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BioAssay: AID 602409

Single concentration confirmation of uHTS hits from a small molecule antagonists of the CXCR6 receptor via a 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
All(704)
 
 
Active(458)
 
 
Inactive(246)
 
 
 Tested Substances
 Tested Substances
All(704)
 
 
Active(458)
 
 
Inactive(246)
 
 
AID: 602409
Data Source: Burnham Center for Chemical Genomics (SBCCG-A811-CXCR6-CP-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-16

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 458
Depositor Specified Assays
AIDNameTypeComment
602244uHTS identification of CXCR6 Inhibitors in a B-arrestin luminescence assayscreening
602249Summary assay for small molecule Inhibitors of CXCR6summary
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.

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 Chemokine CXCR6 receptor in the CHO-K1 PathHunter CHO-K1 CXCR6 b-arrestin cell line in 1536-well plate format in uHTS mode.

B. Materials:
PathHunter CHO-K1 CXCR6 b-arrestin cell line (DiscoveRx, Cat# 93-0205C2)
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)
Cell strainer, 40 um (BD, Cat# 352340)
1536-well, white, solid-bottom, Kalypsys compatible, TC plate (Corning)
CXCCL16 (R&D Systems, Cat# 976-CX)
PathHunter Detection Reagents (DiscoveRx, Cat# 93-0001)
Galacton Star
Emerald 11
Cell Assay Buffer

C. Hit Confirmation Procedures:
Day1 Cell Seeding
1) Plate 800 cells/well in 3 uL of assay media into columns 1-48 of a 1536-well assay plate, using combi dispenser.
2) Centrifuge plates at 500 rpm for 1 minute on a Vspin centrifuge.
3) Put Kalypsis lids on and 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 10 nL from a 10 mM Echo qualified plate containing DPI liquid reordered test compounds into assay plate Col. 5 - 48 (final concentration of test compounds is 20 uM, 0.2% DMSO). Transfer 10 nL of DMSO to positive and negative control wells in Columns 1 - 4.
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.
4) Using the Biotek Dispenser, add 2ul/well of 25 nM CXCL16 (FAC = 10 nM) in assay media to Col. 3-48 for the negative control and test compounds.
5) Centrifuge plates at 1000 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 3 minute on a Vspin centrifuge.
9) Incubate plates for 60 minutes at 25 degrees in the dark.
10) Read plates using the Envision plate reader 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
Same as Growth Media without the selection reagents
Trypsin
Dilute 0.25% Trypsin/EDTA to 0.05% Trypsin/EDTA using DPBS
Negative Control
Growth Media with 10 nM CXCL16
Detection Reagent
Use the following ratio to prepare the detection reagent:
Galacton Star : Emerald II : Assay Buffer = 1 : 5 : 19
Comment
In this assay selected hits from the initial screen (AID 602244) were retested 4 times. Compounds with %activity_mean >40% are defined "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. 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.
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
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Activity at 20 uM_Mean (20μM**)Mean % inhibition in primary screeningFloat%
2%Activity at 20 uM_1 (20μM**)% inhibition in primary of the first replicateFloat%
3%Activity at 20 uM_2 (20μM**)% inhibition in primary of the +second replicateFloat%
4%Activity at 20 uM_3 (20μM**)% inhibition in primary of the third replicateFloat%
5%Activity at 20 uM_4 (20μM**)% inhibition in primary of the fourth replicateFloat%
6Value_1 (20μM**)Value of the first replicateFloatRLU
7Value_2 (20μM**)Value of the second replicateFloatRLU
8Value_3 (20μM**)Value of the third replicateFloatRLU
9Value_4 (20μM**)Value of the fourth replicateFloatRLU
10Mean High_1Mean fluorescent signal of negative controls in the corresponding plate FloatRLU
11Mean High_2Mean fluorescent signal of negative controls in the corresponding plate FloatRLU
12Mean High_3Mean fluorescent signal of negative controls in the corresponding plate FloatRLU
13Mean High_4Mean fluorescent signal of negative controls in the corresponding plate FloatRLU
14STD Deviation High_1Standard deviation (n=64) of negative controls in the corresponding plateFloatRLU
15STD Deviation High_2Standard deviation (n=64) of negative controls in the corresponding plateFloatRLU
16STD Deviation High_3Standard deviation (n=64) of negative controls in the corresponding plateFloatRLU
17STD Deviation High_4Standard deviation (n=64) of negative controls in the corresponding plateFloatRLU
18Mean Low_1Mean fluorescent signal of positive controls in the corresponding plateFloatRLU
19Mean Low_2Mean fluorescent signal of positive controls in the corresponding plateFloatRLU
20Mean Low_3Mean fluorescent signal of positive controls in the corresponding plateFloatRLU
21Mean Low_4Mean fluorescent signal of positive controls in the corresponding plateFloatRLU
22STD Deviation Low_1Standard deviation (n=64) of positive controls in the corresponding plate FloatRLU
23STD Deviation Low_2Standard deviation (n=64) of positive controls in the corresponding plate FloatRLU
24STD Deviation Low_3Standard deviation (n=64) of positive controls in the corresponding plate FloatRLU
25STD Deviation Low_4Standard deviation (n=64) of positive controls in the corresponding plate FloatRLU

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

Data Table (Concise)
Classification
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