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BioAssay: AID 463227

SAR analysis of Antagonists of the GPR35 Receptor using an Image-Based Assay - Set 5

Assay Provider: Dr. Mary Abood, (Temple University, formerly at California Pacific Medical Center Research Institute) ..more
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 Tested Compounds
 Tested Compounds
All(14)
 
 
Active(10)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(14)
 
 
Active(10)
 
 
Inactive(4)
 
 
AID: 463227
Data Source: Burnham Center for Chemical Genomics (BCCG-A427-GPR35-Antagonist-DryPowder-5)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-09-16
Hold-until Date: 2011-05-01
Modify Date: 2011-05-03

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 10
Depositor Specified Assays
AIDNameTypeComment
2058Image-Based HTS for Selective Antagonists of GPR35confirmatory
2079Summary of Image-based HTS for Selective Antagonists of GPR35summarySummary AID.
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1X01 DA026205-01
Assay Provider: Dr. Mary Abood, (Temple University, formerly at California Pacific Medical Center Research Institute)

Addictive behavior stems from abnormal signaling activities in the brain. Thus identification of compounds blocking this modified signaling activity may lead to treatments for addictive behavior. GPR35, a to-date uncharacterized orphan G-Protein Coupled Receptor, is thought to play a role in addiction and has homology to other known receptors of abuse.

This high-content imaging assay is developed and performed to confirm activity of hits originally identified in a high-content screen for antagonists of the GPR35 receptor, "Image-Based HTS for Selective Antagonists of GPR35" (AID 2058) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.

The assay will identify small molecule antagonists of the GPR35 receptor. This imaging assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and the GPR35 receptor. Upon agonist-mediated GPCR activation, the arrestin-GFP redistributes from the cytosolic compartment to the plasma membrane and further into coated pits, which can be quantified as increased local aggregation of the GFP-arrestins.
Protocol
Assay Materials:
1) 384-well plates, black with clear bottom (Greiner# 781091)
2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and GPR35 receptor
3) Culture Media: MEM with L-glutamine, Pen-Strep, 10% Fetal Bovine Serum, 10mM Hepes, and selection antibiotics - 200ug/ml G418 and 50ug/ml Zeocin
4) DMSO Working Solution: 100% DMSO
5) Agonist Working Solution: Zaprinast (Alexis #ALX-430-020-M010,10mg) 5mM stock in DMSO diluted to 100uM in PBS.
6) Test compounds from dry powder working solution: 10mM in 100% DMSO
7) Fixative Working Solution: 6% Paraformaldehyde (PFA) diluted in PBS.
8) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4).

Assay Procedure:
1) 45ul of cell suspension (200,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser.
2) Plates are incubated overnight or approximately 20 hours at 37 degree C and 5% CO2.
3) Serum is removed by media aspiration and replacing with 45ul serum-free MEM prior to addition of compounds.
4) Compound addition was done on the ECHO550 Liquid Handler. The "dose response protocol" was used to dispense corresponding volumes of each 10mM compound on the assay plate.
a. Compounds were added to columns 3 to 22. Final concentration ranged from 62nM to 32uM (ten doses), in duplicate.
b. Agonist working solution (Zaprinast) was added to the entire plate except columns 1 and 24 (DMSO only as positive non-inhibition control). Zaprinast final concentration was 10uM.
c. DMSO was added to column 1, 2 and 23, 24. Final DMSO concentration was 0.31%
d. DMSO was back-filled to each well to achieve a 0.31% final concentration.
5) Plates were incubated for 45 minutes at 37 degrees C and 5% CO2.
6) Media was aspirated leaving 20ul liquid in each well using a Titertek plate washer.
7) 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA.
8) Plates were incubated for 40 minutes at room temperature.
9) Fixative was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
10) 40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100ng/ml. Aluminum plate sealers were applied to each plate.

Image Acquisition and Analysis:
1) Image acquisition was performed on an Opera QEHS (Perkin Elmer) with 45 plate capacity loader/stacker and the following settings:
- 40x 0.6 NA air objective
- Acquisition camera set to 2-by-2 binning for an image size of 688 by 512 pixels
- 2 channels acquired sequentially: Exp1Cam1 = Beta-arrestin GFP using 488 nm laser excitation and 540/70 nm mission filters, Exp2Cam2 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
- 4 fields per well for Hit Confirmation
2) Image analysis was performed using the Acapella Spot Detection Algorithm with the following analysis settings:
NUCLEI DETECTION
- Threshold Adjustment: 4
- Nuclear Splitting Adjustment: 10
- Minimum Nuclear Area: 200
- Minimum Nuclear Contrast: 0

CYTOPLASM DETECTION
- Cytoplasm Threshold Adjustment: 0
- Cytoplasm Individual Threshold Adjustment: 0.05
SPOT DETECTION
- Spot Minimum Distance 3
- Spot Peak Radius 0
- Spot Reference Radius 3
- Spot Minimum Contrast 0.26
- Spot Minimum to Cell Intensity 0.5
3) Metrics calculated from
NUCLEI IMAGES: Cell Count ("NumberofCellsAnalyzed"), Nuclei Area ("AreaoftheNucleus"), Integrated Intensity of the Nuclei ("TotalIntegratedIntensityoftheNucleus"), Average Intensity of the Nuclei ("AverageIntensityoftheNucleus")
GFP IMAGES: Integrated Intensity of the Cytoplasm ("TotalCytoplasmIntensity"), Integrated Intensity of the Detected Spots ("TotalSpotIntensity"), Ratio of the Integrated Spot to Integrated Cytoplasm Intensities ("RatioofSpotIntensitytoCytoplasmintensity"), Number of Spots per Cell ("AverageSpotsPerCell"), Percentage of Cells Positive for Spot Formation ("PercentagePositiveCells")
4) The "RatioofSpotIntensitytoCytoplasmintensity" metric was used to calculate the dose response curves and parameters.

IC50 values were calculated using CBIS software (ChemInnovations) employing a sigmoidal dose-response equation through non-linear regression.
Comment
Compounds with an IC50 < 10 uM are defined as actives in the dose response confirmation.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and is not applicable to this assay

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues.

a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. For the remaining compounds the score is linearly correlated with a compound's inhibitory potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3),
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1_Range1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1IC50 value determined using sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 32 uM_first_point (32μM**)% inhibition at a given concentrationFloat%
9% Activity at 16 uM_first_point (16μM**)% inhibition at a given concentrationFloat%
10% Activity at 8 uM_first_point (8μM**)% inhibition at a given concentrationFloat%
11% Activity at 4 uM_first_point (4μM**)% inhibition at a given concentrationFloat%
12% Activity at 2 uM_first_point (2μM**)% inhibition at a given concentrationFloat%
13% Activity at 1 uM_first_point (1μM**)% inhibition at a given concentrationFloat%
14% Activity at 0.5 uM_first_point (0.5μM**)% inhibition at a given concentrationFloat%
15% Activity at 0.25 uM_first_point (0.25μM**)% inhibition at a given concentrationFloat%
16% Activity at 0.125 uM_first_point (0.125μM**)% inhibition at a given concentrationFloat%
17% Activity at 0.0625 uM_first_point (0.0625μM**)% inhibition at a given concentrationFloat%
18Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
19% Activity at 32 uM_second_point (32μM**)% inhibition at a given concentrationFloat%
20% Activity at 16 uM_second_point (16μM**)% inhibition at a given concentrationFloat%
21% Activity at 8 uM_second_point (8μM**)% inhibition at a given concentrationFloat%
22% Activity at 4 uM_second_point (4μM**)% inhibition at a given concentrationFloat%
23% Activity at 2 uM_second_point (2μM**)% inhibition at a given concentrationFloat%
24% Activity at 1 uM_second_point (1μM**)% inhibition at a given concentrationFloat%
25% Activity at 0.5 uM_second_point (0.5μM**)% inhibition at a given concentrationFloat%
26% Activity at 0.25 uM_second_point (0.25μM**)% inhibition at a given concentrationFloat%
27% Activity at 0.125 uM_second_point (0.125μM**)% inhibition at a given concentrationFloat%
28% Activity at 0.0625 uM_second_point (0.0625μM**)% inhibition at a given concentrationFloat%
29IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2_Range1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
30IC50_2IC50 value determined using sigmoidal dose response equationFloatμM
31Std.Err(IC50)_2Standard Error of IC50 valueFloatμM
32nH_2Hill coefficient determined using sigmoidal dose response equationFloat
33Excluded_Points_third_pointFlags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
34% Activity at 32 uM_third_point (32μM**)% inhibition at a given concentrationFloat%
35% Activity at 16 uM_third_point (16μM**)% inhibition at a given concentrationFloat%
36% Activity at 8 uM_third_point (8μM**)% inhibition at a given concentrationFloat%
37% Activity at 4 uM_third_point (4μM**)% inhibition at a given concentrationFloat%
38% Activity at 2 uM_third_point (2μM**)% inhibition at a given concentrationFloat%
39% Activity at 1 uM_third_point (1μM**)% inhibition at a given concentrationFloat%
40% Activity at 0.5 uM_third_point (0.5μM**)% inhibition at a given concentrationFloat%
41% Activity at 0.25 uM_third_point (0.25μM**)% inhibition at a given concentrationFloat%
42% Activity at 0.125 uM_third_point (0.125μM**)% inhibition at a given concentrationFloat%
43% Activity at 0.0625 uM_third_point (0.0625μM**)% inhibition at a given concentrationFloat%
44Excluded_Points_fourth_pointFlags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
45% Activity at 32 uM_fourth_point (32μM**)% inhibition at a given concentrationFloat%
46% Activity at 16 uM_fourth_point (16μM**)% inhibition at a given concentrationFloat%
47% Activity at 8 uM_fourth_point (8μM**)% inhibition at a given concentrationFloat%
48% Activity at 4 uM_fourth_point (4μM**)% inhibition at a given concentrationFloat%
49% Activity at 2 uM_fourth_point (2μM**)% inhibition at a given concentrationFloat%
50% Activity at 1 uM_fourth_point (1μM**)% inhibition at a given concentrationFloat%
51% Activity at 0.5 uM_fourth_point (0.5μM**)% inhibition at a given concentrationFloat%
52% Activity at 0.25 uM_fourth_point (0.25μM**)% inhibition at a given concentrationFloat%
53% Activity at 0.125 uM_fourth_point (0.125μM**)% inhibition at a given concentrationFloat%
54% Activity at 0.0625 uM_fourth_point (0.0625μM**)% inhibition at a given concentrationFloat%
55IC50_Qualifier_3This qualifier is to be used with the next TID, IC50_3. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
56IC50_3IC50 value determined using sigmoidal dose response equationFloatμM
57Std.Err(IC50)_3Standard Error of IC50 valueFloatμM
58nH_3Hill coefficient determined using sigmoidal dose response equationFloat
59Excluded_Points_fifth_pointFlags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
60% Activity at 32 uM_fifth_point (32μM**)% inhibition at a given concentrationFloat%
61% Activity at 16 uM_fifth_point (16μM**)% inhibition at a given concentrationFloat%
62% Activity at 8 uM_fifth_point (8μM**)% inhibition at a given concentrationFloat%
63% Activity at 4 uM_fifth_point (4μM**)% inhibition at a given concentrationFloat%
64% Activity at 2 uM_fifth_point (2μM**)% inhibition at a given concentrationFloat%
65% Activity at 1 uM_fifth_point (1μM**)% inhibition at a given concentrationFloat%
66% Activity at 0.5 uM_fifth_point (0.5μM**)% inhibition at a given concentrationFloat%
67% Activity at 0.25 uM_fifth_point (0.25μM**)% inhibition at a given concentrationFloat%
68% Activity at 0.125 uM_fifth_point (0.125μM**)% inhibition at a given concentrationFloat%
69% Activity at 0.0625 uM_fifth_point (0.0625μM**)% inhibition at a given concentrationFloat%
70Excluded_Points_sixth_pointFlags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
71% Activity at 32 uM_sixth_point (32μM**)% inhibition at a given concentrationFloat%
72% Activity at 16 uM_sixth_point (16μM**)% inhibition at a given concentrationFloat%
73% Activity at 8 uM_sixth_point (8μM**)% inhibition at a given concentrationFloat%
74% Activity at 4 uM_sixth_point (4μM**)% inhibition at a given concentrationFloat%
75% Activity at 2 uM_sixth_point (2μM**)% inhibition at a given concentrationFloat%
76% Activity at 1 uM_sixth_point (1μM**)% inhibition at a given concentrationFloat%
77% Activity at 0.5 uM_sixth_point (0.5μM**)% inhibition at a given concentrationFloat%
78% Activity at 0.25 uM_sixth_point (0.25μM**)% inhibition at a given concentrationFloat%
79% Activity at 0.125 uM_sixth_point (0.125μM**)% inhibition at a given concentrationFloat%
80% Activity at 0.0625 uM_sixth_point (0.0625μM**)% inhibition at a given concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1X01 DA026205-01

Data Table (Concise)
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