Advances in small molecule selective ligands for heteromeric nicotinic acetylcholine receptors.

The study of nicotinic acetylcholine receptors (nAChRs) has significantly progressed in the last decade, due to a) the improved techniques available for structural studies; b) the identification of ligands interacting at orthosteric and allosteric recognition sites on the nAChR proteins, able to tune channel conformational states; c) the better functional characterization of receptor subtypes/subunits and their therapeutic potential; d) the availability of novel pharmacological agents able to activate or block nicotinic-mediated cholinergic responses with subtype or stoichiometry selectivity. The copious literature on nAChRs is related to the pharmacological profile of new, promising subtype selective derivatives as well as the encouraging preclinical and early clinical evaluation of known ligands. However, recently approved therapeutic derivatives are still missing, and examples of ligands discontinued in advanced CNS clinical trials include drug candidates acting at both neuronal homomeric and heteromeric receptors. In this review, we have selected heteromeric nAChRs as the target and comment on literature reports of the past five years dealing with the discovery of new small molecule ligands or the advanced pharmacological/preclinical investigation of more promising compounds. The results obtained with bifunctional nicotinic ligands and a light-activated ligand as well as the applications of promising radiopharmaceuticals for heteromeric subtypes are also discussed.

Ligand design for human acetylcholinesterase and nicotinic acetylcholine receptors, extending beyond the conventional and canonical.

We detail here distinctive departures from lead classical cholinesterase re-activators, the pyridinium aldoximes, to achieve rapid CNS penetration and reactivation of AChE in the CNS (brain and spinal cord). Such reactivation is consistent with these non-canonical re-activators enhancing survival parameters in both mice and macaques following exposure to organophosphates. Thus, the ideal cholinesterase re-activator should show minimal toxicity, limited inhibitory activity in the absence of an organophosphate, and rapid CNS penetration, in addition to its nucleophilic potential at the target, the conjugated AChE active center. These are structural properties directed to reactivity profiles at the conjugated AChE active center, reinforced by the pharmacokinetic and tissue disposition properties of the re-activator leads. In the case of nicotinic acetylcholine receptor (nAChR) agonists and antagonists, with the many existing receptor subtypes in mammals, we prioritize subtype selectivity in their design. In contrast to nicotine and its analogues that react with panoply of AChR subtypes, the substituted di-2-picolyl amine pyrimidines possess distinctive ionization characteristics reflecting in selectivity for the orthosteric site at the α7 subtypes of receptor. Here, entry to the CNS should be prioritized for the therapeutic objectives of the nicotinic agent influencing aberrant CNS activity in development or in the sequence of CNS ageing (longevity) in mammals, along with general peripheral activities controlling inflammation.

α-Conotoxin as Potential to α7-nAChR Recombinant Expressed in Escherichia coli.

α7 nicotinic acetylcholine receptors (nAChR) is an important nicotinic acetylcholine receptors subtype and closely associated with cognitive disorders, such as Alzheimer's and schizophrenia disease. The mutant ArIB (V11L, V16A) of α-conotoxin ArIB with 17-amino acid residues specifically targets α7 nAChR with no obvious effect on other nAChR subtypes. In the study, the synthetic gene encoding mature peptide of ArIB and mutant ArIB (V11L, V16A) carried a fusion protein Trx and 6 × His-tag was separately inserted in pET-32a (+) vector and transformed into Escherichia coli strain BL21(DE3) pLysS for expression. The expressions of Trx-ArIB-His6 and Trx-ArIB (V11L, V16A)-His6 were soluble in Escherichia coli, which were purified by Ni-NTA affinity chromatography column and cleaved by enterokinase to release rArIB and rArIB (V11L, V16A). Then, rArIB and rArIB (V11L, V16A) were purified by high-performance liquid chromatography (HPLC) and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bioactivity of rArIB and rArIB (V11L, V16A) was assessed by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing human nAChR subtypes. The results indicated that the yield of the fusion proteins was approximately 50 mg/L and rArIB (V11L, V16A) antagonized the α7 nAChR subtype selectively with 8-nM IC50. In summary, this study provides an efficient method to biosynthesize α-conotoxin ArIB and rArIB (V11L, V16A) in Escherichia coli, which could be economical to obtain massively bioactive disulfide-rich polypeptides at fast speed.

Oligoarginine Peptides, a New Family of Nicotinic Acetylcholine Receptor Inhibitors.

Many peptide ligands of nicotinic acetylcholine receptors (nAChRs) contain a large number of positively charged amino acid residues, a striking example being conotoxins RgIA and GeXIVA from marine mollusk venom, with an arginine content of >30%. To determine whether peptides built exclusively from arginine residues will interact with different nAChR subtypes or with their structural homologs such as the acetylcholine-binding protein and ligand-binding domain of the nAChR α9 subunit, we synthesized a series of R3, R6, R8, and R16 oligoarginines and investigated their activity by competition with radioiodinated α-bungarotoxin, two-electrode voltage-clamp electrophysiology, and calcium imaging. R6 and longer peptides inhibited muscle-type nAChRs, α7 nAChRs, and α3ß2 nAChRs in the micromolar range. The most efficient inhibition of ion currents was detected for muscle nAChR by R16 (IC50 = 157 nM) and for the α9α10 subtype by R8 and R16 (IC50 = 44 and 120 nM, respectively). Since the R8 affinity for other tested nAChRs was 100-fold lower, R8 appears to be a selective antagonist of α9α10 nAChR. For R8, the electrophysiological and competition experiments indicated the existence of two distinct binding sites on α9α10 nAChR. Since modified oligoarginines and other cationic molecules are widely used as cell-penetrating peptides, we studied several cationic polymers and demonstrated their nAChR inhibitory activity. SIGNIFICANT STATEMENT: By using radioligand analysis, electrophysiology, and calcium imaging, we found that oligoarginine peptides are a new group of inhibitors for muscle nicotinic acetylcholine receptors (nAChRs) and some neuronal nAChRs, the most active being those with 16 and 8 Arg residues. Such compounds and other cationic polymers are cell-penetrating tools for drug delivery, and we also demonstrated the inhibition of nAChRs for several of the latter. Possible positive and negative consequences of such an action should be taken into account.

Molecular determinants of α-conotoxin potency for inhibition of human and rat α6ß4 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) containing α6 and ß4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain. Rodent models are often used to evaluate the efficacy of analgesic compounds, but species differences may affect the activity of some nAChR ligands. A previous candidate α-conotoxin-based therapeutic yielded promising results in rodent models, but failed in human clinical trials, emphasizing the importance of understanding species differences in ligand activity. Here, we show that human and rat α6/α3ß4 nAChRs expressed in Xenopus laevis oocytes exhibit differential sensitivity to α-conotoxins. Sequence homology comparisons of human and rat α6ß4 nAChR subunits indicated that α6 residues forming the ligand-binding pocket are highly conserved between the two species, but several residues of ß4 differed, including a Leu-Gln difference at position 119. X-ray crystallography of α-conotoxin PeIA complexed with the Aplysia californica acetylcholine-binding protein (AChBP) revealed that binding of PeIA orients Pro13 in close proximity to residue 119 of the AChBP complementary subunit. Site-directed mutagenesis studies revealed that Leu119 of human ß4 contributes to higher sensitivity of human α6/α3ß4 nAChRs to α-conotoxins, and structure-activity studies indicated that PeIA Pro13 is critical for high potency. Human and rat α6/α3ß4 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro13 and residue 119 of the ß4 subunit. These results highlight the potential significance of species differences in α6ß4 nAChR pharmacology that should be taken into consideration when evaluating the activity of candidate human therapeutics in rodent models.

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors.

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT3A receptors (5-HT3ARs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT3AR can be exchanged between the homomeric 5-HT3AR and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT3AR affect the affinity of nicotine, a competitive antagonist of 5-HT3ARs, in a cooperative manner. Using mutant cycle analyses between the 5-HT3A triad, loop A residues Asn-101 and Glu-102, ß9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

Di- and heptavalent nicotinic analogues to interfere with α7 nicotinic acetylcholine receptors.

In the field of nicotinic acetylcholine receptors (nAChRs), recognized as important therapeutic targets, much effort has been dedicated to the development of nicotinic analogues to agonize or antagonize distinct homo- and heteropentamers nAChR subtypes, selectively. In this work we developed di- and heptavalent nicotinic derivatives based on ethylene glycol (EG) and cyclodextrin cores, respectively. The compounds showed a concentration dependent inhibition of acetylcholine-induced currents on α7 nAChR expressed by Xenopus oocytes. Interesting features were observed with the divalent nicotinic derivatives, acting as antagonists with varied inhibitory concentrations (IC50) in function of the spacer arm length. The best divalent compounds showed a 16-fold lowered IC50 compared to the monovalent reference (12 vs 195 µM). Docking investigations provide guidelines to rationalize these experimental findings.

Identification of a Novel O-Conotoxin Reveals an Unusual and Potent Inhibitor of the Human α9α10 Nicotinic Acetylcholine Receptor.

Conotoxins are a pool of disulfide-rich peptide neurotoxins produced by cone snails for predation and defense. They are a rich reservoir of novel ligands for ion channels, neurotransmitter receptors and transporters in the nervous system. In this study, we identified a novel conotoxin component, O-conotoxin GeXXVIIA, from the venom of Conus generalis. The native form of this component is a disulfide-linked homodimer of a 5-Cys-containing peptide. Surprisingly, our electrophysiological studies showed that, in comparison to the folded monomers, the linear peptide of this toxin had the highest inhibitory activity at the human α9α10 nicotinic acetylcholine receptor (nAChR), with an IC50 of 16.2 ± 1.4 nM. The activities of the N-terminal and C-terminal halves of the linear toxin are markedly reduced compared with the full-length toxin, suggesting that the intact sequence is required to potently inhibit the hα9α10 nAChR. α9α10 nAChRs are expressed not only in the nervous system, but also in a variety of non-neuronal cells, such as cochlear hair cells, keratinocytes, epithelial and immune cells. A potent inhibitor of human α9α10 nAChRs, such as GeXXVIIA, would facilitate unraveling the functions of this nAChR subtype. Furthermore, this unusual nAChR inhibitor may lead to the development of novel α9α10 nAChR-targeting drugs.

Genetic Algorithm Managed Peptide Mutant Screening: Optimizing Peptide Ligands for Targeted Receptor Binding.

This study demonstrates the utility of genetic algorithms to search exceptionally large and otherwise intractable mutant libraries for sequences with optimal binding affinities for target receptors. The Genetic Algorithm Managed Peptide Mutant Screening (GAMPMS) program was used to search an α-conotoxin (α-CTx) MII mutant library of approximately 41 billion possible peptide sequences for those exhibiting the greatest binding affinity for the α3ß2-nicotinic acetylcholine receptor (nAChR) isoform. A series of top resulting peptide ligands with high sequence homology was obtained, with each mutant having an estimated ΔGbind approximately double that of the potent native α-CTx MII ligand. A consensus sequence from the top GAMPMS results was subjected to more rigorous binding free energy calculations by molecular dynamics and compared to α-CTx MII and other related variants for binding with α3ß2-nAChR. In this study, the efficiency of GAMPMS to substantially reduce the sample population size through evolutionary selection criteria to produce ligands with higher predicted binding affinity is demonstrated.

Molecular Basis for Differential Sensitivity of α-Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors.

α-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The α3ß2 and α3ß4 nAChR subtypes are expressed in the central and peripheral nervous systems and play a critical role in various pathophysiological conditions ranging from nicotine addiction to the development and progression of lung cancer. Here we used the α4/7-conotoxin RegIIA, a disulfide-bonded peptide from the venom of Conus regius, and its analog [N11A,N12A]RegIIA to probe the specific pharmacological properties of rat and human nAChR subtypes. nAChR subtypes were heterologously expressed in Xenopus oocytes and two-electrode voltage clamp recordings used to investigate the effects of the peptides on nAChR activity. RegIIA potently inhibited currents evoked by acetylcholine (ACh) at rat α3ß2 (IC50 = 10.7 nM), whereas a 70-fold lower potency was observed at human α3ß2 nAChR (IC50 = 704.1 nM). Conversely, there were no species-specific differences in sensitivity to RegIIA at the α3ß4 nAChR. Receptor mutagenesis and molecular dynamics studies revealed that this difference can be attributed primarily to a single amino acid change: Glu198 on the rat α3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species- and subunit-specific receptor-antagonist interaction.

A novel α4/7-conotoxin LvIA from Conus lividus that selectively blocks α3ß2 vs. α6/α3ß2ß3 nicotinic acetylcholine receptors.

This study was performed to discover and characterize the first potent α3ß2-subtype-selective nicotinic acetylcholine receptor (nAChR) ligand. A novel α4/7-conotoxin, α-CTxLvIA, was cloned from Conus lividus. Its pharmacological profile at Xenopus laevis oocyte-expressed rat nAChR subtypes was determined by 2-electrode voltage-clamp electrophysiology, and its 3-dimensional (3D) structure was determined by NMR spectroscopy. α-CTx LvIA is a 16-aa C-terminally-amidated peptide with 2-disulfide bridges. Using rat subunits expressed in Xenopus oocytes, we found the highest affinity of α-CTxLvIA was for α3ß2 nAChRs (IC50 8.7 nM), where blockade was reversible within 2 min. IC50 values were >100 nM at α6/α3ß2ß3, α6/α3ß4, and α3ß4 nAChRs, and ≥3 µM at all other subtypes tested. α3ß2 vs. α6ß2 subtype selectivity was confirmed for human-subunit nAChRs with much greater preference (300-fold) for α3ß2 over α6ß2 nAChRs. This is the first α-CTx reported to show high selectivity for human α3ß2 vs. α6ß2 nAChRs. α-CTxLvIA adopts two similarly populated conformations water: one (assumed to be bioactive) is highly structured, whereas the other is mostly random coil in nature. Selectivity differences with the similarly potent, but less selective, α3ß2 nAChR antagonist α-CTx PeIA probably reside within the three residues, which differ in loop 2, given their otherwise similar 3D structures. α4/7-CTx LvIA is a new, potent, selective α3ß2 nAChR antagonist, which will enable detailed studies of α3ß2 nAChR structure, function, and physiological roles.

Isolation and structural and pharmacological characterization of α-elapitoxin-Dpp2d, an amidated three finger toxin from black mamba venom.

We isolated a novel, atypical long-chain three-finger toxin (TFT), α-elapitoxin-Dpp2d (α-EPTX-Dpp2d), from black mamba (Dendroaspis polylepis polylepis) venom. Proteolytic digestion with trypsin and V8 protease, together with MS/MS de novo sequencing, indicated that the mature toxin has an amidated C-terminal arginine, a posttranslational modification rarely observed for snake TFTs. α-EPTX-Dpp2d was found to potently inhibit α7 neuronal nicotinic acetylcholine receptors (nAChR; IC50, 58 ± 24 nM) and muscle-type nAChR (IC50, 114 ± 37 nM) but did not affect α3ß2 and α3ß4 nAChR isoforms at 1 µM concentrations. Competitive radioligand binding assays demonstrated that α-EPTX-Dpp2d competes with epibatidine binding to the Lymnea stagnalis acetylcholine-binding protein (Ls-AChBP; IC50, 4.9 ± 2.3 nM). The activity profile and binding data are reminiscent of classical long-chain TFTs with a free carboxyl termini, suggesting that amidation does not significantly affect toxin selectivity. The crystal structure of α-EPTX-Dpp2d was determined at 1.7 Å resolution and displayed a dimeric toxin assembly with each monomer positioned in an antiparallel orientation. The dimeric structure is stabilized by extensive intermolecular hydrogen bonds and electrostatic interactions, which raised the possibility that the toxin may exist as a noncovalent homodimer in solution. However, chemical cross-linking and size-exclusion chromatography coupled with multiangle laser light scattering (MALLS) data indicated that the toxin is predominantly monomeric under physiological conditions. Because of its high potency and selectivity, we expect this toxin to be a valuable pharmacological tool for studying the structure and function of nAChRs.

Acetylcholine promotes binding of α-conotoxin MII at α3 ß2 nicotinic acetylcholine receptors.

α-Conotoxin MII (α-CTxMII) is a 16-residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2-Cys8 and Cys3-Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand-binding affinity, homology models of the heteropentameric α3ß2-nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3- and ß2-subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3ß2-nAChR for α-CTxMII with ACh bound to the receptor, and this was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs.

Differential pharmacological activity of JN403 between α7 and muscle nicotinic acetylcholine receptors.

The differential action of the novel agonist JN403 at neuronal α7 and muscle nicotinic receptors (AChRs) was explored by using a combination of functional and structural approaches. Single-channel recordings reveal that JN403 is a potent agonist of α7 but a very low-efficacy agonist of muscle AChRs. JN403 elicits detectable openings of α7 and muscle AChRs at concentrations ~1000-fold lower and ~20-fold higher, respectively, than that for ACh. Single-channel activity elicited by JN403 is very similar to that elicited by ACh in α7 but profoundly different in muscle AChRs, where openings are brief and infrequent and do not appear in clusters at any concentration. JN403 elicits single-channel activity of muscle AChRs lacking the ε subunit, with opening events being more frequent and prolonged than those of wild-type AChRs. This finding is in line with the molecular docking studies predicting that JN403 may form a hydrogen bond required for potent activation at the α-δ but not at the α-ε binding site. JN403 does not elicit detectable Ca²âº influx in muscle AChRs but inhibits (±)-epibatidine-elicited influx mainly by a noncompetitive mechanism. Such inhibition is compatible with single-channel recordings revealing that JN403 produces open-channel blockade and early termination of ACh-elicited clusters, and it is therefore also a potent desensitizing enhancer of muscle AChRs. The latter mechanism is supported by the JN403-induced increase in the level of binding of [³H]cytisine and [³H]TCP to resting AChRs. Elucidation of the differences in activity of JN403 between neuronal α7 and muscle AChRs provides further insights into mechanisms underlying selectivity for α7 AChRs.

Atomic force microscopy to characterize binding properties of α7-containing nicotinic acetylcholine receptors on neurokinin-1 receptor-expressing medullary respiratory neurons.

In the present study, we used atomic force microscopy (AFM) to examine the ligand-binding properties of α7-containing nicotinic acetylcholine receptors (nAChRs) expressed on neurons from the ventral respiratory group. We also determined the effect of acute and prolonged exposure to nicotine on the binding probability of nAChRs. Neurons from neonatal (postnatal day 5-10) and juvenile rats (3-4 weeks old) were cultured. Internalization of Alexa Fluor 488-conjugated substance P was used to identify respiratory neurons that expressed the neurokinin-1 receptor (NK1-R), a recognized marker of ventral respiratory group neurons. To assess functional changes in nAChRs, AFM probes conjugated with anti-α7 subunit nAChR antibody were used to interact cyclically with the surface of the soma of NK1-R-positive neurons. Measurements were made of the frequency of antibody adhesion to the α7 receptor subunit and of the detachment forces between the membrane-attached receptor and the AFM probe tip. Addition of α-bungarotoxin (a specific antagonist of α7 subunit-containing nAChRs) to the cell bath produced a 69% reduction in binding to the α7 subunit (P < 0.05, n = 10), supporting specificity of binding. Acute exposure to nicotine (1 µM added to culture media) produced an 80% reduction in nAChR antibody binding to the α7 subunit (P < 0.05, n = 9). Prolonged incubation (72 h) of the cell culture in nicotine significantly reduced α7 binding in a concentration-dependent manner. Collectively, these findings demonstrate that AFM is a sensitive tool for assessment of functional changes in nAChRs expressed on the surface of living NK1-R-expressing medullary neurons. Moreover, these data demonstrate that nicotine exposure decreases the binding probability of α7 subunit-containing nAChRs.

Serotonergic and cholinergic elements of the hypoxic ventilatory response in developing zebrafish.

The chemosensory roles of gill neuroepithelial cells (NECs) in mediating the hyperventilatory response to hypoxia are not clearly defined in fish. While serotonin (5-HT) is the predominant neurotransmitter in O(2)-sensitive gill NECs, acetylcholine (ACh) plays a more prominent role in O(2) sensing in terrestrial vertebrates. The present study characterized the developmental chronology of potential serotonergic and cholinergic chemosensory pathways of the gill in the model vertebrate, the zebrafish (Danio rerio). In immunolabelled whole gills from larvae, serotonergic NECs were observed in epithelia of the gill filaments and gill arches, while non-serotonergic NECs were found primarily in the gill arches. Acclimation of developing zebrafish to hypoxia (P(O2)=75 mmHg) reduced the number of serotonergic NECs observed at 7 days post-fertilization (d.p.f.), and this effect was absent at 10 d.p.f. In vivo administration of 5-HT mimicked hypoxia by increasing ventilation frequency (f(V)) in early stage (7-10 d.p.f.) and late stage larvae (14-21 d.p.f.), while ACh increased f(V) only in late stage larvae. In time course experiments, application of ketanserin inhibited the hyperventilatory response to acute hypoxia (P(O2)=25 mmHg) at 10 d.p.f., while hexamethonium did not have this effect until 12 d.p.f. Cells immunoreactive for the vesicular acetylcholine transporter (VAChT) began to appear in the gill filaments by 14 d.p.f. Characterization in adult gills revealed that VAChT-positive cells were a separate population of neurosecretory cells of the gill filaments. These studies suggest that serotonergic and cholinergic pathways in the zebrafish gill develop at different times and contribute to the hyperventilatory response to hypoxia.

Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism.

Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.

Bupropion binds to two sites in the Torpedo nicotinic acetylcholine receptor transmembrane domain: a photoaffinity labeling study with the bupropion analogue [(125)I]-SADU-3-72.

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 µM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 µM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, ßV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within ßV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), ßLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.

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.

Micromechanical measurement of AChBP binding for label-free drug discovery.

A potential binding assay based on binding-driven micromechanical motion is described. Acetylcholine binding protein (AChBP) was used to modify a microcantilever. The modified microcantilever was found to bend on application of the naturally occurring agonist (acetylcholine) or the antagonist (nicotine and d-tubocurarine). Control experiments show that microcantilevers modified without AChBP do not respond to acetylcholine, nicotine, and d-tubocurarine. K(d) values obtained for acetylcholine, nicotine, and d-tubocurarine are similar to those obtained from radio-ligand binding assays. These results suggest that the microcantilever system has potential for use in label free, drug screening applications.