qHTS Assay for Inhibitors of HSD17B4, hydroxysteroid (17-beta) dehydrogenase 4
HADH2: Hydroxyacyl-Coenzyme A dehydrogenase, type II, was supplied by researchers at the Structural Genomics Consortium, Oxford University lab (PI Udo Oppermann). The mitochondrial enzyme 17-hydroxysteroid dehydrogenase type 10 (HSD17-10) previously classified as type II hydroxyacyl-CoA dehydrogenase (HADH2) catalyzes the NAD+ dependent oxidation of a variety of substrates including acyl-CoAs, more ..
BioActive Compounds: 5649
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
Structural Genomics Consortium [SGC]
Grant number: None
HADH2: Hydroxyacyl-Coenzyme A dehydrogenase, type II, was supplied by researchers at the Structural Genomics Consortium, Oxford University lab (PI Udo Oppermann). The mitochondrial enzyme 17-hydroxysteroid dehydrogenase type 10 (HSD17-10) previously classified as type II hydroxyacyl-CoA dehydrogenase (HADH2) catalyzes the NAD+ dependent oxidation of a variety of substrates including acyl-CoAs, androgens and neurosteroids as well as bile acids(Shafqat N, Marschall HU, Filling C, et al. Biochemical Journal. 2003;376(Pt 1):49-60). The role in neurodegeneration by binding to amyloid-peptide led to its identification (Oppermann UC, et al FEBS Letters. 1999;451(3):238-242 and Yan SD, et al. Nature. 1997;389(6652):689-695), and mutations in its gene appear to be causative for 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (Ofman R, et al. American Journal of Human Genetics. 2003;72(5):1300-1307). The enzyme was produced by E.coli fermentation as His-tagged recombinant for the purpose of X-ray structure determination, and had been mass-spectrometry characterized.
Inhibition of HADH2 activity was screened by utilizing beta-hydroxybutyryl coenzyme A 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.
Buffer: 100 mM Tris-HCl pH 9.0, containing 0.01 % Tween-20.
Buffer in columns 3 and 4 as negative control (no enzyme).
Substrate/cofactor solution: 1 mM NAD+ and 90 uM beta-hydroxybutyryl coenzyme A (Sigma-Aldrich, St. Louis, MO) final concentrations dispensed throughout the plate.
Enzyme: 20 nM HADH2 final concentration in columns 1, 2, 5-48. Column 1 is neutral (100% activity). Column 2 contained titration of apomorphine (Sigma-Aldrich, A 4393, PubChem CID 2215, top concentration 500 uM, then 1:2 dilution in duplicate)
Three uL of enzyme were dispensed to 1536-well Greiner black solid bottom plates. Compounds and controls (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 every 30 seconds for 4 min 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, MLSCN, MLI, MLSMR, SGC, qHTS, NCGC, hydroxyacyl-Coenzyme A dehydrogenase type II, hadh2, Oxidoreductases, 17beta- Hydroxysteroid Dehydrogenase Type 10
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. ** Test Concentration.
Data Table (Concise)