SAR analysis of antagonists of Angiotensin II Receptor Type 1 to assess selectivity of uHTS small molecule antagonists hits of the APJ receptor - Set 3
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 ..
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: 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-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 aim of this assay is to identify small molecule antagonists of the human angiotensin II receptor type 1 (AT1). The dose response assay is developed and performed to evaluate selectivity of hits originally identified in an uHTS luminescent beta-arrestin assay for antagonists of the APJ receptor. This AT1 antagonist assay utilizes the same luminescent b-Arrestin-based assay readout as the uHTS APJ primary assay. In both assays 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 KOR1 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 ProLin. 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.
This dose response assay is developed and performed to confirm hits originally identified in "uHTS identification of small molecule antagonists of the APJ receptor via a luminescent beta-arrestin assay", AID 2521 and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.
A. Brief Description of the Assay:
The purpose of this assay is to detect antagonists that cause the activation the angiotensin II receptor type 1 (AT1) in the CHO-K1 AGTR-1 b-Arrestin Cell Line in 1536-well plate format in HTS mode.
Angiotensin II receptor type 1 (AGTR-1) Cell Line (DiscoveRx, Cat# 93-0312C2)
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)
Angiotensin II (Sigma-Aldrich, Cat # A9525)
PathHunter Detection Reagents (DiscoveRx, Cat# 93-0001)
Cell Assay Buffer
C. uHTS Procedures:
Day1 - Cell Seeding
1) Plate 500 cells/well in 5 uL of Assay Media into columns 1-48 of a 1536-well assay plate
2) Centrifuge plates at 500 rpm for 1 minute on an Eppendorf 5810 centrifuge
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 an Eppendorf 5810 centrifuge
2) Using LabCyte Echo, transfer varying volumes of 10 mM test compounds in DMSO into assay plate and back fill wells with DMSO (total of 80 nL) to achieve appropriate dose response concentrations and range, and a final DMSO concentration of 1.32%. Test compounds are distributed into Columns 5-44. Transfer 80 nL of DMSO to positive and negative control wells in Columns 1-4 and 45-48.
3) Immediately following compound/DMSO transfer via the Echo, transfer 1 ul/well of Assay Media to the positive control wells in Columns 1-4.
4) Add 1 ul/well of 300 nM Angiotensin II (FAC = 50 nM) in Assay Media to Columns 3-48 containing test compound and positive control wells
5) Centrifuge plates at 500 rpm for 1 minute on an Eppendorf 5810 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 Eppendorf 5810 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: 300 ug/ml Hygromycin B, 800 ug/ml Geneticin
Same as Growth Media, but with 6.0% FBS instead of 10.0%.
Dilute 0.25% Trypsin/EDTA to 0.05% Trypsin/EDTA using DPBS
Angiotensin II (FAC = 50 nM, ~EC80)
Use the following ratio to prepare the detection reagent:
Galacton Star : Emerald II : Assay Buffer = 1 : 5 : 19
Compounds with IC50 < 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 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 the LYP1 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)