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BioAssay: AID 602244

uHTS identification of CXCR6 Inhibitors in a B-arrestin luminescence 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(362050)
 
 
Active(1297)
 
 
Inactive(360755)
 
 
 Tested Substances
 Tested Substances
All(362388)
 
 
Active(1305)
 
 
Inactive(361083)
 
 
AID: 602244
Data Source: Burnham Center for Chemical Genomics (SBCCG-A778-CXCR6-Primary-DR-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-02-03
Modify Date: 2012-03-13

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1297
Related Experiments
Show more
AIDNameTypeComment
602249Summary assay for small molecule Inhibitors of CXCR6Summarydepositor-specified cross reference
602409Single concentration confirmation of uHTS hits from a small molecule antagonists of the CXCR6 receptor via a luminescent beta-arrestin assayScreeningdepositor-specified cross reference
624129Dose Response confirmation of uHTS hits from a small molecule antagonists of the CXCR6 receptor in a screening assayConfirmatorydepositor-specified cross reference
651594Dose Response counterscreen of small molecule antagonists of the CXCR6 receptor using a CXCR5 receptor luminescent beta-arrestin assayConfirmatorydepositor-specified cross reference
651598SAR analysis of small molecule antagonists of the CXCR6 receptor in an APJ-DiscoveRx counter screen panel assayConfirmatorydepositor-specified cross reference
651601SAR analysis of small molecule antagonists of the CXCR6 receptor in a screening panel assayConfirmatorydepositor-specified cross reference
651708SAR analysis of small molecule antagonists of the CXCR6 receptor in a screening panel assay - 3Confirmatorydepositor-specified cross reference
651902SAR analysis of small molecule antagonists of the CXCR6 receptor using a CXCR4 receptor luminescent beta-arrestin counterscreenConfirmatorydepositor-specified cross reference
651919SAR analysis of small molecule antagonists of the CXCR6 receptor in a screening panel assay - 4Confirmatorydepositor-specified cross reference
651940SAR analysis of small molecule antagonists of the hCXCR6 receptor using a mCXCR6 receptor luminescent beta-arrestin selectivity assayConfirmatorydepositor-specified cross reference
651941SAR analysis of small molecule antagonists of the CXCR6 receptor using a CCR6 receptor luminescent beta-arrestin counterscreenConfirmatorydepositor-specified cross reference
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.

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 cause the inhibit the Chemokine CXCR6receptor 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. uHTS 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 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 50 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.
4) Using the Kalypsys 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 Kalypsys 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 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
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
Compounds that demonstrated a normalized or corrected activity of >= 40% at 20 uM concentration are defined as inhibitors of the reaction.

The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.

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
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Activity at 20 uM_Norm (20μM**)Normalized % inhibition in primary screeningFloat%
2%Activity at 20 uM_Corr (20μM**)Genedata pattern corrected % inhibition in primary screeningFloat%
3Value (20μM**)Value of the sample FloatRLU
4Mean HighMean fluorescent signal of negative controls in the corresponding plate FloatRLU
5STD Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatRLU
6Mean LowMean fluorescent signal of positive controls in the corresponding plateFloatRLU
7STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plate FloatRLU

** 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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