Bookmark and Share
BioAssay: AID 2520

uHTS identification of 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 ..
_
   
 Tested Compounds
 Tested Compounds
All(330353)
 
 
Active(347)
 
 
Inactive(330006)
 
 
 Tested Substances
 Tested Substances
All(330436)
 
 
Active(347)
 
 
Inactive(330089)
 
 
AID: 2520
Data Source: Burnham Center for Chemical Genomics (BCCG-A318-APJ-Agonist-Primary-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-12
Modify Date: 2011-01-13

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 347
Related Experiments
Show more
AIDNameTypeProbeComment
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 depositor-specified cross reference
488748Dose Response confirmation of uHTS hits from a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assayConfirmatory depositor-specified cross reference
488811SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assayConfirmatory depositor-specified cross reference
488865Dose Response screen for agonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule agonists hits of the APJ receptorConfirmatory depositor-specified cross reference
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 depositor-specified cross reference
488985SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay - Set 2Confirmatory depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
492985SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay - Set 3Confirmatory depositor-specified cross reference
493183SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay - Set 4Confirmatory depositor-specified cross reference
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 depositor-specified cross reference
504524SAR analysis of a small molecule agonists of the APJ receptor via a luminescent beta-arrestin assay - Set 5Confirmatory depositor-specified cross reference
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 depositor-specified cross reference
485352HTS Dose response counterscreen for assays utilizing the enzyme, beta-galactosidase - Set 2Confirmatory 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 Probe 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.
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
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 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 60 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. 3-48 for the negative control and test compound wells.
4) Using the Kalypsys Dispenser, add 2ul/well of 30 nM Apelin-13 (FAC = 10 nM) 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.
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
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
Due to a low hit rate, zSscore was utilized for selecting hits from the primary screen. Compounds that tested with greater activity than 6-standard deviations above the mean of the plate at 20 uM concentration are defined as actives in the primary 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. The score is correlated with % activity in the assay demonstrated by a compound at 20 uM concentration:
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 (20μM**)% Activity in primary screeningFloat%
2zScore (20μM**)zScore of the corresponding sampleFloat
3Mean HighMean luminescence ratio of positive controls in the corresponding plateFloatRLU
4STD Deviation HighStandard deviation (n=64) of positive controls in the corresponding plateFloatRLU
5Mean LowMean luminescence ratio of negative controls in the corresponding plateFloatRLU
6STD Deviation LowStandard deviation (n=64) of negative controls in the corresponding plateFloatRLU

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

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