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), 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.

Ruthenium-Catalyzed Azide Alkyne Cycloaddition Reaction: Scope, Mechanism, and Applications

“…A diverse set of 1,5-triazole products can be found in these reports which cover a wide range of targets and target classes including various enzyme inhibitors, [203][204][205][206][207][208][209][210][211][212][213] kinases, [214][215][216][217] proteases, 110,112 antivirals 218 (see also chapter 5.3) G-protein coupled receptors (GPCRs), 219 ion channels, [220][221][222] heat shock proteins, 95 and tRNA ligands. 223 1,5-Triazole derivatives have shown activity against numerous cancer cell lines, 205,215,[224][225][226][227] and against the parasites Trypanosoma cruzi 70,228 and Plasmodium falciparum.…”

Metal-catalyzed azide-alkyne “click” reactions: Mechanistic overview and recent trends

“…The concept of "click chemistry" proposed by Kolb, Finn and Sharpless in 2001 [1] has revolutionized molecular engineering including applications to organic and medicinal chemistry [2][3][4][5][6][7][8][9][10][11], polymer science and materials science [12][13][14][15][16][17][18][19][20][21]. Among the various "click" reactions responding to the requirements of this concept, the most generally used one is the copper(I)-catalyzed reaction between terminal alkynes and azides (CuAAC) selectively yielding 1,4-disubstituted 1,2,3-triazoles, that was reported independently by the Sharpless-Fokin [22] and the Meldal groups in 2002 [ 23,24].…”

The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry

“…124 As a member of a superfamily of neurotransmitter ligand-gated ion channels, nAChRs have been investigated as therapeutic targets for medical treatment of central nervous system (CNS) disorders such as schizophrenia, nicotine addiction, and Alzheimer's disease. [125][126][127] However, the development of novel and potent ligands for specific receptor subtypes using classical drug discovery approaches has been difficult because of the nAChR membrane disposition, receptor subtypes diversity, and the dynamic nature of the binding site.…”

“…Grimster et al turned their attention to the in situ click chemistry approach with the acetylcholine binding protein (AChBP) as a structural surrogate for nAChRs. 124 AChBPs are homologous to the N-terminal 210 amino acids in the extracellular receptor domain with flexible subunit interface, thus imitating recognition properties of nAChRs. Initially, screening the triazole library synthesized under standard Cu-catalyzed azide alkyne cycloaddition reaction conditions against AChBPs from Lymnaea stagnalis (Ls), Aplysia californica (Ac), and the Y55W Aplysia californica mutant (AcY55W) revealed compound 26 as the strongest binder to all three nAChR surrogates, with the dissociation constant in the nanomolar range for Ls AChBP (Fig.…”