Generation of selective ligands for nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are members of the cys‐loop receptor family located in synapses of the CNS, neuromuscular junctions, and autonomic ganglia. The α4β2 and α7 subtypes are the most abundant isoforms in the brain and associated with a range of disorders including; smoking addiction, schizophrenia, and Alzheimer's disease making them attractive pharmaceutical targets. Subtype selective ligands have been difficult to design due to the conserved nature of the binding site. The Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins (AChBPs) are structural surrogates for the extracellular domain of nAChRs. We mutated regions of the binding pocket with the Aplysia AChBP towards α7 to obtain pentameric chimeras. We employed a structure‐guided strategy utilizing wildtype and α7‐chimera AChBPs to catalyze in situ azide‐alkyne triazole cycloaddition reactions generating compounds targeted for the α7 nAChR. Responses for α7 and α4β2 nAChRs were characterized using an in vitro fluorescent functional assay using a genetically encoded Ca 2+ indicator. The majority of compounds were α7 agonists with some showing selectivity over α4β2. All compounds with α4β2 activity were identified as antagonists. These results illustrate the utility of in situ click chemistry and its potential expansion into membrane protein templates for generating selective allosteric modulators. Support: R37‐GM18360, UO1‐ DA019372 , NSF GK12 0742551