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1.  Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System 
Analytical chemistry  2013;85(4):2340-2347.
Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a “fast-acting” toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10–6000 μg/kg of shellfish meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins.
doi:10.1021/ac3033432
PMCID: PMC3597463  PMID: 23343192
microsphere-based assay; marine phycotoxins; spirolide; gymnodimine; nicotinic acetylcholine receptor; acetylcholine binding protein; α-bungarotoxin
2.  Generation of Candidate Ligands for Nicotinic Acetylcholine Receptors via In Situ Click Chemistry with a Soluble Acetylcholine Binding Protein Template 
Nicotinic acetylcholine receptors (nAChRs), being responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nA-ChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building block azides and alkynes to form stable 1,2,3-triazoles generated the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product at the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at subunit interfaces of an oligomeric protein and can thus be used as a tool for identification of novel candidate nAChR ligands. The crystal structure of one of the in situ formed triazole–AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.
doi:10.1021/ja3001858
PMCID: PMC3618991  PMID: 22394239
3.  STRUCTURE-GUIDED DRUG DESIGN: CONFERRING SELECTIVITY AMONG NEURONAL NICOTINIC RECEPTOR AND ACETYLCHOLINE BINDING PROTEIN SUBTYPES 
Biochemical Pharmacology  2007;74(8):1164-1171.
Neuronal nicotinic receptors, encoded by nine genes of the alpha and three of the beta type of subunits, and whose gene products assemble in distinct permutations as pentameric molecules, constitute a fertile area for structure-guided drug design. Design strategies are augmented by a wide variety of peptide, alkaloid and terpenoid toxins from various marine and terrestrial species that interact with nicotinic receptors. Also, acetylcholinebinding proteins from mollusks, as structural surrogates of the receptor that mimic its extracellular domain, provide atomic resolution templates for analysis of structure and response. Herein, we describe a structure-guided approach to nicotinic ligand design that employs crystallography of this protein as the basic template, but also takes into consideration the dynamic properties of the receptor molecules in their biological media. We present the crystallographic structures of several complexes of various agonists and antagonists that associate with the agonist site and can competitively block the action of acetylcholine. In so far as the extracellular domain is involved, we identify additional non-competitive sites at those subunit interfaces where agonists do not preferentially bind. Ligand association at these interface sites may modulate receptor function. Ligand binding is also shown by solution-based spectroscopic and spectrometric methods to affect the dynamics of discrete domains of the receptor molecule. The surrogate receptor molecules can then be employed to design ligands selective for receptor subtype through the novel methods of freeze-frame, click chemistry that uses the very structure of the target molecule as a template for synthesis of the inhibitor.
doi:10.1016/j.bcp.2007.07.038
PMCID: PMC3341175  PMID: 17826748
Nicotinic receptor structure; Acetylcholine binding protein; Freeze-frame click chemistry; Crystal structure; Allosteric modulators; Competitive ligand site; Channel vestibule; Fluorescence anisotropy; Isotope exchange
4.  Spectroscopic Analysis of Benzylidene Anabaseine Complexes with Acetylcholine Binding Proteins as Models for Ligand–Nicotinic Receptor Interactions† 
Biochemistry  2006;45(29):8894-8902.
The discovery of the acetylcholine binding proteins (AChBPs) has provided critical soluble surrogates for examining structure and ligand interactions with nicotinic receptors and related pentameric ligand-gated ion channels. The multiple marine and freshwater sources of AChBP constitute a protein family with substantial sequence divergence and selectivity in ligand recognition for analyzing structure–activity relationships. The purification of AChBP in substantial quantities in the absence of a detergent enables one to conduct spectroscopic studies of the ligand–AChBP complexes. To this end, we have examined the interaction of a congeneric series of benzylidene-ring substituted anabaseines with AChBPs from Lymnaea, Aplysia, and Bulinus species and correlated their binding energetics with spectroscopic changes associated with ligand binding. The anabaseines display agonist activity on the α7 nicotinic receptor, a homomeric receptor with sequences similar to those of the AChBPs. Substituted anabaseines show absorbance and fluorescence properties sensitive to the protonation state, relative permittivity (dielectric constant), and the polarizability of the surrounding solvent or the proximal residues in the binding site. Absorbance difference spectra reveal that a single protonation state of the ligand binds to AChBP and that the bound ligand experiences a solvent environment with a high degree of polarizability. Changes in the fluorescence quantum yield of the bound ligand reflect the rigidification of the ring system of the bound ligand. Hence, the spectral properties of the bound ligand allow a description of the electronic character of the bound state of the ligand within its aromatic binding pocket and provide information complementary to that of crystal structures in defining the determinants of interaction.
doi:10.1021/bi060534y
PMCID: PMC3222595  PMID: 16846232
5.  Tryptophan Fluorescence Reveals Conformational Changes in the Acetylcholine Binding Protein* 
The Journal of biological chemistry  2002;277(44):41299-41302.
The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the α-neurotoxins. Since agonist binding occurs in millisecond time frames, and the α-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.
doi:10.1074/jbc.C200462200
PMCID: PMC3191908  PMID: 12235129
6.  Virtual Screening Against α-Cobratoxin 
Journal of biomolecular screening  2009;14(9):1109-1118.
α-Cobratoxin (Cbtx), the neurotoxin isolated from the venom of the Thai cobra Naja kaouthia, causes paralysis by preventing acetylcholine (ACh) binding to nicotinic acetylcholine receptors (nAChRs). In the current study, the region of the Cbtx molecule that is directly involved in binding to nAChRs is used as the target for anticobratoxin drug design. The crystal structure (1YI5) of Cbtx in complex with the acetylcholine binding protein (AChBP), a soluble homolog of the extracellular binding domain of nAChRs, was selected to prepare an α-cobratoxin active binding site for docking. The amino acid residues (Ser182-Tyr192) of the AChBP structure, the binding site of Cbtx, were used as the positive control to validate the prepared Cbtx active binding site (root mean square deviation < 1.2 Å). Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against the Cbtx active site, revealed 39 potential inhibitor candidates. The adapted in vitro radioligand competition assays using [3H]epibatidine and [125I]bungarotoxin against the AChBPs from the marine species, Aplysia californica (Ac), and from the freshwater snails, Lymnaea stagnalis (Ls) and Bolinus truncates (Bt), revealed 4 compounds from the list of inhibitor candidates that had micromolar to nanomolar interferences for the toxin binding to AChBPs. Three hits (NSC42258, NSC121865, and NSC134754) can prolong the survival time of the mice if administered 30 min before injection with Cbtx, but only NSC121865 and NSC134754 can prolong the survival time if injected immediately after injection with Cbtx. These inhibitors serve as novel templates/scaffolds for the development of more potent and specific anticobratoxin.
doi:10.1177/1087057109344617
PMCID: PMC3191909  PMID: 19734437
α-cobratoxin; virtual screening; docking; neurotoxin; nicotinic acetylcholine receptor
7.  Contemporary paradigms for cholinergic ligand design guided by biological structure 
The identification of the various nicotinic receptor subtypes, when coupled with the recent development of three-dimensional structures of surrogate extracellular receptor domains, offers new opportunities to design nicotinic ligands. Conformation and fluctuations in receptor structure are critical to ligand selectivity, and we present here how a flexible receptor template can be used in the development of selective ligands affecting cholinergic neurotransmission.
doi:10.1016/j.bmcl.2003.10.072
PMCID: PMC3191913  PMID: 15050619
8.  Curariform Antagonists Bind in Different Orientations to Acetylcholine-binding Protein* 
The Journal of biological chemistry  2003;278(25):23020-23026.
Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to ~170° rotation and ~30° degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.
doi:10.1074/jbc.M301151200
PMCID: PMC3191914  PMID: 12682067
9.  Characterizing Ligand-Gated Ion Channel Receptors with Genetically Encoded Ca++ Sensors 
PLoS ONE  2011;6(1):e16519.
We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca2+ permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors and a chimera of human α7/mouse 5-HT3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.
doi:10.1371/journal.pone.0016519
PMCID: PMC3030600  PMID: 21305050
10.  Nicotinic Agonist Binding Site Mapped by Methionine- and Tyrosine-Scanning Coupled with Azidochloropyridinyl Photoaffinity Labeling 
Journal of medicinal chemistry  2009;52(12):3735-3741.
Agonists activating nicotinic acetylcholine receptors (nAChR) include potential therapeutic agents and also toxicants such as epibatidine and neonicotinoid insecticides with a chloropyridinyl substituent. Nicotinic agonist interactions with mollusk (Aplysia californica) acetylcholine binding protein, a soluble surrogate of the nAChR extracellular domain, are precisely defined by scanning with 17 methionine and tyrosine mutants within the binding site by photoaffinity labeling with 5-azido-6-chloropyridin-3-yl probes that have similar affinities to their nonazido counterparts. Methionine and tryrosine are the only residues found derivatized, and their reactivity exquisitely depends on the direction of the azido moiety and its apposition to the reactive amino acid side chains.
doi:10.1021/jm900153c
PMCID: PMC2748672  PMID: 19459645
11.  A virtual screening study of the acetylcholine binding protein using a relaxed-complex approach 
The nicotinic acetylcholine receptor (nAChR) is a member of the ligand-gated ion channel family and is implicated in many neurological events. Yet, the receptor is difficult to target without high-resolution structures. In contrast, the structure of the acetylcholine binding protein (AChBP) has been solved to high resolution, and it serves as a surrogate structure of the extra-cellular domain in nAChR. Here we conduct a virtual screening study of the AChBP using the relaxed-complex method, which involves a combination of molecular dynamics simulations (to achieve receptor structures) and ligand docking. The library screened through comes from the National Cancer Institute, and its ligands show great potential for binding AChBP in various manners. These ligands mimic the known binders of AChBP; a significant subset docks well against all species of the protein and some distinguish between the various structures. These novel ligands could serve as potential pharmaceuticals in the AChBP/nAChR systems.
doi:10.1016/j.compbiolchem.2008.12.002
PMCID: PMC2684879  PMID: 19186108
acetylcholine binding protein; nicotinic acetylcholine receptor; relaxed-complex; molecular dynamics; docking; virtual screening

Results 1-11 (11)