Characterization of PCS1055, a novel muscarinic M4 receptor antagonist

Abstract Identification of synthetic ligands selective for muscarinic receptor subtypes has been challenging due to the high sequence identity and structural homology among the five muscarinic acetylcholine receptors. Here, we report the pharmacological characterization of PCS1055, a novel muscarinic M 4 receptor antagonist. PCS1055 inhibited radioligand [ 3 H]-NMS binding to the M 4 receptor with a K i =6.5 nM. Though the potency of PCS1055 is lower than that of pan-muscarinic antagonist atropine, it has better subtype selectivity over previously reported M 4 -selective reagents such as the muscarinic-peptide toxins ( Karlsson et al., 1994 ; Santiago and Potter, 2001a ) at the M 1 subtype, and benzoxazine ligand PD102807 at the M 3 -subtype (Bohme et al., 2002). A detailed head-to-head comparison study using [ 3 H]-NMS competitive binding assays characterizes the selectivity profiles of PCS1055 to that of other potent muscarinic-antagonist compounds PD102807, tropicamide, AF-DX-384, pirenzapine, and atropine. In addition to binding studies, the subtype specificity of PCS1055 is also demonstrated by functional receptor activation as readout by GTP-γ-[ 35 S] binding. These GTP-γ-[ 35 S] binding studies showed that PCS1055 exhibited 255-, 69.1-, 342- and >1000-fold greater inhibition of Oxo-M activity at the M 4 versus the M 1 -, M 2 - , M 3 -or M 5 receptor subtypes, respectively. Schild analyses indicates that PCS1055 acts as a competitive antagonist to muscarinic M 4 receptor, and confirms the affinity of the ligand to be low nanomolar, Kb=5.72 nM. Therefore, PCS1055 represents a new M 4 -preferring antagonist that may be useful in elucidating the roles of M 4 receptor signaling.