Fluorescence-based primary cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).
Name: Fluorescence-based primary cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7). ..more
BioActive Compounds: 3127
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_1X%INH
Name: Fluorescence-based primary cell-based high throughput screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7).
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, 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. 1
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.
GPR7, NPBWR1, G-protein coupled receptor 7, pain, feeding, primary screen, HTS, high throughput screen, 1536, antagonist, antagonism, inhibitor, inhibition, fluorescence, calcium, Fluo-8, fluorescence, dye, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds that inhibit GPR7 activity. Although GPR7 is naturally coupled to Gαi, 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 Gαqi3 (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 singlicate at a final nominal concentration of 4.4 micromolar.
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
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
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.
A mathematical algorithm was used to determine nominally inhibiting compounds in the Primary Screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.
The PubChem Activity Score has been set to 100 for active compounds and 0 for 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, 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 (Aanaspec, part 61653)
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. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. In this assay NPW had an approximate EC50 of 0.8 nM throughout the campaign. 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)