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BioAssay: AID 1452

qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase)

Human lipoxygenase 12hLO is a member of the closely related lipoxygenase family of enzymes which catalyze the site-specific oxidation of arachidonic acid to various hormone precursor molecules and as such is a candidate for drug development in a variety of disease areas, such as cancer and inflammation. ..more
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 Tested Compounds
 Tested Compounds
All(151819)
 
 
Active(181)
 
 
Inactive(149394)
 
 
Inconclusive(2293)
 
 
 Tested Substances
 Tested Substances
All(153607)
 
 
Active(187)
 
 
Inactive(151107)
 
 
Inconclusive(2313)
 
 
AID: 1452
Data Source: NCGC (LPOX867)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2008-12-19

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 181
Depositor Specified Assays
AIDNameTypeComment
2162Confirmation qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase)confirmatory
2163Cuvette-Based Assay for Inhibitors of 12-hLO (12-human lipoxygenase): 8HQ Series - Round 1confirmatory
2164Probe Development Summary of Inhibitors of 12-hLO (12-human lipoxygenase)summary
2584qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase) - Confirmatory and Counterscreen Dataconfirmatory
493209Activators of 12-hLO (12-human lipoxygenase): 12hLO Cuvette-Based Hit Validation Assay of Followup Compoundsconfirmatory
493216Inhibitors of 12-hLO (12-human lipoxygenase): Cuvette-Based Hit Assay for Validation of Followup Compoundsconfirmatory
493219Inhibitors of 12-hLO (12-human lipoxygenase): 15hLO-1 Cuvette-Based Activation Counterscreen Assay for 12hLO Followup Compoundsconfirmatory
493220Inhibitors of 12-hLO (12-human lipoxygenase): 15hLO-1 Cuvette-Based Inhibition Counterscreen Assay for 12hLO Followup Compoundsconfirmatory
Description:
Assay Provider: Holman, T.R., University of California, Santa Cruz
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]

Human lipoxygenase 12hLO is a member of the closely related lipoxygenase family of enzymes which catalyze the site-specific oxidation of arachidonic acid to various hormone precursor molecules and as such is a candidate for drug development in a variety of disease areas, such as cancer and inflammation.
Inhibition of 12hLO activity was screened by utilizing arachidonic acid as a substrate. The extent of hydroperoxide product formation was measured by a secondary chromogenic reaction in which xylenol orange (XO, Sigma-Aldrich, cat. number 39,818-7) reacts with the ferric ions produced from the reaction between the hydroperoxide and ferrous ions. The Fe(III)-XO complex is characterized with red-shifted absorbance at 560 nm. A purified preparation of human 12hLO was supplied by Professor Ted Holman, University of California, Santa Cruz.
Protocol
Assay Protocol:

Buffer: 25 mM Hepes pH 7.0, 0.01% Triton X-100.

Reagents/Controls:
Buffer in columns 3 and 4 as negative control (no enzyme).
Substrate solution: 40 uM arachidonic acid (Sigma-Aldrich, St. Louis, MO) final concentration dispensed throughout the plate.
Enzyme: 150 nM 12hLO final concentration in columns 1, 2, 5-48. Column 1 is neutral (100% activity). Column 2 contained pin-transferred titration of NDGA (nordihidroguaiaretic acid, Sigma-Aldrich, N 5023, top concentration 20 mM in DMSO, then 1:3 dilution in duplicate). (Dilution factor: 23 nL into 4 uL.)
Chromogenic detection reagent (divalent iron/xylenol orange, Fe-XO): 200 uM xylenol orange plus 300 uM ferrous ammonium sulfate prepared freshly in 50 mM sulfuric acid.

Assay Steps:
Three uL of enzyme were dispensed to 1536-well Greiner black clear 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 solution was added to start the reaction. After room temperature incubation for 30 minutes, 4 uL of Fe-XO solution was added to each well and the plates were incubated for 30 min. The absorbance at 405 and 573 nm were measured using ViewLux (Perkin-Elmer) High-throughput CCD imager and absorbance protocol settings. The 573-to-405 absorbance ratio was used to compute reaction progress.
Comment
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. LogAC50 was used for determining relative score and was scaled to each curve class' score range. Acitves in the assay ranged from a score of 40-88.
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 0.731 nM (0.000731429μM**)% Activity at given concentration.Float%
15Activity at 0.732 nM (0.000732μM**)% Activity at given concentration.Float%
16Activity at 1.463 nM (0.00146286μM**)% Activity at given concentration.Float%
17Activity at 1.636 nM (0.001636μM**)% Activity at given concentration.Float%
18Activity at 3.658 nM (0.00365829μM**)% Activity at given concentration.Float%
19Activity at 7.314 nM (0.00731429μM**)% Activity at given concentration.Float%
20Activity at 8.181 nM (0.00818057μM**)% Activity at given concentration.Float%
21Activity at 18.29 nM (0.0182914μM**)% Activity at given concentration.Float%
22Activity at 36.57 nM (0.0365714μM**)% Activity at given concentration.Float%
23Activity at 40.90 nM (0.0408994μM**)% Activity at given concentration.Float%
24Activity at 91.45 nM (0.0914509μM**)% Activity at given concentration.Float%
25Activity at 182.9 nM (0.182857μM**)% Activity at given concentration.Float%
26Activity at 204.5 nM (0.204484μM**)% Activity at given concentration.Float%
27Activity at 457.2 nM (0.457226μM**)% Activity at given concentration.Float%
28Activity at 0.914 uM (0.914286μM**)% Activity at given concentration.Float%
29Activity at 1.022 uM (1.02236μM**)% Activity at given concentration.Float%
30Activity at 2.286 uM (2.28599μM**)% Activity at given concentration.Float%
31Activity at 4.571 uM (4.57143μM**)% Activity at given concentration.Float%
32Activity at 5.111 uM (5.11148μM**)% Activity at given concentration.Float%
33Activity at 11.43 uM (11.4293μM**)% Activity at given concentration.Float%
34Activity at 22.86 uM (22.8571μM**)% Activity at given concentration.Float%
35Activity at 25.56 uM (25.5558μM**)% Activity at given concentration.Float%
36Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
37Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH081283-01A1

Data Table (Concise)
Classification
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