SAR Analysis using an Image-based Vasopressin Antagonist Counter Assay for the Identification of Selective Antagonists of the GPR35 Receptor
The aim of this assay was to characterize selectivity of compounds originally identified in "Image-based HTS for Selective Antagonists of GPR35" (AID 2058) against the unrelated vasopressin receptor. Compounds were either acquired from commercial sources or synthesized by the Sanford-Burnham Center for Chemical Genomics. ..more
Data Source: Dr. Lawrence Barak
Source Affiliation: Duke University
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1X01MH085708-01
Assay Provider: Dr. Lawrence Barak, Duke University
The aim of this assay was to characterize selectivity of compounds originally identified in "Image-based HTS for Selective Antagonists of GPR35" (AID 2058) against the unrelated vasopressin receptor. Compounds were either acquired from commercial sources or synthesized by the Sanford-Burnham Center for Chemical Genomics.
This imaging assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and human vasopressin receptor type IIa. Upon agonist-mediated GPCR activation, the arrestin-GFP redistributes from the cytosolic compartment to the plasma membrane to coated pits. This arrestin-GFP redistribution is measured as increased local concentrations of fluorescent arrestins.
1) 384-well plates, black with clear bottom (MatriCal# MGB101-1-2)
2) U2OS (Human Osteosarcoma) cell line stably expressing the Beta-arrestin 2 GFP and human vasopressin IIa receptor
3) Culture Media: MEM with L-glutamine, Pen-strep, 10% Fetal Bovine Serum and selection antibiotics - 200 ug/ml G418 and 100 ug/ml Zeocin
4) Agonist: Arginine Vasopressin
5) DMSO solution
6) Test Compounds in 100% DMSO
7) Fixative Working Solution: 2% Paraformaldehyde (PFA) diluted in PBS
1) Cells were seeded at 12,000 cells/well in a 384 well plate
2) Plates are incubated overnight at 37 degree C and 5% CO2
3) Serum was removed by media aspiration and replaced with serum-free media
4) Agonist control (Arginine Vasopressin) was added to column 2 at 0, 0.01 uIU/ml, 0.1 uIU/ml, 1 uIU/ml, 10 uIU/ml, 100 uIU/ml, 1 mIU/ml, and 10 mIU/ml (EC80 concentration=16 nM equivalent to 10 uIU/ml)
5) Compounds were added to columns 3 to 22 at 10 uM final concentration and 0.1% DMSO
6) DMSO was added to column 23 for a final concentration of 0.1%
7) Plates were incubated for 15 min and then Arginine Vasopressin was added to columns 3 to 22 at 16 nM. Plates were incubated for additional 40 minutes at 37 degrees C and 5% CO2
8) Fixative solution was added to each well for a final concentration of 1% PFA
9) Plates were incubated overnight at 4 degree C
Image Acquisition and Analysis:
1) Image acquisition was performed on a Zeiss Axiovert 200M fluorescent microscope using the following settings:
Plan-APOCHRMAT 40x /0.95 korr air objective
Acquisition camera providing an image with 1388 by 1040 pixels
1 channel: Beta-arrestin GFP using 488 nm laser excitation and 540/70 nm emission filters
2 fields per well
2) Image analysis was performed using a Wavelet Algorithm using the Batchmode computer software. The number of detected GFP-beta-arrestin aggregates ("# of Spots") was used as the assay read-out.
4) %Activity of the test compounds was calculated based on the number of detected aggregates ("# of Spots") as compared to the negative (0.01 uIU/ml) and positive (10 mIU/ml) controls. Compounds with %Activity > 50% for both datasets at 10 uM were considered active.
Compounds with %Activity > 50% for both datasets at 10 uM were considered active.
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.
2) Second tier (41-80 range) is reserved for dose-response confirmation data.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues.
Compounds that are inactive in this assay are assigned a score of 81.
Compounds that are active in this assay are assigned a score of 90.
** Test Concentration.
Data Table (Concise)