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High-throughput screening of antagonists for the orphan G-protein coupled receptor GPR139

AIM
To discover antagonists of the orphan G-protein coupled receptor GPR139 through high-throughput screening of a collection of diverse small molecules.


METHODS
Calcium mobilization assays were used to identify initial hits and for subsequent confirmation studies.


RESULTS
Five small molecule antagonists, representing 4 different scaffolds, were identified following high-throughput screening of 16 000 synthetic compounds.


CONCLUSION
The findings provide important tools for further study of this orphan G-protein coupled receptor.


Introduction
G-protein coupled receptors (GPCRs) are the largest family of cell surface communicating molecules, and they play crucial roles in numerous physiological processes and disease states. GPCRs share high levels of homology and contain seven transmembrane helices separated by intra-and extracellular loops [1] . They signal via heterotrimeric G proteins composed of Gα, β, and γ subunits, and there are four major Gα protein subfamilies: Gq, Gs, Gi, and G 12/13 . The Gs and Gi subunits regulate adenyl cyclase activity, whereas the Gq subunit regulates phospholipase C and the subsequent production of diacylglycerol and inositol phosphate, which initiates calcium release from intracellular stores. The G 12/13 subunit regulates the activities of the small G protein RhoA [2] .
The human GPR139 gene was identified by searching the GenBank genomic database [2][3][4][5] . The human GPR139 (also known as hGPRg1 or hGPCR12) protein is a 345-amino acid orphan receptor located on chromosome 16p12.3. It shares 96% amino acid homology with murine orthologs [2] . Expression studies in mice have revealed that the transcription of GPR139 is more evident in the brain, both in the adult stage and during development [3] . Human GPR139 mRNA is pre-dominantly expressed in the fetal and adult central nervous system (CNS), especially in the basal ganglia and the hypothalamus [2,3] , which are involved in movement control, regulation of food intake and metabolism [2,3,6] . The consistent expression of GPR139 mRNA in the CNS of different species suggests that it plays specific roles in the modulation of brain functions. Thus, it is implicated as a potential drug target for diabetes, obesity and Parkinson's disease.
Understanding the signaling pathway of a receptor is critical for the development of in vitro bioassays to support drug discovery. The signal transduction pathway of GPR139, however, is still not fully defined due to a lack of natural or synthetic ligands.
To date, three groups have reported small molecule ligands for GPR139 ( Figure 1). Shi et al identified compound 1 as a GPR139 receptor agonist with an EC 50 of 39 nmol/L in a calcium mobilization assay for a CHO-K1 cell line stably expressing the human GPR139 for high-throughput screening (HTS) [7] . Isberg et al discovered dipeptides and L-α-amino acids as GPR139 agonists by building a pharmacophore model based on the characterization of 13 compounds reported by Shi et al [8] . Four dipeptides [TyrTrp (2), TyrPhe, TrpTyr, and TyrHis] consisting of aromatic amino acids were obtained. Among them, TyrTrp (2) and TyrPhe satisfied all five elements of the pharmacophore model [9] and therefore displayed the highest potencies (EC 50 =160 μmol/L for TyrTrp and 180 μmol/L for TyrPhe), whereas TrpTyr lacks a hydrogen bond acceptor element, and TyrHis lacks an aromatic element. In addition, two amino acids, Trp and Phe, and their L-and D-isomers were found to activate GPR139 with an E max similar to compound 1.
Hu et al identified compound 3 as a GPR139 agonist after screening a large library of small molecules [6] . Compound 3 specifically increased cAMP levels by 8-to 10-fold in cells expressing GPR139 but not in cells expressing GPR142, a GPR139-related receptor with 50% amino acid identity. Furthermore, this compound was unable to induce Ca 2+ mobilization in a fluorescent imaging plate reader (FLIPR) based assay, indicating the absence of a Gα q -mediated response. Antagonist screening with an identified agonist yielded two classes of compounds, including the triazolopyrimidine series, represented by compound 4, and the sulfonamide series, represented by compound 5, which specifically inhibited compound 3 induced cAMP accumulation in GPR139-expressing cells with IC 50 values of 0.67 and 0.64 μmol/L, respectively.
In this study, we report the identification of four new scaffolds of GPR139 antagonists following high-throughput screening of 16 000 synthetic compounds using a calcium mobilization assay.

Compound library
The compound library used in this study consists of both synthetic and natural compounds. The structural diversity covers heterocycles, lactams, sulfonates, sulfonamides, amines, sencondary amides and natural product-derived compounds. The compounds were highly purified, and the stock was presolubilized in 100% DMSO prior to application in the HTS campaign, performed with an average concentration of 10 µmol/L for each compound.

HTS campaign
The cells mentioned above were seeded in 96-well microtiter plates (Corning; 14 000 cells per 100 µL growth medium per well) and rested for 45-60 min at room temperature before incubating at 37 °C, 5% CO 2 and 95% humidity chamber for two days. The dye mix solution was prepared from the Fluo-4 NW calcium assay kit according to the manufacturer's protocol by adding 100 mL assay buffer (HBSS/HEPS) and 1 mL probenecid stock solution (250 mmol/L) to the dye and shaking the resultant solution vigorously for 2 min (stored in dark).
Upon removal of growth medium 100 µL of the dye mix solution was added to each well and incubated for 30 min at 37 °C, then at room temperature for an additional 45 min with plate top covered with tinfoil. This was followed by measurement of fluorescence signals in the first column (8 wells) for 10 s to obtain a baseline, using a FlexStation II microplate reader (Molecular Devices, Sunnyvale, CA, USA). The library compounds in DMSO (1 mg/mL) were diluted in the assay buffer and 25 µL of which was dispensed to each well (final concentration was approximately 10 µmol/L in 0.5% DMSO). After the first measurement for 1 min, the plates were incubated for 9 min at room temperature prior to introduction of the agonist compound 1 (25 µL at a final concentration of 100 nmol/L) followed by the second measurement for 1 min. Intracellular calcium change was recorded at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.  Dose-response study Dose-response study was performed essentially as the same as described above except that test compounds were hand-picked (10 mmol/L concentration in DMSO) and serially diluted (1:5) seven times to give a total of eight different concentrations. This reflects a final compound dilution factor ranging from 1:96 to 1:136. For a starting concentration of 10 mmol/L this corresponded to a concentration range between 103.6 and 80.2 µmol/L. Each compound was tested in duplicate and the dose-response curves were fitted by Prism 5 software (Graph-Pad, San Diego, CA, USA) using nonlinear least square regression in a sigmoidal model with variable slope.

Statistical analysis
The results were analyzed using Prism 5 (GraphPad) and representatives of three independent experiments in duplicate.

Results
The GPR139 agonist compound 1 was employed to screen antagonists of GPR139. A total of 16 000 compounds with diverse structures were screened at 10 µmol/L in the presence of 100 nmol/L of compound 1 using a calcium mobilization assay. The Z' of the screening assay was 0.74 with an S/B ratio of 22 and a coefficient of variation (CV) value of 5.0% ( Figure  2). These parameters suggest that the assay system is of high quality and is suitable for HTS [10] . The scatter plots of HTS are shown in Figure 3.
In the primary screening, 162 hits were identified (≥50% inhibition of compound 1 induced activation). Five hits were subsequently confirmed (Figure 4) in dose-response assays.

Inhibition%= ( 1-Signal test -Signal NC ) ×100%
Signal PC -Signal NC   npg Discussion GPR139 was identified by searching the genomic database and has characteristics of the rhodopsin family of GPCRs [3] . It is abundantly expressed in distinct regions of the brain, both in humans and in mice. In the caudate putamen, habenular nucleus, zona incerta and medial mammillary nucleus, the expression of GPR139 is higher than that in the thalamus, amygdala and spinal cord [2,3] , which suggests a significant role of GPR139 in the CNS. GPR139 was first reported as a Gq-coupled receptor [2,3] . Matsuo et al overexpressed GPR139 in 293-EBNA cells and found that it was capable of activating serum response factor mediated transcription. Additionally, this reaction could be inhibited by a G q/11 selective inhibitor [2] . This observation was confirmed through the discovery of a series of GPR139 agonists using calcium mobilization assays [7,8] . Susens et al identified the signal transduction pathway using both Ca 2+ mobilization and luciferase-reporter-gene assays. They proposed that GPR139 was coupled to an inhibitory G-protein and mediated by phospholipase C [3] . However, Hu et al identified GPR139 as a Gs-coupled receptor because overexpressed GPR139 in HEK239 cells could increase basal intracellular cAMP con-centrations [6] . Previous studies have shown that Gq-coupling is the main signaling pathway of GPR139 and might activate other pathways [8] . Furthermore, it was noted that GPR139 appears to be a monomer in HEK-293 cells and a dimer in CHO-K1 cells [3] .
In this study, we described an HTS assay to screen antagonists to GPR139 based on intracellular calcium influx and identified a series of small molecule hits that blocked the activity of GPR139 induced by compound 1. All of the compounds showed reasonable potencies (close or below 2 µmol/L), of which two compounds (NCRW0001-C02 and NCRW0005-F05) possessed the same core region consisting of 3,3-difluoro-4-phenylazetidin-2-one. A preliminary structure-activity study suggested that substitution of electron-donating groups on the phenyl group was beneficial for antagonistic effects. These compounds showed little similarity to the structures of antagonists previously reported. Our findings thus offer novel structures and provide promising tools in the study of both the signaling pathway(s) and physiological significance of this orphan receptor.
In summary, we conducted an HTS study based on intracellular calcium influx and identified five small-molecule antago-