Each data point represents a single well on a well plate. GPCRs have attracted significant attention for drug discovery due to their numerous physiological and pathological roles in mediating cellular responses to hormones and neurotransmitters [ 21 ]. Within the large GPCR family, GPCRs, excluding olfactory receptors, are classified as orphan receptors because their endogenous ligands are still unknown [ 22 ]. GPR88 is one of these orphan GPCRs that has recently attracted considerable interest because of its linkage to a number of basal ganglia-associated disorders. Despite the emerging pharmacological implications, the detailed functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for CNS investigation.
The availability of a simple, rapid and robust assay to monitor GPR88 activity would expedite the search for novel GPR88 ligands. Although the response to 1 R ,2 R -PCCA was potent in our calcium mobilization assay, the concentration-response curve did not reach a top plateau. We believe this is due to the engineered G protein coupling present in the system as 1 R ,2 R -PCCA is a highly potent agonist that reaches a top plateau in our secondary cAMP assay [ 14 , 16 ].
The phenyl analogue 1b was approximately 2.
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However, substitution with an amide group 1d resulted in a loss of activity. Changing the S -configuration of the amino group to the R -configuration 2b led to a 2-fold loss of potency. Moreover, the isohexyl compound 2c was completely inactive, indicating the amino group is critical for activity, possibly because it forms a hydrogen bonding interaction with the receptor binding site.
Finally, the R , R -configuration of the cyclopropyl is also critical for activity as its S , S -isomer resulted in an inactive compound 3. In general, the SAR trends presented herein agree with the data reported from the past studies [ 14 — 16 ]. The well assay was then optimized and adapted for HTS applications by miniaturizing to well and testing multiple variables cell density, solvent tolerance, and no-wash protocol.
Chemogenomic analysis, based on the alignment of 30 critical residues predicted to line the binding cavity of GPCRs, clustered GPR88 with metabotropic glutamate and GABA B receptors, suggesting they may bind to structurally related ligands [ 29 ]. Accordingly, a pilot neurotransmitter library, including natural and synthesized ligands of adrenergic, serotonergic and GABA B receptors, was screened and yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays.
A larger HTS campaign in the future using a more diverse set of compounds that occupy different chemical spaces might lead to the discovery of novel GPR88 agonists. To the best of our knowledge, this is the first report describing the development and validation of a GPR88 HTS assay.
The only other scientific document outlining such efforts is a university thesis available online through the Leiden University Repository [ 30 ]. Recent efforts in our laboratory have focused on the development of cAMP assays for the orphan GPR88 [ 14 , 16 , 18 ]. Because the cAMP assay is an endpoint assay, only one mode of activity can be measured in this format in a single screen. Our laboratory is interested in identifying multiple types of ligands for GPR88, so developing a HTS assay that can measure more than one mode of activity at a time was highly desirable.
Our laboratory has developed a general FLIPR-based calcium mobilization protocol which allows for both agonist and antagonist screening with a single assay plate, thereby cutting screening time in half. Although our work thus far has been aimed at the identification of GPR88 receptor agonists, in theory, this calcium mobilization assay can be used to identify other ligands as well, including competitive and noncompetitive antagonists, inverse agonists and allosteric modulators.
Such efforts are currently under investigation in our laboratory. Kreitzer AC. Physiology and pharmacology of striatal neurons. Annu Rev Neurosci. Dopamine receptors and brain function. Behavioural pharmacology of glutamate receptors in the basal ganglia. Neurosci Biobehav Rev. Civelli O. Orphan GPCRs and neuromodulation.
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G protein-coupled receptor deorphanizations. Annu Rev Pharmacol Toxicol. A novel G-protein-coupled receptor gene expressed in striatum. Striatal GPR88 expression is confined to the whole projection neuron population and is regulated by dopaminergic and glutamatergic afferents. Eur J Neurosci. Lack of GPR88 enhances medium spiny neuron activity and alters motor- and cue-dependent behaviors. Nat Neurosci.
Transcriptome analysis identifies genes with enriched expression in the mouse central extended amygdala. Identification of novel striatal genes by expression profiling in adult mouse brain. GPR88 - a putative signaling molecule predominantly expressed in the striatum: Cellular localization and developmental regulation.
Basal Ganglia. Mol Cell Neurosci. Mice lacking GPR88 show motor deficit, improved spatial learning, and low anxiety reversed by delta opioid antagonist.
Biol Psychiatry. Synthesis, pharmacological characterization, and structure-activity relationship studies of small molecular agonists for the orphan GPR88 receptor.
ACS Chem Neurosci. Bioorg Med Chem Lett. Effect of substitution on the aniline moiety of the GPR88 agonist 2-PCCA: synthesis, structure-activity relationships, and molecular modeling studies.
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Design, synthesis, and evaluation of phenylglycinols and phenyl amines as agonists of GPR Design, synthesis and pharmacological evaluation of 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives as GPR88 agonists. Bioorg Med Chem. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen. Int J High Throughput Screen. Trends in the exploitation of novel drug targets. Nat Rev Drug Discov. Mining the receptorome. J Biol Chem. G16 as a universal G protein adapter: implications for agonist screening strategies.
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G-protein Coupled Receptors: A Potential Candidate for Drug Discovery - SciAlert Responsive Version
Kostenis E. Is Galpha16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors? Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal Biochem. Milligan G, Rees S. Chimaeric G alpha proteins: their potential use in drug discovery.
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