Late-stage luminescence-based cell-based dose response assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Cytotoxicity assay
Name: Late-stage luminescence-based cell-based dose response assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Cytotoxicity assay. ..more
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: HT29CYTOX_ANT_LUMI_384_4XCC50
Name: Late-stage luminescence-based cell-based dose response assay to identify antagonists of the G-protein coupled receptor 7 (GPR7): Cytotoxicity assay.
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.
1. Pan HL., Wu ZZ., Zhou HY., Chen SR., Zhang HM. & Li DP. Modulation of pain transmission by G-protein-coupled receptors. Pharmacology & therapeutics 2008 Jan;117(1):141-61
2. Lagerstrom MC. & Schioth HB. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nature reviews. Drug discovery 2008 Apr;7(4):339-57
3. Thompson MD., Cole DE. & Jose PA. Pharmacogenomics of G protein-coupled receptor signaling: insights from health and disease. Methods in molecular biology (Clifton, N.J.) 2008;448:77-107
4. Bosier B. & Hermans E. Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends in pharmacological sciences 2007 Aug;28(8):438-46
5. Tanaka H., Yoshida T., Miyamoto N., Motoike T., Kurosu H., Shibata K., Yamanaka A., Williams SC., Richardson JA., Tsujino N., Garry MG., Lerner MR., King DS., O'Dowd BF., Sakurai T. & Yanagisawa M. Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8. Proceedings of the National Academy of Sciences of the United States of America 2003 May 13;100(10):6251-6
6. O'Dowd BF., Scheideler MA., Nguyen T., Cheng R., Rasmussen JS., Marchese A., Zastawny R., Heng HH., Tsui LC. & Shi X. 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 Jul 1;28(1):84-91
7. Brezillon S., Lannoy V., Franssen JD., Le Poul E., Dupriez V., Lucchetti J., Detheux M. & Parmentier M. Identification of natural ligands for the orphan G protein-coupled receptors GPR7 and GPR8. The Journal of biological chemistry 2003 Jan 10;278(2):776-83
8. Singh G., Maguire JJ., Kuc RE., Fidock M. & Davenport AP. Identification and cellular localisation of NPW1 (GPR7) receptors for the novel neuropeptide W-23 by [125I]-NPW radioligand binding and immunocytochemistry. Brain research 2004 Aug 13;1017(1-2):222-6
9. Lee DK., Nguyen T., Porter CA., Cheng R., George SR. & O'Dowd BF. Two related G protein-coupled receptors: the distribution of GPR7 in rat brain and the absence of GPR8 in rodents. Brain research. Molecular brain research 1999 Jul 23;71(1):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. & Fujino M. Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7. The Journal of biological chemistry 2002 Sep 13;277(37):34010-6
11. Kelly MA., Beuckmann CT., Williams SC., Sinton CM., Motoike T., Richardson JA., Hammer RE., Garry MG. & Yanagisawa M. Neuropeptide B-deficient mice demonstrate hyperalgesia in response to inflammatory pain. Proceedings of the National Academy of Sciences of the United States of America 2005 Jul 12;102(28):9942-7
12. Ishii M., Fei H. & Friedman JM. Targeted disruption of GPR7, the endogenous receptor for neuropeptides B and W, leads to metabolic defects and adult-onset obesity. Proceedings of the National Academy of Sciences of the United States of America 2003 Sep 2;100(18):10540-5
13. Zaratin PF., Quattrini A., Previtali SC., Comi G., Hervieu G. & Scheideler MA. Schwann cell overexpression of the GPR7 receptor in inflammatory and painful neuropathies. Molecular and cellular neurosciences 2005 Jan;28(1):55-63
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.
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). In this assay, HT29 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. Compounds were tested in triplicate in a 7-point 1:3 dilution series starting at a nominal test concentration of 20 uM.
HT29 cells were grown in Dulbecco's Modified Eagle's Media supplemented with 10% v/v fetal bovine serum, 2 mM L-Glutamine, and 100 U/mL penicillin and streptomycin. Prior to the start of the assay, 2E5 HT29 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.
PubChem Activity Outcome and Score:
Compounds with a CC50 value of <= 10 uM were considered active (cytotoxic). Compounds with a CC50 > 10 uM were considered inactive (nontoxic).
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:
HT29 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)
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.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: HT-29
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)