qHTS Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)
Human 15-Hydroxyprostaglandin dehydrogenase (HPGD) catalyzes the inactivation of prostaglandin E2, and plays a major role in cancer biology by antagonizing the oncogenic potential of cyclooxygenase type 2 (COX2). Assays are available, based on absorbance/fluorescence increase of NADH with the substrate 15-OH prostaglandin E2. ..more
BioActive Compounds: 6428
Human 15-Hydroxyprostaglandin dehydrogenase (HPGD) catalyzes the inactivation of prostaglandin E2, and plays a major role in cancer biology by antagonizing the oncogenic potential of cyclooxygenase type 2 (COX2). Assays are available, based on absorbance/fluorescence increase of NADH with the substrate 15-OH prostaglandin E2.
1. Myung SJ, Rerko RM, Yan M, Platzer P, Guda K, Dotson A, Lawrence E, Dannenberg AJ, Lovgren AK, Luo G, Pretlow TP, Newman RA, Willis J, Dawson D, Markowitz SD. 15-Hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon tumorigenesis. Proc Natl Acad Sci U S A. 2006 Aug 8;103(32):12098-102.
2. Yan M, Rerko RM, Platzer P, Dawson D, Willis J, Tong M, Lawrence E, Lutterbaugh J, Lu S, Willson JK, Luo G, Hensold J, Tai HH, Wilson K, Markowitz SD. 15-Hydroxyprostaglandin dehydrogenase, a COX-2 oncogene antagonist, is a TGF-beta-induced suppressor of human gastrointestinal cancers. Proc Natl Acad Sci U S A. 2004 Dec 14;101(50):17468-73.
3. Yang L, Amann JM, Kikuchi T, Porta R, Guix M, Gonzalez A, Park KH, Billheimer D, Arteaga CL, Tai HH, Dubois R, Carbone DP, Johnson DH. Inhibition of Epidermal Growth Factor Receptor Signaling Elevates 15-Hydroxyprostaglandin Dehydrogenase in Non-Small-Cell Lung Cancer. Cancer Res. 2007 Jun 15;67(12):5587-5593.
Inhibition of HPGD activity was screened by utilizing prostaglandin as an electron donor and NAD+ as an electron acceptor/cofactor. An increase in the fluorescence intensity due to conversion of NAD+ to NADH was used to measure the enzyme activity.
NIH Chemical Genomics Center [NCGC]
Structural Genomics Consortium [SGC]
Buffer: 50 mM Tris-HCl, 0.01% Tween 20, pH 8.
Enzyme Buffer: 50 mM Tris-HCl, 0.5 mM NAD+ and 0.01% Tween 20, pH 8.
Buffer in columns 3 and 4 as negative control (no enzyme).
Substrate/cofactor solution: 1 mM NAD+ and 30 uM prostaglandin (Sigma, prostaglandin E2, # P5640) final concentrations dispensed throughout the plate.
Enzyme: 20 nM HPGD final concentration in columns 1, 2, 5-48. Column 1 and 2 is neutral (100% activity).
A titration of control inhibitor GW5074 (G6416 Sigma-Aldrich, St. Louis, MO) top concentration 10 mM in DMSO, then 1:2 dilution for a total of 16 concentrations in duplicate) was pin-transferred into column 2.
Three uL of enzyme were dispensed to 1536-well Greiner black solid bottom plates. Compounds and control inhibitor (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then 1 uL of substrate/cofactor solution was added to start the reaction. The plates were immediately transferred to and read 5 times every 30 seconds on ViewLux High-throughput CCD imager (Perkin-Elmer) using 360 nm excitation and 450 nm emission fluorescence protocol. The fluorescence intensity difference between the last and the first time points was used to compute reaction progress.
Keywords: NIH Roadmap, MLPCN, MLSCN, MLI, MLSMR, SGC, qHTS, NCGC, hydroxyprostaglandin dehydrogenase 15-(NAD), PGDH; PGDH1; 15-PGDH, HPGD, Human 15-hydroxyprostaglandin dehydrogenase
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)