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BioAssay: AID 588326

Late-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 3

Name: Late-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 3. ..more
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 Tested Compounds
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Inactive(1)
 
 
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Inactive(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 588326
Data Source: The Scripps Research Institute Molecular Screening Center (U2OSCYTOX_ANT_LUMI_384_6XCC50_SET 3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-08-23
Hold-until Date: 2012-03-01
Modify Date: 2012-03-01

Data Table ( Complete ):           All
Tested Compound:
Depositor Specified Assays
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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 Primary screen (GPR7 antagonists in singlicate)
1880Summary of probe development efforts to identify antagonists of the G-protein coupled receptor 7 (GPR7).summary2 Summary (GPR7 antagonists)
1952Fluorescence-based confirmation cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).screening Confirmation screen (GPR& 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 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 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 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 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 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 Dose response counterscreen (Cytotoxicity in triplicate)
485339Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Intracellular calcium releaseconfirmatory Dose response (GPR7 antagonists in triplicate)
504401Late-stage counterscreen panel assay for GPR7 antagonists: Ricerca HitProfilingScreen + CYP450other Late-stage counterscreen panel assay (Ricerca hit profiling screen + CYP450)
504868Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 2confirmatory Dose response (GPR7 antagonists in triplicate)
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 Dose response counterscreen (Cytotoxicity in triplicate)
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 Dose response counterscreen (MCHR1 antagonists in triplicate)
588562Late-stage fluorescence-based cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): percent inhibitionother
588564Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 4confirmatory
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
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: U2OSCYTOX_ANT_LUMI_384_6XCC50_SET 3

Name: Late-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 3.

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, luciferase, luminescence, CellTiter Glo, cell viability, cytotoxicity, plate-based, 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 determine cytotoxicity of a powder sample of a compound identified as active in a previous assay, "Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 2" (AID 504868). In this assay, U2OS cells are incubated with test compound, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compound was tested in 6 replicates in a 7-point 1:3 dilution series starting at a nominal test concentration of 20 uM.

Protocol Summary:

U2OS cells were grown in Dulbecco's Modified Eagle's Media supplemented with 10% v/v fetal bovine serum, 2 mM L-Glutamine, and 100U/mL penicillin and streptomycin. Prior to the start of the assay, 2 x 10^5 U2OS cells in a 20 uL volume of HBSS were dispensed into wells of a 384-well tissue culture-treated microtiter plates. Test compound was diluted 1:100 in growth medium (100 uM final concentration) and then serially diluted 1:3 in growth medium. The assay was started immediately by dispensing 5 uL of test compound, media alone, or rotenone as a positive control (150 uM final concentration) to the appropriate wells. The plates were then incubated for 2 hours at 37 C. Plate was then equilibrated at room temperature for 30 minutes. The assay was stopped by dispensing 25 uL of CellTiter-Glo reagent to each well, followed by incubation in the dark at room temperature for 10 minutes. Well luminescence was measured on the ViewLux plate reader.

The % Cell Viability for each well was then calculated as follows:

%_Cell_Viability = 1 - ( MedianRFU_High_Control - RFU_Test_Compound ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing cells in the presence of test compound.
High_Control is defined as wells containing cells treated with media only (no compound).
Low_Control is defined as wells containing no cells (media only).

Percent Cell Viability was plotted against the log of the compound concentration. The CC50 is reported as "> X uM" (where X = the highest concentration tested for which >50% Cell Viability was observed).

PubChem Activity Outcome and Score:

The following applies to each panel in this assay:

Compounds with a CC50 value of less than 10 uM were considered active (cytotoxic). Compounds with a CC50 value greater than 10 uM were considered inactive (non-cytotoxic).

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 inactive compounds is 0-0. There are no active compounds.

List of Reagents:

U2OS cells (provided by Assay Provider)
DMEM medium (GIBCO, part 25200)
Hank's Balanced Salt Solution (Invitrogen, part 14025-092)
100X Penicillin-Streptomycin mix (Invitrogen, part 15140)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
Rotenone (Sigma R8875)
CellTiter Glo (Promega, part S-G7570)
DMSO (Sigma 472301)
150 mm tissue culture dishes (Corning, part 430599)
384-well plates (Corning, 3570)
Comment
This assay was performed with a synthesized compound. 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 luciferase activity, and compounds that quench or emit luminescence within the well.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data CC50 came from a fitted curve or was determined manually to be less than or greater than its listed CC50 concentration.String
2CC50*The value for concentration at which 50% of surviving cells are observed; CC50 shown in micromolar.FloatμM
3Cell Viability at 20 uM [1] (20μM**)Value of % Cell Viability at 20 uM compound concentration [1]Float%
4Cell Viability at 20 uM [2] (20μM**)Value of % Cell Viability at 20 uM compound concentration [2]Float%
5Cell Viability at 20 uM [3] (20μM**)Value of % Cell Viability at 20 uM compound concentration [3]Float%
6Cell Viability at 20 uM [4] (20μM**)Value of % Cell Viability at 20 uM compound concentration [4]Float%
7Cell Viability at 20 uM [5] (20μM**)Value of % Cell Viability at 20 uM compound concentration [5]Float%
8Cell Viability at 20 uM [6] (20μM**)Value of % Cell Viability at 20 uM compound concentration [6]Float%
9Cell Viability at 6.67 uM [1] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [1]Float%
10Cell Viability at 6.67 uM [2] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [2]Float%
11Cell Viability at 6.67 uM [3] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [3]Float%
12Cell Viability at 6.67 uM [4] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [4]Float%
13Cell Viability at 6.67 uM [5] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [5]Float%
14Cell Viability at 6.67 uM [6] (6.67μM**)Value of % Cell Viability at 6.67 uM compound concentration [6]Float%
15Cell Viability at 2.22 uM [1] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [1]Float%
16Cell Viability at 2.22 uM [2] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [2]Float%
17Cell Viability at 2.22 uM [3] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [3]Float%
18Cell Viability at 2.22 uM [4] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [4]Float%
19Cell Viability at 2.22 uM [5] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [5]Float%
20Cell Viability at 2.22 uM [6] (2.22μM**)Value of % Cell Viability at 2.22 uM compound concentration [6]Float%
21Cell Viability at 0.74 uM [1] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [1]Float%
22Cell Viability at 0.74 uM [2] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [2]Float%
23Cell Viability at 0.74 uM [3] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [3]Float%
24Cell Viability at 0.74 uM [4] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [4]Float%
25Cell Viability at 0.74 uM [5] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [5]Float%
26Cell Viability at 0.74 uM [6] (0.74μM**)Value of % Cell Viability at 0.74 uM compound concentration [6]Float%
27Cell Viability at 0.25 uM [1] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [1]Float%
28Cell Viability at 0.25 uM [2] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [2]Float%
29Cell Viability at 0.25 uM [3] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [3]Float%
30Cell Viability at 0.25 uM [4] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [4]Float%
31Cell Viability at 0.25 uM [5] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [5]Float%
32Cell Viability at 0.25 uM [6] (0.25μM**)Value of % Cell Viability at 0.25 uM compound concentration [6]Float%
33Cell Viability at 0.082 uM [1] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [1]Float%
34Cell Viability at 0.082 uM [2] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [2]Float%
35Cell Viability at 0.082 uM [3] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [3]Float%
36Cell Viability at 0.082 uM [4] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [4]Float%
37Cell Viability at 0.082 uM [5] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [5]Float%
38Cell Viability at 0.082 uM [6] (0.082μM**)Value of % Cell Viability at 0.082 uM compound concentration [6]Float%
39Cell Viability at 0.027 uM [1] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [1]Float%
40Cell Viability at 0.027 uM [2] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [2]Float%
41Cell Viability at 0.027 uM [3] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [3]Float%
42Cell Viability at 0.027 uM [4] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [4]Float%
43Cell Viability at 0.027 uM [5] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [5]Float%
44Cell Viability at 0.027 uM [6] (0.027μM**)Value of % Cell Viability at 0.027 uM compound concentration [6]Float%
45Cell Viability at 0 uM [1] (0μM**)Value of % Cell Viability at 0 uM compound concentration [1]Float%
46Cell Viability at 0 uM [2] (0μM**)Value of % Cell Viability at 0 uM compound concentration [2]Float%
47Cell Viability at 0 uM [3] (0μM**)Value of % Cell Viability at 0 uM compound concentration [3]Float%
48Cell Viability at 0 uM [4] (0μM**)Value of % Cell Viability at 0 uM compound concentration [4]Float%
49Cell Viability at 0 uM [5] (0μM**)Value of % Cell Viability at 0 uM compound concentration [5]Float%
50Cell Viability at 0 uM [6] (0μM**)Value of % Cell Viability at 0 uM compound concentration [6]Float%

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

Data Table (Concise)
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