Image-Based HTS for Selective Antagonists of GPR35
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. The goal of this project is to identify small molecule antagonists of GPR35, which may aid in characterization of this receptor and ultimately further the understanding of the role of GPR35 in addictive behavior. ..more
BioActive Compounds: 153
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1X01MH085708-01
Assay Provider: Dr. Lawrence Barak, Duke University
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. The goal of this project is to identify small molecule antagonists of GPR35, which may aid in characterization of this receptor and ultimately further the understanding of the role of GPR35 in addictive behavior.
This high content 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 to clathrin-coated pits, which can be quantified as increased local aggregation of the GFP-arrestins. When this receptor is blocked by an antagonist, this arrestin-GFP redistribution is inhibited and can be detected as a decrease in local concentrations of fluorescent arrestins as compared to the agonist activity.
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) Positive Control Working Solution: 2.0% DMSO diluted in PBS
5) Agonist Working Solution: Zaprinast (Alexis # ALX-430-020-M010 - 10mg, 5mM stock in DMSO) diluted in PBS to 100uM
6) Fixative Working Solution: 6% Paraformaldehyde (PFA) diluted in PBS.
7) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4).
Primary Screen Procedure:
1) 45ul of cell suspension (133,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser.
2) Plates were incubated overnight or approximately 20 hours at 37 degree C and 5% CO2.
3) Serum was removed by media aspiration and replaced with 45ul serum-free MEM prior to addition of compounds.
4) Compound addition was done on a Biomek FX with 384-head dispenser (Beckman):
a) 5ul of 100uM compound solution was added to columns 3 through 24 of the assay plates for a final assay compound concentration of 10uM and 0.5% DMSO.
b) 5ul of 5% DMSO was added to columns 1 and 2 to balance the volume and DMSO concentration across the plate.
c) 5ul of positive control (2% DMSO) working solution was added to column 1.
5) Plates were incubated for 15 minutes at room temperature.
6) Agonist addition was done on a Biomek FX with 384-head dispenser (Beckman):
a) 5ul of agonist (100uM Zaprinast) working solution was added to columns 2-24. (This also serves as the negative control in column 2.)
7) Plates were incubated for 45 minutes at 37 degrees C and 5% CO2.
8) Media was aspirated leaving 20ul liquid in each well using a Titertek plate washer.
9) 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA.
10) Plates were incubated for 40 minutes at room temperature.
11) Fixative was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
12) 40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100ng/ml. Aluminum plate seals were applied to each plate.
Hit Confirmation Procedure:
A. Single-Concentration Confirmation
1) Same as primary screen, except compound addition was done using the ECHO550 Acoustic Dispenser using 2mM compound stock solution, volumes were adjusted accordingly and the final DMSO concentration was 0.31%.
B. Dose Response Confirmation
1) Same as primary screen steps 1 and 2.
2) Volume after media aspiration and replace (step 3) was 50ul.
3) Compound addition was done on the ECHO555 Acoustic Dispenser. 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 32uM to 500nM (seven doses), performed in duplicate and on 2 different days.
b. For control wells, 177.6nL of 100% DMSO was added to columns 1 and 2 for a final DMSO concentration of 0.32%.
4) Plates were incubated for 15 minutes at room temperature.
5) 5.5ul of agonist (100uM Zaprinast) working solution was added to columns 2-24 using the Biomek FX with 384-head dispenser (Beckman). (This also serves as the negative control in column 2.)
6) 5.5ul of 2% DMSO was manually added to column 1.
7) Primary screen procedure steps 7 to 12 were followed.
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
- 3 fields per well for Primary screen
- 4 fields per well for Hit Confirmation
2) Image analysis was performed using the Acapella Spot Detection Algorithm with the following analysis settings:
- Threshold Adjustment: 4
- Nuclear Splitting Adjustment: 10
- Individual Threshold Adjustment 0.05
- Minimum Nuclear Area: 200
- Minimum Nuclear Contrast: 0
- Cytoplasm Individual Threshold Adjustment: 0
- 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")
Actives from the primary screen were determined using CBIS soft ware (ChemInnovations) by calculating the % Response of the "RatioofSpotIntensitytoCytoplasmIntensity" metric and using a hit criteria of Inhibition >= 50% and "NumberofCellsAnalyzed" >= 30 and "TotalCytoplasmIntensity" <= 10,000,000. Wells with cell counts lower than 30 in the 3 acquired images were flagged "cytotoxic / low cell count". Wells with a very high total GFP intensity ("TotalCytoplasmIntensity" >= 10,000,000) were flagged as artifacts due to autofluorescence. All flagged wells were excluded from hit selection and were assigned an outcome of "inconclusive".
The primary screen "actives" were retested for hit confirmation. Compounds that confirmed in quadruplicate at 10uM concentration were considered "active".
Retested primary screen "actives" were then tested for dose-response hit confirmation. Compounds with an IC50 <10uM were considered "confirmed actives".
To simplify the distinction between the actives of the primary screen and of the confirmatory dose response, a tiered activity scoring system was devised to take into consideration compound efficacy and potential interference with the assay. The outline of the scoring system utilized for the screen is as follows:
1) First tier (0-40 range) is reserved for primary and confirmation screening data. The score is correlated with %activity in the assay demonstrated by a compound at 10 uM concentration:
a. If primary %Inhibition is less than 0%, then the assigned score is 0
b. If primary %Inhibition is greater than 100%, then the assigned score is 40
c. If primary %Inhibition is between 0% and 100%, then the calculated score is (%Inhibition)*0.4
2) Second tier (41-80 range) is reserved for dose-response confirmation data.
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
Score = 44 + 6*(pIC50 - 3)
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior is likely to be an artifact of that assay will generally have lower score values.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)