Contact EN Microplate reader Menu BMG LABTECHResourcesApplication notesAN285 285 Fluorescence polarization based assay suitable for screening H-Prostaglandin D Synthase inhibitors Carl Peters BMG LABTECH 07/2015 Download application note PDF The Prostaglandin D Synthase (hematopoietic-type) FP-Based Inhibitor Screening Assay Kit provides a robust and easy to use assay Both the CLARIOstar® and PHERAstar®FS excel at detection of this ﬂuorescence polarization based assay Table of contents {{ item.content }} Introduction Prostaglandins are a group of lipid compounds that are involved in diverse effects in animal physiology. Prosta-glandin D2 (PGD2) has been characterized for its role in asthma where its concentration has been shown to be 10 times higher in asthma patients leading to bronchial airway contraction after exposure to an allergen. For this and other reasons inhibitors are sought for the hematopoietic prostaglandin D synthase (H-PGDS) which catalyzes the ﬁnal PGD2 biosynthesis step. Here we show the performance of Cayman Chemicals green FP-based inhibitor assay screening kit. The kit was tested with both the CLARIOstar and PHERAstar FS. Settings more suitable for high throughput screening (HTS) were also tested. Assay Principle The Prostaglandin D Synthase FP-Based Inhibitor Screening Assay provides a convenient one step assay. An H-PGDS inhibitor was conjugated to ﬂuorescein and other inhibitors are discovered by their ability to displace this probe from binding with H-PGDS (Figure 1). Fig. 1: H-PGDS Green FP Inhibitor Assay Principle. When a ﬂuorescein conjugated probe interacts with H-PGDS a high FP signal is detected. Inhibitors disrupt binding of the probe and the free probe exhibits a low FP signal. In the absence of an effective inhibitor the probe will remain bound to H-PGDS thus reducing the rotational freedom of the probe, resulting in a high FP signal. Increasing concentrations of an effective inhibitor will lead to an increased amount of displaced probe. The displaced probe has an increased rotational freedom and therefore a lower FP signal. This assay prevents the need for the traditional multistep assay that utilizes the highly unstable PGD2-precursor PGH2. Materials & Methods Prostaglandin D Synthase (hematopoietic-type) FP- Based Inhibitor Screening Assay Kit – Green (Cayman Chemicals # 600007) PHERAstar FS and CLARIOstar microplate readers (BMG LABTECH) Inhibitors and additional reagents were purchased from commercial sources To test kit function 16 point titration curves were prepared for 2 known H-PGDS inhibitors; HQL-79 (provided in the kit) and TFC 007. In addition a similar titration curve was prepared for AT 56 an inhibitor of lipocalin-type PGDS (L-PGDS). For all 2-fold serial dilutions were made in DMSO. Kit reagents were prepared according to kit instructions to create an assay cocktail. 47.5 μl of this cocktail was pipetted into the wells of a 384-well plate. This was followed by 2.5 μl of the appropriate inhibitor concentration or DMSO as a negative inhibition control. The ﬁnal concentration of the 1st point in the titration curve was 2.5 mM for AT 56, 250 μM for HQL-79 and 25 μM for TFC 007. Following an incubation of 90 minutes at room temperature the plates were read using the following settings: CLARIOstar settings Measurement method: Fluorescence Polarization, End Point Filter Settings: 482-16 / LP 504 / 530-40 Settling Time: 0.1 s Number of flashes: 200 Focus and Gain: Adjusted prior to measurement Target mP: 200 mP set on DMSO control PHERAstar FS settings Measurement method: Fluorescence Polarization, End Point Optic Module: FP 485 520 520 Settling Time: 0.3 or 0.1 s Number of flashes: 200 or 10 Focus and Gain: Adjusted prior to measurement Target mP: 200 mP set on DMSO control Results & Discussion We ﬁrst wanted to conﬁrm that the appropriate ﬁlters were selected by performing a spectral scan of the H-PGDS FP Fluorescent Probe – Green (Figure 2). The ﬂuorescein conjugated probe behaves as expected, conﬁrming the selection of CLARIOstar ﬁlters and of PHERAstar FS optic module. Fig. 2: H-PGDS FP Probe - Green Spectral Scan. Probe was scanned using a Fluorescence spectrum measurement. Excitation scan from 420-10 to 520-10 using 548-16 as the emission wavelength. Emission scan from 500-10 to 600-10 using 472-16 as the excitation wavelength. The response to inhibitor treatments detected with the CLARIOstar is shown in Figure 3. The assay exhibits high quality based on the calculated Z’ value of 0.84. Fig. 3: Inhibitor titration curves on CLARIOstar. 4-parameter ﬁt curves for TFC 007 (blue: R2=0.999), HQL-79 (red: R2=0.998) and AT 56 ( green: R2=0.984). From the 4-parameter ﬁt curves we can determine the IC50 for inhibition of H-PGDS. TFC 007 exhibits an IC50 of 45 nM which corresponds well with the reported value of 83 nM1. HQL-79 is reported to have an IC50 of 6 μM2 and in this experiment exhibited an IC50 of 13 μM. AT 56 is an L-PDGS selective inhibitor and had previously been reported to have no effect on H-PGDS at concentrations as high as 100 μM3. The results shown here predict an IC50 of 1.9 mM but would require further analysis at higher concentrations. The PHERAstar FS provides similarly robust detection (Table 1). We therefore tested whether settings more conducive to HTS could be employed. Figure 4 shows results using 10 ﬂashes which indicate that the PHERAstar FS maintains performance predicting similar IC50 values to those seen with the CLARIOstar. Fig. 4: Inhibitor titration curves on PHERAstar FS. 4-parameter ﬁt curves for TFC 007 (blue: R2=0.998, IC50=48 nM), HQL-79 (red: R2=0.997, IC50=15 μM) and AT 56 (green: R2=0.969, IC50=1.25 mM). Table 1 shows that outstanding assay quality is maintained in the PHERAstar FS even though HTS settings are used. Flashes / Settling Time Z‘ 384-well read time 200/0.3 0.82 7 minutes 10/0.1 0.74 1 minute 33 sec Conclusion Both the CLARIOstar and PHERAstar FS exhibit excellent assay quality when used to read Cayman’s Prostaglandin D Synthase FP-Based Inhibitor Screening Assay. Furthermore the PHERAstar FS maintains this assay quality even when using settings more suitable for high throughput. References 1. Nabe T., et al. (2011) Prostaglandins Other Lipid Mediat. 95(1-4) 27-34 2. Artiake K. et al. (2006) J. Biol. Chem. 281(22) 15277-15286. 3. Irikura, D., et al. (2009) J. Biol. Chem. 284(12) 7623-7630. FP AS TRF TR-FRET UV/vis FI Neph Lum Back to Overview Compatible readers CLARIOstar Plus PHERAstar FSX Contact +49 781 96968-0 +49 781 96968-67 sales@bmglabtech.com Company Newsletter Privacy statement Legal notice EXTRAnet TeamViewer Support Legacy Plate Readers Luminometer Fluorometer Spectrophotometer Elisa plate reader High-throughput screening Neurodegenerative diseases Luminescence Social Media Copyright ©2021. All rights reserved.