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BioAssay: AID 652000

Single concentration EBI2 agonist assay to profile uHTS hits from a small molecule antagonists of the EBI2 receptor

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 ..
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 Tested Compounds
 Tested Compounds
All(2431)
 
 
Inactive(2431)
 
 
 Tested Substances
 Tested Substances
All(2440)
 
 
Inactive(2440)
 
 
AID: 652000
Data Source: Burnham Center for Chemical Genomics (SBCCG-A952-EBI2-Agonist-CP-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-01-30

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
651636uHTS identification of small molecule antagonists of the EBI2 receptor via a luminescent beta-arrestin assayscreening
651641Summary assay for small molecule antagonists of the EBI2 receptorsummary
Description:
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 is similar to "uHTS identification of small molecule antagonists of the EBI2 receptor via a luminescent beta-arrestin assay", AID 651636, except that it is performed in an agonist mode to profile the hits identified in EBI2 antagonist primary screening.

References
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.
Protocol
Day1 Cell Seeding
1) Plate 1500 cells/well in 5 uL of assay media into columns 1-48 of a 1536-well assay plate, using Multidrop Combi 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 2.5 nL of 10 mM compound solutions into assay plate col 5 - 48. Transfer 2.5 nL of DMSO to positive and negative control wells in columns 1 - 4.
3) Dispense 10 nL 40 uM 7alpha,25-OHC to columns 1-2 for the positive control wells.
4) Centrifuge plates at 1000 rpm for 1 minute on a Vspin centrifuge and re-lid plates.
5) Incubate plates at 37 degrees, 100% relative humidity, 5% CO2 for 60 minutes.
6) Following 60 minute incubation, deliver 2.5 uL of Detection Reagent solution to each assay plate (Columns 1 - 48) using a Multidrop Combi dispenser.
7) Centrifuge plates at 2000 rpm for 2 minute on a Vspin centrifuge and re-lid plates.
8) Incubate plates for 60 minutes at 25 degrees in the dark.
9) Read plates using the Viewlux or Envision using a luminescence protocol.
Comment
Compounds that demonstrated a % activity of >=50% are defined as active compounds.

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
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 and is not applicable:
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:
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
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Activity at 5 uM_Mean (5μM**)Mean % activity in primary screeningFloat%
2%Activity at 5 uM_1 (5μM**)% activity in primary screening for the first replicateFloat%
3%Activity at 5 uM_2 (5μM**)% activity in primary screening for the second replicateFloat%
4%Activity at 5 uM_3 (5μM**)% activity in primary screening for the third replicateFloat%
5Value_1 (5μM**)Value of the first replicateFloatRLU
6Value_2 (5μM**)Value of the second replicateFloatRLU
7Value_3 (5μM**)Value of the third replicateFloatRLU
8Mean High_1Mean fluorescence of positive controls in the corresponding plateFloatRLU
9Mean High_2Mean fluorescence of positive controls in the corresponding plateFloatRLU
10Mean High_3Mean fluorescence of positive controls in the corresponding plateFloatRLU
11STD Deviation High_1Standard deviation (n=64) of positive controls in the corresponding plateFloatRLU
12STD Deviation High_2Standard deviation (n=64) of positive controls in the corresponding plateFloatRLU
13STD Deviation High_3Standard deviation (n=64) of positive controls in the corresponding plateFloatRLU
14Mean Low_1Mean fluorescence of negative controls in the corresponding plateFloatRLU
15Mean Low_2Mean fluorescence of negative controls in the corresponding plateFloatRLU
16Mean Low_3Mean fluorescence of negative controls in the corresponding plateFloatRLU
17STD Deviation Low_1Standard deviation (n=64) of negative controls in the corresponding plateFloatRLU
18STD Deviation Low_2Standard deviation (n=64) of negative controls in the corresponding plateFloatRLU
19STD Deviation Low_3Standard deviation (n=64) of negative controls in the corresponding plateFloatRLU

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH097522-01

Data Table (Concise)
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