Chemical modification of the M(1) agonist VU0364572 reveals molecular switches in pharmacology and a bitopic binding mode.

Abstract

We previously reported the discovery of VU0364572 and VU0357017 as M(1)-selective agonists that appear to activate M(1) through actions at an allosteric site. Previous studies have revealed that chemical scaffolds for many allosteric modulators contain molecular switches that allow discovery of allosteric antagonists and allosteric agonists or positive allosteric modulators (PAMs) based on a single chemical scaffold. Based on this, we initiated a series of studies to develop selective M(1) allosteric antagonists based on the VU0364572 scaffold. Interestingly, two lead antagonists identified in this series, VU0409774 and VU0409775, inhibited ACh-induced Ca(2+) responses at rat M(1-5) receptor subtypes, suggesting they are nonselective muscarinic antagonists. VU0409774 and VU0409775 also completely displaced binding of the nonselective radioligand [(3)H]-NMS at M(1) and M(3) mAChRs with affinities similar to their functional IC(50) values. Finally, Schild analysis revealed that these compounds inhibit M(1) responses through a fully competitive interaction at the orthosteric binding site. This surprising finding prompted further studies to determine whether agonist activity of VU0364572 and VU0357017 may also engage in previously unappreciated actions at the orthosteric site on M(1). Surprisingly, both VU0364572 and VU0357017 completely displaced [(3)H]-NMS binding to the orthosteric site of M(1)-M(5) receptors at high concentrations. Furthermore, evaluation of agonist activity in systems with varying levels of receptor reserve and Furchgott analysis using a cell line expressing M(1) under control of an inducible promotor was consistent with an action of these compounds as weak orthosteric partial agonists of M(1). However, consistent with previous studies suggesting actions at a site that is distinct from the orthosteric binding site, VU0364572 or VU0357017 slowed the rate of [(3)H]-NMS dissociation from CHO-rM(1) membranes. Together, these results suggest that VU0364572 and VU0357017 act as bitopic ligands and that novel antagonists in this series act as competitive orthosteric site antagonists.