Journal of the American Chemical Society, Vol.139, No.10, 3676-3684, 2017
Substituted 2-Aminopyrimidines Selective for alpha 7-Nicotinic Acetylcholine Receptor Activation and Association with Acetylcholine Binding Proteins
Through studies with ligand binding to the acetylcholine binding protein (AChBP), we previously identified a series of 4,6-substituted 2-aminopyrimidines that associate with this soluble surrogate of the nicotinic acetylcholine receptor (nAChR) in a cooperative fashion, not seen for classical nicotinic agonists and antagonists. To examine receptor interactions of this structural family on ligand-gated ion channels, we employed HEK cells transfected with cDNAs encoding three requisite receptor subtypes: alpha 7-nAChR, alpha 4 beta 2-nAChR, and a serotonin receptor (5-HT3AR), along with a fluorescent reporter. Initial screening of a series of over 50 newly characterized 2-aminopyrimidines with affinity for AChBP showed only two to be agonists on the alpha 7-nAChR below 10 mu M concentration. Their unique structural features were incorporated into design of a second subset of 2-aminopyrimidines yielding several congeners that elicited alpha 7 activation with EC50 values of 70 nM and K-d values for AChBP in a similar range. Several compounds within this series exhibit specificity for the a7-nAChR, showing no activation or antagonism of alpha 4 beta 2-nAChR or 5-HT3AR at concentrations up to 10 mu M, while others were weaker antagonists (or partial agonists) on these receptors. Analysis following cocrystallization of four ligand complexes with AChBP show binding at the subunit interface, but with an orientation or binding pose that differs from classical nicotinic agonists and antagonists and from the previously analyzed set of 2-aminopyrimidines that displayed distinct cooperative interactions with AChBP. Orientations of aromatic side chains of these complexes are distinctive, suggesting new modes of binding at the agonist-antagonist site and perhaps an allosteric action for heteromeric nAChRs.