Fluorescence-based cell-based primary high throughput confirmation assay to identify antagonists of the Galanin Receptor 3 (GalR3)
Name: Fluorescence-based cell-based primary high throughput confirmation assay to identify antagonists of the Galanin Receptor 3 (GalR3). ..more
BioActive Compounds: 916
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Patricia McDonald, The Scripps Research Institute
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS067631-01
Grant Proposal PI: Patricia McDonald, The Scripps Research Institute
External Assay ID: GALR3_ANT_CNGC_1536_3X%INH CRUN
Name: Fluorescence-based cell-based primary high throughput confirmation assay to identify antagonists of the Galanin Receptor 3 (GalR3).
Galanin, a 29 amino acid neuropeptide (30 residues in humans), is cleaved from preprogalanin and is involved in many physiological processes including nervous system development, feeding, metabolism and reproduction, and regulation of neurotransmitter and hormone release (1, 2). The physiologic response to galanin is mediated in part by three G protein-coupled metabotropic 7-transmembrane receptor subtypes, GalR1, GalR2 and GalR3. These receptors are expressed throughout the peripheral and central nervous systems as well as the endocrine system. Both GalR1 and GalR2 are widely expressed in the CNS whereas GalR3 is the least abundantly expressed of the galanin receptor subtypes. The GalR3 in particular has been strongly implicated in addiction and mood related disorders such as anxiety and depression. It has been the target of many drug discovery programs within the pharmaceutical industry but despite the significant resources and effort devoted to discovery of galanin receptor subtype selective small molecule modulators, there have been very few reports for the discovery of such molecules and in addition, all access to primary screening data remains proprietary information. As a result the identification of novel GalR3 antagonists would serve as useful tools for understanding galanin biology and for possible investigations into the role of GalR3 in addiction and mood disorders (3-5).
1. Walton, K. M., Chin, J. E., Duplantier, A. J., and Mather, R. J. Galanin function in the central nervous system. Current opinion in drug discovery & development, 2006. 9(5): p. 560-570.
2. Wrenn, C. C., and Holmes, A. The role of galanin in modulating stress-related neural pathways. Drug news & perspectives, 2006. 19(8).
3. Swanson, C.J., et al., Anxiolytic- and antidepressant-like profiles of the galanin-3 receptor (Gal3) antagonists SNAP 37889 and SNAP 398299. Proc Natl Acad Sci U S A, 2005. 102(48): p. 17489-94.
4. Packiarajan, M, - 2,4,6-Triaminopyrimidines for the treatment of depression and/or anxiety. US Patent 6936607, 2005.
5. Packiarajan, M, Use of GAL3 antagonist for treatment of depression and/or anxiety and compounds useful in such methods. US Patent 7465750, 2008.
CRUN, confirmation, CNG, ActOne, Galanin receptor, anxiety, addiction, mood, pain, CNS, receptor, galanin, cAMP, fluor, fluorescence, forskolin, membrane potential, dye, FLIPR, Gi, G-protein, biosensor, GALR3, antagonist, inhibitor, HTS, primary, dose response screen, G protein coupled receptor, GPCR, 1536, inhibit, decrease, high throughput screen, 1536, 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 GALR3 activity. This assay employs HEK293 cells stably transfected with a Gal3/1ctR chimera construct, where "1ctR" refers to the carboxy tail of Gal1R, was generated by in-frame fusion of the wildtype GALR3 (residues1-299) and the carboxy terminus of the wildtype Gal1R (299-350). The purpose of the chimera was to eliminate the endoplasmic retention motifs in the carboxy tail of wildtype GALR3 in order to rescue cell surface expression. In this assay HEK293 cells stably expressing Gal3/1ctR chimera construct and a modified cyclic nucleotide gated (CNG) acting as a biosensor for cAMP. Changes in cAMP are measured with a combination of membrane potential dye are incubated either with in the presence of test compounds, or with forskolin (EC90) (HIGH control wells) or forskolin (EC90)/galanin peptide (EC90) (LOW control wells). This assay employs the cAMP ACTone biosensor assay. As designed, a compound that inhibits galanin-induced GALR3 activity will decrease Gi-coupled signaling activity will increase galanin inhibited forskolin stimulated cAMP levels, leading to an increase in the fluorescence of the membrane potential dye. Compounds are tested in triplicate at a final nominal concentration of 3 uM.
The GALR3/1 HEK293-CNG cells were routinely cultured in T-175 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, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 400 ug/mL Geneticin, 200 ug/ml hygromycin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
Prior to the start of the assay, 3,000 GALR3/1 HEK293-CNG cells in a 3 uL volume of growth media were dispensed into each well of 1536-well black clear bottom tissue culture-treated microtiter plates. Next, the plates were incubated for 24 hours at 37 C, 5% CO2 and 95% relative humidity (RH). The assay was started by dispensing two uL per well of 1X concentrated probe loading dye with 25uM PDEI into all wells, and the plates were incubated at room temperature for 3 hours. Following incubation, the compounds are added at 15nL and allowed to equilibrate for 15 minutes. Then, the first fluorescence measurement was performed (510-545 nm excitation and 565-625 nm emission) on the FLIPR Tetra (Molecular Devices), then the cells were challenged by pinning 30 nL of forskolin EC90/Galanin EC90 in DMSO. The plates were then incubated for 45 minutes at room temperature before the final fluorescence measurement with the same instrument settings.
The following mathematical expression was used to normalize data:
Ratio = T45 / T0
T0 represents the measured fluorescence emission intensity before the addition of stimulus and challenge.
T45 represents the measured fluorescence emission intensity 45 minutes post addition of the challenge.
The percent inhibition for each compound was calculated 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, forskolin and galanin.
Low_Control is defined as wells with DMSO, forskolin and galanin.
High_Control is defined as wells with DMSO and forskolin.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition 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 % inhibition than the cutoff parameter was declared active. This confirmation assay uses that primary screen cutoff.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-9, and for inactive compounds 9-0.
List of Reagents:
DMEM media (Life Technologies, part 11995-073)
TrypLE(Life Technologies, part 12563-029)
Heat Inactivated Fetal Bovine Serum (Invitrogen, part 10082-147)
Hygromycin B (Invitrogen, part 10687-010)
Geneticin (Invitrogen, part 10131-027)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Nunc, part 159910)
Forskolin (MP Biomedicals, part 190669)
Galanin (American Peptide, part 46-1-10)
1536-well plates (Corning, part 7338)
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. 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, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)