Modulators of the EP2 prostaglandin E2 receptor - Primary Screening
Injury of the brain is a major cause of death and morbidity after the prolonged seizures termed status epilepticus (SE). Studies in rodents have demonstrated that cyclooxygenase 2 (COX2) activation by ischemia and SE generally contributes to neuronal injury, but multiple downstream COX2 signaling pathways, and our data, suggest that the mechanisms promoting and opposing brain injury are complex. ..more
BioActive Compounds: 2141
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
Injury of the brain is a major cause of death and morbidity after the prolonged seizures termed status epilepticus (SE). Studies in rodents have demonstrated that cyclooxygenase 2 (COX2) activation by ischemia and SE generally contributes to neuronal injury, but multiple downstream COX2 signaling pathways, and our data, suggest that the mechanisms promoting and opposing brain injury are complex.
Value of allosteric prostanoid receptor modulators. Given the broad roles of COX2 in inflammation and neurodegeneration, and the recognition that interrupting the entire signaling cascade with a COX2 blocker prevents generation of protective as well as harmful prostanoids, it is surprising that there have not been larger advances in the development of agents that selectively target different prostanoid receptors. Merck has had some success in development of EP1, EP3 and DP1 antagonists. However, only one selective agonist for EP2 is known (butaprost), and there are no selective EP2 or DP2 antagonists available. An approach to developing highly selective compounds that has been very successful for ligand-gated ion channels and G-protein coupled receptors is to search for allosteric regulators of receptor function. Allosteric modulators provide spatial and temporal context dependence because they only affect receptor activation in the presence of the agonist. This is an important feature for our purpose because the endogenous prostanoids are typically released in a pulsatile fashion in response to a stimulus. Allosteric regulators typically bind to a site on the receptor different from the agonist binding site and so do not target the typically highly conserved agonist binding pocket. Allosteric regulators are also insensitive to the concentration of agonist and therefore are effective even in the presence of the high concentrations of released prostaglandins expected during and after seizures. Finally, allosteric modulators used as therapeutics provide an additional level of safety in cases of overdose because their response typically saturates.
EP2 is a Gs-coupled GPCR that, when activated, causes an increase in the cellular content of cAMP. 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 IBMX. 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, IBMX, 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 6000 to 9000 cells/well in a 384-well plate yielded optimum screening metrics, with Z' >0.75 and S:N > 15. The assay tolerates up to about 2% DMSO. The natural agonist, PGE2, is approximately 7-fold more potent than butaprost, and will be used in HTS because the action of allosteric modulators is frequently influenced by the agonist chosen. Finally, over the two month optimization period the Z' and S:N remained sufficiently robust for screening.
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 supernatant
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 different 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 control 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 refrigerator before reading
Step 13: Read plates using 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.
Percent induction is then calculated based on a comparison with wells containing 1 uM PGE2:
% induction = (Pct Inhibition Compound / Pct Inhibition 1 uM PGE2) * 100
The PubChem Activity Score is calculated by rounding the Pct Induction 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 > 60 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)