Abstract

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.