uHTS identification of small molecule antagonists of the EBI2 receptor via a luminescent beta-arrestin assay
Epstein-Barr virus-induced gene 2 (EBI2) (1-4), also known as GPR183, is expressed on B cells and is highly induced upon activation (5). Recent gene targeting experiments revealed that EBI2-/- B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses (1, 2). Most recently, two research teams made the unlikely discovery that oxysterol compounds, more ..
BioActive Compounds: 2946
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 Number: 1 R03 MH097522-01
Assay Provider: Robert Rickert, Ph.D., Sanford-Burnham Medical Research Institute
Epstein-Barr virus-induced gene 2 (EBI2) (1-4), also known as GPR183, is expressed on B cells and is highly induced upon activation (5). Recent gene targeting experiments revealed that EBI2-/- B cells exhibited defective migration, resulting in strongly impaired T cell-dependent antibody responses (1, 2). Most recently, two research teams made the unlikely discovery that oxysterol compounds, previously known to bind nuclear receptors, are the physiologic ligands for EBI2 (3, 4). Although other closely related oxysterols showed some potency and binding capability to EBI2, the most potent endogenous EBI2 receptor ligand and activator was 7alpha,25-dihydroxycholesterol (7alpha,25-OHC) (4). EBI2-/- B cells did not bind 7alpha,25-OHC and mice deficient for cholesterol 25-hydroxylase that is necessary to generate 7alpha,25-OHC display a phenotype similar to that of EBI2-/- mice (3). The recent publication in July 2011 of the deorphanizing of this receptor is a major step forward towards understanding the role of EBI2 in immunobiology. In order to investigate the importance of EBI2 in immune processes and potential as a drug target, selective potent chemical probes that antagonize this receptor need to be identified and made freely available to the research community.
This cell-based luminescent assay utilizes the HitHunter DiscoveRx approach to screen the MLPCN chemical library to identify the first small molecule antagonists of EBI2.
1. Gatto D, Paus D, Basten A, Mackay CR, Brink R. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses. Immunity. 2009;31(2):259-69.
2. Pereira JP, Kelly LM, Xu Y, Cyster JG. EBI2 mediates B cell segregation between the outer and centre follicle. Nature. 2009;460(7259):1122-6. PMCID: 2809436.
3. Hannedouche S, Zhang J, Yi T, Shen W, Nguyen D, Pereira JP, et al. Oxysterols direct immune cell migration via EBI2. Nature. 2011;475(7357):524-7.
4. Liu C, Yang XV, Wu J, Kuei C, Mani NS, Zhang L, et al. Oxysterols direct B-cell migration through EBI2. Nature. 2011;475(7357):519-23.
5. Chan TD, Gardam S, Gatto D, Turner VM, Silke J, Brink R. In vivo control of B-cell survival and antigen-specific B-cell responses. Immunological Reviews. 2010;237(1):90-103.
Day1 Cell Seeding
1) Plate 600 cells/well in 4 uL of assay media into columns 1-48 of a 1536-well assay plate, using straight tip dispense on a Bioraptr dispenser.
2) Centrifuge plates at 1000 rpm for 1 minute on a Vspin centrifuge. Re-lid with Kalypsys metal lids.
3) Incubate overnight at 37 degrees, 100% relative humidity, 5% CO2.
Day2 Compound Addition
1) Centrifuge compound plates at 500 rpm for 1 minute on a Vspin centrifuge.
2) Using LabCyte Echo, transfer 12.5 nL of 2 mM compound solutions into assay plate col 5 - 48. Transfer 12.5 nL of DMSO to positive and negative control wells in columns 1 - 4.
3) Dispense 2mul/well of assay media to columns 1-2 for the positive control wells.
4) Using the Bioraptr Dispenser, add 2mul/well of 400 nM 7a,25-OHC (FAC = 80 nM) in assay media to columns 3-48 for the negative control and test compound wells.
5) Centrifuge plates at 1000 rpm for 1 minute on a Vspin centrifuge and re-lid plates.
6) Incubate plates at 37 degrees, 100% relative humidity, 5% CO2 for 60 minutes.
7) Following 60 minute incubation, deliver 2.5 muL of Detection Reagent solution to each assay plate (Columns 1 - 48) using a Bioraptr dispenser.
8) Centrifuge plates at 2000 rpm for 2 minute on a Vspin centrifuge and re-lid plates.
9) Incubate plates for 60 minutes at 25 degrees in the dark.
10) Read plates using the Viewlux or Envision using a luminescence protocol.
Compounds that demonstrated a corrected % activity of >=50% are defined as inhibitors of the reaction.
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
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.
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. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
** Test Concentration.
Data Table (Concise)