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BioAssay: AID 651594

Dose Response counterscreen of small molecule antagonists of the CXCR6 receptor using a CXCR5 receptor luminescent beta-arrestin assay

Prostate cancer (PCa) is the second leading cause of cancer death in American men and its morbidity has increased globally in recent years. The high mortality rate is closely associated with the spread of malignant cells to various tissues including bone. Nearly 10% of patients whose conditions are diagnosed as PCa initially present with bone metastasis and almost all patients who die of prostate more ..
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 Tested Compounds
 Tested Compounds
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Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
AID: 651594
Data Source: Burnham Center for Chemical Genomics (SBCCG-A906-CXCR5-Antagonist-DMSO-DR-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-09-21
Modify Date: 2012-10-05

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeComment
602244uHTS identification of CXCR6 Inhibitors in a B-arrestin luminescence assayScreeningdepositor-specified cross reference
602249Summary assay for small molecule Inhibitors of CXCR6Summarydepositor-specified cross reference
493098uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assayScreeningsame project related to Summary assay
493121Summary assay for selective small molecule antagonists of the CCR6 receptorSummarysame project related to Summary assay
504712HTS Dose response counterscreen for assays utilizing the enzyme, beta-galactosidase - Set 3Confirmatorysame project related to Summary assay
504728Dose Response confirmation of small molecule antagonists of the CCR6 receptor: a luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
540334SAR analysis of small molecule antagonists of the CCR6 receptor: a luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
540340SAR analysis counterscreen of small molecule antagonists of the CCR6 receptor using a CXCR5 receptor luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
588359SAR analysis counterscreen of small molecule antagonists of the CCR6 receptor using an APJ receptor luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
493098uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assayScreeningsame project related to Summary assay
493159Summary assay for small molecule antagonists of the CCR6 receptorSummarysame project related to Summary assay
504712HTS Dose response counterscreen for assays utilizing the enzyme, beta-galactosidase - Set 3Confirmatorysame project related to Summary assay
504728Dose Response confirmation of small molecule antagonists of the CCR6 receptor: a luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
540334SAR analysis of small molecule antagonists of the CCR6 receptor: a luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
540340SAR analysis counterscreen of small molecule antagonists of the CCR6 receptor using a CXCR5 receptor luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
588359SAR analysis counterscreen of small molecule antagonists of the CCR6 receptor using an APJ receptor luminescent beta-arrestin assayConfirmatorysame project related to Summary assay
602409Single concentration confirmation of uHTS hits from a small molecule antagonists of the CXCR6 receptor via a luminescent beta-arrestin assayScreeningsame project related to Summary assay
624129Dose Response confirmation of uHTS hits from a small molecule antagonists of the CXCR6 receptor in a screening assayConfirmatorysame project related to Summary assay
651598SAR analysis of small molecule antagonists of the CXCR6 receptor in an APJ-DiscoveRx counter screen panel assayConfirmatorysame project related to Summary assay
651601SAR analysis of small molecule antagonists of the CXCR6 receptor in a screening panel assayConfirmatorysame project related to Summary assay
651708SAR analysis of small molecule antagonists of the CXCR6 receptor in a screening panel assay - 3Confirmatorysame project related to Summary assay
651902SAR analysis of small molecule antagonists of the CXCR6 receptor using a CXCR4 receptor luminescent beta-arrestin counterscreenConfirmatorysame project related to Summary assay
651919SAR analysis of small molecule antagonists of the CXCR6 receptor in a screening panel assay - 4Confirmatorysame project related to Summary assay
651940SAR analysis of small molecule antagonists of the hCXCR6 receptor using a mCXCR6 receptor luminescent beta-arrestin selectivity assayConfirmatorysame project related to Summary assay
651941SAR analysis of small molecule antagonists of the CXCR6 receptor using a CCR6 receptor luminescent beta-arrestin counterscreenConfirmatorysame 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: 1R03MH095589-01 (Cycle 18)
Assay Provider: Gregory Roth Ph.D., Sanford Burnham Medical Research Institute.

Prostate cancer (PCa) is the second leading cause of cancer death in American men and its morbidity has increased globally in recent years. The high mortality rate is closely associated with the spread of malignant cells to various tissues including bone. Nearly 10% of patients whose conditions are diagnosed as PCa initially present with bone metastasis and almost all patients who die of prostate cancers have skeletal involvement. Identifying new mechanisms that control bone metastasis is of great consequence to facilitate the design of therapeutics aimed at decreasing metastatic risk and/or its complications. To address this unmet medical need, our team is actively engaged in exploring the chemical biology, medicinal chemistry, and therapeutic significance of modulating tumor cell trafficking and metastasis via chemokine receptor inhibition. The primary objective of this proposal is to use high throughput screening methods to identify small molecule antagonist probes that selectively inhibit CXCR6. Our team intends to address a key hypothesis: The CXCR6/CXCL16 axis significantly contributes to PCa cell metastasis and subsequent bone invasion. A small molecule antagonist would block cancer cell trafficking; hence mediate a metastatic event and disease progression to bone. Thus, access to pharmacologically available small molecule antagonists will ultimately enable our studies in disease relevant models and allow for a more seamless translational advance to clinical applications.

The project goal is to identify a chemical probe of chemokine CXCR6 receptor function that inhibits the receptor. An antagonist of this receptor would provide a novel research tool to evaluate cancer cell trafficking and bone metastasis in prostrate and other cancers

In this description 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 CXCR6 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.

This assay is a follow-up to "uHTS identification of CXCR6 Inhibitors in a B-arrestin luminescence assay", AID 602244. Compounds were tested in the primary assay against the CXCR6 receptor. Compounds considered active in this assay are non-selective, non-specific and/or assay artifacts of the primary assay and are not desired.

REFERENCES
Hu, W; Zhen, X; Xiong, B; Wang, B; Zhang, W; Zhou, W CXCR6 is expressed in human prostate cancer in vivo and is involved in the in vitro invasion of PC3 and LNCap cells. Cancer Sci 2008, 99, 1362-1369.

Matloubian, M; David, A; Engel, S; Ryan, JE; Cyster, JG A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo. Nature Immunol 2000, 1, 298-304.

Chandrasekar B, Bysani S, Mummidi S. CXCL16 signals via Gi, phosphatidylinositol 3-kinase, Akt, I kappa B kinase, and nuclear factor-kappa B and induces cell-cell adhesion and aortic smooth muscle cell proliferation. J Biol Chem 2004, 279, 3188-3196.
Protocol
A. Brief Description of the Assay:
The purpose of this assay is to detect antagonists that inhibit the activation of the CXCR5 receptor in the CHO-K1 beta-Arrestin Cell Line in 1536-well plate format in uHTS mode.
B. Materials:
PathHunter CHO-K1 CXCR5 b-arrestin cell line (DiscoveRx, Cat# 93-0204C2)
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)
384-well white solid bottom, TC plate (Griener)
CXCL13 peptide (R&D Systems, Cat# 801-CX)
PathHunter Detection Reagents (DiscoveRx, Cat# 93-0001)
Galacton Star
Emerald 11
Cell Assay Buffer
C. uHTS Procedures:
Day1 Cell Seeding
1) Plate 2500 cells/well in 20 uL of assay media into columns 1-24 of a 384-well assay plate, using Biotek dispenser.
2) Centrifuge plates at 500 rpm for 1 minute on a Vspin centrifuge.
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 200 nL of DMSO to positive and negative control wells in columns 1 - 2 and 23-24, respectively. Using a dose response protocol, transfer compounds from 10mM and 0.312 mM Echo qualified plates into assay plate columns -3-22. (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 10ul/well of Assay media to Col. 1-2 for the positive control wells.
and 10ul/well of 225 nM CXCL13 (FAC = 75 nM) in assay media to Col. 3-24 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 15 uL of Detection Reagent solution to each assay plate (Columns 1 - 24) 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.
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 with IC50_Mean <= 50 uM are defined as active 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 and is not applicable in this assay.
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with a compound's 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- or pathway-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 the 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 all the items discussed above is
Score = 44 + 6*(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 50 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.
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 ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Qualifier_MeanThis qualifier is to be used with the next TID, IC50_Mean. If the qualifier is ""="", the IC50 result equals the value in that column. If the qualifier is "">"", the IC50 result is greater than that value. If the qualifier is ""<"", the IC50 result is smaller than that valueString
2IC50_Mean*IC50 value determined using a sigmoidal dose response equationFloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
4IC50_1IC50 value determined using a sigmoidal dose response equationFloatμM
5Std.Err(IC50)_1Standard Error of the IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7Excluded_Points_first_pointFlags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
8% Activity at 66.6 uM_first_point (66.6μM**)% Activity at the test concentrationFloat%
9% Activity at 33.3 uM_first_point (33.3μM**)% Activity at the test concentrationFloat%
10% Activity at 16.65 uM_first_point (16.65μM**)% Activity at the test concentrationFloat%
11% Activity at 8.325 uM_first_point (8.325μM**)% Activity at the test concentrationFloat%
12% Activity at 4.1625 uM_first_point (4.1625μM**)% Activity at the test concentrationFloat%
13% Activity at 2.08125 uM_first_point (2.08125μM**)% Activity at the test concentrationFloat%
14% Activity at 1.040625 uM_first_point (1.04062μM**)% Activity at the test concentrationFloat%
15% Activity at 0.5203125 uM_first_point (0.520312μM**)% Activity at the test concentrationFloat%
16% Activity at 0.2601562 uM_first_point (0.260156μM**)% Activity at the test concentrationFloat%
17% Activity at 0.1300781 uM_first_point (0.130078μM**)% Activity at the test concentrationFloat%
18% Activity at 0.06503906 uM_first_point (0.0650391μM**)% Activity at the test concentrationFloat%
19% Activity at 0.03251953 uM_first_point (0.0325195μM**)% Activity at the test concentrationFloat%
20IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2. If the qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that valueString
21IC50_2IC50 value determined using a sigmoidal dose response equationFloatμM
22Std.Err(IC50)_2Standard Error of the IC50 valueFloatμM
23nH_2Hill coefficient determined using sigmoidal dose response equationFloat
24Excluded_Points_second_pointFlags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
25% Activity at 66.6 uM_second_point (66.6μM**)% Activity at the test concentrationFloat%
26% Activity at 33.3 uM_second_point (33.3μM**)% Activity at the test concentrationFloat%
27% Activity at 16.65 uM_second_point (16.65μM**)% Activity at the test concentrationFloat%
28% Activity at 8.325 uM_second_point (8.325μM**)% Activity at the test concentrationFloat%
29% Activity at 4.1625 uM_second_point (4.1625μM**)% Activity at the test concentrationFloat%
30% Activity at 2.08125 uM_second_point (2.08125μM**)% Activity at the test concentrationFloat%
31% Activity at 1.040625 uM_second_point (1.04062μM**)% Activity at the test concentrationFloat%
32% Activity at 0.5203125 uM_second_point (0.520312μM**)% Activity at the test concentrationFloat%
33% Activity at 0.2601562 uM_second_point (0.260156μM**)% Activity at the test concentrationFloat%
34% Activity at 0.1300781 uM_second_point (0.130078μM**)% Activity at the test concentrationFloat%
35% Activity at 0.06503906 uM_second_point (0.0650391μM**)% Activity at the test concentrationFloat%
36% Activity at 0.03251953 uM_second_point (0.0325195μM**)% Activity at the test concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH095589-01 (Cycle 18)

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