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BioAssay: AID 2157

Confirmation qHTS Assay for Inhibitors of 15-hLO (15-human lipoxygenase)

Human lipoxygenase 15hLO-1 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 15hLO-1 activity was screened by utilizing arachidonic acid as a substrate. more ..
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 Tested Compounds
 Tested Compounds
All(54)
 
 
Active(12)
 
 
Inactive(27)
 
 
Inconclusive(15)
 
 
 Tested Substances
 Tested Substances
All(54)
 
 
Active(12)
 
 
Inactive(27)
 
 
Inconclusive(15)
 
 
AID: 2157
Data Source: NCGC (LPOX956)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-11-24

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 12
Related Experiments
AIDNameTypeComment
887qHTS Assay for Inhibitors of 15-hLO (15-human lipoxygenase)Confirmatorydepositor-specified cross reference: primary qHTS assay for 15hLO-1
2169Probe Development Summary of Inhibitors of 15-hLO-1 (15-human lipoxygenase 1)Summarydepositor-specified cross reference
2155Cuvette-Based Assay fo Inhibitors of 15-hLO-1 (15-human lipoxygenase 1): Series 1 - Round 1Confirmatorysame project related to Summary assay
493220Inhibitors of 12-hLO (12-human lipoxygenase): 15hLO-1 Cuvette-Based Inhibition Counterscreen Assay for 12hLO Followup CompoundsConfirmatorysame project related to Summary assay
Description:
Assay Provider: Holman, T.R., University of California, Santa Cruz
Screening Center PI: Austin, Christopher P.
Screening Center: NIH Chemical Genomics Center

Human lipoxygenase 15hLO-1 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 15hLO-1 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 15hLO-1 was supplied by Professor Ted Holman, University of California, Santa Cruz.

This assay includes some confirmation and follow-up compounds for related primary screening assay AID 887.
Protocol
Assay Protocol:

Buffer: 25 mM Hepes pH 7.5, 0.01% Triton X-100.

Reagents/Controls:
Buffer in columns 3 and 4 as negative control (no enzyme). Substrate solution: 50 uM arachidonic acid (Sigma-Aldrich, St. Louis, MO) final concentration dispensed throughout the plate. Enzyme: 40 nM 15hLO-1 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 10 mM in DMSO, then 1:2 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 6 minutes, 4 uL of Fe-XO solution was added to each well followed by a short spin (1000 rpm, 15 sec) and the plates were then 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
Keywords: NIH Roadmap, MLSCN, MLPCN, MLI, MLSMR, qHTS, NCGC, human
lipoxygenase, 15hLO1, 15-hLO-1

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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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%
4Curve_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
5Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
6Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
7Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
8Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
9Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
10Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
11Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
12Compound QCSource of compound quality controlString

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

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