Orphan GPR52 is emerging as a promising neurotherapeutic target. Optimization of previously reported lead 4a employing an iterative drug design strategy led to identification of a series of unique GPR52 agonists, such as 10a (PW0677), 15b (PW0729) and 24f (PW0866), with improved potency and efficacy. Intriguingly, compounds 10a and 24f showed greater bias for G protein/cAMP signaling and induced significantly less in vitro desensitization than parent compound 4a, indicating that reducing GPR52 β-arrestin activity with biased agonism results in sustained GPR52 activation. Further exploration of compounds 15b and 24f indicated improved potency and efficacy, excellent target selectivity, but limited brain exposure warranting further optimization. These balanced and biased GPR52 agonists provide important pharmacological tools to study GPR52 activation, signaling bias, and therapeutic potential for neuropsychiatric and neurological diseases.

GPR52 is a highly conserved, brain-enriched, Gs/olf-coupled orphan G protein-coupled receptor (GPCR) that controls various cyclic AMP (cAMP)-dependent physiological and pathological processes. Stimulation of GPR52 activity might be beneficial for the treatment of schizophrenia, psychiatric disorders and other human neurological diseases, whereas inhibition of its activity might provide a potential therapeutic approach for Huntington’s disease. Excitingly, HTL0048149 (HTL’149), an orally available GPR52 agonist, has been advanced into phase I human clinical trials for the treatment of schizophrenia. In this concise review, we summarize the current understanding of GPR52 receptor distribution as well as its structure and functions, highlighting the recent advances in drug discovery efforts towards small-molecule GPR52 ligands. The opportunities and challenges presented by targeting GPR52 for novel therapeutics are also briefly discussed.

ID 27887 Poster Board 537 GPR52, recently identified by GWAS as a schizophrenia risk gene, is a brain orphan G protein-coupled receptor. GPR52 is primarily expressed in D2 medium spiny neurons in the human striatum, particularly the nucleus accumbens. This unique expression profile of GPR52 suggests that the receptor may functionally regulate cAMP signaling to oppose the activity of dopamine D2 receptors. This distinguishes GPR52 as an attractive target for numerous psychiatric disorders, including schizophrenia and substance use disorders. Here we report our efforts to elucidate GPR52 neuronal signaling and to discover selective agonists for this receptor. In molecular signaling studies, expression of low levels of human GPR52 in wildtype HEK293 cells elevated basal cAMP levels over 100-fold, with further elevation of cAMP in response to the agonist FTBMT. This cAMP response was eliminated by stable knockout of Gs/olf proteins using CRISPR/Cas9 genome editing. Iterative medicinal chemistry design and pharmacological evaluation of novel small molecules led to the optimized GPR52 agonist PW0787. PW0787 increased GPR52 cAMP signaling with good potency (EC50: 135nM) and efficacy (300% over basal), while exhibiting excellent target selectivity, brain penetrance, and serum concentration. Molecular docking of PW0787 into the GPR52 crystal structure suggested compound binding with extracellular loop 2 (ECL2) and an allosteric mode of action. In a whole cell patch clamp study using mouse brain slices, PW0787 increased the frequency and number of evoked action potentials in D2, but not D1, medium spiny neurons of the nucleus accumbens. In this study, PW0787 also rescued neuronal excitability to basal levels after treatment with the dopamine D2 receptor agonist quinpirole. Dose-dependent testing of PW0787 revealed 3 and 10 mg/kg treatments in mice significantly reduced amphetamine-induced hyperlocomotion, indicating antipsychotic-like activity. Together, these findings indicate that GPR52, via Gs/olf cAMP signaling, is a highly constitutively active, excitatory receptor selectively expressed in D2 medium spiny neurons. Our drug discovery effort has resulted in novel GPR52 activators with PW0787 being a potent, selective, orally bioavailable, brain-penetrant agonist that excites D2 medium spiny neurons and shows antipsychotic-like activity. Functional alterations of striatal cAMP signaling by GPR52 may help explain why the receptor is a schizophrenia risk gene. These findings further support that GPR52 is a druggable target with therapeutic potential for treating psychiatric disorders. Acknowledgements: The UTMB Center for Addiction Research, NIDA 1U18DA052543-01 (JAA), and 2022 PhRMA Foundation Pre-Doctoral Fellowship in Drug Discovery (REM).

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.

The G protein-coupled receptor 52 (GPR52) is an orphan receptor that is selectively expressed in the striatum and regulates various brain functions through activation of cAMP-dependent pathways. GPR52 has been identified as a promising therapeutic target for central nervous system disorders including schizophrenia and substance use disorders. Here, a series of novel GPR52 agonists were designed, synthesized, and evaluated based on compound 4. Several potent and efficacious GPR52 agonists (12c, 23a, 23d, 23e, 23f, and 23h) were identified with nanomolar range potency based on a systematic structure-activity relationship exploration. Further studies of 12c indicate enhanced efficacy, excellent target selectivity, and pharmacokinetic properties including good brain permeability. In vivo proof-of-concept investigations revealed that 12c displayed antipsychotic-like activity by significantly inhibiting amphetamine-induced hyperlocomotor behavior in mice. Collectively, our findings have resulted in an efficacious, brain-penetrant GPR52 agonist as a valuable pharmacological tool for investigating the physiological and therapeutic potential of GPR52 activation.