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BioAssay: AID 2251

Fluorescence-based dose response cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).

Name: Fluorescence-based dose response cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7). ..more
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 Tested Compounds
 Tested Compounds
All(126)
 
 
Active(19)
 
 
Inactive(107)
 
 
 Tested Substances
 Tested Substances
All(126)
 
 
Active(19)
 
 
Inactive(107)
 
 
AID: 2251
Data Source: The Scripps Research Institute Molecular Screening Center (GPR7_ANT_FLUO8_1536_3X%IC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-01-14

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 19
Related Experiments
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AIDNameTypeProbeComment
1880Summary of probe development efforts to identify antagonists of the G-protein coupled receptor 7 (GPR7).Summary2 depositor-specified cross reference: Summary AID.
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: Screening assay (GPR7 antagonists).
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
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
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
485339Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Intracellular calcium releaseConfirmatory depositor-specified cross reference
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
1861Fluorescence-based primary cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).Screening same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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_1536_3X%IC50

Name: Fluorescence-based dose response cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).

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:

GPR7, NPBWR1, G-protein coupled receptor 7, pain, feeding, dose response, HTS, high throughput screen, 1536, antagonist, antagonism, inhibitor, inhibition, fluorescence, calcium, Fluo-8, fluorescence, dye, plate-based, Scripps, Scripps Florida, 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 determine dose response curves for compounds that confirmed activity in a previous set of experiments entitled, "Fluorescence-based confirmation cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7)" (AID 1952). 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 compounds were assayed in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 44 micromolar.
Protocol Summary:
The hGPR7 HEK293T/Gqi3 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees 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 micrograms/mL Hygromycin-B, 200 micrograms/mL Geneticin, 0.625 microgram/mL Puromycin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
The day before the assay 1500 cells in 3 microliters of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 23 hours. Next, 2 microliters 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 1 hour at 37 degrees C, 5% CO2, and 95 % RH, 22 nL of test compound in DMSO, or DMSO alone were dispensed to the appropriate wells. The assay was started after an additional 30 minute incubation at room temperature, by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of GPR7 agonist (2 nM final concentration) in DMSO, or DMSO alone were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 180 seconds of the assay.
A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:
Ratio = I_Max / I_Min
Where:
I_Max represents the maximum measured fluorescence emission intensity over the 185 second read and I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.
Percent inhibition was calculated from the median ratio as follows:
% Inhibition = ( 1 - ( ( Ratio_Test_Compound - Median_Ratio_High_Control ) / ( Median_Ratio_Low_Control - Median_Ratio_High_Control ) ) ) * 100
Where:
Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO, NPW.
High_Control is defined as wells containing DMSO.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). 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. In cases where the highest concentration tested (i.e. 44 micromolar) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 44 uM. Compounds with an IC50/EC50 greater than 10 uM were considered inactive. Compounds with an IC50/EC50 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, with the most potent compounds assigned the highest activity scores.
The activity score range for active compounds is 100-83, for inactive 81-0.
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, part FB-02)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Aurora, part 19326)
hNPW-23 (Anaspec, part 61653)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate cAMP and CNG activity or membrane potential, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
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
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentration.Float
4Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
5Hill S0Y-min of the curve.Float
6Hill SinfY-max of the curve.Float
7Hill dSThe range of Y.Float
8Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
9RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
10Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
11Inhibition at 2.2 nM (0.0022μM**)Value of %inhibition at 2.2 nanomolar inhibitor concentration; average of triplicate measurement.Float%
12Inhibition at 6.7 nM (0.0067μM**)Value of %inhibition at 6.7 nanomolar inhibitor concentration; average of triplicate measurement.Float%
13Inhibition at 20.0 nM (0.02μM**)Value of %inhibition at 20.0 nanomolar inhibitor concentration; average of triplicate measurement.Float%
14Inhibition at 60.1 nM (0.0601μM**)Value of %inhibition at 60.1 nanomolar inhibitor concentration; average of triplicate measurement.Float%
15Inhibition at 180.3 nM (0.1803μM**)Value of %inhibition at 180.3 nanomolar inhibitor concentration; average of triplicate measurement.Float%
16Inhibition at 540.8 nM (0.5408μM**)Value of %inhibition at 540.8 nanomolar inhibitor concentration; average of triplicate measurement.Float%
17Inhibition at 1.6 uM (1.6μM**)Value of %inhibition at 1.6 micromolar inhibitor concentration; average of triplicate measurement.Float%
18Inhibition at 4.9 uM (4.9μM**)Value of %inhibition at 4.9 micromolar inhibitor concentration; average of triplicate measurement.Float%
19Inhibition at 14.6 uM (14.6μM**)Value of %inhibition at 14.6 micromolar inhibitor concentration; average of triplicate measurement.Float%
20Inhibition at 43.8 uM (43.8μM**)Value of %inhibition at 43.8 micromolar inhibitor concentration; average of triplicate measurement.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
Classification
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