Primary Cell Based High Throughput Screening Assay for Agonists of GALR2
Galanin is a 29 amino acid neuropeptide primarily expressed on the central and peripheral nervous systems and the endocrine system (Vrontakis, 2002). It is often found to be co-localized with neurotransmitters and is highly up-regulated upon nerve injury (Lundstrom et al., 2005). There are 3 G protein-coupled galanin receptor subtypes (GALR1-3). Galanin has been implicated in cognition, memory, more ..
BioActive Compounds: 79
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Steven Brown, TSRI
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1R21NS057101-01
PI: Steven Brown
External Assay ID: GALR2_Agonist_BLAReporter_MaybridgeHitFinder_PrimaryScreen
Name: Primary Cell Based High Throughput Screening Assay for Agonists of GALR2
Galanin is a 29 amino acid neuropeptide primarily expressed on the central and peripheral nervous systems and the endocrine system (Vrontakis, 2002). It is often found to be co-localized with neurotransmitters and is highly up-regulated upon nerve injury (Lundstrom et al., 2005). There are 3 G protein-coupled galanin receptor subtypes (GALR1-3). Galanin has been implicated in cognition, memory, depression, mood regulation, pain processing, hormone secretion, feeding behavior, and cancer (Lang et al., 2007; Berger et al., 2005). Galanin receptor 2 (GALR2) has been reported to play a role in neurite outgrowth, inflammatory pain behaviour, and protection from neuronal damage (Hobson et al., 2006; Elliott-Hunt et al., 2007). A GALR2 agonist has been shown to reduce glutamate toxicity in primary neuronal hippocampal cells (Pirondi et al., 2005).
Probe compound agonists and antagonists of GALR2 will be useful in exploring the biology of galanin receptor signaling, and a subtype-specific agonist/antagonist will help us differentiate distinct roles of each galanin receptor (Walton et al., 2006). Such compounds may serve as starting points for development of therapeutic application to treat Alzheimer's disease, depression, cancer, diabetes, and eating disorders (Ogren et al., 2006).
1. Berger, A., R. Santic, et al. (2005). "Galanin and galanin receptors in human cancers." Neuropeptides 39(3): 353-9.
2. Elliott-Hunt, C. R., R. J. Pope, et al. (2007). "Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage." J Neurochem 100(3): 780-9.
3. Hobson, S. A., F. E. Holmes, et al. (2006). "Mice deficient for galanin receptor 2 have decreased neurite outgrowth from adult sensory neurons and impaired pain-like behaviour." J Neurochem 99(3): 1000-10.
4. Lang, R., A. L. Gundlach, et al. (2007). "The galanin peptide family: receptor pharmacology, pleiotropic biological actions, and implications in health and disease." Pharmacol Ther 115(2): 177-207.
5. Lundstrom, L., A. Elmquist, et al. (2005). "Galanin and its receptors in neurological disorders." Neuromolecular Med 7(1-2): 157-80.
6. Ogren, S. O., E. Kuteeva, et al. (2006). "Galanin receptor antagonists : a potential novel pharmacological treatment for mood disorders." CNS Drugs 20(8): 633-54.
7. Pirondi, S., M. Fernandez, et al. (2005). "The galanin-R2 agonist AR-M1896 reduces glutamate toxicity in primary neural hippocampal cells." J Neurochem 95(3): 821-33.
8. Vrontakis, M. E. (2002). "Galanin: a biologically active peptide." Curr Drug Targets CNS Neurol Disord 1(6): 531-41.
9. Walton, K. M., J. E. Chin, et al. (2006). "Galanin function in the central nervous system." Curr Opin Drug Discov Devel 9(5): 560-70.
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NFAT-BLA-CHO cells with the beta-lactamase (BLA) reporter-gene under control of the nuclear factor of activated T-cells (NFAT) promoter were stably transfected with plasmids expressing the rat GALR2. When cells were agonized with galanin/agonist, transcription of the NFAT-BLA gene occurred. The amount of BLA activity was proportional to the concentration of agonist. BLA activity was measured with a fluorescent BLA substrate. Test compounds were screened for activation of GALR2 as measured by BLA activity. Hit compounds were counterscreened to eliminate direct BLA activation.
The 5H9 GALR2-NFAT-BLA-CHO cells were cultured in 100 mm tissue culture dish (Corning, part 430167) at 37C and 95% RH. The growth media consisted of Dulbecco's Modified Eagle's Media with GlutaMAX-I (Invitrogen, part 10566-024) containing 10% v/v dialyzed fetal bovine serum (Hyclone, part SH30079.03), 0.1 mM NEAA (Invitrogen, part 1114-050), 1 mM Sodium Pyruvate (Invitrogen, part 11360-070), 25 mM HEPES (Invitrogen, part 15630-080), 1 mg/mL Geneticin (Invitrogen, part 10131-027), 100 ug/mL Zeocin (Invitrogen, part 45-0430) and 1X penicillin-streptomycin (Invitrogen, part 15140-163).
Prior to assay, cells were suspended to a concentration of 0.3 million/mL in the assay media (Dulbecco's Modified Eagle's Media with GlutaMAX-I containing 10% v/v dialyzed fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, and 25 mM HEPES).
The HTS assay began by dispensing 10 uL of cell suspension to each well of the assay plates (Griener LV 384-well, part 788092) and incubating for 20 hours. Then 1.1 uL of test compound, DMSO control (for negative control wells) or 10X galanin (10 uM, for positive control wells) was added. The plates were incubated at 37C for 4 hrs and then loaded with 1 uM CCF4/AM. The plates were incubated for 2 hrs at room temperature in dark. Thereafter, the plates were read using the EnVision multi-label reader (PerkinElmer Lifesciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 460 nm & 590 nm. The ratio of 460 nm/590 nm fluorescence emission was calculated.
Percent activation was calculated from the median ratio as follows:
% activation= ((test_compound - median_negative_control)/(median_postitive_control - median_negative_control))*100
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.
The reported Pubchem_Activity_Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on the microtiter plate, and compounds that quench or emit fluorescence.
Data Table (Concise)