Inhibitors of the EP2 Prostaglandin E2 Receptor - Primary Screen
Prostaglandin E2 that is produced by COX2 in response to cellular injury is involved in a multimodal inflammatory response in many tissues, including the brain. Studies in rodents have demonstrated that cyclooxygenase 2 (COX2) activation following ischemia and status epilepticus generally contributes to neuroinflammation, possibly in part via activation of EP2, a Gs-coupled GPCR that, when more ..
BioActive Compounds: 1304
NIH Molecular Libraries Screening Centers Network [MLSCN]
Emory Chemical Biology Discovery Center in MLSCN
Assay provider: Dr. Raymond Dingledine, Emory University
MLSCN Grant: 5-U01NS058158-02
Prostaglandin E2 that is produced by COX2 in response to cellular injury is involved in a multimodal inflammatory response in many tissues, including the brain. Studies in rodents have demonstrated that cyclooxygenase 2 (COX2) activation following ischemia and status epilepticus generally contributes to neuroinflammation, possibly in part via activation of EP2, a Gs-coupled GPCR that, when activated, causes an increase in the cellular content of cAMP. However, EP2 inhibitors that could be used to test this hypothesis are not available. We created a rat C6 glioma cell line that stably expresses human EP2 prostanoid receptors under control of the CMV promoter. Thirteen subclones were expanded and tested for the ability of 1 uM butaprost (a selective EP2 agonist) and 20 uM forskolin (strong activator of adenylate cyclase) to cause cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram. The subclone chosen for further study responded to 1 uM butaprost with a 1000-fold increase in cAMP levels (from 0.057 to 58 fmoles / 6000 cells in a 384 well plate). The parent C6 glioma does not respond to butaprost or the endogenous ligand, PGE2 (not shown).
The primary cell-based assay depends upon competition by cell-derived cAMP for binding of a cAMP-labeled FRET acceptor to a cAMP antibody, which is itself labeled with a FRET donor (HTRF assay from CisBio). The FRET signal decreases as cAMP concentration rises. Incubation of cells with butaprost in the presence of the phosphodiesterase inhibitor, rolipram, reduces the FRET signal in a concentration-dependent manner. Importantly, the assay was stable when this incubation was carried out for 10 or 40 min at room temperature, which provides a comfortable time window for manipulating cell culture plates in this assay. The FRET signal itself was stable for at least 12 hours after lysing the cells.
A cell density of 2000 to 4000 cells/well in a 1536-well plate yielded optimum screening metrics. The assay tolerates up to about 2% DMSO. The natural agonist, PGE2, is approximately 7-fold more potent than butaprost, and was used in HTS because the action of allosteric modulators is frequently influenced by the agonist chosen. The primary screen involved incubation of cells with 20 uM of library compounds, followed by a 30-40 min incubation with a saturating concentration (1 uM) of PGE2. The percent inhibition of the PGE2-induced reduction in TR-FRET signal was measured.
Cell culture medium: 500 ml DMEM high glucose medium, 50 ml FBS (10% heat-inactivated), 5 ml Pen Strept, 15 ml 50 mg/ml Geneticin
1. Grow cells:
Cells were grown over a week in T-175 flasks and split when appropriate to reach the final volume of 40-50 T-175 flasks necessary for screening.
2. Harvest cells:
1. Aspirate medium from T-175 flasks, rinse with HBSS without Ca-Mg.
2. Add 3 ml /flask Versene (EDTA, 0.2 mg/ml) (BioWhittaker Cat# 17-711E)
3. Tap flasks vigorously
4. Add 10 ml/flask HBSS (+Ca+Mg) and combine the cells in 4 centrifuge tubes (50ml)
5. Centrifuge cells at 800g for 8 min
6. Discard supernant
7. Resuspend cells in about 100 ml HBSS(+Ca+Mg)
8. Count cell number
3. Cell density optimization
Make dilutions of PGE2 in HBSS to arrive at testing concentrations of PGE2.
Make dilutions of cells to arrive at testing concentrations of cells.
Add 1 ul of d2-conjugate (1:9 in HBSS) and incubate at RT for 10 - 40 min
Add 2 ul of anti-cAMP Europium conjugate (1:18), centrifuge plate and incubate at RT for 20min to 2 hr
Read FRET signal using Envision (100us delay)
Analyze data and calculate the EC50s at differen cell density
Choose cell density for screening (EC50 of PGE2 is 1 - 3 nM)
Step 1: make 250 ml of cells (for 40 1536-well plates), add 250 ul of 10 mM Rolipram
Step 2: dispense 4 ul of cells to column 3 - 46
Step 3: dispense 1 ul of HBSS to column 3 - 4
Step 4: add 0.1 ul of 1 mM compound (final compound concentration: 20 uM) using Pin-tool (Beckman NX)
Step 5: add 1 ul of 6 uM PGE2 to PGE2 control wells (final 1 uM)
Step 6: add 1 ul of 120 uM Forsklin to forsklin contrl wells (final 20 uM)
Step 7: dispense 1 ul of d2-conjugate (1:9) to column 4
Step 8: make mixture of d2-conjugate and PGE2:
Step 9: dispense 2 ul of d2-conjugate and PGE2 mixture to column 5 - 46
step 10: shake plates well, centrifuge plates at 1500g for 10min, incubate at RT for around 10mins (no longer that 20 min)
Step 11: dispense 2 ul of anti-cAMP Europium conjugate (1: 18 dilution in lysis buffer) to column 3 - 46
Step 12: shake plates well, centrifuge plates at 1500g for 10 min and incubate at RT for 10 min. Put plates to 4C refregrator before reading
Step 13: Read plates uisng Envision with Twister II robot. Run 5 plates/batch from refrigerator.
5. Data analysis:
1. FRET signals are expressed as FRET ratios:
FRET ratio = F665 nm / F615 nm * 10000
F665 nm: Fluorescence counts at 665 nm emission (units: cps)
F620 nm: Fluorescence counts at 615 nm emission (units: cps)
2. Assay data are analyzed using BioAssay software from CambridgeSoft. Percentage of inhibition is calculated with the following equation based on normalized data from each plate.
Normalized FRET signal = ((FRET compound well - FRET blank(compound added)) / ((FRET compound well - FRET background) (no compound added))
% of activity = (Normalized FRET signal from compound well / average normalized FRET signal from control wells) * 100
% of inhibition = 100 - % of activity
Where FRET compound well is the FRET ratio from a well with a test compound, FRET background is an average FRET ratio from wells with cells, Anti-cAMP-Eu and HBSS buffer only; FRET control is an average FRET ratio from wells containing 0.3 nM PGE2 that defines maximum FRET signal.
The PubChem Activity Score is calculated by rounding the Pct Inhibition to 0 decimal places. Negative scores are changed to 0, and scores higher than 100 are changed to 100.
Compounds with a PubChem Activity Score > 40 are defined as active.
1. Artifacts of this assay could result from, but are not limited to, intrinsic fluorescence of some compounds, compounds that can quench fluorescence, dust or lint.
2. All data reported were normalized on a per-plate basis.
** Test Concentration.
Data Table (Concise)