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BioAssay: AID 485339

Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Intracellular calcium release

Name: Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Intracellular calcium release. ..more
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AID: 485339
Data Source: The Scripps Research Institute Molecular Screening Center (GPR7_ANT_FLUO8_96_IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-10-01
Hold-until Date: 2011-04-14
Modify Date: 2011-04-14

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
Related Experiments
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AIDNameTypeProbeComment
1861Fluorescence-based primary cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).Screening depositor-specified cross reference: Primary screen (GPR7 antagonists in singlicate)
1880Summary of probe development efforts to identify antagonists of the G-protein coupled receptor 7 (GPR7).Summary2 depositor-specified cross reference: Summary (GPR7 antagonists)
1952Fluorescence-based confirmation cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).Screening depositor-specified cross reference: Confirmation screen (GPR7 antagonists in triplicate)
2148Fluorescence-based counterscreen for antagonists of the G-protein coupled receptor 7 (GPR7): cell-based high throughput screening assay to identify antagonists of the melanin-concentrating hormone receptor 1 (MCHR1).Screening depositor-specified cross reference: Counterscreen (MCHR1 antagonists in triplicate)
2251Fluorescence-based dose response cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).Confirmatory depositor-specified cross reference: Dose response (GPR7 antagonists in triplicate)
2257Fluorescence-based counterscreen for antagonists of the G-protein coupled receptor 7 (GPR7): cell-based high throughput dose response assay to identify antagonists of the melanin-concentrating hormone receptor 1 (MCHR1).Confirmatory depositor-specified cross reference: Dose response counterscreen (MCHR1 antagonists in triplicate)
463250Late-stage fluorescence-based counterscreen for antagonists of the G-protein coupled receptor 7 (GPR7): cell-based dose response assay to identify antagonists of the melanin-concentrating hormone receptor 1 (MCHR1)Confirmatory depositor-specified cross reference: Late-stage dose response counterscreen (MCHR1 antagonists in triplicate)
463251Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7)Confirmatory depositor-specified cross reference: Late-stage dose response (GPR7 antagonists in triplicate)
463253Late-stage luminescence-based cell-based dose response assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Cytotoxicity assayConfirmatory depositor-specified cross reference: Late-stage dose response counterscreen (Cytotoxicity in triplicate)
504401Late-stage counterscreen panel assay for GPR7 antagonists: Ricerca HitProfilingScreen + CYP450Other depositor-specified cross reference
504868Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 2Confirmatory depositor-specified cross reference
504886Late-stage results from the probe development effort to identify antagonists of the G-protein coupled receptor 7 (GPR7): luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of antagonist compounds Set 2Confirmatory depositor-specified cross reference
504889Late-stage fluorescence-based counterscreen for antagonists of the G-protein coupled receptor 7 (GPR7): cell-based dose response assay to identify antagonists of the melanin-concentrating hormone receptor 1 (MCHR1) Set 2Confirmatory depositor-specified cross reference
540345Late-stage counterscreen panel assay for GPR7 antagonists: Ricerca HitProfilingScreen + CYP450: Set 2Other depositor-specified cross reference
540365Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 3Confirmatory depositor-specified cross reference
540371Late-stage fluorescence-based counterscreen for antagonists of the G-protein coupled receptor 7 (GPR7): cell-based dose response assay to identify antagonists of the melanin-concentrating hormone receptor 1 (MCHR1) Set 3Confirmatory depositor-specified cross reference
588326Late-stage results from the probe development effort to identify antagonists of the G-protein coupled receptor 7 (GPR7): luminescence-based cell-based dose response counterscreen assay to determine cytotoxicity of antagonist compounds Set 3Confirmatory depositor-specified cross reference
588562Late-stage fluorescence-based cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): percent inhibitionOther depositor-specified cross reference
588563Late-stage fluorescence-based counterscreen for antagonists of the G-protein coupled receptor 7 (GPR7): cell-based dose response assay to identify antagonists of the melanin-concentrating hormone receptor 1 (MCHR1) Set 5Confirmatory depositor-specified cross reference
588564Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 4Confirmatory depositor-specified cross reference
588566Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 5Confirmatory depositor-specified cross reference
588568Late-stage fluorescence-based counterscreen for antagonists of the G-protein coupled receptor 7 (GPR7): cell-based dose response assay to identify antagonists of the melanin-concentrating hormone receptor 1 (MCHR1) Set 4Confirmatory depositor-specified cross reference
588581Late-stage fluorescence-based counterscreen for antagonists of the G-protein coupled receptor 7 (GPR7): cell-based dose response assay to identify antagonists of the melanin-concentrating hormone receptor 1 (MCHR1) Set 6Confirmatory depositor-specified cross reference
588583Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 6Confirmatory depositor-specified cross reference
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Olivier Civelli, University of California, Irvine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-R03-DA026557-01
Grant Proposal PI: Olivier Civelli
External Assay ID: GPR7_ANT_FLUO8_96_IC50

Name: Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Intracellular calcium release.

Description:

Heterotrimeric G-protein coupled receptors (GPCRs) are major targets for disease therapeutics, due in part to their broad tissue distribution, structural diversity, varied modes of action, and disease-associated mutations (1-4). For example, targeting of opiod receptors by opiates such as morphine is a widespread clinical application for GPCR modulation in pain management. The recently de-orphanized GPR7 (5) is localized predominantly in the cerebellum and prefrontal cortex (6), with additional expression in the pituitary, hippocampus, amygdala, and spinal cord (7-9). GPR7 is highly conserved in humans and rodents (6), and exhibits structural features of both GPCRs and somatostatin receptors (7). Studies identifying the energy-regulating neuropeptides Neuropeptide W (NPW) and Neuropeptide B (NPB) as endogenous ligands of GPR7 (5, 10), and the development of hyperphagia and obesity in male GPR7 knockout mice (11, 12), implicate GPR7 in feeding behavior. Additional studies identifying GPR7 expression in peripheral Schwann cells (13) and increased GPR7 expression in rat models and human patients with inflammation-associated neuropathic pain (11, 13), suggest a role for GPR7 in mediating the inflammatory pain response. The identification of modulators of GPR7 will provide useful tools to elucidate the diverse roles of this receptor in central neuropeptide signaling and nociception in general.

References:

1. Pan, H.L., Wu, Z.Z., Zhou, H.Y., Chen, S.R., Zhang, H.M., and Li, D.P., Modulation of pain transmission by G-protein-coupled receptors. Pharmacol Ther, 2008. 117(1): p. 141-61.
2. Lagerstrom, M.C. and Schioth, H.B., Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov, 2008. 7(4): p. 339-57.
3. Thompson, M.D., Cole, D.E., and Jose, P.A., Pharmacogenomics of G protein-coupled receptor signaling: insights from health and disease. Methods Mol Biol, 2008. 448: p. 77-107.
4. Bosier, B. and Hermans, E., Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol Sci, 2007. 28(8): p. 438-46.
5. Tanaka, H., Yoshida, T., Miyamoto, N., Motoike, T., Kurosu, H., Shibata, K., Yamanaka, A., Williams, S.C., Richardson, J.A., Tsujino, N., Garry, M.G., Lerner, M.R., King, D.S., O'Dowd, B.F., Sakurai, T., and Yanagisawa, M., Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8. Proc Natl Acad Sci U S A, 2003. 100(10): p. 6251-6.
6. O'Dowd, B.F., Scheideler, M.A., Nguyen, T., Cheng, R., Rasmussen, J.S., Marchese, A., Zastawny, R., Heng, H.H., Tsui, L.C., Shi, X., and et al., The cloning and chromosomal mapping of two novel human opioid-somatostatin-like receptor genes, GPR7 and GPR8, expressed in discrete areas of the brain. Genomics, 1995. 28(1): p. 84-91.
7. Brezillon, S., Lannoy, V., Franssen, J.D., Le Poul, E., Dupriez, V., Lucchetti, J., Detheux, M., and Parmentier, M., Identification of natural ligands for the orphan G protein-coupled receptors GPR7 and GPR8. J Biol Chem, 2003. 278(2): p. 776-83.
8. Singh, G., Maguire, J.J., Kuc, R.E., Fidock, M., and Davenport, A.P., Identification and cellular localisation of NPW1 (GPR7) receptors for the novel neuropeptide W-23 by [125I]-NPW radioligand binding and immunocytochemistry. Brain Res, 2004. 1017(1-2): p. 222-6.
9. Lee, D.K., Nguyen, T., Porter, C.A., Cheng, R., George, S.R., and O'Dowd, B.F., Two related G protein-coupled receptors: the distribution of GPR7 in rat brain and the absence of GPR8 in rodents. Brain Res Mol Brain Res, 1999. 71(1): p. 96-103.
10. Fujii, R., Yoshida, H., Fukusumi, S., Habata, Y., Hosoya, M., Kawamata, Y., Yano, T., Hinuma, S., Kitada, C., Asami, T., Mori, M., Fujisawa, Y., and Fujino, M., Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7. J Biol Chem, 2002. 277(37): p. 34010-6.
11. Kelly, M.A., Beuckmann, C.T., Williams, S.C., Sinton, C.M., Motoike, T., Richardson, J.A., Hammer, R.E., Garry, M.G., and Yanagisawa, M., Neuropeptide B-deficient mice demonstrate hyperalgesia in response to inflammatory pain. Proc Natl Acad Sci U S A, 2005. 102(28): p. 9942-7.
12. Ishii, M., Fei, H., and Friedman, J.M., Targeted disruption of GPR7, the endogenous receptor for neuropeptides B and W, leads to metabolic defects and adult-onset obesity. Proc Natl Acad Sci U S A, 2003. 100(18): p. 10540-5.
13. Zaratin, P.F., Quattrini, A., Previtali, S.C., Comi, G., Hervieu, G., and Scheideler, M.A., Schwann cell overexpression of the GPR7 receptor in inflammatory and painful neuropathies. Mol Cell Neurosci, 2005. 28(1): p. 55-63.

Keywords:

Late stage, late stage AID, powders, GPR7, NPBWR1, G-protein coupled receptor 7, pain, feeding, dose response, 384, antagonist, antagonism, inhibitor, inhibition, fluorescence, calcium, calcium release, Fluo-8, fluorescence, dye, cell-based, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to identify compounds that inhibit GPR7 activity. Although GPR7 is naturally coupled to Gai, which decreases cAMP levels upon activation, this assay employs a chimeric cell line that forces the receptor to use Gqi3, and therefore the assay readout is calcium release. In this assay HEK cells stably co-transfected with the human GPR7 receptor and Gaqi3 (hGPR7 HEK293T/Gqi3 cell line) are treated with test compounds, followed by measurement of intracellular calcium as monitored by the FLUO-8 fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as GPR7 antagonists will decrease calcium mobilization, resulting in decreased relative fluorescence of the indicator dye, and thus decreased well fluorescence. Test compound was assayed in triplicate in an 8-point 1:3 dilution series starting at a nominal test concentration of 25 uM.
Protocol Summary:
The hGPR7 HEK293T/Gqi3 cell line was routinely cultured in T-75 sq cm flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 25 mM HEPES, 200 ug/mL Hygromycin-B, 200 ug/mL G418, 0.625 ug/mL Puromycin, and 1X antibiotic mix (penicillin and streptomycin).
The day before the assay 70,000 cells in 100 uL of growth media were seeded into each well of 96 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 100 uL of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer's protocol) was added to each well. After incubation for 50 minutes at 37 C, 5% CO2, and 95 % relative humidity, a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 1 second on the FLIPR1 (Molecular Devices). 50 uL of test compound in FLIPR buffer, or buffer alone, were dispensed to the appropriate wells. After an additional 10 minute incubation at 37 C, 50 uL of GPR7 agonist NPB (10 nM final concentration) in FLIPR buffer (HBSS/20 mM Hepes/0.1% BSA) was dispensed to the appropriate wells. Then real time fluorescence was continuously monitored for the remaining 180 seconds of the assay.
Percent inhibition was calculated as follows:
% Inhibition = ( I_Max - I_Min ) / I_Min
Where:
I_Max represents the maximum measured fluorescence emission intensity after the compound was added.
I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.
The data was then plotted against compound concentration. The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value using GraphPad Prism software.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds
List of Reagents:
hGPR7 HEK293T/Gqi3 cell line (provided by Assay Provider)
Fluo-8 No Wash Calcium Assay Kit (ABD Bioquest, part 36316)
Trypan red plus (ABD Bioquest, part 2456)
DMEM (Invitrogen, part 11965)
Geneticin (Invitrogen, part 10131-027)
Hygromycin-B (Invitrogen, part 10687-010)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Omega Scientific FB-02, Lot 10061-3))
100X Penicillin-Streptomycin mix (Invitrogen, part 15070-063)
T-75 tissue culture flasks (NUNC, part 178905)
96-well plates (ISC BioExpress T-3073-46)
Neuropeptide B-23 (NPB-23)/L8 (Human) (Phoenix Pharmaceuticals 005-53)
Comment
Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that quench or emit fluorescence within the well. This assay was performed by the Assay Provider with synthesized compound provided by the Scripps Research Institute Molecular Screening Center.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK 293T
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) averaged from three calculated curves and shown in micromolar.FloatμM
2Standard DeviationThe standard deviation of the value of the IC50.Float
3Inhibition at 0.001 uM [1] (0.001μM**)Value of % inhibition at 0.001 micromolar inhibitor concentration; replicate one.Float%
4Inhibition at 0.034 uM [1] (0.034μM**)Value of % inhibition at 0.034 micromolar inhibitor concentration; replicate one.Float%
5Inhibition at 0.103 uM [1] (0.103μM**)Value of % inhibition at 0.103 micromolar inhibitor concentration; replicate one.Float%
6Inhibition at 0.309 uM [1] (0.309μM**)Value of % inhibition at 0.309 micromolar inhibitor concentration; replicate one.Float%
7Inhibition at 0.926 uM [1] (0.926μM**)Value of % inhibition at 0.926 micromolar inhibitor concentration; replicate one.Float%
8Inhibition at 2.8 uM [1] (2.8μM**)Value of % inhibition at 2.8 micromolar inhibitor concentration; replicate one.Float%
9Inhibition at 8.3 uM [1] (8.3μM**)Value of % inhibition at 8.3 micromolar inhibitor concentration; replicate one.Float%
10Inhibition at 25 uM [1] (25μM**)Value of % inhibition at 25 micromolar inhibitor concentration; replicate one.Float%
11Inhibition at 0.001 uM [2] (0.001μM**)Value of % inhibition at 0.001 micromolar inhibitor concentration; replicate two.Float%
12Inhibition at 0.034 uM [2] (0.034μM**)Value of % inhibition at 0.034 micromolar inhibitor concentration; replicate two.Float%
13Inhibition at 0.103 uM [2] (0.103μM**)Value of % inhibition at 0.103 micromolar inhibitor concentration; replicate two.Float%
14Inhibition at 0.309 uM [2] (0.309μM**)Value of % inhibition at 0.309 micromolar inhibitor concentration; replicate two.Float%
15Inhibition at 0.926 uM [2] (0.926μM**)Value of % inhibition at 0.926 micromolar inhibitor concentration; replicate two.Float%
16Inhibition at 2.8 uM [2] (2.8μM**)Value of % inhibition at 2.8 micromolar inhibitor concentration; replicate two.Float%
17Inhibition at 8.3 uM [2] (8.3μM**)Value of % inhibition at 8.3 micromolar inhibitor concentration; replicate two.Float%
18Inhibition at 25 uM [2] (25μM**)Value of % inhibition at 25 micromolar inhibitor concentration; replicate two.Float%
19Inhibition at 0.001 uM [3] (0.001μM**)Value of % inhibition at 0.001 micromolar inhibitor concentration; replicate three.Float%
20Inhibition at 0.034 uM [3] (0.034μM**)Value of % inhibition at 0.034 micromolar inhibitor concentration; replicate three.Float%
21Inhibition at 0.103 uM [3] (0.103μM**)Value of % inhibition at 0.103 micromolar inhibitor concentration; replicate three.Float%
22Inhibition at 0.309 uM [3] (0.309μM**)Value of % inhibition at 0.309 micromolar inhibitor concentration; replicate three.Float%
23Inhibition at 0.926 uM [3] (0.926μM**)Value of % inhibition at 0.926 micromolar inhibitor concentration; replicate three.Float%
24Inhibition at 2.8 uM [3] (2.8μM**)Value of % inhibition at 2.8 micromolar inhibitor concentration; replicate three.Float%
25Inhibition at 8.3 uM [3] (8.3μM**)Value of % inhibition at 8.3 micromolar inhibitor concentration; replicate three.Float%
26Inhibition at 25 uM [3] (25μM**)Value of % inhibition at 25 micromolar inhibitor concentration; replicate three.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1-R03-DA026557-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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