SAR analysis of small molecule antagonists of the CCR6 receptor: 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 ..
BioActive Compounds: 13
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.
The goal of this assay is to validate hits obtained in "uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assay", AID 493098 and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
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.
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.
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)
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 Biotek 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 555, transfer 40 nL of DMSO to positive and negative control wells in columns 1 - 4 and 45-48, respectively. Using a dose response protocol, transfer compounds from 10mM and 0.312 mM Echo qualified plates into assay plate columns 5 - 45. (Final concentrations range 66 uM to 0.128 uM, 10 doses, with 0.66% DMSO.)
3) Immediately following compound/DMSO transfer via the Echo, using the Biotek Dispenser, transfer 2ul/well of Assay media to Col. 1-2 for the positive control wells.
4) Using the Biotek 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 Biotek 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 Envision using a luminescence protocol.
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
Dilute 0.25% Trypsin/EDTA to 0.05% Trypsin/EDTA using DPBS
Growth Media with 14 nM MIP3a/CCL20
Use the following ratio to prepare the detection reagent:
Galacton Star : Emerald II : Assay Buffer = 1 : 5 : 19
Compounds that demonstrated an IC50 < 20 uM 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 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 and is not applicable in this assay.
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
a. Inactive compounds of the confirmatory stage are assigned a score value equal 81.
b. The score is linearly correlated with a compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
c. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in this assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
d. Summary equation that takes into account the items discussed above is
Score = 82 + 3*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)