Molecular modeling of the α9α10 nicotinic acetylcholine receptor subtype

This study reports the comparative molecular modeling, docking and dynamic simulations of human α9α10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and α-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary α10(?) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal α9(+), and Asp114 in the complementary α10(?) subunit, might confer the potency and selectivity to α-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.     

Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus

Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken β2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 μM) and choline (100 μM). Rsanα1/β2 was insensitive to both imidacloprid (100 μM) and spinosad (100 μM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides.     

α7 nicotinic acetylcholine receptor (α7nAChR) expression in bone marrow-derived non-T cells is required for the inflammatory reflex

The immune response to infection or injury coordinates host defense and tissue repair, but also has the capacity to damage host tissues. Recent advances in understanding protective mechanisms have found neural circuits that suppress release of damaging cytokines. Stimulation of the vagus nerve protects from excessive cytokine production and ameliorates experimental inflammatory disease. This mechanism, the inflammatory reflex, requires the α7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel expressed on macrophages, lymphocytes, neurons and other cells. To investigate cell-specific function of α7nAChR in the inflammatory reflex, we created chimeric mice by cross-transferring bone marrow between wild-type (WT) and α7nAChR-deficient mice. Deficiency of α7nAChR in bone marrow-derived cells significantly impaired vagus nerve-mediated regulation of tumor necrosis factor (TNF), whereas α7nAChR deficiency in neurons and other cells had no significant effect. In agreement with recent work, the inflammatory reflex was not functional in nude mice, because functional T cells are required for the integrity of the pathway. To investigate the role of T-cell α7nAChR, we adoptively transferred α7nAChR-deficient or WT T cells to nude mice. Transfer of WT and α7nAChR-deficient T cells restored function, indicating that α7nAChR expression on T cells is not necessary for this pathway. Together, these results indicate that α7nAChR expression in bone marrow-derived non-T cells is required for the integrity of the inflammatory reflex.     

Consequences of linker length alteration of the α7 nicotinic acetylcholine receptor (nAChR) agonist, SEN12333

A series of ligands based on SEN12333, containing either contracted or elongated alkyl chains, were synthesized and evaluated in molecular docking studies against a homology model of the α7 nicotinic acetylcholine receptor (nAChR) subtype. The predicted binding of all ligands was highly similar, with the exception of the analog containing a 5 methylene unit spacer. However, in vitro competition binding assays revealed that the ligands possessed dissimilar binding affinities, with a K(i) range of more than an order of magnitude (K(i)=0.50 to >10 μM), and only SEN12333 itself exhibited functional activity at the α7 nAChR.     

SAR of α7 nicotinic receptor agonists derived from tilorone: exploration of a novel nicotinic pharmacophore

The well-known interferon-inducer tilorone was found to possess potent affinity for the agonist site of the α7 neuronal nicotinic receptor (K(i)=56 nM). SAR investigations determined that both basic sidechains are essential for potent activity, however active monosubstituted derivatives can also be prepared if the flexible sidechains are replaced with conformationally rigidified cyclic amines. Analogs in which the fluorenone core is replaced with either dibenzothiophene-5,5-dioxide or xanthenone also retain potent activity.     

Detection of a CfoI polymorphism within exon 5 of the human neuronal nicotinic acetylcholine receptor α4 subunit gene (CHRNA4)

The gene coding for the 4 subunit of the neuronal nicotinic acetylcholine receptor (CHRNA4) has been recently mapped in the candidate region for benign familial neonatal convulsions (BFNC) on chromosome 20q13.2–q13.3. The region is only partially covered with polymorphic markers, and so far no PCR-based polymorphisms have been described in the critical region for BFNC. We now report the first polymorphic marker in the coding region of CHRNA4. The new marker, which is detected by PCR, will be useful for evaluation of the role of CHRNA4 as a candidate gene for BFNC. It will further enable the investigation of this important brain-specific gene in association studies with different types of epileptic diseases and other neurological disorders.     

Carbamoylcholine analogs as nicotinic acetylcholine receptor agonists—Structural modifications of 3-(dimethylamino)butyl dimethylcarbamate (DMABC)

Compounds based on the 3-(dimethylamino)butyl dimethylcarbamate (DMABC) scaffold were synthesized and pharmacologically characterized at the α₄β₂, α₃β_4, α₄β₄ and α₇ neuronal nicotinic acetylcholine receptors (nAChRs). The carbamate functionality and a small hydrophobic substituent in the C-3 position were found to be vital for the binding affinity to the nAChRs, whereas the carbamate nitrogen substituents were important for nAChR subtype selectivity. Finally, the compounds were found to be agonists at the α₃β₄ nAChR.     

Gene-based analysis suggests association of the nicotinic acetylcholine receptor β1 subunit (CHRNB1) and M1 muscarinic acetylcholine receptor (CHRM1) with vulnerability for nicotine dependence

Based on our previously identified linkage regions for nicotine dependence (ND), we selected six and five single nucleotide polymorphisms (SNPs) in the muscarinic cholinergic receptor subtype M1 (CHRM1) and nicotinic cholinergic receptor β1 (CHRNB1), respectively, to determine the association of the two genes with ND in a total of 2,037 subjects from 602 nuclear families of either African-American (AA) or European-American (EA) origin. Individual SNP- and/or haplotype-based analyses indicated that the CHRNB1 was significantly associated with ND, which was assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI), and the Fagerström Test for ND (FTND), in both ethnic samples. The association of rs2302763 in the CHRNB1 was significant with adjusted SQ in the EA sample after correction for multiple testing (P = 0.013). Haplotype A-T-A formed by SNPs rs2302765, rs2302762, and rs9217 in the CHRNB1 was significantly associated with the high risk allele for all the three ND measures (minimum P = 0.009, 0.006, and 0.008 for SQ, HSI and FTND, respectively) in the AA sample while haplotype A-T-A formed by rs2302765, rs2302763, and rs9217 was significantly positively associated with ND (minimum P = 0.005, 0.016, and 0.016 for SQ, HSI and FTND, respectively) in the EA sample. The CHRM1 exhibited significant protective associations of haplotype C-C-A-T-G-G formed by all six SNPs of this gene with at least one ND measure in the AA sample after Bonferroni correction (minimum P = 0.008, 0.013, and 0.009 for SQ, HSI and FTND, respectively), but no significant association was found in the EA sample. The significant associations, together with their location of linked region to ND, suggest that the CHRNB1 and CHRM1 are likely candidates for further investigation.     

Purification of the nicotinic acetylcholine receptor protein by affinity chromatography using a regioselectively modified and reversibly immobilized α-toxin from Naja nigricollis

A new method of affinity chromatography purification of the detergent-solubilized nicotinic acetylcholine receptor protein (nAChR) is presented, based on the reversible coupling of a chemically monomodified α-toxin from Naja nigricollis to a resin. The α-toxin was monothiolated on the ϵ-amino group of its lysine-15 by reaction with N-succinimidyl-3-(2-pyridyldithio)propionate and was covalently linked in a reversible manner to a thiopropyl-activated agarose resin by thiol-disulfide exchange. We found that 50% of the immobilized toxin molecules were effective for purifying nAChR, indicating a high accessibility of resin-bound toxins to their binding sites on the receptor protein. Purified α-toxin/nAChR complexes were eluted with nearly 100% recovery by reduction of disulfide bridges with dithiothreitol. nAChR solutions of high purity were obtained, as shown by polyacrylamide gel electrophoresis. A comparison was made with two other procedures of affinity chromatography using: (1) α-bungarotoxin from Bungarus multicinctus polymodified on several amines and covalently linked to a resin in a reversible manner, and (2) a commercial agarose resin bearing irreversibly immobilized α-cobrotoxin from Naja naja kaouthia. We conclude that: (1) the use of a selected regioselective linking of a peptidic ligand to a chromatography resin results in an increased efficiency of protein binding, and (2) a high yield of protein recovery is obtained via reversible covalent linking.     

The structures of the human neuronal nicotinic acetylcholine receptor β2- and α3-subunit genes (CHRNB2 and CHRNA3)

The α4-subunit gene (CHRNA4) of the neuronal nicotinic acetylcholine receptor (nAChR) subunit family has recently been identified in two families as the gene responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), a rare monogenic idiopathic epilepsy. As a result of this finding, other subunits of the neuronal nAChR gene family are being considered as candidate genes for ADNFLE in families not linked to CHRNA4 and for other idiopathic epilepsies. α4-subunitsoften assemble together with β2-subunits (gene symbol CHRNB2) to build heteromeric nAChRs. The gene encoding another abundant AChR subunit, the α3-subunit gene (CHRNA3), is present with those encoding two other subunits, CHRNB4 and CHRNA5, in a gene cluster whose functional role is still unclear. Here we provide the information on the genomic structures of both the CHRNB2 and the CHRNA3 genes that is necessary for comprehensive mutational analyses, and we refine the genomic assignment of CHRNB2 on chromosome 1. Received: 5 August 1998 / Accepted: 13 October 1998     

Computational studies of novel carbonyl-containing diazabicyclic ligands interacting with α4β2 nicotinic acetylcholine receptor (nAChR) reveal alternative binding modes

We have carried out computational studies on interactions of diazabicyclic amide analogs with α4β2 nAChR using homology modeling, docking and pharmacophore elucidation techniques. We have found alternative ligand binding modes in most cases. All these diverse poses exhibit the quintessential hydrogen-bonding interaction between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the highly conserved Trp-149. This hydrogen bond was always found to be shorter than the one contracted by the ligand carbonyl group and a second hydrogen-bond made by the cationic center with Tyr-93 of the principal face of the protein. In most of the poses observed, cation–π interactions involved three aromatic residues located in the principal face of the protein: Trp-149, Tyr-190 and Tyr-197. The latter amino acid residue appears to often donate a hydrogen-bond to the ligand carbonyl oxygen atom. We also describe two rings of alternative receptor-based hydrogen-bond donor features equidistantly separated from the carbonyl oxygen of the highly conserved Trp-149 approximately by 5 and 8 Å, respectively. These findings could be exploited to design diverse and selective novel chemical libraries for the treatment of diseases and conditions where the α4β2 nAChR is disrupted, such as Alzheimer disease, Parkinson’s disease and l-dopa-induced dyskinesia (LID).     

Function of human α3β4α5 nicotinic acetylcholine receptors is reduced by the α5(D398N) variant

Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR α5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of α3β4α5 nAChR in Xenopus oocytes. α5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common α5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked α3, β4, and α5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric α3β4* nAChRs. α5 subunit incorporation reduces α3β4* nAChR function after coinjection with unlinked α3 and β4 subunits but increases that of α3β4α5 versus α3β4-only concatemers. α5 subunit incorporation into α3β4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of α5 subunits, free α3 and β4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit α3β4-only subtypes are dissimilar both to each other and to those of α3β4α5 nAChR. The α5 variant-induced change in α3β4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.     

Computational neural network analysis of the affinity of N-n-alkylnicotinium salts for the α4β2* nicotinic acetylcholine receptor

Based on an 85 molecule database, linear regression with different size datasets and an artificial neural network approach have been used to build mathematical relationships to fit experimentally obtained affinity values (K_i) of a series of mono- and bis-quaternary ammonium salts from [³H]nicotine binding assays using rat striatal membrane preparations. The fitted results were then used to analyze the pattern among the experimental K_i values of a set of N-n-alkylnicotinium analogs with increasing n-alkyl chain length from 1 to 20 carbons. The affinity of these N-n-alkylnicotinium compounds was shown to parabolically vary with increasing numbers of carbon atoms in the n-alkyl chain, with a local minimum for the C₄ (n-butyl) analogue. A decrease in K_i value between C₁₂ and C₁₃ was also observed. The statistical results for the best neural network fit of the 85 experimental K_i values are r² = 0.84, rmsd = 0.39; r_cv² = 0.68, and loormsd = 0.56. The generated neural network model with the 85 molecule training set may also be of value for future predictions of K_i values for new virtual compounds, which can then be identified, subsequently synthesized, and tested experimentally.     

3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate the design of a novel selective antagonist of human α4β2 neuronal nicotinic acetylcholine receptors

Subtype selective molecules for α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4β2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3β4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4β2 nAChRs and human α3β4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4β2 nAChRs.