Identification of a series of 1,3,4-oxadiazol-2-amines as potent alpha-7 agonists with efficacy in the novel object recognition model of cognition

A series of α7 nicotinic acetylcholine receptor full-agonists with a 1,3,4-oxadiazol-2-amine core has been discovered. Systematic exploration of the structure-activity relationships for both α7 potency and selectivity with respect to interaction with the hERG channel are described. Further profiling led to the identification of compound 22, a potent full agonist showing efficacy in the novel object recognition model of cognition enhancement.     

The alpha7 nicotinic acetylcholine receptor: molecular modelling, electrostatics, and energetics

The structure of a homopentameric alpha7 nicotinic acetylcholine receptor is modelled by combining structural information from two sources: the X-ray structure of a water soluble acetylcholine binding protein from Lymnea stagnalis, and the electron microscopy derived structure of the transmembrane domain of the Torpedo nicotinic receptor. The alpha7 nicotinic receptor model is generated by simultaneously optimising: (i) chain connectivity, (ii) avoidance of stereochemically unfavourable contacts, and (iii) contact between the beta1-beta2 and M2-M3 loops that have been suggested to be involved in transmission of conformational change between the extracellular and transmembrane domains. A Gaussian network model was used to predict patterns of residue mobility in the alpha7 model. The results of these calculations suggested a flexibility gradient along the transmembrane domain, with the extracellular end of the domain more flexible that the intracellular end. Poisson-Boltzmann (PB) energy calculations and atomistic (molecular dynamics) simulations were used to estimate the free energy profile of a Na+ ion as a function of position along the axis of the pore-lining M2 helix bundle of the transmembrane domain. Both types of calculation suggested a significant energy barrier to exist in the centre of the (closed) pore, consistent with a "hydrophobic gating" model. Estimations of the PB energy profile as a function of ionic strength suggest a role of the extracellular domain in determining the cation selectivity of the alpha7 nicotinic receptor. These studies illustrate how molecular models of members of the nicotinic receptor superfamily of channels may be used to study structure-function relationships.     

Discovery of non-peptide, small molecule antagonists of α9α10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain

A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of α9α10 nAChRs and greater selectivity over α7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.     

Gamma-lactams—A novel scaffold for highly potent and selective α7 nicotinic acetylcholine receptor agonists

A novel class of α7 nicotinic acetylcholine receptor (nAChR) agonists has been discovered through high-throughput screening. The cis γ-lactam scaffold has been optimized to reveal highly potent and selective α7 nAChR agonists with in vitro activity and selectivity and with good brain penetration in mice.     

Structure-based design, synthesis and structure-activity relationships of dibenzosuberyl- and benzoate-substituted tropines as ligands for acetylcholine-binding protein

Using structure-based optimization procedures on in silico hits, dibenzosuberyl- and benzoate substituted tropines were designed as ligands for acetylcholine-binding protein (AChBP). This protein is a homolog to the ligand binding domain of the nicotinic acetylcholine receptor (nAChR). Distinct SAR is observed between two AChBP species variants and between the α7 and α4β2 nAChR subtype. The AChBP species differences are indicative of a difference in accessibility of a ligand-inducible subpocket. Hereby, we have identified a region that can be scrutinized to achieve selectivity for nicotinic receptor subtypes.     

Discovery of novel α7 nicotinic receptor antagonists

Two distinct families of small molecules were discovered as novel α7 nicotinic acetylcholine receptor (nAChR) antagonists by pharmacophore-based virtual screening. These novel antagonists exhibited selectivity for the neuronal α7 subtype over other nAChRs and good brain penetration. Neuroprotection was demonstrated by representative compounds 7i and 8 in a mouse seizure-like behavior model induced by the nerve agent diisopropylfluorophosphate (DFP). These novel nAChR antagonists have potential use as antidote for organophosphorus nerve agent intoxication.     

Molecular interactions of type I and type II positive allosteric modulators with the human α7 nicotinic acetylcholine receptor: an in silico study

The binding site locations and structural components for type I and type II positive allosteric modulators (PAMs) of the α7 nicotinic acetylcholine receptor (nAChR) have not been fully characterized yet. In this regard, homology models of the human α7 nAChR and hα7/m5-HT_3A chimera, built using the crystal structure of the serotonin type 3A receptor (5-ΗΤ_3ΑR), were used for molecular docking and molecular dynamics simulations to study the molecular interactions of selected type I (5-hydroxyindol, NS-1738, and LY-2087101) and type II (PNU-120596, PAM-2, and TBS-516) PAMs. The docking results indicate: (1) a site located in the extracellular domain (ECD) for type I PAMs such as NS-1738 and LY-2087101, but not for 5-HI; (2) an overlapping site in the ECD–transmembrane domain (TMD) junction for all studied PAMs. Additional docking results on the hα7/m5-HT_3A chimera supported experimental results indicating that the ECD site might be relevant for type I PAM activity; and (3) two TMD sites, an intrasubunit site that recognizes type II PAMs, and an intersubunit pocket with high specificity for 5-HI (type I PAM). The in silico α7TSLMF mutant results support the view that M1–Ser223 and M3–Ile281 are key residues for the interaction of PAM-2 and PNU-120596 with the intrasubunit cavity. Our in silico results are in agreement with experimental data showing that the intrasubunit cavity is relevant for the activity of type II PAMs, and suggest that the ECD–TMD junction and intersubunit sites could be significant for the activity of type I PAMs.     

Creating an α7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses

Determining the structure of the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) has been a long standing goal in the design of selective drugs useful in implicated diseases for this prevalent receptor family. Acetylcholine-binding proteins have proven to be valuable surrogates with structural similarity and sequence identity to the extracellular domain of the nicotinic receptor, yet these soluble proteins have their unique features and do not serve as exact replicates of the nAChRs of interest. Here we systematically modify the sequence of these proteins toward the homomeric human α7 nAChR. These chimeric proteins exhibit a shift in affinities to reflect α7 binding characteristics yet maintain expression levels and stability conducive for crystallization. We also present a pentameric humanoid nAChR extracellular domain with the structural determination of the α7 nAChR glycosylation site.     

Design and synthesis of a novel series of 4-heteroarylamino-1′-azaspiro[oxazole-5,3′-bicyclo[2.2.2]octanes as α7 nicotinic receptor agonists 2. Development of 4-heteroaryl SAR

Quinuclidine-containing spirooxazolines, as described in the previous report in this series, were demonstrated to have utility as α7 nicotinic acetylcholine receptor (α7 nAChR) partial agonists. In this work, the SAR of this chemotype was expanded to include an array of diazine heterocyclic substitutions. Many of the heterocyclic analogs were potent partial agonists of the α7 receptor, selective against other nicotinic receptors and the serotinergic 5HT_3A receptor. (1′S,3′R,4′S)-N-(6-phenylpyrimidin-4-yl)-4H-1′-azaspiro[oxazole-5,3′-bicyclo[2.2.2]octan]-2-amine, a potent and selective α7 nAChR partial agonist, was demonstrated to improve cognition in the mouse novel object recognition (NOR) model of episodic memory.     

The effect of amantadine on nicotinic acetylcholine receptor (nAchR) in reconstituted membranes

The antiviral drug amantadine is also a potent neuromuscular blocking agent. When the nicotinic receptor from a Torpedinidae species is reconstituted into soybean liposomes, the binding of α-bungarotoxin is not altered although the carbamylcholine induced radioactive cation influx is blocked.By studying cation fluxes in amantadine preincubated membranes previously exposed to different concentrations of carbamylcholine for different periods of time, we have shown that the drug accelerates the conversion of the nicotinic acetylcholine receptor from a state of low affinity to a state of high affinity for carbamyalcholine, a phenomenon correlated with receptor desensitization. The drug did not induce such a shift by itself.The present data and those by Earnest et al. (Biochemistry22, 5523–5535, 1984) show that the nicotinic acetylcholine receptor reconstituted into liposomes is a good model for studying the effects of noncompetitive blockers of nicotinic acetylcholine receptor function.     

An extracellular RRR motif flanking the M1 transmembrane domain governs the biogenesis of homomeric neuronal nicotinic acetylcholine receptors

We have previously demonstrated that the highly conserved R209, that flanks the M1 transmembrane segment of nicotinic acetylcholine (ACh) receptors, is required for the transport of assembled homomeric neuronal α7 nicotinic ACh receptors to the cell surface. In the present paper we show that basic residues at positions 208 and 210 are necessary for the assembly of α7 receptors. On the contrary, a basic residue at position 210 of α3 subunit decreases the assembly of heteromeric neuronal α3β4 nicotinic ACh receptors. A basic residue at position 210 of the β4 subunit slightly decreases α3β4 receptor expression. We conclude that a pre-M1 RRR motif is necessary for the biogenesis of homomeric α-bungarotoxin-sensitive neuronal α7 nicotinic ACh receptors.     

Activation of α-7 Nicotinic Acetylcholine Receptor Attenuates Cardiac Inflammation Through NLRP3/Caspase-1/IL-18 Pathway

Biochemical Genetics - Activation of α-7 nicotinic acetylcholine receptor (α7nAChR) receptor might induce cardiac inflammation, cardiac remodeling, and dysfunction. In this regard, this...     

Structural characterization and agonist binding to human α4β2 nicotinic receptors

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition – particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.     

Three-dimensional structure of α-conotoxin EI determined by 1H NMR spectroscopy H resonance assignments of α-conotoxin EI

α-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other α-conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other α4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other α4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these α4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI.     

Acetylcholine binding protein (AChBP) as template for hierarchical in silico screening procedures to identify structurally novel ligands for the nicotinic receptors

Hierarchical in silico screening protocols against the agonist bound acetylcholine binding protein (AChBP) crystal structure were efficient in identifying novel chemotypes for AChBP and the human α7 receptor. Two hit structures were cocrystallized with AChBP revealing intermolecular cation-π interactions with loop C but lacking intermolecular hydrogen bonding. The compounds act as competitive α7 receptor antagonists and as non-competitive α4β2 receptor inhibitors. These results underline the usability of AChBP in structure-based in silico screening strategies in finding novel scaffolds for the α7 receptor, but also illustrates some limitations of using AChBP as bait to find competitive α4β2 receptor ligands and α7 receptor agonists.     

Modulation of inflammatory pathways by the immune cholinergic system

Research done in the past years pointed to a novel function of cholinergic transmission. It has been shown that cholinergic transmission can modulate various aspects of the immune function, whether innate or adaptive. Cholinergic transmission affects immune cell proliferation, cytokine production, T helper differentiation and antigen presentation. Theses effects are mediated by cholinergic muscarinic and nicotinic receptors and other cholinergic components present in immune cells, such as acetylcholinesterase (AChE) and cholineacetyltransferase. The α7 nicotinic acetylcholine receptor was designated anti-inflammatory activity and has shown promise in pre-clinical models of inflammatory disorders. We herein describe the various components of the immune cholinergic system, and specifically the immune suppressive effects of α7 activation. This activation can be accomplished either by direct stimulation or indirectly, by inhibition of AChE. Thus, the presence of the immune cholinergic system can pave the way for novel immunomodulatory agents, or to the broadening of use of known cholinergic agents.     

Limitations of RNAi of α6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

Abstract Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The α6 nicotinic acetylcholine receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6. RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4‐UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants. These Dα6‐deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Dα6 was reduced 60%–75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6. RNAi of Tcasα6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed.     

A novel α-conotoxin MII-sensitive nicotinic acetylcholine receptor modulates [(3) H]-GABA release in the superficial layers of the mouse superior colliculus

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. [(125) I]-Epibatidine competition-binding studies revealed that the α3β2* and α6β2* nicotinic subtype-selective peptide α-conotoxin MII-blocked binding to 40 ± 5% of SuSC nAChRs. Acetylcholine-evoked [(3) H]-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 [(3) H]-GABA release is inhibited by α-conotoxin MII. However, the highly α6β2*-subtype-selective α-conotoxin PIA did not affect [(3) H]-GABA release. Nicotinic subunit-null mutant mouse experiments revealed that ACh-stimulated SuSC [(3) H]-GABA release is entirely β2 subunit-dependent. α4 subunit deletion decreased total function by >90%, and eliminated α-conotoxin MII-resistant release. ACh-stimulated SuSC [(3) H]-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-conotoxin MII-sensitive α3α4β2 nAChR. The remaining α-conotoxin MII-resistant, nAChR agonist-evoked SuSC GABA release appears to be mediated via α4β2* subtype nAChRs.     

Nicotinic Acetylcholine Receptors have Ligand-Specific Attachment Point Patterns

Abstract

Employing a panel of synthetic peptides as representative structural elements of the nicotinic acetylcholine receptor from Torpedo electric organ, we recently identified three sequence regions of the receptor (α55–74, α134–153 and α181–200) serving as subsites for the binding of high molecular weight antagonists of acetylcholine (Conti-Tronconi et al. 1990). The relative binding affinities to these subsites of α-bungarotoxin and three competitive antibodies varied in a ligand-specific fashion. Employing a set of homologous synthetic peptides differing from α181–200 by the exchange of single amino acid residues along the sequence, we now find that ligand binding crucially depends on the presence of particular amino acids within the subsite while others influence binding only marginally if at all. The existence of ligand-specific attachment points may account for the wide range in binding and kinetic parameters, pharmacological specificity and distinct mean open times of the receptor-integral cation channel observed for cholinergic ligands.     

Nine Residues Influence the Binding of α-Bungarotoxin in α-Subunit Region 185–200 of Human Muscle Acetylcholine Receptor

Abstract: Identification of residues in the skeletal muscle nicotinic acetylcholine receptor (AChR) that bind snake venom a-neurotoxin antagonists of acetylcholine [e.g., α-bungarotoxin (α-BTx)] provides structural information about the neurotransmitter binding region of the receptor. Using synthetic peptides of the human AChR α-subunit region 177–208, we previously localized a pharmacologically specific binding site for α-BTx in segment 185–199. To define in more detail the residues that influence the binding of α-BTx to this region, we prepared 16 peptide analogues of the α-subunit segment 185–200, with the amino acid Lalanine sequentially replacing each native amino acid. Circular dichroism spectroscopy did not reveal changes in the secondary structure of the peptides except for the analogue in which Pro¹⁹⁴ was substituted with alanine. This implies that any change in α-BTx binding could be attributed to replacement of the native residue's side chain by alanine's methyl group, rather than to a change in the structure of the peptide. The influence of each substitution with alanine was determined by comparing the analogue to the parental sequence α 185–200 in solution-phase competition with native human AChR for binding of ¹²⁵I-labeled α-BTx. The binding of α-BTx by analogue peptides with alanine substituted for Tyr¹⁹⁰, Cys¹⁹², or Cys¹⁹³ was greatly diminished. Binding of α-BTx to peptides containing alanine replacements at Val¹⁸⁸, Thr¹⁸⁹, Pro¹⁹⁴, Asp¹⁹⁵, or Tyr¹⁹⁸ was also reduced significantly (p < 0.003). An unanticipated finding was that substitution of alanine for Ser¹⁹¹ significantly increased α-BTx binding (p < 0.003). The data imply that these nine amino acids influence the binding of the antagonist, α-BTx, to the nicotinic acetylcholine receptor of human skeletal muscle, and confirm previous reports for certain contact residues for α-BTX that were found in region α181-200 of the Torpedo AChR.