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BioAssay: AID 588359

SAR analysis counterscreen of small molecule antagonists of the CCR6 receptor using an APJ receptor luminescent beta-arrestin assay

This assay was developed and performed as a counter assay for the primary assay originally identified as "uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assay", AID 493098. Compounds are either acquired from commercial sources or synthesized internally. A probe that is selective against APJ (within equipotent range as defined by a >10-fold difference in IC50) is desired. ..more
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 Tested Compounds
 Tested Compounds
All(19)
 
 
Active(11)
 
 
Inactive(8)
 
 
 Tested Substances
 Tested Substances
All(19)
 
 
Active(11)
 
 
Inactive(8)
 
 
AID: 588359
Data Source: Burnham Center for Chemical Genomics (SBCCG-A685-CCR6-Antagonist-DryPowder-CTR-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-09-14
Hold-until Date: 2011-10-24
Modify Date: 2011-10-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 11
Related Experiments
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AIDNameTypeComment
493098uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assayScreeningdepositor-specified cross reference
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 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
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
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

This assay was developed and performed as a counter assay for the primary assay originally identified as "uHTS identification of small molecule antagonists of the CCR6 receptor via a luminescent beta-arrestin assay", AID 493098. Compounds are either acquired from commercial sources or synthesized internally. A probe that is selective against APJ (within equipotent range as defined by a >10-fold difference in IC50) is desired.

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 APJ 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.
Protocol
A. Brief Description of the Assay:
The purpose of this assay is to detect antagonists that inhibit the activation of the Angiotensin II receptor-like 1 (Apelin receptor) in the CHO-K1 AGTRL-1 beta-Arrestin Cell Line in 1536-well plate format in uHTS mode.

B. Materials:
Angiotensin II receptor-like 1 (AGTRL-1) Cell Line (DiscoveRx, Cat# 93-0250C2)
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)
Apelin-13 (Sigma-Aldrich, Cat# A6469)
PathHunter Detection Reagents (DiscoveRx, Cat# 93-0001)
Galacton Star
Emerald 11
Cell Assay Buffer

C. uHTS Procedures:
Day1 Cell Seeding
1) Plate 1000 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.
3) Incubate overnight at 37 degrees, 100% relative humidity, 5% CO2 for 16-18 hours. Use Kalypsys metal lids.

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. 1-4 for the positive control wells.
4) Using the Biotek Dispenser, add 2ul/well of 30 nM Apelin-13 (FAC = 10 nM) in assay media to Col. 5-48 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 3 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
Same as Growth Media without the selection reagents and 2.5% FBS
Positive Control
Growth media only
Negative Control
Growth Media with 10 nM Apelin-13
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 < 20 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. Its utilization for the assay is described below.

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 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
From ChEMBL:
Assay Type: Functional
Assay Cell Type: NULL
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis 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_Mean_Qualifier_CCR6This 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
4IC50_Mean_CCR6IC50 value determined using a sigmoidal dose response equationFloatμM
5IC50_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
6IC50_1IC50 value determined using a sigmoidal dose response equationFloatμM
7Std.Err(IC50)_1Standard Error of the IC50 valueFloatμM
8nH_1Hill coefficient determined using sigmoidal dose response equationFloat
9Excluded_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
10% Activity at 66.6 uM_first_point (66.6μM**)% inhibition at the test concentrationFloat%
11% Activity at 33.3 uM_first_point (33.3μM**)% inhibition at the test concentrationFloat%
12% Activity at 16.65 uM_first_point (16.65μM**)% inhibition at the test concentrationFloat%
13% Activity at 8.325 uM_first_point (8.325μM**)% inhibition at the test concentrationFloat%
14% Activity at 4.1625 uM_first_point (4.1625μM**)% inhibition at the test concentrationFloat%
15% Activity at 2.08125 uM_first_point (2.08125μM**)% inhibition at the test concentrationFloat%
16% Activity at 1.040625 uM_first_point (1.04062μM**)% inhibition at the test concentrationFloat%
17% Activity at 0.5203125 uM_first_point (0.520312μM**)% inhibition at the test concentrationFloat%
18% Activity at 0.2601562 uM_first_point (0.260156μM**)% inhibition at the test concentrationFloat%
19% Activity at 0.1300781 uM_first_point (0.130078μM**)% inhibition at the test concentrationFloat%
20Excluded_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
21% Activity at 66.6 uM_second_point (66.6μM**)% inhibition at the test concentrationFloat%
22% Activity at 33.3 uM_second_point (33.3μM**)% inhibition at the test concentrationFloat%
23% Activity at 16.65 uM_second_point (16.65μM**)% inhibition at the test concentrationFloat%
24% Activity at 8.325 uM_second_point (8.325μM**)% inhibition at the test concentrationFloat%
25% Activity at 4.1625 uM_second_point (4.1625μM**)% inhibition at the test concentrationFloat%
26% Activity at 2.08125 uM_second_point (2.08125μM**)% inhibition at the test concentrationFloat%
27% Activity at 1.040625 uM_second_point (1.04062μM**)% inhibition at the test concentrationFloat%
28% Activity at 0.5203125 uM_second_point (0.520312μM**)% inhibition at the test concentrationFloat%
29% Activity at 0.2601562 uM_second_point (0.260156μM**)% inhibition at the test concentrationFloat%
30% Activity at 0.1300781 uM_second_point (0.130078μM**)% inhibition at the test concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R21 NS064746-01A

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