Acetylcholine Promotes Binding of α‐Conotoxin MII at α3β2 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 α₃β₂‐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 β₂‐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 α₃β₂‐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.     

Molecular dynamics studies of AChBP with nicotine and carbamylcholine: the role of water in the binding pocket

The acetylcholine-binding protein (AChBP) is homologous to the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) and other members of the Cys-loop family of neurotransmitter receptors. The high-resolution X-ray structures of AChBP mean it has been used as a model from which to understand agonist and antagonist binding to nAChRs. We present here a molecular dynamics (MD) study of AChBP with nicotine and carbamylcholine bound. Our results suggest that the ligand imposes rigidity on the binding pocket residues. The simulations also suggest that the protein undergoes breathing motions with respect to the five-fold axis, a motion that has been postulated to be related to gating in the nAChR. We analyzed the behaviour of the water molecules in and around the binding site and found that they occupied five distinct sites within the binding pocket. Water occupied these sites in the absence of ligand, but the presence of ligand increased the probability that a water molecule would be found in these sites. Finally, we demonstrate how the positions of these waters might be used in the design of new ligands by comparing the positions of these sites with other recent structures.     

Central nicotinic receptors: structure, function, ligands, and therapeutic potential

The growing interest in nicotinic receptors, because of their wide expression in neuronal and non-neuronal tissues and their involvement in several important CNS pathologies, has stimulated the synthesis of a high number of ligands able to modulate their function. These membrane proteins appear to be highly heterogeneous, and still only incomplete information is available on their structure, subunit composition, and stoichiometry. This is due to the lack of selective ligands to study the role of nAChR under physiological or pathological conditions; so far, only compounds showing selectivity between alpha4beta2 and alpha7 receptors have been obtained. The nicotinic receptor ligands have been designed starting from lead compounds from natural sources such as nicotine, cytisine, or epibatidine, and, more recently, through the high-throughput screening of chemical libraries. This review focuses on the structure of the new agonists, antagonists, and allosteric ligands of nicotinic receptors, it highlights the current knowledge on the binding site models as a molecular modeling approach to design new compounds, and it discusses the nAChR modulators which have entered clinical trials.     

Chemistry and Pharmacology of Nicotinic Ligands Based on 6‐[5‐(Azetidin‐2‐ylmethoxy)pyridin‐3‐yl]hex‐5‐yn‐1‐ol (AMOP‐H‐OH) for Possible Use in Depression

AMOP‐H‐OH (sazetidine‐A; 6‐[5‐(azetidin‐2‐ylmethoxy)pyridin‐3‐yl]hex‐5‐yn‐1‐ol) and some sulfur‐bearing analogues were tested for their activities in vitro against human α4β2‐, α4β4‐, α3β4*‐ and α1*‐nicotinic acetylcholine receptors (nAChRs). AMOP‐H‐OH was also assessed in an antidepressant efficacy model. AMOP‐H‐OH and some of its analogues have high potency and selectivity for α4β2‐nAChRs over other nAChR subtypes. Effects are manifested as partial agonism, perhaps reflecting selectivity for high sensitivity (α4)₃(β2)₂‐nAChRs. More prolonged exposure to AMOP‐H‐OH and its analogues produces inhibition of subsequent responses to acute challenges with full nicotinic agonists, again selectively for α4β2‐nAChRs over other nAChR subtypes. The inhibition is mediated either via antagonism or desensitization of nAChR function, but the degree of inhibition of α4β2‐nAChRs is limited by the partial agonist activity of the drugs. Certain aspects of the in vitro pharmacology suggest that AMOP‐H‐OH and some of its analogues have a set of binding sites on α4β2‐nAChRs that are distinct from those for full agonists. The in vitro pharmacological profile suggests that peripheral side effects of AMOP‐H‐OH or its analogues would be minimal and that their behavioral effects would be dominated by central nAChR actions. AMOP‐H‐OH also has profound and high potency antidepressant‐like effects in the forced swim test. The net action of prolonged exposure to AMOP‐H‐OH or its analogues, as for nicotine, seems to be a selective decrease in α4β2‐nAChR function. Inactivation of nAChRs may be a common neurochemical endpoint for nicotine dependence, its treatment, and some of its manifestations, including relief from depression.     

Fluorescent agonists for the Torpedo nicotinic acetylcholine receptor

We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC-Gly-C6-choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl- and NBD-acylcholine analogues. Binding of DEAC-Gly-C6-choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the alphadelta-binding site and 15.0 nM for the alphagamma-binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug-screening studies.     

The Human-Restricted Isoform of the α7 nAChR,CHRFAM7A: A Double-Edged Sword in Neurological and Inflammatory Disorders

CHRFAM7A is a relatively recent and exclusively human gene arising from the partial duplication of exons 5 to 10 of the α7 neuronal nicotinic acetylcholine receptor subunit (α7 nAChR) encoding gene, CHRNA7. CHRNA7 is related to several disorders that involve cognitive deficits, including neuropsychiatric, neurodegenerative, and inflammatory disorders. In extra-neuronal tissues, α7nAChR plays an important role in proliferation, differentiation, migration, adhesion, cell contact, apoptosis, angiogenesis, and tumor progression, as well as in the modulation of the inflammatory response through the “cholinergic anti-inflammatory pathway”. CHRFAM7A translates the dupα7 protein in a multitude of cell lines and heterologous systems, while maintaining processing and trafficking that are very similar to the full-length form. It does not form functional ion channel receptors alone. In the presence of CHRNA7 gene products, dupα7 can assemble and form heteromeric receptors that, in order to be functional, should include at least two α7 subunits to form the agonist binding site. When incorporated into the receptor, in vitro and in vivo data showed that dupα7 negatively modulated α7 activity, probably due to a reduction in the number of ACh binding sites. Very recent data in the literature report that the presence of the duplicated gene may be responsible for the translational gap in several human diseases. Here, we will review the studies that have been conducted on CHRFAM7A in different pathologies, with the intent of providing evidence regarding when and how the expression of this duplicated gene may be beneficial or detrimental in the pathogenesis, and eventually in the therapeutic response, to CHRNA7-related neurological and non-neurological diseases.     

Alpha7 nicotinic acetylcholine receptor is a target in pharmacology and toxicology

Alpha7 nicotinic acetylcholine receptor (α7 nAChR) is an important part of the cholinergic nerve system in the brain. Moreover, it is associated with a cholinergic anti-inflammatory pathway in the termination of the parasympathetic nervous system. Antagonists of α7 nAChR are a wide group represented by conotoxin and bungarotoxin. Even Alzheimer's disease drug memantine acting as an antagonist in its side pathway belongs in this group. Agonists of α7 nAChR are suitable for treatment of multiple cognitive dysfunctions such as Alzheimer's disease or schizophrenia. Inflammation or even sepsis can be ameliorated by the agonistic acting compounds. Preparations RG3487, SEN34625/WYE-103914, SEN12333, ABT-107, Clozapine, GTS-21, CNI-1493, and AR-R17779 are representative examples of the novel compounds with affinity toward the α7 nAChR. Pharmacological, toxicological, and medicinal significance of α7 nAChR are discussed throughout this paper.     

Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor

A novel series of 30 symmetric bispyridinium and related N‐heteroaromatic bisquaternary salts with a propane‐1,3‐diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4‐tert‐butyl‐substituted bispyridinium salt MB327 was previously identified as a nAChR re‐sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N‐alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)‐based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pK_i=4.73±0.03). Notably, the newly prepared lipophilic 4‐tert‐butyl‐3‐phenyl‐substituted bispyridinium salt PTM0022 ( 3 h ) was found to have significantly higher binding affinity, with a pK_i value of 5.16±0.07, thus representing considerable progress toward the development of more potent nAChR re‐sensitizers.     

Design, synthesis, and pharmacological characterization of novel spirocyclic quinuclidinyl-Δ2-isoxazoline derivatives as potent and selective agonists of α7 nicotinic acetylcholine receptors

A set of racemic spirocyclic quinuclidinyl‐Δ²‐isoxazoline derivatives was synthesized using a 1,3‐dipolar cycloaddition‐based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (α7) and heteromeric (α4β2) nicotinic acetylcholine receptors. Δ²‐Isoxazolines 3 a (3‐Br), 6 a (3‐OMe), 5 a (3‐Ph), 8 a (3‐OnPr), and 4 a (3‐Me) were the ligands with the highest affinity for the α7 subtype (K_i values equal to 13.5, 14.2, 25.0, 71.6, and 96.2 nM , respectively), and showed excellent α7 versus α4β2 subtype selectivity. These compounds, tested in electrophysiological experiments against human α7 and α4β2 receptors stably expressed in cell lines, behaved as partial α7 agonists with varying levels of potency. The two enantiomers of (±)‐3‐methoxy‐1‐oxa‐2,7‐diaza‐7,10‐ethanospiro[4.5]dec‐2‐ene sesquifumarate 6 a were prepared using (+)‐dibenzoyl‐L ‐ or (?)‐dibenzoyl‐D ‐tartaric acid as resolving agents. Enantiomer (R)‐(?)‐ 6 a was found to be the eutomer, with K_i values of 4.6 and 48.7 nM against rat and human α7 receptors, respectively.     

Mode of Action of Neonicotinoid Insecticides Imidacloprid and Thiacloprid to the Cockroach Pameα7 Nicotinic Acetylcholine Receptor

The functional expression of the cockroach Pameα7 nicotinic acetylcholine receptor subunit has been previously studied, and was found to be able to form a homomeric receptor when expressed in Xenopus laevis oocytes. In this study, we found that the neonicotinoid insecticide imidacloprid is unable to activate the cockroach Pameα7 receptor, although thiacloprid induces low inward currents, suggesting that it is a partial agonist. In addition, the co-application or 5 min pretreatment with 10 µM imidacloprid increased nicotine current amplitudes, while the co-application or 5 min pretreatment with 10 µM thiacloprid decreased nicotine-evoked current amplitudes by 54% and 28%, respectively. This suggesting that these two representatives of neonicotinoid insecticides bind differently to the cockroach Pameα7 receptor. Interestingly, the docking models demonstrate that the orientation and interactions of the two insecticides in the cockroach Pameα7 nAChR binding pocket are very similar. Electrophysiological results have provided evidence to suggest that imidacloprid and thiacloprid could act as modulators of the cockroach Pameα7 receptors.     

Synthesis and application of the first radioligand targeting the allosteric binding pocket of chemokine receptor CXCR3

Strategies for the identification of allosteric modulators of chemokine receptors largely rely on various cell‐based functional assays. Radioligand binding assays are typically not available for allosteric binding sites. We synthesized, purified, and applied the first tritium‐labeled allosteric modulator of the human chemokine receptor CXCR3 (RAMX3, [³H]N‐{1‐[3‐(4‐ethoxyphenyl)‐4‐oxo‐3,4‐dihydropyrido[2,3‐d]pyrimidin‐2‐yl]ethyl}‐2‐[4‐fluoro‐3‐(trifluoromethyl)phenyl]‐N‐[(1‐methylpiperidin‐4‐yl)methyl]acetamide). RAMX3 is chemically derived from 8‐azaquinazolinone‐type allosteric modulators and binds to the CXCR3 receptor with a K_d value of 1.08 nM (specific activity: 80.4 Ci mmol^?1). Radioligand displacement assays showed potent negative cooperativity between RAMX3 and chemokine CXCL11, providing a basis for the use of RAMX3 to investigate other potential allosteric modulators. Additionally, the synthesis and characterization of a number of other full and truncated 8‐azaquinazoline analogues were used to validate the binding properties of RAMX3. We demonstrate that RAMX3 can be efficiently used to facilitate the discovery and characterization of small molecules as allosteric modulators of the CXCR3 receptor.     

Dynamic structural investigations on the torpedo nicotinic acetylcholine receptor by time-resolved photoaffinity labeling

An increasing number of high-resolution structures of membrane-embedded ion channels (or soluble homologues) have emerged during the last couple of years. The most pressing need now is to understand the complex mechanism underlying ion-channel function. Time-resolved photoaffinity labeling is a suitable tool for investigating the molecular function of membrane proteins, especially when high-resolution structures of related proteins are available. However until now this methodology has only been used on the Torpedo nicotinic acetylcholine receptor (nAChR). nAChRs are allosteric cation-selective receptor channels that are activated by the neurotransmitter acetylcholine (ACh) and implicated in numerous physiological and pathological processes. Time-resolved photoaffinity labeling has already enabled local motions of nAChR subdomains (i.e. agonist binding sites, ion channel, subunit interface) to be understood at the molecular level, and has helped to explain how small molecules can exert their physiological effect, an important step toward the development of drug design. Recent analytical and technical improvements should allow the application of this powerful methodology to other membrane proteins in the near future.     

Acute Lung Injury in Cholinergic-Deficient Mice Supports Anti-Inflammatory Role of α7 Nicotinic Acetylcholine Receptor

(1) Background: The lung cholinergic pathway is important for controlling pulmonary inflammation in acute lung injury, a condition that is characterized by a sudden onset and intense inflammation. This study investigated changes in the expression levels of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) in the lung during acute lung injury. (2) Methods: acute lung injury (ALI) was induced in wild-type and cholinergic-deficient (VAChT-KD^HOM) mice using intratracheal lipopolysaccharide (LPS) instillation with or without concurrent treatment with nicotinic ligands. Bronchoalveolar lavage fluid was collected to evaluate markers of inflammation, and then the lung was removed and processed for isolation of membrane fraction and determination of acetylcholine receptors level using radioligand binding assays. (3) Results: LPS-induced increase in lung inflammatory markers (e.g., neutrophils and IL-1β) was significantly higher in VAChT-KD^HOM than wild-type mice. In contrast, LPS treatment resulted in a significant increase in lung’s α7 nicotinic receptor level in wild-type, but not in VAChT-KD^HOM mice. However, treatment with PNU 282987, a selective α7 nicotinic receptor agonist, restored VAChT-KD^HOM mice’s ability to increase α7 nicotinic receptor levels in response to LPS-induced acute lung injury and reduced lung inflammation. LPS also increased muscarinic receptors level in VAChT-KD^HOM mice, and PNU 282987 treatment reduced this response. (4) Conclusions: Our data indicate that the anti-inflammatory effects of the lung cholinergic system involve an increase in the level of α7 nicotinic receptors. Pharmacological agents that increase the expression or the function of lung α7 nicotinic receptors have potential clinical uses for treating acute lung injury.     

Homology modeling of NR2B modulatory domain of NMDA receptor and analysis of ifenprodil binding

NMDA receptors are glutamate-gated ion channels (iGluRs) that are involved in several important physiological functions such as neuronal development, synaptic plasticity, learning, and memory. Among iGluRs, NMDA receptors have been perhaps the most actively investigated for their role in chronic neurodegeneration such as Alzheimer's, Parkinson's, and Huntington's diseases. Recent studies have shown that the NTD of subunit NR2B modulates ion channel gating through the binding of allosteric modulators such as the prototypical compound ifenprodil. In the present paper, the construction of a three-dimensional model for the NR2B modulatory domain is described and docking calculations allow, for the first time, definition of the ifenprodil binding pose at an atomic level and fully explain all the available structure-activity relationships. Moreover, in an attempt to add further insight into the ifenprodil mechanism of action, as it is not completely clear if it binds and stabilizes an open or a closed conformation of the NR2B modulatory domain, a matter, which is fundamental for the rational design of NMDA antagonists, MD simulations followed by an MM-PBSA analysis were performed. These calculations reveal that the closed conformation of the R1-R2 domain, rather than the open, constitutes the high affinity binding site for ifenprodil and that a profound stabilization of the closed conformation upon ifenprodil binding occurs. Thus, for a rational design and/or for virtual screening experiments, the closed conformation of the R1-R2 domain should be taken into account and our 3D model can provide valuable hints for the design of NR2B-selective antagonists.     

Modeling the Combining Site of the Human Asialoglycoprotein Receptor

A model for the carbohydrate recognition domain (CRD) and combining site of the human asialoglycoprotein (ASGP) receptor has been computed on the basis of the close sequence homology with the mannose-binding lectin (MBP), whose three-dimensional structure in complex with a ligand has been determined by crystallographic methods (Weis, W.I.; Drickamer, K.; Hendrickson, W.A. Nature 1992, 360: 127). Within the limitations of modeling methods, the model is compatible with data on ligand binding of the family C-type lectins, of which the MBP and the ASGP receptor are members. The model derived can serve as a guide for designing site-directed mutagenesis experiments in order to further elucidate the origins of specificity of the ASGP receptor toward galactose. In particular, the model focuses attention on the possible role of position 207 (MBP sequence numbering) in promoting galactose binding.     

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K_D=42 and 84 μM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K_D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.     

Ligand-binding modes in cationic biogenic amine receptors

The binding site in G protein-coupled cationic biogenic amine receptors is formed in the cleft of the seven transmembrane segments. Upon binding the ligand, the receptors are activated or inactivated through the conformational changes of the transmembrane segments. G protein-coupled receptors bind four functionally distinct ligands; inverse agonists, antagonists, partial agonists, and full agonists. Hence, putative structural models for biogenic amine receptors corresponding to the ligand function (inverse agonist-, antagonist-, partial agonist-, and full agonist-bound receptor models) were built by using photointermediate models in the rhodopsin photocascade (M. Ishiguro et al. ChemBioChem. 2004, 5, 298-310). The ligand-receptor recognition of each was examined by modeling receptor-ligand complexes with functional ligands. The complex models suggested that each functional ligand binds the corresponding receptor structure and that ligand-specific interactions contribute to stabilization of the corresponding receptor structure.     

The Third Extracellular Loop of Mammalian Odorant Receptors Is Involved in Ligand Binding

Mammals recognize chemicals in the air via G protein-coupled odorant receptors (ORs). In addition to their orthosteric binding site, other segments of these receptors modulate ligand recognition. Focusing on human hOR1A1, which is considered prototypical of class II ORs, we used a combination of molecular modeling, site-directed mutagenesis, and in vitro functional assays. We showed that the third extracellular loop of ORs (ECL3) contributes to ligand recognition and receptor activation. Indeed, site-directed mutations in ECL3 showed differential effects on the potency and efficacy of both carvones, citronellol, and 2-nonanone.     

A Structural Rationale for N-Methylbicuculline Acting as a Promiscuous Competitive Antagonist of Inhibitory Pentameric Ligand-Gated Ion Channels

Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABA_A receptors and affects other pentameric ligand-gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence-quenching assay and isothermal titration calorimetry to record low-micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine-binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Å resolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABA_A receptor–bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures, but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand-gated ion channels in the CNS.