Synthesis and pharmacological validation of a novel radioligand for the orphan GPR88 receptor.

GPR88 is an orphan G protein-coupled receptor which has been implicated in a number of striatal-associated disorders. Herein we describe the synthesis and pharmacological characterization of the first GPR88 radioligand, [3H]RTI-33, derived from a synthetic agonist RTI-13951-33. [3H]RTI-33 has a specific activity of 83.4 Ci/mmol and showed one-site, saturable binding (KD of 85 nM) in membranes prepared from stable PPLS-HA-hGPR88-CHO cells. A competition binding assay was developed to determine binding affinities of several known GPR88 agonists. This radioligand represents a powerful tool for future mechanistic and cell-based ligand-receptor interaction studies of GPR88.

The Orphan GPCR Receptor, GPR88, Interacts with Nuclear Protein Partners in the Cerebral Cortex.

GPR88 is an orphan G-protein-coupled receptor (GPCR) highly expressed in striatal medium spiny neurons (MSN), also found in cortical neurons at low level. In MSN, GPR88 has a canonical GPCR plasma membrane/cytoplasmic expression, whereas in cortical neurons, we previously reported an atypical intranuclear localization. Molecular size analysis suggests that GPR88, expressed in plasma membrane of MSN or in nuclear compartment of cortical neurons, corresponds to the full-length protein. By transfection of cortical neurons, we showed that GPR88 fluorescent chimeras exhibit a nuclear localization. This localization is contingent on the third intracytoplasmic loop and C-terminus domains, even though these domains do not contain any known nuclear localization signals (NLS). Using yeast two-hybrid screening with these domains, we identified the nuclear proteins ATRX, TOP2B, and BAZ2B, all involved in chromatin remodeling, as potential protein partners of GPR88. We also validated the interaction of GPR88 with these nuclear proteins by proximity ligation assay on cortical neurons in culture and coimmunoprecipitation experiments on cortical extracts from GPR88 wild-type (WT) and knockout (KO) mice. The identification of GPR88 subcellular partners may provide novel functional insights for nonclassical modes of GPCR action that could be relevant in the maturating process of neocortical neurons.

The GPR88 agonist RTI-13951-33 reduces alcohol drinking and seeking in mice.

GPR88 is an orphan G-protein-coupled receptor that is considered a potential target to treat neuropsychiatric disorders, including addiction. Most knowledge about GPR88 function stems from knockout mouse studies, and in vivo pharmacology is still scarce. Here we examine the effects of the novel brain-penetrant agonist RTI-13951-33 on several alcohol-related behaviours in the mouse. In the intermittent-access-two-bottle-choice paradigm, the compound reduced excessive voluntary alcohol drinking, while water drinking was intact. This was observed for C57BL/6 mice, as well as for control but not Gpr88 knockout mice, demonstrating efficacy and specificity of the drug in vivo. In the drinking-in-the-dark paradigm, RTI-13951-33 also reduced binge-like drinking behaviour for control but not Gpr88 knockout mice, confirming the alcohol consumption-reducing effect and in vivo specificity of the drug. When C57BL/6 mice were trained for alcohol self-administration, RTI-13951-33 decreased the number of nose-pokes over a 4-h session and reduced the number of licks and bursts of licks, suggesting reduced motivation to obtain alcohol. Finally, RTI-13951-33 did not induce any place preference or aversion but reduced the expression of conditioned place preference to alcohol, indicative of a reduction of alcohol-reward seeking. Altogether, data show that RTI-13951-33 limits alcohol intake under distinct conditions that require consummatory behaviour, operant response or association with contextual cues. RTI-13951-33 therefore is a promising lead compound to evaluate GPR88 as a therapeutic target for alcohol use disorders. More broadly, RTI-13951-33 represents a unique tool to better understand GPR88 function, disentangle receptor roles in development from those in the adult and perhaps address other neuropsychiatric disorders.

Delta Opioid Receptors: Learning and Motivation.

Delta opioid receptor (DOR) displays a unique, highly conserved, structure and an original pattern of distribution in the central nervous system, pointing to a distinct and specific functional role among opioid peptide receptors. Over the last 15 years, in vivo pharmacology and genetic models have allowed significant advances in the understanding of this role. In this review, we will focus on the involvement of DOR in modulating different types of hippocampal- and striatal-dependent learning processes as well as motor function, motivation, and reward. Remarkably, DOR seems to play a key role in balancing hippocampal and striatal functions, with major implications for the control of cognitive performance and motor function under healthy and pathological conditions.

Development and validation of a high-throughput calcium mobilization assay for the orphan receptor GPR88.

BACKGROUND: GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions. METHODS: In this paper, we describe the development of a CHO-Gαqi5-GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gαqi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader. RESULTS: The CHO-Gαqi5-GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z'-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays. CONCLUSIONS: We have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.

Design, synthesis and pharmacological evaluation of 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives as GPR88 agonists.

The orphan receptor GPR88 is an attractive therapeutic target because of its implications in a number of basal ganglia-associated disorders. To date, pharmacological characterization of GPR88 has been limited due to the lack of potent and selective agonists and antagonists appropriate for CNS investigations. We have previously reported that GPR88 couples to Gαi proteins and modulates cAMP levels upon treatment with a small molecule agonist 2-PCCA. Recently, another chemotype of GPR88 agonist, represented by 2-AMPP [(2S)-N-((1R)-2-amino-1-(4-(2-methylpentyloxy)-phenyl)ethyl)-2-phenylpropanamide], has also been discovered. In this report, a new series of 2-AMPP structurally related 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives have been designed and evaluated for agonist activity at GPR88. The structure-activity relationship (SAR) studies suggest that the amine group in 2-AMPP can be replaced by hydroxyl, ester and amide groups, resulting in analogues with good to moderate potency, whereas the phenyl group on the amide cap is essential for activity and has limited size, shape and electronic tolerance.

Possible involvement of orphan receptors GPR88 and GPR124 in the development of hypertension in spontaneously hypertensive rat.

Hypertension (HBP) is a chronic disease characterized by increased blood pressure, which despite several treatments maintains a high morbi-mortality, which suggests that there are other mechanisms involved in this pathology, within which the orphan receptors could be candidates for the treatment of the HBP; these receptors are called orphan receptors because their ligand is unknown. These receptors have been suggested to participate in some pathologies because they are associated with various systems such as GPR88, which has been linked to the dopaminergic system, and GPR124 with angiogenesis, suggesting that these receptors could take part in HBP. Hence, the aim of this work was to study the expression of orphan receptors GPR88 and GPR124 in various tissues of normotensive and hypertensive rats. We used Wistar Kyoto (WKY) and spontaneously hypertensive rat (SHR) of 6-8 and 10-12 weeks of age and we determined systolic blood pressure (SBP), heart rate, as well as mRNA of GPR88 and GPR124 receptors by reverse transcription polymerase chain reaction (RT-PCR) in the aorta, heart, kidney, and brain. Our results showed that GPR88 and GPR124 were expressed in all analyzed tissues, but their expression is dependent on the age and development of HBP because their expression tends to be modified as HBP is established. Therefore, we conclude that GPR88 and GPR124 receptors may be involved in the development or maintenance of high blood pressure.

The discovery of potent agonists for GPR88, an orphan GPCR, for the potential treatment of CNS disorders.

Modulating GPR88 activity is suggested to have therapeutic utility in the treatment of CNS disorders, such as schizophrenia. This Letter will describe the discovery and SAR development of a class of potent GPR88 agonists.

Design, synthesis, and evaluation of phenylglycinols and phenyl amines as agonists of GPR88.

Small molecule modulators of GPR88 activity (agonists, antagonists, or modulators) are of interest as potential agents for the treatment of a variety of psychiatric disorders including schizophrenia. A series of phenylglycinol and phenylamine analogs have been prepared and evaluated for their GPR88 agonist activity and pharmacokinetic (PK) properties.

Novel Therapeutic GPCRs for Psychiatric Disorders.

G protein-coupled receptors (GPCRs) are the most common targets of the neuropharmacological drugs in the central nervous system (CNS). GPCRs are activated by manifold neurotransmitters, and their activation in turn evokes slow synaptic transmission. They are deeply involved in multiple neurological and psychiatric disorders such as Parkinson's disease and schizophrenia. In the brain, the striatum is strongly innervated by the ventral tegmental area (VTA) and plays a central role in manifestation of psychiatric disorders. Recently, anatomical and comprehensive transcriptome analysis of the non-odorant GPCR superfamily revealed that the orphan GPCRs GPR88, GPR6, and GPR52, as well as dopamine D1 and D2 receptors and the adenosine A2a receptor, are the most highly enriched in the rodent striatum. Genetically engineered animal models and molecular biological studies have suggested that these striatally enriched GPCRs have a potential to be therapeutic psychiatric receptors. This review summarizes the current understanding of the therapeutic GPCR candidates for psychiatric disorders.

Design, Synthesis, and Structure-Activity Relationship Studies of Novel GPR88 Agonists (4-Substituted-phenyl)acetamides Based on the Reversed Amide Scaffold.

The development of synthetic agonists for the orphan receptor GPR88 has recently attracted significant interest, given the promise of GPR88 as a novel drug target for psychiatric and neurodegenerative disorders. Examination of structure-activity relationships of two known agonist scaffolds 2-PCCA and 2-AMPP, as well as the recently resolved cryo-EM structure of 2-PCCA-bound GPR88, led to the design of a new scaffold based on the "reversed amide" strategy of 2-AMPP. A series of novel (4-substituted-phenyl)acetamides were synthesized and assessed in cAMP accumulation assays as GPR88 agonists, which led to the discovery of several compounds with better or comparable potencies to 2-AMPP. Computational docking studies suggest that these novel GPR88 agonists bind to the same allosteric site of GPR88 that 2-PCCA occupies. Collectively, our findings provide structural insight and SAR requirement at the allosteric site of GPR88 and a new scaffold for further development of GPR88 allosteric agonists.

Orphan peptide and G protein-coupled receptor signalling in alcohol use disorder.

Neuropeptides and G protein-coupled receptors (GPCRs) have long been, and continue to be, one of the most popular target classes for drug discovery in CNS disorders, including alcohol use disorder (AUD). Yet, orphaned neuropeptide systems and receptors (oGPCR), which have no known cognate receptor or ligand, remain understudied in drug discovery and development. Orphan neuropeptides and oGPCRs are abundantly expressed within the brain and represent an unprecedented opportunity to address brain function and may hold potential as novel treatments for disease. Here, we describe the current literature regarding orphaned neuropeptides and oGPCRs implicated in AUD. Specifically, in this review, we focus on the orphaned neuropeptide cocaine- and amphetamine-regulated transcript (CART), and several oGPCRs that have been directly implicated in AUD (GPR6, GPR26, GPR88, GPR139, GPR158) and discuss their potential and pitfalls as novel treatments, and progress in identifying their cognate receptors or ligands.

Gpr88 Deletion Impacts Motivational Control Without Overt Disruptions to Striatal Dopamine.

Background: Disrupted motivational control is a common-but poorly treated-feature of psychiatric disorders, arising via aberrant mesolimbic dopaminergic signaling. GPR88 is an orphan G protein-coupled receptor that is highly expressed in the striatum and therefore well placed to modulate disrupted signaling. While the phenotype of Gpr88 knockout mice suggests a role in motivational pathways, it is unclear whether GPR88 is involved in reward valuation and/or effort-based decision making in a sex-dependent manner and whether this involves altered dopamine function. Methods: In male and female Gpr88 knockout mice, we used touchscreen-based progressive ratio, with and without reward devaluation, and effort-related choice tasks to assess motivation and cost/benefit decision making, respectively. To explore whether these motivational behaviors were related to alterations in the striatal dopamine system, we quantified expression of dopamine-related genes and/or proteins and used [18F]DOPA positron emission tomography and GTPγ[35S] binding to assess presynaptic and postsynaptic dopamine function, respectively. Results: We showed that male and female Gpr88 knockout mice displayed greater motivational drive than wild-type mice, which was maintained following reward devaluation. Furthermore, we showed that cost/benefit decision making was impaired in male, but not female, Gpr88 knockout mice. Surprisingly, we found that Gpr88 deletion had no effect on striatal dopamine by any of the measures assessed. Conclusions: Our results highlight that GPR88 regulates motivational control but that disruption of such behaviors following Gpr88 deletion occurs independently of gross perturbations to striatal dopamine at a gene, protein, or functional level. This work provides further insights into GPR88 as a drug target for motivational disorders.

Orphan receptor GPR88 as a potential therapeutic target for CNS disorders - an in silico approach.

The G-protein-coupled receptors are a part of the largest and most physiologically relevant family of membrane proteins. One-third of the medications, now on the market, target the GPCR receptor family, which is one of the most important therapeutic targets for many disorders. In the reported work, we have focussed on orphan GPR88 receptor which is a part of the GPCR protein family and a potential target for central nervous system disorders. GPR88 is known to show the highest expression in the striatum, which is a key region in motor control and cognitive functions. Recent studies have reported that GPR88 is activated by two agonists, 2-PCCA and RTI-13951-33. In this study, we have predicted the three-dimensional protein structure for the orphan GPR88 using the homology modeling approach. We then used shape-based screening techniques based on known agonists and structure-based virtual screening methods employing docking to uncover novel GPR88 ligands. The screened GPR88-ligand complexes were further subjected to molecular dynamics simulation studies. The selected ligands could fasten the development of novel treatments for the vast list of movement and central nervous system disorders.Communicated by Ramaswamy H. Sarma.

Genetic deletion of GPR88 enhances the locomotor response to L-DOPA in experimental parkinsonism while counteracting the induction of dyskinesia.

Parkinson's disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons and treated with the dopamine precursor, 3,4-dihydroxy-l-phenylalanine (L-DOPA). Prolonged L-DOPA treatment is however associated with waning efficacy and the induction of L-DOPA induced dyskinesia (LID). GPR88 is an orphan G-protein Coupled Receptor (GPCR) expressed in dopaminoceptive striatal medium spiny neurons (MSNs) and their afferent corticostriatal glutamatergic neurons. Here, we studied the role of GPR88 in experimental parkinsonism and LID. Chronic L-DOPA administration to male GPR88 KO mice, subjected to unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle, resulted in more rotations than in their WT counterparts. Conversely, GPR88 KO mice had a lower abnormal involuntary movements (AIMs) score. These behavioral responses were accompanied by altered transcription of L-DOPA upregulated genes in lesioned GPR88 KO compared to WT striata. In accordance with a role for serotonin neurons in LID development, WT but not GPR88 KO striata exhibited 5-hydroxytryptamine displacement upon repeated L-DOPA treatment. Intact male GPR88 KO mice showed diminished tacrine-induced PD-like tremor and spontaneous hyperlocomotion. Dopamine and its metabolites were not increased in male GPR88 KO mice, but biosensor recordings revealed increased spontaneous/basal and evoked glutamate release in striata of male GPR88 KO mice. In conclusion, genetic deletion of GPR88 promotes l-DOPA-induced rotation and spontaneous locomotion yet suppresses the induction of LIDs and also reduces tremor. These data provide behavioral, neurochemical and molecular support that GPR88 antagonism may favour motor relief in PD patients without aggravating the induction of motor side effects.

Orphan Receptor GPR88 as an Emerging Neurotherapeutic Target.

Although G protein-coupled receptors (GPCRs) are recognized as pivotal drug targets involved in multiple physiological and pathological processes, the majority of GPCRs including orphan GPCRs (oGPCRs) are unexploited. GPR88, a brain-specific oGPCR with particularly robust expression in the striatum, regulates diverse brain and behavioral functions, including cognition, mood, movement control, and reward-based learning, and is thus emerging as a novel drug target for central nervous system disorders including schizophrenia, Parkinson's disease, anxiety, and addiction. Nevertheless, no effective GPR88 synthetic ligands have yet entered into clinical trials, and GPR88 endogenous ligands remain unknown. Despite the recent discovery and early stage study of several GPR88 agonists, such as 2-PCCA, RTI-13951-33, and phenylglycinol derivatives, further research into GPR88 pharmacology, medicinal chemistry, and chemical biology is urgently needed to yield structurally diversified GPR88-specific ligands. Drug-like pharmacological tool function and relevant signaling elucidation will also accelerate the evaluation of this receptor as a viable neurotherapeutic target.

Knock-Down of GPR88 in the Dorsal Striatum Alters the Response of Medium Spiny Neurons to the Loss of Dopamine Input and L-3-4-Dyhydroxyphenylalanine.

The effects of L-3-4-dyhydroxyphenylalanine (L-DOPA) treatment for replacing the dopamine (DA) loss in Parkinson's disease (PD) progressively wear off and are hindered by the development of dyskinesia, prompting the search for new treatments. The orphan G protein-coupled receptor 88 (Gpr88) represents a potential new target, as it is highly and almost exclusively expressed in the projecting gamma-Aminobutyric Acid-ergic (GABAergic) medium spiny neurons of the striatum, is implicated in motor activity, and is downregulated by 6-hydroxydopamine (6-OHDA) lesions, an effect that is reversed by L-DOPA. Thus, to evaluate Gpr88 as a potential target for the management of PD and L-DOPA-induced dyskinesia (LID), we inactivated Gpr88 by lentiviral-mediated knock-down with a specifically designed microRNA (miR) (KD-Gpr88) in a 6-OHDA rat model of hemiparkinsonism. Then, we investigated the effects of the KD-Gpr88 in the DA-deprived dorsal striatum on circling behavior and LID as well as on specific markers of striatal neuron activity. The KD-Gpr88 reduced the acute amphetamine-induced and increased L-DOPA-induced turning behavior. Moreover, it normalized the upregulated expression of striatal Gad67 and proenkephalin provoked by the 6-OHDA lesion. Finally, despite promoting ΔFosB accumulation, the KD-Gpr88 was associated neither with the upregulation of prodynorphin, which is causally linked to the severity of LID, nor with the aggravation of LID following chronic L-DOPA treatment in 6-OHDA-lesioned rats. These results thus justify further evaluation of Gpr88 as a potentially novel target for the management of PD as an alternative to L-DOPA therapy.

Lack of anticipatory behavior in Gpr88 knockout mice showed by automatized home cage phenotyping.

Mouse models are widely used to understand genetic bases of behavior. Traditional testing typically requires multiple experimental settings, captures only snapshots of behavior and involves human intervention. The recent development of automated home cage monitoring offers an alternative method to study mouse behavior in their familiar and social environment, and over weeks. Here, we used the IntelliCage system to test this approach for mouse phenotyping, and studied mice lacking Gpr88 that have been extensively studied using standard testing. We monitored mouse behavior over 22 days in 4 different phases. In the free adaptation phase, Gpr88 -/- mice showed delayed habituation to the home cage, and increased frequency of same corner returns behavior in their alternation pattern. In the following nose-poke adaptation phase, non-habituation continued, however, mutant mice acquired nose-poke conditioning similar to controls. In the place learning and reversal phase, Gpr88-/- mice developed preference for the water/sucrose corner with some delay, but did not differ from controls for reversal. Finally, in a fixed schedule-drinking phase, control animals showed higher activity during the hour preceding water accessibility, and reduced activity after access to water was terminated. Mutant mice did not show this behavior, showing lack of anticipatory behavior. Our data therefore confirm hyperactivity, non-habituation and altered exploratory behaviors that were reported previously. Learning deficits described in other settings were barely detectable, and a novel phenotype was discovered. Home cage monitoring therefore extends previous findings and shows yet another facet of GPR88 function that deserves further investigation.

Mapping GPR88-Venus illuminates a novel role for GPR88 in sensory processing.

GPR88 is an orphan G-protein coupled receptor originally characterized as a striatal-enriched transcript and is a potential target for neuropsychiatric disorders. At present, gene knockout studies in the mouse have essentially focused on striatal-related functions and a comprehensive knowledge of GPR88 protein distribution and function in the brain is still lacking. Here, we first created Gpr88-Venus knock-in mice expressing a functional fluorescent receptor to fine-map GPR88 localization in the brain. The receptor protein was detected in neuronal soma, fibers and primary cilia depending on the brain region, and remarkably, whole-brain mapping revealed a yet unreported layer-4 cortical lamination pattern specifically in sensory processing areas. The unique GPR88 barrel pattern in L4 of the somatosensory cortex appeared 3 days after birth and persisted into adulthood, suggesting a potential function for GPR88 in sensory integration. We next examined Gpr88 knockout mice for cortical structure and behavioral responses in sensory tasks. Magnetic resonance imaging of live mice revealed abnormally high fractional anisotropy, predominant in somatosensory cortex and caudate putamen, indicating significant microstructural alterations in these GPR88-enriched areas. Further, behavioral analysis showed delayed responses in somatosensory-, visual- and olfactory-dependent tasks, demonstrating a role for GPR88 in the integration rather than perception of sensory stimuli. In conclusion, our data show for the first time a prominent role for GPR88 in multisensory processing. Because sensory integration is disrupted in many psychiatric diseases, our study definitely positions GPR88 as a target to treat mental disorders perhaps via activity on cortical sensory networks.

Increased Alcohol Seeking in Mice Lacking Gpr88 Involves Dysfunctional Mesocorticolimbic Networks.

BACKGOUND: Alcohol use disorder (AUD) is devastating and poorly treated, and innovative targets are actively sought for prevention and treatment. The orphan G protein-coupled receptor GPR88 is enriched in mesocorticolimbic pathways, and Gpr88 knockout mice show hyperactivity and risk-taking behavior, but a potential role for this receptor in drug abuse has not been examined. METHODS: We tested Gpr88 knockout mice for alcohol-drinking and -seeking behaviors. To gain system-level understanding of their alcohol endophenotype, we also analyzed whole-brain functional connectivity in naïve mice using resting-state functional magnetic resonance imaging. RESULTS: Gpr88 knockout mice showed increased voluntary alcohol drinking at both moderate and excessive levels, with intact alcohol sedation and metabolism. Mutant mice also showed increased operant responding and motivation for alcohol, while food and chocolate operant self-administration were unchanged. Alcohol place conditioning and alcohol-induced dopamine release in the nucleus accumbens were decreased, suggesting reduced alcohol reward in mutant mice that may partly explain enhanced alcohol drinking. Seed-based voxelwise functional connectivity analysis revealed significant remodeling of mesocorticolimbic centers, whose hallmark was predominant weakening of prefrontal cortex, ventral tegmental area, and amygdala connectional patterns. Also, effective connectivity from the ventral tegmental area to the nucleus accumbens and amygdala was reduced. CONCLUSIONS: Gpr88 deletion disrupts executive, reward, and emotional networks in a configuration that reduces alcohol reward and promotes alcohol seeking and drinking. The functional connectivity signature is reminiscent of alterations observed in individuals at risk for AUD. The Gpr88 gene, therefore, may represent a vulnerability/resilience factor for AUD, and a potential drug target for AUD treatment.