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BioAssay: AID 488748

Dose Response confirmation of uHTS hits from a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay

Currently there are no small molecule tools to investigate the biological functions of apelin and its receptor. Apelin is the endogenous peptide ligand for the G-protein coupled receptor (GPCR) APJ (angiotensin II receptor-like 1, AGTRL-1 and APLNR). Until the discovery of apelin, APJ was an orphan GPCR. APJ is coupled to Gai, and has been shown in cell culture to inhibit adenylate cyclase. The more ..
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AID: 488748
Data Source: Burnham Center for Chemical Genomics (SBCCG-A450-APJ-Agonist-DR-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
2520uHTS identification of small molecule agonists of the APJ receptor via a luminescent beta-arrestin assayScreening depositor-specified cross reference
2580Summary assay for small molecule agonists of the APJ receptorSummary1 depositor-specified cross reference
2764Single concentration confirmation of uHTS hits from a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assayScreening same project related to Summary assay
485352HTS Dose response counterscreen for assays utilizing the enzyme, beta-galactosidase - Set 2Confirmatory same project related to Summary assay
488811SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assayConfirmatory same project related to Summary assay
488865Dose Response screen for agonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule agonists hits of the APJ receptorConfirmatory same project related to Summary assay
488881SAR analysis of small molecule agonists of the Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule agonists hits of the APJ receptorConfirmatory same project related to Summary assay
488985SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay - Set 2Confirmatory same project related to Summary assay
488986SAR analysis of small molecule agonists of the Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule agonists hits of the APJ receptor - Set 2Confirmatory same project related to Summary assay
492982SAR analysis of small molecule agonists of the Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule agonists hits of the APJ receptor - Set 3Confirmatory same project related to Summary assay
492985SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay - Set 3Confirmatory same project related to Summary assay
493183SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay - Set 4Confirmatory same project related to Summary assay
493202SAR analysis of small molecule agonists of the Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule agonists hits of the APJ receptor - Set 4Confirmatory same project related to Summary assay
504524SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay - Set 5Confirmatory same project related to Summary assay
504532SAR analysis of small molecule agonists of the Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule agonists hits of the APJ receptor - Set 5Confirmatory same 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)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1R21NS059422-01
Assay Provider: Dr. Layton Smith, Sanford-Burnham Medical Research Institute

Currently there are no small molecule tools to investigate the biological functions of apelin and its receptor. Apelin is the endogenous peptide ligand for the G-protein coupled receptor (GPCR) APJ (angiotensin II receptor-like 1, AGTRL-1 and APLNR). Until the discovery of apelin, APJ was an orphan GPCR. APJ is coupled to Gai, and has been shown in cell culture to inhibit adenylate cyclase. The APJ gene encodes a receptor that most closely resembles the angiotensin receptor AT1. However, the APJ receptor does not bind angiotensin II. Underscoring the emerging importance of the apelin/APJ system, recent studies have shown that apelin reduces the extent of atherosclerotic lesions in ApoE-/- mice, and opposes the development of abdominal aortic aneurysms. Additional research has revealed that APJ forms a heterodimer with the Ang II receptor AT1, and that this complex facilitates antagonism of Ang II signaling by apelin. Despite these exciting results, there remains a multitude of unanswered questions regarding the role of apelin and APJ in physiology and pathology.
The project goal is to identify a chemical probe of apelin receptor function that transiently and reversibly activates the receptor. An agonist or potentiator of this receptor would provide a novel research tool to evaluate the role of apelin in cardiovascular and metabolic disease pathology.
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.

The purpose of this assay confirm hits from "uHTS identification of small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay", AID 2520.
Protocol
A. Brief Description of the Assay:
The purpose of this assay is to detect agonists that cause the activation 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
D. Dose Response 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 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 varying volumes from a 2 mM Echo qualified plate containing test compounds into assay plate Col. 5 - 48 to achieve the desired dose response concentrations and range. Backfill with DMSO to equalize final DMSO concentrations in all test wells. Transfer equal volume 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. 3-48 for the negative control and test compound wells.
4) Using the Kalypsys Dispenser, add 2ul/well of 30 nM Apelin-13 in assay media to Col. 1-2 for the positive control .
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 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.
E. 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
Positive Control
Growth Media with 30 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 an EC50_Mean < 20 are considered to be active
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 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 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 the items discussed above is
Score = 44 + 6*(pEC50 - 3)*QC,
where pEC50 is a negative log(10) of the EC50 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
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50_Mean_QualifierThis qualifier is to be used with the next TID, EC50_Mean. If qualifier is "=", the EC50 result equals the value in that column. If the qualifier is ">", the EC50 result is greater than that value. If the qualifier is "<", the EC50 result is smaller than that valueString
2EC50_Mean*EC50 value determined using sigmoidal dose response equationFloatμM
3EC50_Qualifier_1This qualifier is to be used with the next TID, EC50_1. If qualifier is "=", the EC50 result equals the value in that column. If the qualifier is ">", the EC50 result is greater than that value. If the qualifier is "<", the EC50 result is smaller than that valueString
4EC50_1Standard Error of EC50 valueFloatμM
5Std.Err(EC50)_1Standard Error of EC50 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 100 uM_first_point (100μM**)% Activity at a given concentrationFloat%
9% Activity at 50 uM_first_point (50μM**)% Activity at a given concentrationFloat%
10% Activity at 25 uM_first_point (25μM**)% Activity at a given concentrationFloat%
11% Activity at 12.5 uM_first_point (12.5μM**)% Activity at a given concentrationFloat%
12% Activity at 6.25 uM_first_point (6.25μM**)% Activity at a given concentrationFloat%
13% Activity at 3.125 uM_first_point (3.125μM**)% Activity at a given concentrationFloat%
14% Activity at 1.5625 uM_first_point (1.5625μM**)% Activity at a given concentrationFloat%
15% Activity at 0.78125 uM_first_point (0.78125μM**)% Activity at a given concentrationFloat%
16Excluded_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
17% Activity at 100 uM_second_point (100μM**)% Activity at a given concentrationFloat%
18% Activity at 50 uM_second_point (50μM**)% Activity at a given concentrationFloat%
19% Activity at 25 uM_second_point (25μM**)% Activity at a given concentrationFloat%
20% Activity at 12.5 uM_second_point (12.5μM**)% Activity at a given concentrationFloat%
21% Activity at 6.25 uM_second_point (6.25μM**)% Activity at a given concentrationFloat%
22% Activity at 3.125 uM_second_point (3.125μM**)% Activity at a given concentrationFloat%
23% Activity at 1.5625 uM_second_point (1.5625μM**)% Activity at a given concentrationFloat%
24% Activity at 0.78125 uM_second_point (0.78125μM**)% Activity at a given concentrationFloat%
25Excluded_Points_third_pointFlags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
26% Activity at 100 uM_third_point (100μM**)% Activity at a given concentrationFloat%
27% Activity at 50 uM_third_point (50μM**)% Activity at a given concentrationFloat%
28% Activity at 25 uM_third_point (25μM**)% Activity at a given concentrationFloat%
29% Activity at 12.5 uM_third_point (12.5μM**)% Activity at a given concentrationFloat%
30% Activity at 6.25 uM_third_point (6.25μM**)% Activity at a given concentrationFloat%
31% Activity at 3.125 uM_third_point (3.125μM**)% Activity at a given concentrationFloat%
32% Activity at 1.5625 uM_third_point (1.5625μM**)% Activity at a given concentrationFloat%
33% Activity at 0.78125 uM_third_point (0.78125μM**)% Activity at a given concentrationFloat%
34Excluded_Points_fourth_pointFlags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
35% Activity at 100 uM_fourth_point (100μM**)% Activity at a given concentrationFloat%
36% Activity at 50 uM_fourth_point (50μM**)% Activity at a given concentrationFloat%
37% Activity at 25 uM_fourth_point (25μM**)% Activity at a given concentrationFloat%
38% Activity at 12.5 uM_fourth_point (12.5μM**)% Activity at a given concentrationFloat%
39% Activity at 6.25 uM_fourth_point (6.25μM**)% Activity at a given concentrationFloat%
40% Activity at 3.125 uM_fourth_point (3.125μM**)% Activity at a given concentrationFloat%
41% Activity at 1.5625 uM_fourth_point (1.5625μM**)% Activity at a given concentrationFloat%
42% Activity at 0.78125 uM_fourth_point (0.78125μM**)% Activity at a given concentrationFloat%
43EC50_Qualifier_2This qualifier is to be used with the next TID, EC50_2. If qualifier is "=", the EC50 result equals the value in that column. If the qualifier is ">", the EC50 result is greater than that value. If the qualifier is "<", the EC50 result is smaller than that valueString
44EC50_2Standard Error of EC50 valueFloatμM
45Std.Err(EC50)_2Standard Error of EC50 valueFloatμM
46nH_2Hill coefficient determined using sigmoidal dose response equationFloat
47Excluded_Points_fifth_pointFlags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
48% Activity at 100 uM_fifth_point (100μM**)% Activity at a given concentrationFloat%
49% Activity at 50 uM_fifth_point (50μM**)% Activity at a given concentrationFloat%
50% Activity at 25 uM_fifth_point (25μM**)% Activity at a given concentrationFloat%
51% Activity at 12.5 uM_fifth_point (12.5μM**)% Activity at a given concentrationFloat%
52% Activity at 6.25 uM_fifth_point (6.25μM**)% Activity at a given concentrationFloat%
53% Activity at 3.125 uM_fifth_point (3.125μM**)% Activity at a given concentrationFloat%
54% Activity at 1.5625 uM_fifth_point (1.5625μM**)% Activity at a given concentrationFloat%
55% Activity at 0.78125 uM_fifth_point (0.78125μM**)% Inhibition at a given concentrationFloat%
56Excluded_Points_sixth_pointFlags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
57% Activity at 100 uM_sixth_point (100μM**)% Activity at a given concentrationFloat%
58% Activity at 50 uM_sixth_point (50μM**)% Activity at a given concentrationFloat%
59% Activity at 25 uM_sixth_point (25μM**)% Activity at a given concentrationFloat%
60% Activity at 12.5 uM_sixth_point (12.5μM**)% Activity at a given concentrationFloat%
61% Activity at 6.25 uM_sixth_point (6.25μM**)% Activity at a given concentrationFloat%
62% Activity at 3.125 uM_sixth_point (3.125μM**)% Activity at a given concentrationFloat%
63% Activity at 1.5625 uM_sixth_point (1.5625μM**)% Activity at a given concentrationFloat%
64% Activity at 0.78125 uM_sixth_point (0.78125μM**)% Activity at a given concentrationFloat%
65Excluded_Points_seventh_pointFlags to indicate which of the seventh dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
66% Activity at 100 uM_seventh_point (100μM**)% Activity at a given concentrationFloat%
67% Activity at 50 uM_seventh_point (50μM**)% Activity at a given concentrationFloat%
68% Activity at 25 uM_seventh_point (25μM**)% Activity at a given concentrationFloat%
69% Activity at 12.5 uM_seventh_point (12.5μM**)% Activity at a given concentrationFloat%
70% Activity at 6.25 uM_seventh_point (6.25μM**)% Activity at a given concentrationFloat%
71% Activity at 3.125 uM_seventh_point (3.125μM**)% Activity at a given concentrationFloat%
72% Activity at 1.5625 uM_seventh_point (1.5625μM**)% Activity at a given concentrationFloat%
73% Activity at 0.78125 uM_seventh_point (0.78125μM**)% Activity at a given concentrationFloat%
74Excluded_Points_eighth_pointFlags to indicate which of the eighth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.String
75% Activity at 100 uM_eighth_point (100μM**)% Activity at a given concentrationFloat%
76% Activity at 50 uM_eighth_point (50μM**)% Activity at a given concentrationFloat%
77% Activity at 25 uM_eighth_point (25μM**)% Activity at a given concentrationFloat%
78% Activity at 12.5 uM_eighth_point (12.5μM**)% Activity at a given concentrationFloat%
79% Activity at 6.25 uM_eighth_point (6.25μM**)% Activity at a given concentrationFloat%
80% Activity at 3.125 uM_eighth_point (3.125μM**)% Activity at a given concentrationFloat%
81% Activity at 1.5625 uM_eighth_point (1.5625μM**)% Activity at a given concentrationFloat%
82% Activity at 0.78125 uM_eighth_point (0.78125μM**)% Activity at a given concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R21NS059422-01

Data Table (Concise)
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