Fluorescence-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).
Fluorescence-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).
Dose response counterscreen (MCHR1 antagonists in triplicate)
Late-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)
Dose response counterscreen (MCHR1 antagonists in triplicate)
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 2
Dose response counterscreen (Cytotoxicity in triplicate)
Late-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 2
Dose response counterscreen (MCHR1 antagonists in triplicate)
Late-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 4
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
Name: Late-stage fluorescence-based dose response cell-based screening assay to identify antagonists of the G-protein coupled receptor 7 (GPR7) Set 3.
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. 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.
Late stage, late stage AID, powders, GPR7, NPBWR1, G-protein coupled receptor 7, pain, feeding, dose response, 384, antagonist, antagonism, inhibitor, inhibition, fluorescence, calcium, Fluo-8, fluorescence, dye, 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 dose response curves for synthesized compounds in a 384-well plate format for antagonism of GPR7. 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 an 8-point 1:3 dilution series starting at a nominal test concentration of 20 uM.
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 Geneticin, 0.625 ug/mL Puromycin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
The day before the assay 10,000 cells in 25 uL of growth media were seeded into each well of 384 well microtiter plates and allowed to incubate at 37 C, 5% CO2, and 95 % RH for 23 hours. Next, 25 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, 100 nL of test compound in DMSO, or DMSO alone, were dispensed to the appropriate wells. The assay was started after an additional 15-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, 5.5 nL of GPR7 agonist (20 nM final concentration) in FLIPR buffer (HBSS/20 mM Hepes/0.1% BSA) was 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. I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.
Percent inhibition was calculated from the median ratio as follows:
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 activation was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc) normalized from 0 to 100 for each assay. The software-generated IC50 values were reported. In cases where the highest concentration tested (i.e. 20 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 20 uM.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
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.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
Experiment 1 Score: The PubChem Activity Score range for active compounds is 100-78, and for inactive compounds 1-0.
Experiment 2 Score: The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.
Experiment 3 Score: The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.
Overall Outcome and Score:
Compounds that were active in all experiments were considered active, otherwise they were considered inactive.
The overall score is 0 if the compound was inactive, otherwise the score is taken as the fraction of panels where the compound is active, multiplied by 100.
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
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 (Invitrogen, part 26140-079) 100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055) T-75 tissue culture flasks (NUNC, part 178905) 384-well plates (Greiner, part 781964) Neuropeptide W-23 (NPW-23)/L8 (Human) (Phoenix Pharmaceuticals 005-60)
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. This assay was performed with compounds synthesized as powders.