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BioAssay: AID 493098

uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assay

Currently there are no published patents or studies specifically describing small molecule antagonists of the chemokine receptor CCR6. CCL20 (MIP-3 alpha) is the endogenous peptide ligand for the G-protein coupled receptor (GPCR) CCR6. The receptor ligand pair is responsible for the chemoattraction of immature dendritic cells, effector/memory T cells, B cells, and also plays a role at skin and more ..
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 Tested Compounds
 Tested Compounds
All(340685)
 
 
Active(1654)
 
 
Inactive(339035)
 
 
 Tested Substances
 Tested Substances
All(340929)
 
 
Active(1656)
 
 
Inactive(339273)
 
 
AID: 493098
Data Source: Burnham Center for Chemical Genomics (SBCCG-A556-CCR6-Antagonist-Primary-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-01-27

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1654
Related Experiments
Show more
AIDNameTypeComment
493121Summary assay for selective small molecule antagonists of the CCR6 receptorSummarydepositor-specified cross reference
493159Summary assay for small molecule antagonists of the CCR6 receptorSummarydepositor-specified cross reference
504712HTS Dose response counterscreen for assays utilizing the enzyme, beta-galactosidase - Set 3Confirmatorydepositor-specified cross reference
504728Dose Response confirmation of small molecule antagonists of the CCR6 receptor: a luminescent beta-arrestin assayConfirmatorydepositor-specified cross reference
540334SAR analysis of small molecule antagonists of the CCR6 receptor: a luminescent beta-arrestin assayConfirmatorydepositor-specified cross reference
540340SAR analysis counterscreen of small molecule antagonists of the CCR6 receptor using a CXCR5 receptor luminescent beta-arrestin assayConfirmatorydepositor-specified cross reference
588359SAR analysis counterscreen of small molecule antagonists of the CCR6 receptor using an APJ receptor luminescent beta-arrestin assayConfirmatorydepositor-specified cross reference
651641Summary assay for small molecule antagonists of the EBI2 receptorSummarydepositor-specified cross reference
743408On Hold
743410On Hold
743411On Hold
651636uHTS identification of small molecule antagonists of the EBI2 receptor via a luminescent beta-arrestin assayScreeningsame project related to Summary assay
651997Single concentration confirmation of uHTS hits from a small molecule antagonists of the EBI2 receptor via a luminescent beta-arrestin assayScreeningsame project related to Summary assay
652000Single concentration EBI2 agonist assay to profile uHTS hits from a small molecule antagonists of the EBI2 receptorScreeningsame project related to Summary assay
652176Single concentration validation of small molecule antagonists of the EBI2 receptor via a luminescent beta-galactosidase assayScreeningsame project related to Summary assay
652193Dose response counterscreen for EBI2 assay utilizing the enzyme, b-galactosidaseConfirmatorysame project related to Summary assay
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: R21 NS064746-01A
Assay Provider: Dr. Greg Roth, Sanford-Burnham Medical Research Institute

Currently there are no published patents or studies specifically describing small molecule antagonists of the chemokine receptor CCR6. CCL20 (MIP-3 alpha) is the endogenous peptide ligand for the G-protein coupled receptor (GPCR) CCR6. The receptor ligand pair is responsible for the chemoattraction of immature dendritic cells, effector/memory T cells, B cells, and also plays a role at skin and mucosal surfaces. The CCR6 receptor is expressed by B cells, subsets of T cells, and dendritic cells (DC). The link between the CCR6lCCL20 axis and cancer cell metastasis is a recent finding. There are two key studies that describe a relation between CCR6 and colorectal liver metastasis. The association between CCR6 expression levels in 64 primary tumor specimens in primary CRC and synchronous liver metastases suggests that CCR6 and CCL20 are involved in the metastatic spread to the liver. A small molecule tool would address a key hypothesis: Modulation of the CCR6/CCL20 axis will regulate pathogenic activities of B cells in a variety of diseases including hematopoietic malignancy and cancer metastasis.

The project goal is to identify a chemical probe of CCR6 receptor that can specifically act as 'chemical modulator' of CCR6 through inhibition (antagonism) of functional response. An important objective of this research program is to provide new insight into the regulation of cancer metastasis modulated by the CCR6/CCL20 (MIP-3 alpha) axis.

In this assay we utilize enzyme-fragment complementation to directly measure GPCR activation. Unlike imaging or other second messenger assays, the DiscoveRx b-Arrestin assay allows for a direct measure of GPCR activation by detection of b-Arrestin binding to the CCR6 receptor. In this system, b-Arrestin is fused to an N-terminal deletion mutant of b-gal (termed the enzyme acceptor of EA) and the GPCR of interest is fused to a smaller (42 amino acids), weakly complementing fragment termed ProLink. In cells that stably express these fusion proteins, ligand stimulation results in the interaction of b-Arrestin and the Prolink-tagged GPCR, forcing the complementation of the two b-gal fragments and resulting in the formation of a functional enzyme that converts substrate to detectable signal. Antagonists would be expected to inhibit agonist activation of the receptor resulting in the inhibition of signal formation in this assay.

References:
Schutyser, E.; Struyf, S.; Van Oamme, J. "The CC chemokine CCL20 and its receptor CCR6" Cytokine and Growth Factor Rev. 2003, 14,409-426.

Ghadjar P, Coupland SE, Na IK, Noutsias M, Letsch A, Stroux A, Bauer S, Buhr HJ, Thiel E, Scheibenbogen C, Keilholz U. "Chemokine receptor CCR6 expression level and liver metastasis In colorectal cancer" J. Clin. Oneal. 2006, 24, 1010-1016.

Rubie C, Oliveira V, Kempf K, Wagner M, Tilton B, Rau B, Kruse B, Konig J, Schilling M. "Involvement of chemokine receptor CCR6 in colorectal cancer metastasis" Tumour Biology 2006,27, 166-174.
Protocol
A. Brief Description of the Assay:
The purpose of this assay is to detect antagonists that inhibit the activation of the CCR6 receptor in the CHO-K1 beta-Arrestin Cell Line in 1536-well plate format in uHTS mode.

B. Materials:
PathHunter CHO-K1 CCR6 b-arrestin cell line (DiscoveRx, Cat# 93-0194C2)
F12 nutrient mix HAMs (Invitrogen, Cat# 11765)
Fetal Bovine Serum, heat-inactivated (Hyclone, Cat# SH30396)
100X Penicillin/Streptomycin Solution (Invitrogen, Cat#15140-122)
Hygromycin B (Roche, Cat# 10843555001)
Geneticin (MPBiomedicals, Cat # 1672548)
Trypsin-EDTA 0.25% (Invitrogen, Cat# 25200-056)
Cell Dissociation Buffer (Invitrogen, Cat# 13151)
DPBS (Hyclone, Cat# 30028.02)
T225 TC Flask (Nunc, Cat# 159934)
1536-well, white, solid-bottom, Kalypsys compatible, TC plate (Aurora)
MIP-3a/CCL20 peptide (R&D Systems, Cat# 360MP)
PathHunter Detection Reagents (DiscoveRx, Cat# 93-0001)
Galacton Star
Emerald 11
Cell Assay Buffer

C. uHTS Procedures:
Day1 Cell Seeding
1) Plate 800 cells/well in 4 uL of assay media into columns 1-48 of a 1536-well assay plate, using straight tip dispense on a Kalypsys dispenser.
2) Centrifuge plates at 500 rpm for 1 minute on a Vspin centrifuge. Use Kalypsys metal lids.
3) Incubate overnight at 37 degrees, 100% relative humidity, 5% CO2 for 16-18 hours.
Day2 Compound Addition
1) Centrifuge compound plates at 500 rpm for 1 minute on a Vspin centrifuge.
2) Using LabCyte Echo, transfer 60 nL from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48 (final concentration of test compounds is 20 uM, 1% DMSO). Transfer 60 nL of DMSO to positive and negative control wells in Columns 1 - 4.
3) Immediately following compound/DMSO transfer via the Echo, using the Kalypsys Dispenser, transfer 2ul/well of Assay media to Col. 1-2 for the positive control wells.
4) Using the Kalypsys Dispenser, add 2ul/well of 42 nM MIP3a/CCL20 (FAC = 14 nM) in assay media to Col. 3-48 for the negative control and test compound wells.
5) Centrifuge plates at 500 rpm for 1 minute on a Vspin centrifuge.
6) Incubate plates at 25 degrees in the dark for 90 minutes.
7) Following 90 minute incubation, deliver 3.0 uL of Detection Reagent solution to each assay plate (Columns 1 - 48) using a Kalypsys dispenser.
8) Centrifuge plates at 2000 rpm for 2 minute on a Vspin centrifuge.
9) Incubate plates for 60 minutes at 25 degrees in the dark.
10) Read plates using the Viewlux using a luminescence protocol.

D. Recipes:
Growth Media
F12 nutrient mix HAMs supplemented with 10% hi-FBS, 1X Penicillin/Streptomycin; selection reagents: 300ug/ml Hygromycin B, 800ug/ml Geneticin
Assay Media/Positive Control
Same as Growth Media without the selection reagents and only 2.5% hi-FBS
Trypsin
Dilute 0.25% Trypsin/EDTA to 0.05% Trypsin/EDTA using DPBS
Negative Control
Growth Media with 14 nM MIP3a/CCL20
Detection Reagent
Use the following ratio to prepare the detection reagent:
Galacton Star : Emerald II : Assay Buffer = 1 : 5 : 19
Comment
Compounds that demonstrated an inhibition of >= 50% at 20 uM concentration are defined as actives in this assay.

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:
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
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: CHO-K1
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Inhibition at 20 uM (20μM**)inhibition in primary screeningFloat%
2Value (20μM**)Value of the sampleFloatRLU
3Mean LowMean luminescence ratio of positive controls in the corresponding plateFloatRLU
4Std Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatRLU
5Mean HighMean luminescence ratio of negative controls in the corresponding plateFloatRLU
6Std Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatRLU

** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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