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BioAssay: AID 687013

Fluorescence-based cell-based primary high throughput dose response assay to identify antagonists of the Galanin Receptor 3 (GalR3).

Name: Fluorescence-based cell-based primary high throughput dose response assay to identify antagonists of the Galanin Receptor 3 (GalR3). ..more
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 Tested Compounds
 Tested Compounds
All(235)
 
 
Active(184)
 
 
Inactive(51)
 
 
 Tested Substances
 Tested Substances
All(236)
 
 
Active(185)
 
 
Inactive(51)
 
 
AID: 687013
Data Source: The Scripps Research Institute Molecular Screening Center (GALR3_ANT_CNGC_1536_3XIC50 DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-05-31

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 184
Related Experiments
AIDNameTypeComment
651719Fluorescence-based cell-based primary high throughput screening assay to identify antagonists of the Galanin Receptor 3 (GalR3)Screeningdepositor-specified cross reference: Primary assay (GALR3 ANT in singlicate)
651727Summary of the probe development effort to identify antagonists of the Galanin Receptor 3 (GALR3)Summarydepositor-specified cross reference: Summary (GALR3 ANT)
652245Fluorescence-based cell-based primary high throughput confirmation assay to identify antagonists of the Galanin Receptor 3 (GalR3)Screeningdepositor-specified cross reference: Confirmation assay(GALR3 in triplicate)
743092On Hold
743095On Hold
652268Counterscreen for antagonists of the galanin Receptor 3 (GalR3): Fluorescence-based cell-based high throughput screening assay to identify non specific agonists of the parental HEK-CNG cellsScreeningsame project related to Summary assay
687015Counterscreen for antagonists of the galanin Receptor 3 (GalR3): Fluorescence-based cell-based high throughput dose response assay to identify non specific activators of the parental HEK-CNG cells.Confirmatorysame project related to Summary assay
Description:
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_3XIC50 DRUN

Name: Fluorescence-based cell-based primary high throughput dose response assay to identify antagonists of the Galanin Receptor 3 (GalR3).

Description:

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).

References:

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.

Keywords:

DRUN, Dose Response, 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, 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.
Protocol
Assay Overview:

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.

Protocol Summary:

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, 200ug/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. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 29.9 uM.

The following mathematical expression was used to normalize data:

Ratio = T45 / T0

Where:

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

Where:

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.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 29.9 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 29.9 uM.

PubChem Activity Outcome and Score:

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.

The PubChem Activity Score range for active compounds is 100-41, and for inactive compounds 40-0.

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)
Comment
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
Assay: Dictionary: Version: 0.1

Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]

Assay: CurveFit [1]: Mask: Excluded Points

Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
8Response RangeThe range of Y.Float
9Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
10RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
11Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Inhibition at 0.0015 uM [1] (0.0015μM**)Value of %inhibition at 0.0015 micromolar inhibitor concentration; replicate [1]Float%
14Inhibition at 0.0015 uM [2] (0.0015μM**)Value of %inhibition at 0.0015 micromolar inhibitor concentration; replicate [2]Float%
15Inhibition at 0.0015 uM [3] (0.0015μM**)Value of %inhibition at 0.0015 micromolar inhibitor concentration; replicate [3]Float%
16Inhibition at 0.0045 uM [1] (0.0045μM**)Value of %inhibition at 0.0045 micromolar inhibitor concentration; replicate [1]Float%
17Inhibition at 0.0045 uM [2] (0.0045μM**)Value of %inhibition at 0.0045 micromolar inhibitor concentration; replicate [2]Float%
18Inhibition at 0.0045 uM [3] (0.0045μM**)Value of %inhibition at 0.0045 micromolar inhibitor concentration; replicate [3]Float%
19Inhibition at 0.014 uM [1] (0.014μM**)Value of %inhibition at 0.014 micromolar inhibitor concentration; replicate [1]Float%
20Inhibition at 0.014 uM [2] (0.014μM**)Value of %inhibition at 0.014 micromolar inhibitor concentration; replicate [2]Float%
21Inhibition at 0.014 uM [3] (0.014μM**)Value of %inhibition at 0.014 micromolar inhibitor concentration; replicate [3]Float%
22Inhibition at 0.041 uM [2] (0.041μM**)Value of %inhibition at 0.041 micromolar inhibitor concentration; replicate [2]Float%
23Inhibition at 0.041 uM [3] (0.041μM**)Value of %inhibition at 0.041 micromolar inhibitor concentration; replicate [3]Float%
24Inhibition at 0.041 uM [1] (0.041μM**)Value of %inhibition at 0.041 micromolar inhibitor concentration; replicate [1]Float%
25Inhibition at 0.12 uM [3] (0.12μM**)Value of %inhibition at 0.12 micromolar inhibitor concentration; replicate [3]Float%
26Inhibition at 0.12 uM [1] (0.12μM**)Value of %inhibition at 0.12 micromolar inhibitor concentration; replicate [1]Float%
27Inhibition at 0.12 uM [2] (0.12μM**)Value of %inhibition at 0.12 micromolar inhibitor concentration; replicate [2]Float%
28Inhibition at 0.37 uM [3] (0.37μM**)Value of %inhibition at 0.37 micromolar inhibitor concentration; replicate [3]Float%
29Inhibition at 0.37 uM [1] (0.37μM**)Value of %inhibition at 0.37 micromolar inhibitor concentration; replicate [1]Float%
30Inhibition at 0.37 uM [2] (0.37μM**)Value of %inhibition at 0.37 micromolar inhibitor concentration; replicate [2]Float%
31Inhibition at 1.1 uM [3] (1.1μM**)Value of %inhibition at 1.1 micromolar inhibitor concentration; replicate [3]Float%
32Inhibition at 1.1 uM [2] (1.1μM**)Value of %inhibition at 1.1 micromolar inhibitor concentration; replicate [2]Float%
33Inhibition at 1.1 uM [1] (1.1μM**)Value of %inhibition at 1.1 micromolar inhibitor concentration; replicate [1]Float%
34Inhibition at 3.3 uM [2] (3.3μM**)Value of %inhibition at 3.3 micromolar inhibitor concentration; replicate [2]Float%
35Inhibition at 3.3 uM [3] (3.3μM**)Value of %inhibition at 3.3 micromolar inhibitor concentration; replicate [3]Float%
36Inhibition at 3.3 uM [1] (3.3μM**)Value of %inhibition at 3.3 micromolar inhibitor concentration; replicate [1]Float%
37Inhibition at 10 uM [3] (10μM**)Value of %inhibition at 10 micromolar inhibitor concentration; replicate [3]Float%
38Inhibition at 10 uM [2] (10μM**)Value of %inhibition at 10 micromolar inhibitor concentration; replicate [2]Float%
39Inhibition at 10 uM [1] (10μM**)Value of %inhibition at 10 micromolar inhibitor concentration; replicate [1]Float%
40Inhibition at 29.9 uM [1] (29.9μM**)Value of %inhibition at 29.9 micromolar inhibitor concentration; replicate [1]Float%
41Inhibition at 29.9 uM [2] (29.9μM**)Value of %inhibition at 29.9 micromolar inhibitor concentration; replicate [2]Float%
42Inhibition at 29.9 uM [3] (29.9μM**)Value of %inhibition at 29.9 micromolar inhibitor concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R21 NS067631-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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