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BioAssay: AID 2427

Thermal Shift 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). Studies to assess the stability of human 15-PGDH in presence of compounds was performed as described (Niesen et al., 2007) with variations. ..more
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 Tested Compounds
 Tested Compounds
All(42)
 
 
Active(22)
 
 
Inactive(2)
 
 
Inconclusive(18)
 
 
 Tested Substances
 Tested Substances
All(42)
 
 
Active(22)
 
 
Inactive(2)
 
 
Inconclusive(18)
 
 
AID: 2427
Data Source: NCGC (HPGD478)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2010-02-25
Modify Date: 2010-03-05

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 22
Depositor Specified Assays
AIDNameTypeComment
894qHTS Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)confirmatoryPrimary qHTS Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)
2407Probe Development Summary for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)summary
Description:
Assay Overview:

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). Studies to assess the stability of human 15-PGDH in presence of compounds was performed as described (Niesen et al., 2007) with variations.

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.

4. Niesen FH, Berglund H, Vedadi M: The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature protocols 2007; 2:2212-2221

NIH Chemical Genomics Center [NCGC]
Structural Genomics Consortium [SGC]
Protocol
HPGD was diluted to a final assay concentration of 1 uM in 100 mM Tris buffer, pH 8.0, containing 0.01% Tween-20 and 1:1000 SYPRO orange dye. The final assay volume is 10 ul, with or without 200 uM of either NAD+ or NADH. Compounds were added 1:5 from appropriate stock solutions in 10% (v/v) DMSO to comprise either 50 or 250 uM final concentrations. Heat denaturation curves were recorded using a RT-PCR instrument (Mx3005p, Agilent) applying a temperature gradient of 2 degree C/min. Analysis of the data was performed using DSF Analysis vers. 2.5 (ftp://ftp.sgc.ox.ac.uk/pub/biophysics) and Prism 5.0 (GraphPad Software, Inc.)
Comment
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, SGC, qHTS, NCGC, hydroxyprostaglandin dehydrogenase 15-(NAD), PGDH; PGDH1; 15-PGDH, HPGD, Human 15-hydroxyprostaglandin dehydrogenase

Compounds were ranked based on the Delta_Tm_NAD+*10. The larger the Tm shift, the greater the compound's effect on enzyme stabilization. Thermal shifts greater than 3 degree C are considered active, greater than 1 are inconclusive and remaining are considered inactive as far as this thermal shift assay is considered.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Delta_Tm_NAD+ (250μM**)thermal shift in degrees Celcius, in presence of NAD+FloatCelcius
2Delta_Tm_NADH (250μM**)thermal shift in degrees Celcius, in presence of NADHFloatCelcius
3Compound_QCSource of Compound QCString

** Test Concentration.
Additional Information
Grant Number: U54 CDP

Data Table (Concise)
Classification
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