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| | Human PTGES Biochemical Enzyme Inhibition Assay |
| Description: | Recombinant proteins (human isoforms) of PTGES containing a FLAG tag, expressed in baculovirus infected insect cells (Hi-5) and purified by affinity chromatography was used as enzyme in the assay. Substrate was prostaglandin H2 (Cayman Chemicals).For the assay, 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black microtiter plate (384 or 1536, Greiner Bio-One, Frickenhausen, Germany). 4 ul of a solution of human PTGES in assay buffer [100 mM sodium phosphate pH 7.2, 2.5 mM Glutathion reduced, 1 mM EDTA, 0.01% BSA, 0.4 mM DTT, 0.15 mM n-dodecylmaltoside] was added to the well containing test compound and incubated for 15-20 minutes to allow binding of the compound to the enzyme prior to the enzymatic reaction. The reaction was started by addition of 1 ul of an ice cold solution containing PGH2 (40 nM in assay buffer resulting in a final concentration of 8 nM PGH2 in the assay). Reaction time of the mix was 60 seconds at room temperature (PGH2 in aqueous solution quickly converts nonenyzmatically to PGE2 with a short half-life). The concentration of each isoform of PTGES was adapted to the activity of the respective enzyme preparation to maintain linear reaction properties within the reaction time. Typical concentration was around 0.75 nM. The reaction was stopped by addition of 1 ul of a solution containing 15 mM SnCl2 and 400 mM KF in water. SnCl2 converts the remaining unstable PGH2 to stable PGF2alpha. Then, 3 ul of the a first detection solution containing PGE2-D2 (Cisbio Bioassays, TR-FRET reagent, diluted according to the manufacturer's recommendation, typically 1:20 in reconstitution buffer) was added. Finally, 3 μl of the second detection solution containing Lanthanide-kryptate labelled anti-PGE2 antibody (Cisbio Bioassays, diluted according to the manufacturer's recommendation, typically 1:20 in reconstitution buffer) was added to the mix. The resulting mix was incubated overnight at 4° C. to allow the formation of a complex of PGE2 and the detection reagents. The amount of PGE2 that had been produced by PTGES from PGH2 was then determined by testing resonance energy transfer of the Lanthanide-kryptate labelled anti-PGE2 antibody to PGE2-D2. Hereby the fluorescent emissions at 620 nm and 665 nm were measured after excitation at 337-350 nm in a TR-FRE compatible microplate reader (typically BMG Pherastar or Perkin-Elmer ViewLux). The ratio of the emissions at 665 nm and 620 nm was used to determine the amount of PGE2 that was catalyzed by the enzyme. Data were normalized (enzyme reaction without inhibitor=0% inhibition, assay setup without enzyme=100% inhibition). Compounds were tested in duplicates at up to 10 concentrations (for example 20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM and 0.073 nM). Dilution series were made prior to the assay in a 100 fold concentrated form by serial dilution. IC50 values were calculated by 4-Parameter fitting. |
| Affinity data for this assay | |
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