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BioAssay: AID 893

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 ..
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 Tested Compounds
 Tested Compounds
All(73910)
 
 
Active(5649)
 
 
Inactive(62883)
 
 
Inconclusive(5463)
 
 
 Tested Substances
 Tested Substances
All(75028)
 
 
Active(5711)
 
 
Inactive(63840)
 
 
Inconclusive(5477)
 
 
AID: 893
Data Source: NCGC (HSDB130)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-17
Modify Date: 2010-07-06

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 5649
Related Experiments
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AIDNameTypeProbeComment
2407Probe Development Summary for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)Summary3 depositor-specified cross reference
886qHTS Assay for Inhibitors of HADH2 (Hydroxyacyl-Coenzyme A Dehydrogenase, Type II)Confirmatory same project related to Summary assay
894qHTS Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)Confirmatory same project related to Summary assay
1030qHTS Assay for Inhibitors of Aldehyde Dehydrogenase 1 (ALDH1A1)Confirmatory same project related to Summary assay
2427Thermal Shift Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)Confirmatory same project related to Summary assay
2429Confirmation qHTS Assay for Inhibitors of HPGD (15-Hydroxyprostaglandin Dehydrogenase)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
743196Extended Characterization of HPGD Inhibitors: SummarySummary same project related to Summary assay
Description:
Assay Overview:
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.
Protocol
Assay Protocol:

Buffer: 100 mM Tris-HCl pH 9.0, containing 0.01 % Tween-20.

Reagents/Controls:

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)

Assay Steps:
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.
Comment
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, SGC, qHTS, NCGC, hydroxyacyl-Coenzyme A dehydrogenase type II, hadh2, Oxidoreductases, 17beta- Hydroxysteroid Dehydrogenase Type 10

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.
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%
14Activity at 0.0007000000 uM (0.0007μM**)% Activity at given concentration.Float%
15Activity at 0.00160 uM (0.0016μM**)% Activity at given concentration.Float%
16Activity at 0.00370 uM (0.0037μM**)% Activity at given concentration.Float%
17Activity at 0.00820 uM (0.0082μM**)% Activity at given concentration.Float%
18Activity at 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
19Activity at 0.041 uM (0.0409μM**)% Activity at given concentration.Float%
20Activity at 0.092 uM (0.0915μM**)% Activity at given concentration.Float%
21Activity at 0.205 uM (0.2045μM**)% Activity at given concentration.Float%
22Activity at 0.457 uM (0.4572μM**)% Activity at given concentration.Float%
23Activity at 1.022 uM (1.0224μM**)% Activity at given concentration.Float%
24Activity at 2.286 uM (2.286μM**)% Activity at given concentration.Float%
25Activity at 5.112 uM (5.1115μM**)% Activity at given concentration.Float%
26Activity at 11.43 uM (11.4293μM**)% Activity at given concentration.Float%
27Activity at 25.56 uM (25.5558μM**)% Activity at given concentration.Float%
28Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
29Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

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