SAR Analysis for the identification of Selective Antagonists of GPR55 using an Image-Based Screen
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 the project is to identify small molecule agonists 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: 3
Data Source: Dept. of Cell Biology, Duke University
Source Affiliation: Duke University (Durham, NC)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1X01 DA026205-01
Assay Provider: Dr. Larry Barak/Yushi Bai
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 the project is to identify small molecule agonists 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 imaging assay utilizes HEK293 transiently expressing a PKC II GFP biosensor and a wild type GPR55 receptor. Upon antagonist treatment, the GPR55-mediated G-protein signaling is measured by PKC II GFP recruitment to the plasma membrane and formation of widespread plasma membrane remodeling. This PKC recruitment is shown as increased fluorescence on plasma membrane and development of blebs. The results with or without GPR55 shows the specificity of the bleb formation to the receptor.
This assay is developed and performed to confirm hits originally identified in "Image-based HTS for Selective Antagonists of GPR55" (AID 2013) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally
1) 384-well plates, black with clear bottom (MatriCal# MGB101-1-2).
2) HEK (Human Embryonic Kidney) cell line transiently expressing the PKC betaII GFP and GPR55 receptor.
3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum.
4) Positive Control Working Solution: AM251 (Sigma A6226, 10mM stock in DMSO) diluted in PBS (Ca+, Mg+) to desired concentrations.
5) Negative Control Working Solution: 100% DMSO.
6) Test Compounds Working Solution: 10mM in 100% DMSO.
7) Fixative Working Solution: 2% Paraformaldehyde (PFA) in PBS.
1) 40 ul of cell suspension (~1,000,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Thermo-Multidrop.
2) Plates are incubated overnight at 37 degrees C and 5% CO2.
3) Serum is removed by media aspiration and replacing with 30ul serum-free clear MEM prior to addition of compounds.
4) Compound addition was performed using multi-channel pipette.
a. Compounds were added to columns 3 to 22. Final concentration ranged from 10 uM to 100 uM (four doses), in duplicate.
b. Negative control was added to column 23. DMSO final concentration was 0.1%.
5) Plates were incubated for 15 minutes at 37 degrees C and 5% CO2.
6) AM251 working solution was added to all compound wells for a final concentration of 10uM. The plate was again incubated for 30 minutes.
7) 40 ul of fixative working solution was added to each well using a Thermo-Multidrop for a final concentration of 1% PFA.
8) Plates were incubated overnight at 4 degrees C.
Image Acquisition and Analysis:
Image acquisition was performed on either Zeiss Axiovert 200M fluorescent microscope
setting --- Plan-APOCHRMAT 40x /0.95 korr air objective;
Acquisition camera set to 1388x1040 standard mono.
or LSM-510 con-focal fluorescent microscope
setting --- 100x /1.4 oil objective;
Using the time stack acquisition mode to follow GFP membrane recruitment in living cells.
Image analysis was performed by eye ---
'-' means no GFP membrane recruitment or blebs formation
'+' means ~1% GFP membrane recruitment or blebs formation
'++' means ~10% GFP membrane recruitment or blebs formation
'+++' means ~30% GFP membrane recruitment or blebs formation
'++++' means ~60% GFP membrane recruitment or blebs formation
Compounds with <= 1% GFP Membrane recruitment are considered "active" in this assay.
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 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.
Compounds that are "inactive" in this assay are given a score of 81.
Compounds that are "active" in this assay are given a score of 90.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)