidine’s unique structure and its potent nicotinic agonist activity have had a tremendous impact on nicotine receptor research. This research has led to a better understanding of the nicotinic acetylcholine receptor (nAChR) pharmacophore and to epibatidine analogues with potential as pharmacotherapies for treating various CNS disorders. In this study, we report the synthesis, receptor binding ([3H]epibatidine and [125I]iodoMLA), and in vivo pharmacological properties (mouse tail flick, hot plate, hypothermia, and spontaneous activity) of a series of 3′-(substituted phenyl)epibatidine analogues (5a–m). Results from these studies have added to the understanding of the nAChR pharmacophore and led to nicotinic partial agonists that may have potential for smoking cessation. All the analogues had affinities for the α4β2 nAChR similar to epibatidine (1). 3′-(3-Dimethylaminophenyl)epibatidine (5m) has a nicotinic partial agonist pharmacological profile similar to the smoking cessation drug varenicline. Other analogues are partial agonists with varying degrees of nicotinic functional agonist and antagonist activity. 3′-(3-Aminophenyl)epibatidine (5j) is a more potent functional agonist and antagonist in all tests than varenicline. 3′-(3-Fluorophenyl)epibatidine and 3′-(3-chlorophenyl)epibatidine (5c and 5e) are more potent than varenicline when tested as agonists in four pharmacological tests and antagonists when evaluated against nicotine in the analgesia hot-plate test.
PMCID: PMC2846203  PMID: 20038125
10.  Quantification of Smoking-Induced Occupancy of β2-Nicotinic Acetylcholine Receptors:Estimation of Nondisplaceable Binding 
5-123I-iodo-85380 (123I-5-IA) is used to quantitate high-affinity nicotinic acetylcholine receptors (β2-nAChRs) on human SPECT scans. The primary outcome measure is VT/fP, the ratio at equilibrium between total tissue concentration (free, nonspecifically bound, and specifically bound) and the free plasma concentration. Nondisplaceable uptake (free plus nonspecific) of 123I-5-IA has not been measured in human subjects. Nicotine has high affinity for β2*-nAChRs (nAChRs containing the β2* subunit, for which * represents other subunits that may also be part of the receptor) and displaces specifically bound 123I-5-IA. In this study, we measured nicotine occupancy and nondisplaceable binding in healthy smokers after they had smoked to satiety.
Eleven nicotine-dependent smokers (mean age ± SD, 35.6 ± 14.4 y) completed the study. One subject was excluded from subsequent analyses because of abnormal blood nicotine levels. Subjects abstained from tobacco smoke for 5.3 ± 0.9 d and participated in a 15- to 17-h SPECT scanning day. 123I-5-IA was administered by bolus plus constant infusion, with a total injected dose of 361 ± 20 MBq. At approximately 6 h after the start of the infusion, three 30-min SPECT scans and a 15-min transmission–emission scan were acquired to obtain baseline β2*-nAChR availability. Subjects then smoked to satiety (2.4 ± 0.7 cigarettes), and arterial (first 40 min) and venous (until study completion) plasma nicotine and cotinine levels were collected. About 1 h after subjects had smoked to satiety, up to six 30-min SPECT scans were acquired. VT/fP data, computed from the tissue and plasma radioactivity measurements from the presmoking baseline and postsmoking scans, were analyzed using the Lassen plot method.
Receptor occupancy after subjects had smoked to satiety was 67% ± 9% (range, 55%–80%). Nondisplaceable uptake was estimated as 19.4 ± 5.8 mL·cm−3 (range, 15–28 mL·cm−3). Thus, in the thalamus, where mean VT/fP is 93 mL·cm−3, nondisplaceable binding represents approximately 20% of the total binding.
These results are in agreement with previous findings and suggest that when satiating doses of nicotine are administered to smokers, imaging of receptor availability can yield valuable data, such as quantifiable measures of nondisplaceable binding.
PMCID: PMC3707518  PMID: 20660383
SPECT; 5-123I-iodo-85380; smoking to satiety; human smokers
11.  Free-energy Landscapes of Ion-channel Gating Are Malleable: changes in the number of bound ligands are accompanied by changes in the location of the transition state in acetylcholine-receptor channels† 
Biochemistry  2003;42(50):14977-14987.
Acetylcholine-receptor channels (AChRs) are allosteric membrane proteins that mediate synaptic transmission by alternatively opening and closing (‘gating’) a cation-selective transmembrane pore. Although ligand binding is not required for the channel to open, the binding of agonists (for example, acetylcholine) increases the closed ⇌ open equilibrium constant because the ion-impermeable → ion-permeable transition of the ion pathway is accompanied by a low → high affinity change at the agonist-binding sites. The fact that the gating conformational change of muscle AChRs can be kinetically modeled as a two-state reaction has paved the way to the experimental characterization of the corresponding transition state, which represents a snapshot of the continuous sequence of molecular events separating the closed and open states. Previous studies of fully (di-) liganded AChRs, combining single-channel kinetic measurements, site-directed mutagenesis, and data analysis in the framework of the linear free-energy relationships of physical organic chemistry, have suggested a transition-state structure that is consistent with channel opening being an asynchronous conformational change that starts at the extracellular agonist-binding sites and propagates towards the intracellular end of the pore. In this paper, I characterize the gating transition state of unliganded AChRs, and report a remarkable difference: unlike that of diliganded gating, the unliganded transition state is not a hybrid of the closed- and open-state structures but, rather, is almost indistinguishable from the open state itself. This displacement of the transition state along the reaction coordinate obscures the mechanism underlying the unliganded closed ⇌ open reaction but brings to light the malleable nature of free-energy landscapes of ion-channel gating.
The muscle acetylcholine receptor channel (AChR)1 is the neurotransmitter-gated ion channel that mediates neuromuscular synaptic transmission in vertebrates (1). Although the structure of this large pentameric transmembrane protein (∼470 residues per subunit) is not known with atomic resolution, a wealth of structural information exists, mainly from mutational studies, affinity labeling, chemical modification of specific residues, electron microscopy, and crystallography (reviewed in ref. 2). As is the case of any other allosteric protein, the dynamic behavior of this receptor-channel can be understood in the framework of thermodynamic cycles, with conformational changes and ligand-binding events as the elementary steps (3-5). Thus, the AChR can adopt a variety of different conformations that can interconvert (closed, open, and desensitized ‘states’), and each conformation has a distinct ligand-binding affinity (low affinity in the closed state and high affinity in the open and desensitized states) and a particular ‘catalytic efficiency’ (ion-impermeable in the closed and desensitized states, and ion-permeable in the open state). To meet the physiological requirement of a small closed ⇌ open (‘gating’) equilibrium constant for the unliganded receptor, and a large gating equilibrium constant for the ACh-diliganded receptor, the affinity of the AChR for ACh must be higher in the open than in the closed conformation (4-6). This follows from the notion that the equilibrium constants governing the different reaction steps (ligand binding and gating) of these cyclic reaction schemes are constrained by the principle of detailed balance.
Hence, irrespective of whether the receptor is diliganded, monoliganded or unliganded, two changes must take place in going from the closed state (low ligand affinity and ion-impermeable) to the open state (high ligand affinity and ion-permeable): a) the pore becomes permeable to ions, and b) the transmitter-binding sites, some 50 Å away from the pore domain (7), increase their affinity for the ligand (with the reverse changes taking place during closing). The apparent lack of stable intermediates between the closed and open conformations, inferred from kinetic modeling of the diliganded-gating reaction (8), suggests that these two changes occur as a result of a one-step, global conformational change. The question, then, arises as to whether this concerted conformational change proceeds synchronously (i.e., every residue of the protein moves ‘in unison’) or asynchronously (i.e., following a sequence of events; ref. 9) and, if the latter were the case, whether multiple, few, or just one sequence of events is actually traversed by the channel to ‘connect’ the end states.
Analysis of the correlation between rate and equilibrium constants of gating in diliganded AChRs has allowed us to address some of these issues by probing the structure of the transition state (8, 10-12), that is, the intermediate species between the end states of a one-step reaction that can be most easily studied. Interpretation of these results in the framework of the classical rate-equilibrium free-energy relationships of physical organic chemistry (13, 14), revealed that AChR diliganded gating is a highly asynchronous reaction, and suggested that the transition-state ensemble is quite homogeneous, as if the crossing of the energy barrier were confined to a narrow pass at the top of the energy landscape. In the opening direction, the conformational rearrangement that leads to the low-to-high affinity change at the extracellular binding sites precedes the conformational rearrangement of the pore that renders the channel ion-permeable. This propagated global conformational change, which we have referred to as a ‘conformational wave’ (11), must reverse during channel closing so that closing starts at the pore and propagates all the way to the binding sites.
It is not at all obvious why the diliganded-gating conformational change starts at the binding sites when the channel opens, nor even why the conformational change propagates at all through the receptor, instead of taking place synchronously throughout the protein. Is there any correlation between the location of the domain that binds agonist and the location of the initiation site for the opening conformational change? Could the latter have started from the intracellular end of the pore, for example, and have propagated to the (extracellular) transmitter-binding sites? What difference does it make to be liganded or unliganded as far as the mechanism of the gating conformational change is concerned? To address these issues, I set out to explore the mechanism of gating in unliganded AChRs by probing the structure of the corresponding transition state using kinetic measurements, site-directed mutagenesis, and the concepts of rate-equilibrium free-energy relationships and Φ-value analysis.
Briefly, a Φ-value can be assigned to any position in the protein by estimating the slope of a ‘Brönsted plot’2 [log (gating rate constant) versus log (gating equilibrium constant)] where each point corresponds to a different amino-acid substitution at that given position. More coarsegrained Φ-values can also be obtained by using different agonists or different transmembrane potentials, for example, as a means of altering the rate and equilibrium constants of gating. Very often, rate-equilibrium plots are linear, and 0 < Φ < 1. A value of Φ = 0 suggests that the position in question (in the case of a mutation series) experiences a closed-state-like environment at the transition state whereas a value of Φ = 1 suggests an open-state-like environment. A fractional Φ-value suggests an environment that is intermediate between those experienced in the closed and open states (16).
Earlier results indicated that the Φ-values obtained by varying the transmembrane potential are different in diliganded and unliganded AChRs. These Φ-values, which are a measure of the closed-state-like versus open-state-like character of the channel’s voltage-sensing elements at the transition state, are 0.070 ± 0.060 in diliganded receptors (17), and 1.025 ± 0.053 in unliganded AChRs (11, 18). The present study reveals that residues at the transmitter-binding sites (Figure 1), the extracellular loop that links the second (M2) and third (M3) transmembrane segments (M2-M3 linker), and the upper and lower half of M2, which during diliganded gating have Φ-values of ∼1 (ref. 11), ∼0.7 (ref. 10), ∼0.35 (refs 8, 11, 12), and ∼0 (ref. 12), respectively, have also Φ-values very close to 1 during unliganded gating. This generalized shift in Φ-values suggests that the diliganded → unliganded perturbation deforms the energy landscape of gating in such a way that the ‘new’ transition state occurs very close to the open state, to such an extent that all tested positions experience an open-state-like environment at the transition state of unliganded gating. Thus, the transition state occurs so ‘late’ (i.e., so close to the open state) that its inferred structure does not provide any clues as to the intermediate stages of this reaction.
Hence, the mechanism of unliganded gating remains obscure. The change in the position of the transition state along a reaction coordinate, as a result of perturbations to the energy landscape, is a very well known phenomenon in organic chemistry (e.g., refs 20-26), and protein folding (e.g., refs 27-34). In this paper, I show that this phenomenon can also take place in the case of allosteric transitions and, therefore, that the structure of the transition state of a global conformational change need not be fixed; rather, it can change depending on the experimental conditions.
PMCID: PMC1463891  PMID: 14674774
12.  Modeling Multiple Species of Nicotine and Deschloroepibatidine Interacting with α4β2 Nicotinic Acetylcholine Receptor: From Microscopic Binding to Phenomenological Binding Affinity 
Journal of the American Chemical Society  2005;127(41):14401-14414.
A variety of molecular modeling, molecular docking, and first-principles electronic structure calculations were performed to study how the α4β2 nicotinic acetylcholine receptor (nAChR) binds with different species of two typical agonists, S-(−)-nicotine and R-(−)-deschloroepibatidine, each of which are distinguished by different free bases and protonation states. Based on these results, predictions were made regarding the corresponding microscopic binding free energies. Hydrogen bonding and cation-π interactions between the receptor and the respective ligands were found to be the dominant factors differentiating the binding strengths of different microscopic binding species. The calculated results and analyses demonstrate that for each agonist, all the species are interchangeable and can quickly achieve a thermodynamic equilibration in solution and at the nAChR binding site. This allows quantitation of the equilibrium concentration distributions of the free ligand species and the corresponding microscopic ligand-receptor binding species, their pH-dependence, and their contributions to the phenomenological binding affinity. The predicted equilibrium concentration distributions, pKa values, absolute phenomenological binding affinities and their pH-dependence are all in good agreement with available experimental data, suggesting that the computational strategy from the microscopic binding species and affinities to the phenomenological binding affinity is reliable for studying α4β2 nAChR-ligand binding. This should provide valuable information for future rational design of drugs targeting nAChRs. The general strategy of the “from-microscopic-to-phenomenological” approach for studying interactions of α4β2 nAChR with S-(−)-nicotine and R-(−)-deschloroepibatidine may also be useful in studying other types of ligand-protein interactions involving multiple molecular species of a ligand and in associated rational drug design.
PMCID: PMC3182463  PMID: 16218635
13.  3’-Fluoro substitution in the pyridine ring of epibatidine improves selectivity and efficacy for α4β2 versus α3β4 nAChRs 
Neuropharmacology  2008;55(8):1287-1292.
The analog of epibatidine having a fluoro- substituent at the 3’ position of the pyridine ring has been recently developed and shown to possess binding affinity in the pM range to α4β2 nAChRs and in the nM range to α7 nAChRs and to exhibit potent agonist activity in nicotine-induced analgesia tests. Here we used patch-clamp technique in a whole-cell configuration to compare functional activity of 3’-fluoroepibatidine to that of epibatidine by itself on recombinant α4β2, α7 and α3β4 neuronal nAChRs. The agonist effect of (±)-epibatidine was partial and yielded comparable EC50s of 0.012 µM (72% efficacy) and 0.027 µM (81% efficacy) at α4β2 and α3β4 nAChRs, respectively, but was full at α7 nAChRs with an EC50 of 4.8 µM. Testing of the analog at different concentrations revealed that it acts as a full agonist with an EC50 of 0.36 µM at α4β2 nAChRs and induces partial agonist effect (66% efficacy) at α7 nAChRs with an EC50 of 9.8 µM and an IC50 corresponding to 225 µM. In contrast, the analog caused only 24% maximal activation at the range of concentrations from 0.1–100 µM and, in addition, induced an inhibition of α3β4 nAChR function with an IC50 of 8.3 µM. Our functional data, which are in agreement with previous binding and behavioral findings, demonstrate that 3’-fluoro substitution in the pyridine ring of epibatidine results in an improved pharmacological profile as observed by an increased efficacy and selectivity for α4β2 versus α3β4 nAChRs.
PMCID: PMC2669717  PMID: 18775444
recombinant neuronal nicotinic receptors; epibatidine; epibatidine analogs; whole-cell; nicotine-induced analgesia; binding
14.  Effects of chronic neonatal nicotine exposure on nAChR binding, cell death and morphology in hippocampus and cerebellum 
Neuroscience  2007;146(4):1854-1868.
Nicotine, the major psychoactive ingredient in tobacco interacting with nicotinic acetylcholine receptors (nAChR), is believed to have neuroprotective and neurotoxic effects on the developing brain. Neurotoxicity has been attributed to activation of homomeric α7 nAChRs, neuroprotection to heteromeric α4β2 nAChRs. Thus, developmental nicotine could have opposite effects in different brain regions, depending on nAChR subtype expression. Here, we determined if chronic neonatal nicotine exposure (CNN), during a period of brain growth corresponding to the third human trimester, differentially regulates nAChR expression, cell death, and morphological properties in hippocampus and cerebellum, two structures maturing postnatally. Rat pups were orally treated with 6 mg/kg/day nicotine from postnatal day (P)1 to P7. On P8, expression for α4, α7 and β2 mRNA was determined by in situ hybridization; nAChR binding sites in by receptor autoradiography, dying neurons by TUNEL and Fluoro-Jade staining and morphological properties by analysis of Cresyl-Violet stained sections. In control cerebellum, strong expression of α4, β2 mRNA and heteromeric nAChRs labeled with [125I]-Epibatidine were found in granule cells, and α7 mRNA and homomeric nAChRs labeled with [125I]-αBungarotoxin in the external germinal layer. In control hippocampus, low expression of α4 mRNA and heteromeric nAChRs and high expression of α7 mRNA and homomeric nAChRs were detected. CNN increased heteromeric nAChR binding in hippocampus but not cerebellum and significantly decreased neuronal soma size and increased packing density in hippocampal principal cells but not in cerebellum. CNN did not increase the number of dying cells in any area, but significantly fewer TUNEL-labeled cells were found in CA3 strata oriens and radiatum and cerebellar granule layer. Thus, the hippocampus seems to be more sensitive than the cerebellum to CNN which could result from different nAChR subtype expression and might explain long-lasting altered cognitive functions correlated with gestational nicotine exposure due to changes in hippocampal cell morphology.
PMCID: PMC2001269  PMID: 17434679
apoptosis; development; nicotinic; receptor; neuronal density
15.  Decreased α4β2 nicotinic receptor number in the absence of mRNA changes suggest post-transcriptional regulation in the spontaneously hypertensive rat model of ADHD 
Journal of neurochemistry  2011;119(1):240-250.
The spontaneously hypertensive rat (SHR) is widely used as a model of attention-deficit/hyperactivity disorder (ADHD). Deficits in central nicotinic receptors (nAChRs) have previously been observed in SHRs, which is interesting since epidemiological studies have identified an association between smoking and ADHD symptoms in humans. Here we examine whether nAChR deficits in SHRs compared to Wistar Kyoto rat (WKY) controls are nAChR subtype-specific and whether these deficits correlate with changes at the level of mRNA transcription in specific brain regions. Levels of binding sites (Bmax) and dissociation constants (Kd) for nAChRs were determined from saturation curves of high-affinity [3H]epibatidine- and [3H]MLA binding to membranes from cortex, striatum, hippocampus and cerebellum. In additional brain regions, nAChRs were examined by autoradiography with [125I]A-85380 and [125I]α-bungarotoxin. Levels of mRNA encoding nAChR subunits were measured using quantitative real-time PCR (qPCR). We show that the number of α4β2 nAChR binding sites is lower globally in the SHR brain compared to WKY in the absence of significant differences in mRNA levels, with the exception of lower α4 mRNA in cerebellum of SHR compared to WKY. Further, nAChR deficits were subtype- specific because no strain difference was found in α7 nAChR binding or α7 mRNA levels. Our results suggest that the lower α4β2 nAChR number in SHR compared to WKY may be a consequence of dysfunctional post-transcriptional regulation of nAChRs.
PMCID: PMC3171636  PMID: 21824140
Attention-deficit/hyperactivity disorder; mRNA; nicotinic receptor; post translational; spontaneous hypertensive rat; Wistar Kyoto rat
16.  A novel α-conotoxin MII sensitive nicotinic acetylcholine receptor modulates [3H]-GABA release in the superficial layers of the mouse superior colliculus 
Journal of neurochemistry  2012;122(1):48-57.
Mouse superficial superior colliculus (SuSC) contains dense GABAergic innervation and diverse nicotinic acetylcholine receptor subtypes. Pharmacological and genetic approaches were used to investigate the subunit compositions of nicotinic acetylcholine receptors (nAChR) expressed on mouse SuSC GABAergic terminals. [125I]-Epibatidine competition binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxinMII blocked binding to 40 +/- 5% of SuSC nAChRs. Acetylcholine-evoked [3H]-GABA release from SuSC crude synaptosomal preparations is calcium dependent, blocked by the voltage-sensitive calcium channel blocker, cadmium, and the nAChR antagonist mecamylamine, but is unaffected by muscarinic, glutamatergic, P2X and 5-HT3 receptor antagonists. Approximately 50% of nAChR-mediated SuSC [3H]-GABA release is inhibited by α-conotoxinMII. However, the highly-α6β2*-subtype-selective α-conotoxinPIA did not affect [3H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [3H]-GABA release is entirely β2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxinMII-resistant release. ACh-stimulated SuSC [3H]-GABA release was unaffected by β3, α5 or α6 nicotinic subunit deletions. Together, these data suggest that a significant proportion of mouse SuSC nicotinic agonist-evoked GABA-release is mediated by a novel, α-conotoxinMII-sensitive α3α4β2 nAChR. The remaining α-conotoxinMII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4β2* subtype nAChRs.
PMCID: PMC4026281  PMID: 22506481
Nicotinic acetylcholine receptors; GABA; synaptosome; subunit-null mutant; α-conotoxinMII; superior colliculus
17.  86Rb+ Efflux Mediated by α4β2*-Nicotinic Acetylcholine Receptors with High and Low Sensitivity to Stimulation by Acetylcholine Display Similar Agonist-Induced Desensitization 
Biochemical pharmacology  2010;80(8):1238-1251.
The nicotinic acetylcholine receptors (nAChR) assembled from α4 and β2 subunits are the most densely expressed subtype in the brain. Concentration-effect curves for agonist activation of α4β2*-nAChR are biphasic. This biphasic agonist sensitivity is ascribed to differences in subunit stoichiometry. The studies described here evaluated desensitization elicited by low concentrations of epibatidine, nicotine, cytisine or methylcarbachol of brain α4β2-nAChR function measured with acetylcholine stimulated 86Rb+ efflux from mouse thalamic synaptosomes. Each agonist elicited concentration-dependent desensitization. The agonists differed in potency. However, IC50 values for each agonist for desensitization of 86Rb+ efflux both with high (EC50≈3 μM) and low (EC50≈ 150 μM) acetylcholine sensitivity were not significantly different. Concentrations required to elicit desensitization were higher that their respective KD values for receptor binding. Even though the two components of α4β2*-nAChR mediated 86Rb+ efflux from mouse brain differ markedly in EC50 values for agonist activation, they are equally sensitive to desensitization by exposure to low agonist concentrations. Mice were also chronically treated with nicotine by continuous infusion of 0, 0.5 or 4.0 mg/kg/hr and desensitization induced by nicotine was evaluated. Consistent with previous results, chronic nicotine treatment increased the density of epibatidine binding sites. Acute exposure to nicotine also elicited concentration-dependent desensitization of both high sensitivity and low sensitivity acetylcholine-stimulated 86Rb+ efflux from cortical and thalamic synaptosomes. Although chronic nicotine treatment reduced maximal 86Rb+ efflux from thalamus, IC50 values in both brain regions were unaffected by chronic nicotine treatment.
PMCID: PMC2935307  PMID: 20599770
nicotinic acetylcholine receptor; desensitization; nicotine; epibatidine; cytisine; methylcarbachol
18.  Synthesis and Nicotinic Acetylcholine Receptor In Vitro and In Vivo Pharmacological Properties of 2'-Fluoro-3'-(substituted phenyl)deschloroepibatidine Analogues of 2'-Fluoro-3'-(4-nitrophenyl)deschloroepibatidine (4-Nitro-PFEB or RTI-7527-102) 
Journal of medicinal chemistry  2012;55(14):6512-6522.
Herein, we report the synthesis and nicotinic acetylcholine receptor (nAChR) in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines 5b–g, analogues of 3'-(4-nitrophenyl) compound 5a. All compounds had high affinity for the α4β2-nAChR and low affinity for α7-nAChR. Initial electrophysiological studies showed that all analogues were antagonists at α4β2-, α3β4-, and α7-nAChRs. The 4-carbamoylphenyl analogue 5g was highly selective for α4β2-nAChR over α3β4- and α7-nAChRs. All the analogues were antagonists of nicotine-induced antinociception in the tail-flick test. Molecular modeling docking studies using agonist-bound form of the X-ray crystal structure of the acetylcholine binding protein suggested several different binding modes for epibatidine, varenicline, and 5a–5g. In particular, a unique binding mode for 5g was suggested by these docking simulations. The high binding affinity, in vitro efficacy, and selectivity of 5g for α4β2-nAChR combined with its nAChR functional antagonist properties suggest that 5g will be a valuable pharmacological tool for studying the nAChR and may have potential as a pharmacotherapy for addiction and other CNS disorders.
PMCID: PMC3431023  PMID: 22742586
Nicotinic receptors; epibatidine; nicotinic antagonist; α4β2-nAChR selectivity; electrophysiological studies; tail-flick; hot-plate; molecular modeling
19.  Gene Targeting Demonstrates That α4 Nicotinic Acetylcholine Receptor Subunits Contribute to Expression of Diverse [3H]Epibatidine Binding Sites and Components of Biphasic 86Rb+ Efflux With High and Low Sensitivity to Stimulation by Acetylcholine 
Neuropharmacology  2007;53(3):390-405.
[3H]Epibatidine binds to nAChR subtypes in mouse brain with higher (KD≈0.02 nM) and lower affinity (KD≈7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of α7, β2 or β4 subunits. Most, but not all, higher and lower affinity binding sites require β2 (Marks et al., 2006). Effects of functional α4 gene deletion are reported here. Deletion of α4 virtually eliminated cytisine-sensitive, higher-affinity [3H]epibatidine binding as did β2 deletion, confirming that these sites are α4β2*-nAChR. Cytisine-resistant, higher-affinity [3H]epibatidine binding sites are diverse and some of these sites require α4 expression. Lower affinity [3H]epibatidine binding sites are also heterogeneous and can be subdivided into α-bungarotoxin-sensitive and - resistant components. Deleting α4 did not affect the α-bungarotoxin-sensitive component, but markedly reduced the α-bungarotoxin–resistant component. This effect was similar, but not quite identical, to the effect of β2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of α4 subunits. The effects of α4 gene targeting on receptor function were measured using a 86Rb+ efflux assay. Concentration-effect curves for ACh-stimulated 86Rb+ efflux are biphasic (EC50 values = 3.3 µM and 300 µM). Targeting α4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of β2. Thus, α4β2*–nAChRs mediate a significant fraction of both phases of ACh stimulated 86Rb+ efflux.
PMCID: PMC2577786  PMID: 17631923
Null mutant mice; Nicotinic acetylcholine receptor; Epibatidine; 86Rb+ efflux; Cytisine; Dihydro-β-erythroidine
20.  Galanthamine and non-competitive inhibitor binding to ACh-binding protein: evidence for a binding site on non α-subunit interfaces of heteromeric neuronal nicotinic receptors 
Journal of molecular biology  2007;369(4):895-901.
Rapid neurotransmission is mediated through a superfamily of Cys-loop receptors, that includes the nicotinic acetylcholine (nAChR), γ-aminobutyric-acid (GABAA/C), serotonin (5-HT3) and glycine receptors. A class of ligands, including galanthamine, local anesthetics and certain toxins, interact with nAChRs non-competitively. Suggested modes of action include blockade of the ion-channel, modulation from as yet undefined extracellular sites, stabilization of desensitized states, and association with annular or boundary lipid. Alignment of mammalian Cys-loop receptors show aromatic residues, found in the acetylcholine or ligand binding pocket of nAChRs, are conserved in all subunit interfaces of neuronal nAChRs, including subunit interfaces that are not formed by α subunits on the principal side of the transmitter binding site. The amino terminal domain containing the ligand recognition site is homologous to the soluble acetylcholine binding protein (AChBP) from mollusks, an established structural and functional surrogate. Herein we assess ligand specificity and employ X-ray crystallography with AChBP to demonstrate ligand interactions at subunit interfaces lacking vicinal cysteines (i.e., the non-α subunit interfaces in nAChRs). Non-competitive nicotinic ligands bind AChBP with high affinity (KD’s of 0.015 to 6 μM). We mutated the vicinal cysteines in loop C of AChBP to mimic the non-alpha subunit interfaces of neuronal nAChRs and other Cys loop receptors. Classical nicotinic agonists show a 10 to 40-fold reduction in binding affinity, whereas binding of ligands known to be non-competitive are not affected. X-ray structures of cocaine and galanthamine bound to AChBP (1.8 and 2.9 Å resolution respectively) reveal interactions deep within the subunit interface and the absence of a contact surface with the tip of loop C. Hence, in addition to channel blocking, non-competitive interactions with heteromeric neuronal nAChR appear to occur at the non-alpha subunit interface, a site presumed to be similar to that of modulating benzodiazepines on GABAA receptors.
PMCID: PMC2031909  PMID: 17481657
non-competitive inhibitors; nicotinic acetylcholine receptors; acetylcholine binding protein; benzodiazepine; galanthamine; cocaine
21.  Photoaffinity Labeling the Agonist Binding Domain of α4β4 and α4β2 Neuronal Nicotinic Acetylcholine Receptors with [125I]Epibatidine and 5[125I]A-85380 
Biochimica et biophysica acta  2009;1788(9):1987-1995.
The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, hold promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [125I]epibatidine and 5[125I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [125I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [125I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [125I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.
PMCID: PMC2744590  PMID: 19545536
Cys-loop receptor; neuronal nicotinic receptor; HEK-293 cell; affinity-purification; photoaffinity labeling; protein sequencing
22.  Genetic Background Influences the Effects of Withdrawal from Chronic Nicotine on Learning and High-Affinity Nicotinic Acetylcholine Receptor Binding in the Dorsal and Ventral Hippocampus 
Psychopharmacology  2012;225(1):201-208.
The effects of nicotine on cognitive processes may play an important role in nicotine addiction. Nicotine withdrawal impairs hippocampus-dependent learning and genetic factors influence this effect. However, the neural changes that contribute to these impairments are unknown. Chronic nicotine upregulates hippocampal nicotinic acetycholine receptors (nAChRs), which may contribute to cognitive deficits when nicotine administration ceases. If nAChR upregulation underlies withdrawal-deficits in learning, then strains of mice exhibiting withdrawal-deficits in hippocampus-dependent learning should also show upregulation of hippocampal nAChRs.
Here, we examined the effects of nicotine withdrawal on fear conditioning and [3H]epibatidine binding in the dorsal and ventral hippocampus in two inbred mouse strains and their F1 hybrids.
Male C57BL/6NTac, 129S6/SvEvTac, and B6129SF1/Tac mice were administered chronic nicotine (18 mg/kg/d) for 12 days through osmotic pumps and then were trained and tested in fear conditioning 24 hours after cessation of nicotine treatment.
Nicotine withdrawal impaired hippocampus-dependent contextual conditioning in C57BL/6NTac mice but not 129S6/SvEvTac or B6129SF1/Tac mice; no changes were observed in hippocampus-independent cued fear conditioning. Upregulated [3H]epibatidine binding was found in the dorsal, but not ventral, hippocampus of C57BL/6NTac mice and in the ventral hippocampus of B6129SF1/Tac mice after chronic nicotine.
Upregulation of high-affinity binding sites in the dorsal hippocampus of C57BL/6NTac mice, the only strain that exhibited nAChR upregulation in this region and withdrawal-deficits in contextual conditioning, suggests that upregulation of high-affinity binding sites in the dorsal hippocampus mediates, in part, nicotine withdrawal-deficits in contextual conditioning and genetic background modulates these effects.
PMCID: PMC3755015  PMID: 22836371
Nicotine; addiction; genetics; learning; withdrawal; acetylcholine
23.  Expression of nicotinic acetylcholine receptors and subunit messenger RNAs in the enteric nervous system of the neonatal rat 
Neuroscience  2008;158(4):1521-1529.
In the enteric nervous system (ENS) excitatory nicotinic cholinergic transmission is mediated by neuronal nicotinic acetylcholine receptors (nAChR) and is critical for the regulation of gastric motility. nAChRs are ligand-gated pentameric ion channels found in the central and peripheral nervous systems. The expression of heteromeric nAChR and receptor subunit mRNAs was investigated in the neonatal rat ENS using receptor autoradiography with the radiolabeled ligand 125I-Epibatidine, and in situ hybridization with subtype specific probes for ligand binding alpha (α2, α3, α4, α5, α6) and structural beta (β2, β3, β4) subunits. The results showed strong nicotine sensitive binding of 125I-Epibatidine around the stomach, and small and large intestines. The binding was partially displaced by A85380, a nicotinic ligand which differentiates between different heteromeric nAChR subtypes, suggesting a mixed receptor population. Radioactive in situ hybridization detected expression of α3, α5, α7, β2 and β4 mRNA in the myenteric plexus of the stomach, and small and large intestines. In the submucosal plexus of the small and large intestines expression of α3, α5 and β4 was found in some ganglia. There was no signal for α4, α6 and β3 in the ENS but positive hybridization signal for α2 transcripts was seen in some areas of the small intestines. However, the signal was not associated with any ganglion cells. The results confirm the presence of heteromeric nAChRs in the ENS similar to those found in the peripheral nervous system, with the majority being composed of α3(α5)β4, and a few α3β2 nAChRs. In addition, homomeric α7 nAChRs could be present.
PMCID: PMC2669225  PMID: 19095047
gastrointestinal; nicotine; myenteric plexus; submucosal; in situ hybridization; epibatidine
24.  Positive allosteric modulators as an approach to nicotinic acetylcholine receptor- targeted therapeutics: advantages and limitations 
Biochemical pharmacology  2011;82(8):915-930.
Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.
PMCID: PMC3162128  PMID: 21575610
Alzheimer’s disease; schizophrenia; drug development; electrophysiology; modeling
25.  Synthesis and evaluation of new radioligands [11C]A-833834 and [11C]A-752274 for positron-emission tomography of α7-nicotinic acetylcholine receptors 
Nuclear medicine and biology  2013;40(3):395-402.
α7-nicotinic acetylcholine receptor (α7-nAChR) is one of the major neuronal nAChR subtypes. α7-nAChR is involved in variety of neuronal processes and disorders including schizophrenia and Alzheimer’s disease. A number of α7-nAChR PET radioligands have been developed, but a quality radiotracer remains to be discovered.
High binding affinity α7-nAChR ligands A-833834 and A-752274 were radiolabeled with 11C. Baseline and blockade biodistribution studies in the mouse brain of [11C]A-833834 (5-(6-(5-[11C]methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-1H-indole) and [11C]A-752274 (2-(6-[11C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)-7-(6-methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)-9H-fluoren-9-one) were performed. [11C]A-752274 was evaluated in a baseline baboon PET study.
[11C]A-833834 and [11C]A-752274 were synthesized by radiomethylation of corresponding des-methyl precursors. The radioligands were prepared with radiochemical yield of 12–32%, high specific radioactivity (330–403 GBq/μmol) and radiochemical purity > 95%. Dissection studies with [11C]A-833834 demonstrated low specific α7-nAChR binding in the mouse brain. [11C]A-752274 specifically (~50%) labeled α7-nAChR in the mouse thalamus. However, [11C]A-752274 exhibited low brain uptake in baboon (%SUV<100).
Two novel α7-nAChR ligands radioligands were synthesized and studied in animals. Specific binding of [11C]A-833834 in the mouse brain is low due to the insufficient binding affinity of the radioligand. The very high binding affinity [11C]A-752274 exhibited good specific binding in the α7-nAChR – rich mouse brain regions. The low uptake of [11C]A-752274 in the baboon brain is due to its high hydrophilicity, rapid metabolism or other properties. Future development of α7-nAChR PET radioligands will be based on compounds with high binding affinities and good blood-brain barrier permeability.
PMCID: PMC3596482  PMID: 23294899
positron emission tomography; PET; radioligand; nicotinic acetylcholine receptor; α7-nAChR

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