Chemical Modification of the M₁ Agonist VU0364572 Reveals Molecular Switches in Pharmacology and a Bitopic Binding Mode

Abstract

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 CHOrM 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.

Effects of Selective Activation of M₁ and M₄ Muscarinic Receptors on Object Recognition Memory Performance in Rats

“…Three activators of mAChRs were administered: VU0364572, an M 1 -selective bitopic agonist [10,19], BQCA, an M 1 -selective PAM [12] and VU0152100, an M 4 -selective PAM [20]. The three doses for each drug were selected based on previous behavioral studies with these drugs [8,11,12,21].…”

Opportunities and challenges in the discovery of allosteric modulators of GPCRs for treating CNS disorders

“…Early studies suggested that M 1 -receptor allosteric agonists may have major advantages over orthosteric M 1 -receptor agonists as they are comparatively more selective for the M 1 -subtype receptors 32–36 ; however, most M 1 -receptor allosteric agonists are so-called ‘bitopic ligands’ that bind simultaneously both to an allosteric site and to the orthosteric site 37–39 and require engagement of the orthosteric site for their activity. In these cases, the compounds bind to the orthosteric sites across all mAChR subtypes, but exhibit functional selectivity for certain subtypes 38 . Most M 1 -receptor bitopic agonists tend to act as weak partial agonists at M 1 receptors and exhibit only weak agonist activity in systems where M 1 receptors are expressed with low receptor reserve.…”

“…Most M 1 -receptor bitopic agonists tend to act as weak partial agonists at M 1 receptors and exhibit only weak agonist activity in systems where M 1 receptors are expressed with low receptor reserve. Owing to this weak partial agonist activity, allosteric agonists can suffer from the same problems as do traditional M 1 -receptor orthosteric agonists and it can be difficult to achieve high selectivity 38 . Although it is not yet clear whether the same loss of selectivity would be observed with allosteric agonists of other receptor subtypes, these findings present major issues for the optimization of M 1 -receptor bitopic agonists as drug candidates owing to dose-limiting off-target activity; however, this problem could be avoided by M 1 -receptor PAMs or ago-PAMs, as long as they do not engage the acetylcholine (ACh) binding site.…”

Drugs for Allosteric Sites on Receptors

“…Such switches are by no means limited to mGlu 5 ; significant examples have been reported for the group II and III metabotropic glutamate receptors as well (68, 69). Indeed, the phenomenon of mode switching appears to be relatively widespread across a diverse set of targets for allosteric ligands, including muscarinic receptors, chemoattractant receptors, and voltage-gated potassium channels (70-73). This research has generated a large body of SAR data, but a unified vision of the underlying ligand-receptor interactions that cause mode switching has yet to be elucidated for these targets.…”