The orexin system, acting through OX1R and OX2R receptors, plays a key role in the regulation of sleep, motivation, and reward-related processes, making it a promising therapeutic target. While dual OXR antagonists are approved for the treatment of insomnia, the selective modulation of individual receptor subtypes remains poorly understood. In particular, the role of OX1R in neuropsychiatric disorders is still largely unexplored. In this study, we combined fragment-based and structure-based drug design approaches to investigate the structural determinants of underlying subtype selectivity in orexin receptor ligands. Through the exploration of novel aryl(amine) moieties, the modulation of aza-cyclic linker geometry, and the optimization of aryl amide cores, we identified key conformational features and substitution patterns governing receptor selectivity. This approach led to the discovery of antagonists with >10-30-fold selectivity for OX1R, and/or >30-100-fold selectivity for OX2R, as demonstrated by radioligand binding and Gq-mediated functional assays (IP1 and Ca2+ signaling). Given the close interplay among the orexin, dopamine and opioid systems in reward and motivation pathways, herein we further highlight the compatibility of dopaminergic and opioid-related scaffolds in expanding the chemical space for OXR ligand design.

GPR3, GPR6, and GPR12 form a subfamily of Class A orphan G protein-coupled receptors (oGPCRs), for which endogenous ligands have not been identified. Despite their high sequence similarity, each receptor exhibits unique expression profiles in human tissues. Their physiological roles and therapeutic potential are gradually being understood, indicating their critical involvement in various diseases, including central nervous system (CNS) disorders, metabolic diseases, and cancer. Notably, the GPR6 inverse agonist CVN424 is currently in Phase III clinical trials for the treatment of Parkinson's disease (PD). The recent determination of high-resolution structures of GPR3, GPR6, and GPR12 has significantly enhanced their attractiveness as emerging therapeutic targets for drug discovery. Herein, we summarize the current understanding of the structural and functional characteristics of GPR3, GPR6, and GPR12. We further highlight recent progress in relevant ligand discovery and discuss the key challenges and opportunities in developing potent and selective modulators targeting these orphan receptors.

Recent advancements in the study of orphan G protein-coupled receptors (oGPCRs) have revealed a large number with high levels of constitutive G protein signaling. Structural studies have suggested a new paradigm in which many constitutively active oGPCRs are auto-activated by their own intrinsic protein motifs, which act as auto-agonists. This includes extracellular loop 2 acting as auto-agonist to promote active-state conformations and G protein signaling. In some cases, the oGPCRs lack inhibitory microswitches that may drive a high level of constitutive activity. In this brief review, we discuss oGPCR constitutive activity, highlighting the auto-activating orphan receptors and overview structural underpinnings of constitutive activity. A discussion into the pharmacological and cell signaling implications of oGPCR constitutive activity is provided. We also propose a new concept in which orphan GPCR constitutive activity sets the baseline tone for cellular signaling and allows for dynamic regulation of cAMP signaling. Taken together, recent mechanistic studies with many oGPCRs indicate high constitutive activity is a common phenomenon that modulates cellular signaling and that can be tuned with pharmacology.

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, previously developed D1R selective agonists possess a catechol moiety which displays poor pharmacokinetic properties. The first selective non-catechol D1R agonists were recently discovered and unexpectedly many of these ligands showed G protein biased signaling. Here, we investigate both catechol and non-catechol D1R agonists to validate potential biased signaling and examine if this impacts agonist-induced D1R endocytosis. We determined that most, but not all, non-catechol agonists display G protein biased signaling at the D1R and have reduced or absent Beta-arrestin recruitment. A notable exception was compound (Cmpd) 19, a non-catechol agonist with full efficacy at both D1R-G protein or D1R Beta-arrestin pathways. In addition, the catechol ligand A-77636 was a highly potent, super agonist for D1R Beta-arrestin activity. When examined for agonist-induced D1R endocytosis, balanced agonists SKF-81297 and Cmpd 19 induced robust D1R endocytosis while the G protein biased agonists did not. The Beta-arrestin super agonist, A-77636, showed significantly increased D1R endocytosis. Moreover, Beta-arrestin recruitment efficacy of tested agonists strongly correlated with total D1R endocytosis. Taken together, these results indicate the degree of D1R signaling functional selectivity profoundly impacts D1R endocytosis regardless of pharmacophore. The range of functional selectivity of these D1R agonists will provide valuable tools to further investigate D1R signaling, trafficking and therapeutic potential.

Recent advancements in the study of orphan G protein-coupled receptors (oGPCRs) have revealed a large number with high levels of constitutive G protein signaling. Structural studies have suggested a new paradigm in which many constitutively active oGPCRs are auto-activated by their own intrinsic protein motifs, which act as auto-agonists. This includes extracellular loop 2 acting as auto-agonist to promote active-state conformations and G protein signaling. In some cases, the oGPCRs lack inhibitory microswitches that may drive a high level of constitutive activity. In this brief review, we discuss oGPCR constitutive activity, highlighting the auto-activating orphan receptors and overview structural underpinnings of constitutive activity. A discussion into the pharmacological and cell signaling implications of oGPCR constitutive activity is provided. We also propose a new concept in which orphan GPCR constitutive activity sets the baseline tone for cellular signaling and allows for dynamic regulation of cAMP signaling. Taken together, recent mechanistic studies with many oGPCRs indicate high constitutive activity is a common phenomenon that modulates cellular signaling and that can be tuned with pharmacology. SIGNIFICANCE STATEMENT: Recent literature describes a subset of orphan Class A G protein-coupled receptors with high constitutive signaling that auto-activate by their own intrinsic protein motifs. Herein, a new concept is proposed in which oGPCR constitutive activity allows dynamic regulation of cAMP signaling. Recently solved structures and functional studies of constitutively active oGPCRs provide fresh insights into oGPCR signaling with relevance for both health and disease.

Abstract ID 130118Poster Board 294GPR52 is an orphan G protein-coupled receptor (GPCR) that is a promising target for neuropsychiatric disorders including schizophrenia and substance abuse disorders. GPR52 is a constitutively active receptor that couples to Gs/olf G proteins to stimulate adenylyl cyclase and increase intracellular cAMP. As with most understudied GPCRs, there is a paucity of structural diversity among known GPR52 ligands. To expand the chemical space of GPR52 ligands, we utilized a proprietary ligand based-pharmacophore model and virtual screening method to identify new potential compounds for GPR52. Greater than 500k synthesis-ready compounds were screened in silico for their structural similarity to the GPR52 agonist c17. This screening provided a compound priority list of 400 predicted GPR52 ligands. These priority compounds were obtained and subsequently screened at 10 μM using a Glosensor cAMP assay in HEK293 cells expressing human GPR52. The primary screen showed 21 hit compounds activated GPR52 cAMP signaling at > 40% of the maximal response to the positive control agonist 4-(3-(3-fluoro-5-(trifluoromethyl) benzyl)-5-methyl-1 H-1,2,4-triazol-1-yl)-2-methylbenzamide (FTBMT). Subsequent re-testing confirmed activities, and the five most active compounds were evaluated in concentration responses to determine potency (EC50) and efficacy (Emax). All five compounds exhibited low micromolar potency (ranging from 1.6 to 4.0 μM) and were partial agonists when compared to FTBMT. Two promising validated compounds included 2-B3 (N-(5-{[3-(trifluoromethyl)phenyl]methyl}-1,3-thiazol-2-yl)cyclopropanecarboxamide) and 5-E1 (ethyl 2-benzyl-4-methyl-1,3-thiazole-5-carboxylate). Compound 2-B3 showed lower partial agonist activity (Emax = 39 ± 2%) with a potency (pEC50) of 5.80 ± 0.07 [1.6 μM]. Compound 5-E1 showed moderate partial agonist activity (Emax = 71 ± 2%) and potency (pEC50) of 5.47 ± 0.02 [3.4 μM]. 2-B3 was docked into the previously solved GPR52 crystal structure (PBD 6LI0) using AMDock program. 2-B3 docked into a similar binding site as the agonist c17, with potential hydrogen bonding at the ECL2 amino acid D188. Structural derivatives of agonist 2-B3 are being actively screened to define structure activity relationships important for GPR52 potency and efficacy. Taken together, this virtual screening with pharmacological validation provides a viable method for the discovery of ligands for orphan GPCRs. Compounds 2-B3 and 5-E1 are promising GPR52 agonist ligands amenable to further chemical optimization to expand ligand diversity for GPR52.This work was supported in part by grant# T32DA007287 from the National Institutes of Health and the UTMB Center for Addiction Sciences and Therapeutics.

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.

Abstract ID 102371Poster Board 561GPR52, identified by GWAS as a schizophrenia risk gene, is a class-A orphan G protein-coupled receptor primarily expressed in D2 medium spiny neurons in the human striatum, where it activates the Gs/cAMP signaling pathway. Recent cryo-EM and x-ray crystallography studies have suggested that GPR52 is a self-activating receptor occurring through a unique conformation of its extracellular loop 2 (ECL2) domain that interacts with a canonical orthosteric ligand-binding pocket. Here we utilize receptor mutagenesis, functional cAMP assays, and computational docking models to determine the mechanisms for both GPR52 self-activation and agonism by novel ligands. In real-time cell-based studies using the GloSensor assay, GPR52 is highly constitutively active for increasing Gs/cAMP signaling. Expression of low levels of wildtype human GPR52 in HEK293 cells increases basal cAMP levels over 100-fold, with further elevation more than 300% over basal in response to saturating concentrations of GPR52 tool agonist FTBMT and an optimized lead agonist PW0787. Molecular docking studies of both ligands into the recently solved GPR52 crystal structure identified a conserved binding mode in an allosteric side-pocket, stabilizing interactions between the ECL2, TM1, TM3, TM7, and N-terminus domains. Mutation of key residues in the receptor structure have profound effects on both constitutive GPR52 cAMP activity and agonism in HEK293 cells. Alanine-span mutation of the “agonist-like” motif of the ECL2 (residues 182-190) decreases basal cAMP by more than 80% while also eliminating all agonist activity, indicating an allosteric mechanism of agonism that requires self-activation of the receptor by the ECL2 domain. A disulfide bond from TM3 C114 to ECL2 C193 locks the ECL2 “lid” motif onto the canonical class-A orthosteric site, and disruption of this unique cysteine bond by point mutation decreases basal cAMP by more than 90%. However, agonism by FTBMT and PW0787 rescues cAMP activity back to wildtype GPR52 basal levels in these mutant receptors, supporting a model of ECL2 self-activation that can be stabilized independently by either the receptor ECL2-TM3 disulfide bond or allosterically by the agonist ligands. Mutagenesis of coordinating residues in our ligand binding model significantly impede or eliminate agonist activity for both FTBMT and PW0787, independent of effects on GPR52 basal cAMP activity. This effect is most profound in disrupting hydrogen-bonding interactions between these agonists and the ECL2, in which we find that interaction with residues 188-191 is essential for the ECL2-stabilizing agonist mechanism of action. These docking and mutagenesis studies have been used to inform our continuing drug discovery program for iterative, structure-guided design of novel small molecule GPR52 allosteric agonists with potential therapeutic use for numerous psychiatric disorders, including schizophrenia and substance use disorders.Acknowledgements: The UTMB Center for Addiction Sciences and Therapeutics, NIDA 1U18DA052543-01 (JAA), and 2022 PhRMA Foundation Pre-Doctoral Fellowship in Drug Discovery (REM).

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 55957 Poster Board 487 Andrew Frazier and John A. Allen Center for Addiction Research, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX USA Corresponding author: John A. Allen, Ph.D., Department of Pharmacology and Toxicology, 301 University Blvd., University of Texas Medical Branch, Galveston, TX, Email: joaallen@utmb.edu GPR37 is a class-A orphan G protein-coupled receptor that is most highly expressed in the human spinal cord with lesser expression in other brain regions including the amygdala, basal ganglia, hippocampus, and frontal cortex. GPR37 has been identified as a promising target for multiple conditions such as Parkinson’s disease, inflammatory pain, ischemic stroke, and some forms of cancer. GPR37 is suggested to modulate cellular signaling through the activation of Gi/o signaling pathways; however, this has not been fully verified, and a natural ligand for GPR37 has yet to be identified. Here, we sought to elucidate GPR37 cellular signaling pathways by assessing basal or agonist-stimulated changes in cAMP levels using the Glosensor assay, release or influx of calcium using a real-time fluorescence imaging plate reader (FLIPR) assay, and β-arrestin2 recruitment using the Tango assay. Through these assays, we discovered that transient expression of wild-type human GPR37 in HEK293 cells significantly decreases cAMP levels in a pertussis toxin-insensitive manner, suggesting a potential signaling mechanism involving the activation of Gz but not Gi/o. When expressed in HEK293 cells, or in human oligodendroglioma (HOG) cells, wildtype human GPR37 and the mature N-terminally truncated form of GPR37, equally reduced cAMP levels. In addition, we determined that GPR37 expression strongly recruited β-arrestin2. These cellular studies also determined two previously suggested GPR37 agonists, TX14A and NPD1, fail to induce calcium signaling using the robust FLIPR assay, in opposition to previous reports. TX14A and NPD1 also failed to show a robust activation or inhibition of cAMP signaling, but both compounds decreased basal GPR37 β-arrestin2 recruitment. The information attained from this work suggests that human GPR37 is highly constitutively active to reduce cAMP signaling and to recruit β-arrestin. Further research is needed to confirm additional GPR37 signaling mechanisms and to verify if TX14A and NPD1 are activators or inhibitors of this orphan receptor. Acknowledgments: The UTMB Center for Addiction Research Support/Funding Information: NIH NINDS R61NS12728

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).

ID 56096 Poster Board 138 Serotonin 5-HT2A receptors (5-HT2ARs) regulate mood and perception in the central nervous system, and are a molecular target for psychedelic hallucinogens, atypical antipsychotics, antidepressants, and anxiolytics. The 5-HT2AR is a seven transmembrane, G protein-coupled receptor (GPCR) that primarily signals via the Gaq family of heterotrimeric G proteins. Activation of the 5-HT2AR ultimately results in the intracellular release of Ca2+ following Gaq-mediated activation of phospholipase C (PLC) and the formation of inositol phosphates. In addition to G-protein dependent signaling, many GPCRs are now known to signal through G protein independent pathways. β-Arrestins are intracellular effector proteins that may mediate G protein independent signaling and are known to regulate G protein dependent signaling via receptor endocytosis and recycling at the plasma membrane. However, when compared to other GPCRs, the importance of β-arrestins for controlling the efficacy and duration of 5-HT2AR signaling is less defined. Live cell confocal imaging utilizing a FLAG-5-HT2AR and β-arrestin2-GFP was utilized to determine if agonist activation of 5-HT2AR receptors resulted in the recruitment of β-arrestin to the plasma membrane. Treating cells with either 5-HT (10mM) or the selective 5-HT2R agonist and hallucinogen DOI (10mM) induced a robust and rapid (within 30 secs) translocation of β-arrestin2-GFP from the cytoplasm to the plasma membrane, where it colocalized with FLAG-5-HT2AR. To determine the contributions of β-arrestin isoforms in 5-HT2AR signaling and trafficking, we utilized CRISPR/Cas9 genome editing to stably knockout (KO) β-arrestins 1 and 2. Western blots confirmed a complete loss of the β-arrestin 1 and 2 proteins in KO cells versus parent cells (WT). Using a receptor cell surface ELISA assay, we confirmed a DOI treatment (5 min) resulted in a rapid loss (∼35%) of receptors from the plasma membrane in WT cells. By comparison, 5-HT2AR endocytosis (3 min to 45 min) was significantly reduced in β-arrestin 1/2 KO cells. Kinetic live-cell Ca2+ release by the 5-HT2AR agonists (5-HT and DOI) was measured using a FLIPR assay. β-arrestin 1/2 KO cells exhibited a prolonged duration of Ca2+ signaling when compared to WT cells. Additionally, the maximal effect (Emax) of 5-HT and DOI was significantly increased (45% and 46%, respectively) in KO cells, although agonist potency was unchanged. Re-expression of β-arrestin 1 and 2 in KO cells reduced elevated agonist-mediated Ca2+ responses to that of WT cells. In addition, knockout of β-arrestin1/2 increased and prolonged the duration of 5-HT2AR agonist-mediated ERK phosphorylation. Taken together, these data indicate rapid 5-HT2AR endocytosis following activation a serotonin or hallucinogen agonist is dependent on β-arrestins, and that β-arrestins rapidly interact with 5-HT2AR receptors to limit both the intensity and duration of Gaq-mediated signal transduction. Taken together, these studies suggest an essential role of β-arrestins in regulating 5-HT2AR pharmacodynamics and the signaling responses to both serotonin and a psychedelic hallucinogen.

G protein-coupled receptors (GPCRs) are successful druggable targets, making up around 35% of all FDA-approved medications. However, a large number of receptors remain orphaned, with no known endogenous ligand, representing a challenging but untapped area to discover new therapeutic targets. Among orphan GPCRs (oGPCRs) of interest, G protein-coupled receptor 37 (GPR37) is highly expressed in the central nervous system (CNS), particularly in the spinal cord and oligodendrocytes. While its cellular signaling mechanisms and endogenous receptor ligands remain elusive, GPR37 has been implicated in several important neurological conditions, including Parkinson's disease (PD), inflammation, pain, autism, and brain tumors. GPR37 structure, signaling, emerging physiology, and pharmacology are reviewed while integrating a discussion on potential therapeutic indications and opportunities.