TY - JOUR T1 - Characterization of the Novel Positive Allosteric Modulator, LY2119620, at the Muscarinic M<sub>2</sub> and M<sub>4</sub> Receptors JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.114.091751 SP - mol.114.091751 AU - Carrie H. Croy AU - Douglas A. Schober AU - Hongling Xiao AU - Anne Quets AU - Arthur Christopoulos AU - Christian C. Felder Y1 - 2014/01/01 UR - http://molpharm.aspetjournals.org/content/early/2014/05/07/mol.114.091751.abstract N2 - The M4 receptor is a compelling therapeutic target as this receptor modulates neural-circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both anti-psychotic and pro-cognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. Here we present the pharmacological characterization of LY2119620, an M2/M4 receptor selective positive-allosteric modulator (PAM), chemically evolved from hits identified through an M4-allosteric functional screen. Though unsuitable as a therapeutic due to M2 receptor cross-reactivity and thus potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity, and broadens research capabilities through its development into a radio-tracer. Characterization of LY2119620 revealed evidence of probe-dependence in both binding and functional assays. GTP-γ-S assays displayed differential potentiation depending on the orthosteric-allosteric pairing with the largest cooperativity observed for Oxo-M-LY2119620. Further [3H]-Oxo-M saturation binding, including studies with GppNHp, suggest that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M4 receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately it was the M2 receptor pharmacology and PAM activity with iperoxo which made LY2119620 the most suitable allosteric partner for the M2 active-state structure recently solved (Kruse et al. 2013); a structure which provides crucial insights into the mechanisms of orthosteric activation and allosteric modulation of muscarinic receptors. ER -