title: Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein template.
abstract: Nicotinic acetylcholine receptors (nAChRs), which are 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 nAChRs 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 the 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 was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for 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 the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying 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.
authors.affiliation: Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Neil P
authors.initials: NP
authors.lastname: Grimster
authors.forename: Bernhard
authors.initials: B
authors.lastname: Stump
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Fotsing
authors.forename: Timo
authors.initials: T
authors.lastname: Weide
authors.forename: Todd T
authors.initials: TT
authors.lastname: Talley
authors.forename: John G
authors.initials: JG
authors.lastname: Yamauchi
authors.forename: Ákos
authors.initials: Á
authors.lastname: Nemecz
authors.forename: Choel
authors.initials: C
authors.lastname: Kim
authors.forename: Kwok-Yiu
authors.initials: KY
authors.lastname: Ho
authors.forename: K Barry
authors.initials: KB
authors.lastname: Sharpless
authors.forename: Palmer
authors.initials: P
authors.lastname: Taylor
authors.forename: Valery V
authors.initials: VV
authors.lastname: Fokin
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D018678:Cholinergic Agents / Q000138:chemical synthesis*; D057930:Click Chemistry; D008024:Ligands; D011978:Receptors, Nicotinic / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D018678:Cholinergic Agents; D008024:Ligands; D011978:Receptors, Nicotinic; D000109:Acetylcholine