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
BioActive Compounds: 22
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.
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]
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.)
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.
** Test Concentration.
Data Table (Concise)