Discovery of Balanced and Novel G Protein Biased Agonists for the Orphan Receptor GPR52
Authors: Murphy Ryan E. , Felsing Daniel E. , Wang Pingyuan , Chen Haiying , Zhou Jia , Allen John A. 
Published: May 2022

GPR52 is an orphan class‐A G protein‐coupled receptor that activates the Gs/cAMP signaling pathway and is primarily co‐expressed in the human striatum with the dopamine D2 receptor. The unique expression profile of GPR52 has distinguished this orphan receptor as a promising drug target for psychiatric disorders including schizophrenia, Huntington’s disease, and substance use disorders. We recently synthesized and pharmacologically evaluated a series of novel indoline‐carboxamide based GPR52 agonists in which the lead compound shows antipsychotic‐like activity to inhibit amphetamine‐induced hyperlocomotion in mice (Wang, Felsing et al J. Med. Chem. 2020 Nov 25;63(22):13951‐13972). Here, we describe the evaluation of a new series of novel aniline‐carboxamide GPR52 agonists with G protein biased activity relative to the parent indoline‐carboxamide compound. In a HEK293 cell‐based cAMP assay, we observed substantial increases in efficacy (>200%) over the parent compound with the opening of the indoline ring. Substitutions around the aniline and lower aromatic moieties are amenable to medicinal chemistry and modulate both potency and efficacy. We then tested a selection of the most potent compounds (EC50: ~30‐200 nM) in a cell‐based β‐arrestin TANGO recruitment assay. The opening of the indoline ring in the parent compound yields over 20‐fold decrease in potency for β‐arrestin activation, with further modulation of potency resulting from modifications to the aniline and lower aromatic moieties. Preliminary results also indicate several of the G protein biased agonists induce less GPR52 desensitization for cAMP signaling when compared to balanced agonists. To identify variations in binding modes that might confer this bias for G protein signaling, we docked the parent compound, along with both a highly biased compound and a balanced compound, into the recently discovered GPR52 crystal structure. The compounds display a conserved position within the binding pocket with no obvious alterations to the protein‐ligand interactions to explain any differences in functional selectivity. GPR52 has been suggested to be self‐activating through its extracellular loop 2 domain (ECL2). The binding mode of our agonists supports an allosteric mode of action, potentiating the activity induced by ECL2 interactions with the typical class‐A GPCR orthosteric pocket. To assess the necessity of the ECL2 interaction for agonist activity, we generated a mutated GPR52 in which the ECL2 was replaced with an equivalent span of alanine residues. In our cell‐based cAMP assay, this mutant GPR52 greatly reduces constitutive signaling of the receptor and eliminates agonist response entirely. Together, our studies have resulted in novel GPR52 agonists with optimized potency and efficacy, demonstrated potential for optimization of functional selectivity for G protein signaling, and supplied evidence of GPR52 self‐activation and an allosteric mechanism of action for these novel GPR52 agonists.