The proposed study has grown from a collaboration of the Sharpless & Taylor laboratories to employ novel """"""""click chemistry"""""""" to develop selective ligands for nicotinic acetylcholine receptor using the acetylcholine binding protein as the reaction vessel. The precursors are anchored to two sites on the receptor and contain extended azide and acetylene moieties that react by cycloaddition to form a triazole. The precursor ligands have modest affinity for the protein and react extremely slowly in solution. On the protein surface, the apposition of the reactant groups and the amphiphilic environment of the protein forces a rapid reaction leading to formation of high affinity complex. Synthesis employs combinatorial reactants, and a single or predominant product is formed that is characterized by DIOS mass spectrometry. Interestingly, this """"""""freezeframe"""""""" procedure catches the complex in unanticipated conformations such that the high affinity regioisomer forms from a low abundance conformation. Initially acetylcholine binding proteins from Lymnaea and Aplysia are employed as templates to form selective nicotinic agents. By mutagenesis the template is modified to incorporate binding determinants of the a7 receptor and then the heterologous receptor subtypes found in the brain. Since this is effectively a """"""""freeze-frame"""""""" reaction, conformational flexibility of the binding templates will be analyzed by decay of fluorescence anisotropy in separate studies. Complexes also offer the potential for X-ray crystallographic studies. Partal agonists and antagonists will be generated from combinatorial libraries using nicotine, epibatidine or other heterocycles or bicyclic rings as the active center and peripheral site anchoring loci. Cycloaddition products will be examined for affinity and specificity for the respective binding templates. Their selectivities will then be examined on nicotinic receptors formed from transfected receptor subunit combinations. The final stages of investigation will include studies in rodents to determine efficacy and toxicity.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01DA019372-04
Application #
7242582
Study Section
Special Emphasis Panel (ZMH1-BRB-S (09))
Program Officer
Hillery, Paul
Project Start
2004-09-30
Project End
2009-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
4
Fiscal Year
2007
Total Cost
$491,341
Indirect Cost
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Yamauchi, John G; Gomez, Kimberly; Grimster, Neil et al. (2012) Synthesis of selective agonists for the ?7 nicotinic acetylcholine receptor with in situ click-chemistry on acetylcholine-binding protein templates. Mol Pharmacol 82:687-99
Nemecz, Akos; Taylor, Palmer (2011) Creating an ?7 nicotinic acetylcholine recognition domain from the acetylcholine-binding protein: crystallographic and ligand selectivity analyses. J Biol Chem 286:42555-65
Yamauchi, John G; Nemecz, Ákos; Nguyen, Quoc Thang et al. (2011) Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors. PLoS One 6:e16519
Sine, Steven M; Wang, Hai-Long; Hansen, Scott et al. (2010) On the origin of ion selectivity in the Cys-loop receptor family. J Mol Neurosci 40:70-6
Bourne, Yves; Radic, Zoran; Taylor, Palmer et al. (2010) Conformational remodeling of femtomolar inhibitor-acetylcholinesterase complexes in the crystalline state. J Am Chem Soc 132:18292-300
Nguyen, Quoc-Thang; Schroeder, Lee F; Mank, Marco et al. (2010) An in vivo biosensor for neurotransmitter release and in situ receptor activity. Nat Neurosci 13:127-132
Hibbs, Ryan E; Sulzenbacher, Gerlind; Shi, Jianxin et al. (2009) Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal alpha7 nicotinic acetylcholine receptor. EMBO J 28:3040-51
Luo, Jie; Taylor, Palmer; Losen, Mario et al. (2009) Main immunogenic region structure promotes binding of conformation-dependent myasthenia gravis autoantibodies, nicotinic acetylcholine receptor conformation maturation, and agonist sensitivity. J Neurosci 29:13898-908
Rana, Brinda K; Wessel, Jennifer; Mahboubi, Vafa et al. (2009) Natural variation within the neuronal nicotinic acetylcholine receptor cluster on human chromosome 15q24: influence on heritable autonomic traits in twin pairs. J Pharmacol Exp Ther 331:419-28
Hansen, Scott B; Wang, Hai-Long; Taylor, Palmer et al. (2008) An ion selectivity filter in the extracellular domain of Cys-loop receptors reveals determinants for ion conductance. J Biol Chem 283:36066-70

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