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BioAssay: AID 894

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
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 Tested Compounds
 Tested Compounds
All(148487)
 
 
Active(6428)
 
 
Inactive(124865)
 
 
Inconclusive(17408)
 
 
 Tested Substances
 Tested Substances
All(150839)
 
 
Active(6496)
 
 
Inactive(126771)
 
 
Inconclusive(17572)
 
 
AID: 894
Data Source: NCGC (HPGD335)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-17
Modify Date: 2010-03-01

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 6428
Related Experiments
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AIDNameTypeProbeComment
2407Probe Development Summary for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)Summary3 depositor-specified cross reference: Probe Development Summary for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)
2427Thermal Shift Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)Confirmatory depositor-specified cross reference: Thermal Shift Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)
2429Confirmation qHTS Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)Confirmatory depositor-specified cross reference: Confirmation qHTS Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)
886qHTS Assay for Inhibitors of HADH2 (Hydroxyacyl-Coenzyme A Dehydrogenase, Type II)Confirmatory same project related to Summary assay
893qHTS Assay for Inhibitors of HSD17B4, hydroxysteroid (17-beta) dehydrogenase 4Confirmatory same project related to Summary assay
1030qHTS Assay for Inhibitors of Aldehyde Dehydrogenase 1 (ALDH1A1)Confirmatory same project related to Summary assay
493210Inhibitors of Aldehyde Dehydrogenase 1 (ALDH1A1): Followup Confirmation and CounterscreenConfirmatory same project related to Summary assay
504587Inhibitors of HPGD: Efflux Ratio ProfilingOther same project related to Summary assay
504606Inhibitors of HPGD: Aqueous Solubility ProfilingOther same project related to Summary assay
504629Inhibitors of HPGD: Caco-2 Cell Permeability ProfilingOther same project related to Summary assay
504638Inhibitors of HPGD: Mouse Liver Microsome ProfilingOther same project related to Summary assay
504844Inhibitors of HPGD: Mouse Plasma Stability ProfilingOther same project related to Summary assay
743171Extended Characterization of HPGD Inhibitors: Activity in Primary Assay (HPGD)Confirmatory same project related to Summary assay
743196Extended Characterization of HPGD Inhibitors: SummarySummary same project related to Summary assay
Description:
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.

References
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]
Protocol
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.
Reagents/Controls:
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.
Assay Steps:
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.
Comment
Keywords: NIH Roadmap, MLPCN, MLSCN, MLI, MLSMR, SGC, qHTS, NCGC, hydroxyprostaglandin dehydrogenase 15-(NAD), PGDH; PGDH1; 15-PGDH, HPGD, Human 15-hydroxyprostaglandin dehydrogenase
Compound Ranking:
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
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Max_Read1First kinetic read (raw fluorescence unit) of sample's highest concentration testedFloatRFU
15Compound QCSource of compound QCString

* Activity Concentration.
Additional Information
Grant Number: U54 CDP

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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