Image-Based HTS for Selective Antagonists for GPR55
The cannabinoid and endocannabinoid system has been implicated in the pathophysiology of drug dependence and addiction disorders. GPR55, an orphan G-Protein Coupled Receptor, has been reported to be a cannabinoid receptor, but its status as such remains unresolved due to conflicting results from pharmacological studies. The goal of this project is to identify small molecule antagonists of GPR55, which may aid in the deorphanization efforts of this receptor and ultimately further the understanding of the role of GPR55 in drug addiction. ..more
BioActive Compounds: 370
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: 1X01 DA026205-01
Assay Provider: Dr. Mary Abood, California Pacific Medical Center Research Institute
The cannabinoid and endocannabinoid system has been implicated in the pathophysiology of drug dependence and addiction disorders. GPR55, an orphan G-Protein Coupled Receptor, has been reported to be a cannabinoid receptor, but its status as such remains unresolved due to conflicting results from pharmacological studies. The goal of this project is to identify small molecule antagonists of GPR55, which may aid in the deorphanization efforts of this receptor and ultimately further the understanding of the role of GPR55 in drug addiction.
This high content imaging assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and an enhanced GPR55 receptor. Upon agonist-mediated GPCR activation, the arrestin-GFP redistributes from the cytosolic compartment to the plasma membrane to coated pits and further into endosomal vesicles, which can be quantified as increased local aggregation of the GFP-arrestins. The GPR55 antagonistic action of a compound in the presence of the GPR55 agonist Lysophosphatidylinositol will be detected as a decrease in local aggregation of the GFP arrestins
1) 384-well plates, black with clear bottom (Greiner# 781091)
2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin GFP and GPR55 receptor
3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 100ug/ml G418 and 50ug/ml Zeocin
4) Agonist Working Solution: Lysophosphatidylinositol (Avanti Polar #850090P, 5mM stock in DMSO) diluted to 50uM in water.
5) Controls Working Solution: 2.5% DMSO diluted in water (for positive control wells: no agonist will be added to mimic 100% inhibition of agonist-mediated response; for negative control wells: LPI agonist will be added to mimic 0% inhibition of agonist-mediated response).
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 (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 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 0.25% DMSO.
b) 5ul of control (2.5% DMSO) working solution was added to column 1 and 2 for a final concentration of 0.25% DMSO.
c) Plates were pre-incubated with compound for 30 minutes at 37 degree C and 5% CO2 prior to addition of LPI.
d) After 30-minutes, 5ul of the agonist (50uM LPI) working solution was added to entire plate except column 1 for a final compound concentration of 5uM (~EC80).
5) Plates were incubated for 75 minutes at 37C 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.
Hit Confirmation Procedure:
A. Single-concentration Confirmation
1)Same as primary screen, except compound addition was done using the ECHO55 Acoustic Dispenser.
B. Dose Response Confirmation
1) Same as primary screen steps 1 to 3.
2) Compound addition was done on the ECHO550 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, 160nL of 100% DMSO was added to columns 1 and 2 for a final DMSO concentration of 0.31%.
c. Plates were pre-incubated with compound for 30 minutes at 37 degree C and 5% CO2 prior to addition of LPI.
d. After 30-minutes, 5ul of the agonist working solution was added to entire plate except column 1 using the Biomek FX . Final LPI concentration was 5uM.
3) Primary screen procedure steps 5 to 10 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:
- 20x 0.45 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 emisssion filters, Exp2Cam2 = DAPI (nuclei) using 365 nm Xenon lamp excitation and 450/50 nm emission filters
- 2 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: 1.5
- Nuclear Splitting Adjustment: 7
- Minimum Nuclear Area: 70
- Minimum Nuclear Contrast: 0.1
- Cytoplasm Threshold Adjustment: 0.45
- Cytoplasm Individual Threshold Adjust.: 0.15
- Spot Minimum Distance: 3
- Spot Peak Radius: 0
- Spot Reference Radius: 2
- Spot Minimum Contrast: 0.25
- Spot Minimum to Cell Intensity: 1
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) Actives from the primary screen were determined using CBIS software (ChemInnovations) by calculating the %activation of the "AverageSpotsPerCells" metric and using a hit criteria of Activity > 60% and "Number of Cells Analyzed" >100 and "TotalCytoplasmIntensity" < 500,000. Wells with cell counts lower than 100 in the 2 acquired images were flagged "cytotoxic / low cell count". Wells with a very high total GFP intensity ("TotalCytoplasmIntensity" > 500,000) were flagged artifacts due to autofluorescence. All flagged wells were excluded from hit selection.
Actives from the primary screen were determined by calculating the %inhibition of the "AverageSpotsPerCells" metric and using a hit criteria of Activity > 60% and "Number of Cells Analyzed" >100 and "TotalCytoplasmIntensity" < 500,000. Wells with cell counts lower than 100 in the 2 acquired images were flagged "cytotoxic / low cell count". Wells with a very high total GFP intensity ("TotalCytoplasmIntensity" > 500,000) were flagged artifacts due to autofluorescence. All flagged wells were excluded from hit selection
and are assigned an outcome of "inconclusive".
The primary screen "actives" were retested for hit confirmation. Compounds that confirmed in duplicate as active at 10uM concentration were then tested in dose response. For the 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 Inhibition 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 screening data. The score is correlated with % Inhibition 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)