Investigating the hydrogen-bond acceptor site of the nicotinic pharmacophore model: a computational and experimental study using epibatidine-related molecular probes (original) (raw)
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Journal of Medicinal Chemistry, 2005
The homology models of the extracellular domains of the neuronal alpha4beta2 (pdb code: 1ole) and ganglionic alpha3beta4 (pdb code: 1olf) rat nicotinic acetylcholine receptor (nAChR) subtypes were refined and energetically minimized. In this work, a series of nAChR ligands (1-15) were docked into the modeled binding cavity of both receptors. High-affinity, toxic ligands such as epibatidine (1) and dechloroepibatidine (2) docked into cluster 1 with the charged tertiary amino group, forming a pi-cation interaction with Trp 147 on the (+) side of the alpha4 subunit and establishing a characteristic H-bond with the Lys 77 on the (-) side of the beta2 subunit. The nontoxic ligands such as 33bMet (3), (S)-A-85380 (4), and acetylcholine (6) docked into cluster 2 with the same pi-cation interaction but with the rest of the molecule occupying a different moiety of the binding pocket. Molecular docking into the alpha3beta4 subtype showed that both enantiomers of 1 (1a and 1b) are representative templates for ligands with affinity toward this ganglionic nAChR subtype. The ranking scores of the docked molecules confirm the existence of structure-dependent subtype selectivity and shed light on the design of specific and selective alpha4beta2 nAChR subtype ligands.
Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor
Biochemical and Biophysical Research Communications, 2008
The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabelled ligand binding techniques, we show that the binding of [ 3 H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 µM. These classes of sites exist in approximately equal numbers and all [ 3 H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.
European Journal of Medicinal Chemistry, 2006
Neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane ligand-gated ion channels. Recent research demonstrated that selective nAChR ligands may have therapeutic potential in a number of CNS diseases and disorders. The alkaloid epibatidine is a highly potent nonopioid analgesic and nAChR agonist, but too toxic to be a useful ligand. To develop ligands selective for distinct nAChR subtypes and with reduced toxicity, a series of epibatidine and homoepibatidine analogues were synthesized. (±)-8-Methyl-3-(pyridin-3-yl)-8-azabicyclo[3,2,1]oct-2-ene, showed high affinity towards α4β2 (K i = 2 nM), subtype selectivity (α4β2/α7 affinity ratio > 100) and relatively low toxicity in mice and can be labeled with 11 C and 18 F as positron emission tomography (PET) tracers for imaging of nAChRs.
Neuronal nicotinic receptor agonists: A multi-approach development of the pharmacophore
Journal of Computer-Aided Molecular Design, 2001
Based on the results obtained with different automated computational approaches as applied to the study of eleven high-affinity agonists of the neuronal nicotine acetylcholine receptor (nAChR), belonging to different chemical classes, new relevant features were detected which complement the existing pharmacophores. Convergent results from DISCO (Distance Comparison), QXP (Quick Explore), Catalyst/HipHop, and MIPSIM (Molecular Interaction Potential Similarity) allowed us to identify and locate, in a well defined spatial arrangement, three geometrically independent key structural features: (i) a positively charged nitrogen atom for ionic or hydrogen bond interactions, (ii) a lone pair of the pyridine nitrogen or a specific lone pair of a carbonyl oxygen, as a hydrogen bond acceptor, and (iii) a centre of a hydrophobic area generally occupied by aliphatic cycles. The pharmacophore presented herein, along with predictive 2D and 3D QSAR models recently developed in our group, could represent valuable computational tools for the design of new nAChR agonists having therapeutical potential.
Structural Basis for Epibatidine Selectivity at Desensitized Nicotinic Receptors
Molecular Pharmacology, 2005
The agonist binding sites of the fetal muscle nicotinic acetylcholine receptor are formed at the interfaces of ␣-subunits and neighboring ␥and ␦-subunits. When the receptor is in the nonconducting desensitized state, the ␣-␥ site binds the agonist epibatidine 200-fold more tightly than does the ␣-␦ site. To determine the structural basis for this selectivity, we constructed ␥/␦-subunit chimeras, coexpressed them with complementary wild-type subunits in HEK 293 cells, and determined epibatidine affinity of the resulting complexes. The results reveal three determinants of epibatidine selectivity: ␥104-117/␦106-␦119, ␥164-171/␦166-177, and ␥Pro190/␦Ala196. Point mutations reveal that three sequence differences within the ␥104-117/␦106-␦119 region are determinants of epibatidine selectivity: ␥Lys104/␦Tyr106, ␥Ser111/ ␦Tyr113, and ␥Tyr117/␦Tyr119. In the ␦-subunit, simultaneous mutation of these residues to their ␥ equivalent produces high affinity, ␥-like epibatidine binding. However, converting ␥ to ␦ affinity requires replacement of the ␥104-117 segment with ␦ sequence, suggesting interplay of residues in this region. The structural basis for epibatidine selectivity is explained by computational docking of epibatidine to a homology model of the ␣-␥ binding site.
ACS Chemical Biology, 2012
The nicotinic acetylcholine receptors (nAChRs) are a family of closely related but pharmacologically distinct neurotransmitter-gated ion channels. They are therapeutic targets for a wide range of neurological disorders, and a key issue in drug development is selective targeting among the greater than 20 subtypes of nAChRs that are known. The present work evaluates a proposed hydrogen bonding interaction involving a residue known as the "loop B glycine" that distinguishes receptors that are highly responsive to ACh and nicotine from those that are much less so. We have performed structure-function studies on the loop B site, including unnatural amino acid mutagenesis, in three different nAChR subtypes and found that the correlation between agonist potency and this residue is strong. Low potency receptor subtypes have a glycine at this key site, and mutation to a residue with a side chain converts a low potency receptor to a high potency receptor. Innately high potency receptors have a lysine at the loop B site and show a decrease in potency for the reverse mutation (i.e. introducing a glycine). This residue lies outside of the agonist binding site, and studies of other residues at the agonist binding site show that the details of how changes at the loop B glycine site impact agonist potency vary for differing receptor subtypes. This suggests a model in which the loop B residue influences the global shape of the agonist binding site rather than modulating any specific interaction.
Molecular pharmacology, 1995
Pharmacological properties of the (+)- and (-)-isomers of synthetic epibatidine, exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.1]heptane, were compared with nicotine and acetylcholine on several subtypes of chicken and human nicotinic acetylcholine receptors (AChRs). Both isomers of epibatidine behaved as extremely potent full agonists on chicken (alpha 3 beta 2, alpha 3 beta 4, alpha 4 beta 2, alpha 7, and alpha 8) and human (alpha 3 beta 2, alpha 3 beta 4, and alpha 7) neuronal AChRs expressed in Xenopus oocytes. Currents induced by epibatidine were effectively blocked by the nicotinic antagonists hexamethonium and mecamylamine. Apparent affinity was 100 to 1000-fold higher for epibatidine than for nicotine or acetylcholine. EC50 values ranged from 1 nM (for homomeric chicken alpha 8) to 2 microM (for homomeric chicken alpha 7). Epibatidine showed comparatively lower affinity for muscle-type AChRs from Torpedo and humans (EC50 values, 1.6 and 16 microM respectively). In binding ass...
The unusual nature of epibatidine responses at the α4β2 nicotinic acetylcholine receptor
Neuropharmacology, 2000
The identification of an equatorial frog toxin, epibatidine, as a potent non-morphinic analgesic, selective for neuronal nicotinic acetylcholine receptors, provoked a marked renewal in our understanding of pain and its mechanisms. In this work we have examined the effects of epibatidine at the major brain rat α4β2 nicotinic acetylcholine receptor expressed in a cell line. Fast drug applications obtained with a modified liquid filament system were used for the analyses of the currents evoked by acetylcholine, nicotine and epibatidine. Characterized by a slow onset and offset, epibatidine responses were of smaller amplitude to those evoked by acetylcholine or nicotine. About a thousand times more sensitive to epibatidine than acetylcholine, the α4β2 receptor also displayed a more pronounced apparent desensitization to this compound. Finally, overnight exposure to 1 nM epibatidine failed to produce the functional upregulation observed with nicotine. These data indicate that, at the rat α4β2 receptor, epibatidine acts as a partial agonist causing a pronounced inhibition of agonist evoked currents at concentrations that do not activate the receptors.
Journal of Thermodynamics & Catalysis, 2012
Nicotinic acetylcholine receptors (AChRs) are the best characterized ion channels representing the Cys-loop ligand-gated ion channel superfamily. Studies using Torpedo AChRs in the closed and open states and acetylcholine binding proteins (AChBPs) from different origins have elucidated the most important structural and functional features of the agonist/competitive antagonist binding sites. The first step in recognizing the neurotransmitter ACh and other agonists is fundamental in the process of agonist-induced activation, including the opening of the intrinsic cation channel. The AChBP studies demonstrated that Loop C is an important structural feature that is modified by ligand binding. These studies defined important pharmacologic features of AChR ligands, including the differences between full and partial agonists, agonists and competitive antagonists, peptidic and non-peptidic ligands, and between high affinity and high selectivity. The studies showing the structural mechanisms by which specific ligands can activate, inhibit, and potentiate different AChR subtypes could be of therapeutic importance.