Dose Response confirmation of uHTS hits from a small molecule antagonists 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 ..
BioActive Compounds: 214
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford- Sanford-Burnham Medical Research Institute(SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: 1R21NS059422-01
Assay Provider: Dr. Layton Smith, Sanford- 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 antagonist or inhibitor receptor activation 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. Antagonists would be expected to inhibit agonist activation of the receptor resulting in the inhibition of signal formation in this assay.
This assay is a follow-up to "uHTS identification of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay", AID 2521 and Single concentration confirmation of uHTS hits from a small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay
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.
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)
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. 1-2 for the positive control wells.
4) Using the Kalypsys Dispenser, add 2ul/well of 30 nM Apelin-13 (FAC = 10 nM) in assay media to Col. 3-48 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 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.
F12 nutrient mix HAMs supplemented with 10% hi-FBS, 1X Penicillin/Streptomycin; selection reagents: 300ug/ml Hygromycin B, 800ug/ml Geneticin
Same as Growth Media without the selection reagents
Dilute 0.25% Trypsin/EDTA to 0.05% Trypsin/EDTA using DPBS
Growth Media with 30 nM Apelin-13
Use the following ratio to prepare the detection reagent:
Galacton Star : Emerald II : Assay Buffer = 1 : 5 : 19
In this assay selected hits from the initial screen (AID 2521) were retested over one or more ranges.
Range1 0-40 uM
Range2 0-20 uM
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 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.
e. Compounds with activity "< 1.25 uM" are given a score of 75.
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
* Activity Concentration. ** Test Concentration.
Data Table (Concise)