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BioAssay: AID 2163

Cuvette-Based Assay for Inhibitors of 12-hLO (12-human lipoxygenase): 8HQ Series - Round 1

The enzyme activity of 12-hLO was determined by a direct measurement of product formation by monitoring the absorbance at 234 nm in a 2 mL cuvette. IC50 values of inhibitors were obtained by measuring the enzymatic rate at a variety of concentrations (Carroll et al, Segraves et al). ..more
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 Tested Compounds
 Tested Compounds
All(35)
 
 
Active(21)
 
 
Inactive(14)
 
 
 Tested Substances
 Tested Substances
All(35)
 
 
Active(21)
 
 
Inactive(14)
 
 
AID: 2163
Data Source: NCGC (LPOX812)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-11-25
Hold-until Date: 2010-05-14
Modify Date: 2011-02-14

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 21
Depositor Specified Assays
Show more
AIDNameTypeComment
1452qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase)confirmatoryprimary qHTS assay for 12hLO
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
2584qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase) - Confirmatory and Counterscreen DataconfirmatoryqHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase) - Confirmatory and Counterscreen Data
2164Probe Development Summary of Inhibitors of 12-hLO (12-human lipoxygenase)summaryProbe Development Summary of Inhibitors of 12-hLO (12-human lipoxygenase)
493220Inhibitors of 12-hLO (12-human lipoxygenase): 15hLO-1 Cuvette-Based Inhibition Counterscreen Assay for 12hLO Followup Compoundsconfirmatory
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
Description:
Assay Provider: Holman, T.R., University of California, Santa Cruz
Screening Center PI: Austin, Christopher P.
Screening Center: NIH Chemical Genomics Center

The enzyme activity of 12-hLO was determined by a direct measurement of product formation by monitoring the absorbance at 234 nm in a 2 mL cuvette. IC50 values of inhibitors were obtained by measuring the enzymatic rate at a variety of concentrations (Carroll et al, Segraves et al).
Protocol
For control experiments, 2 mL of substrate buffer (10 uM arachidonic acid / 25 mM HEPES / 0.01% (v/v) Triton X-100, pH 8.0) was aliquoted in a cuvette with a magnetic stir bar. After equilibrium was ensured, an aliquot of inhibitor solvent was added (DMSO), and equilibrium was once again assured. The reaction was started by adding enzyme to the cuvette and the reaction was followed until completed. The inhibition experiments were performed as above, except the actual inhibitory compound was added instead of vehicle. To achieve an IC50, typically 5 concentrations of the inhibitor were studied. If the inhibitor concentration was constant, then five different reaction volumes were used.

Step \ Parameter \ Value \ Description
1; UV-Vis; 234 nm; Equilibration
2; Reagent; 2 mL; Substrate buffer
3; UV-Vis; 234 nm; Equilibration
4; Reagent; varies; Compound
5; UV-Vis; 234 nm; Equilibration
6; Reagent; 50-200 nM; Enzyme 12-hLO
7; UV-Vis; 234 nm; Enzymatic reaction

Step \ Notes
1; Blank cuvette to air.
2; 10 uM AA, 25 mM HEPES, 0.01% Triton X-100, pH 8.
3; No change in absorbance ensured.
4; Compound vehicle or compound in various concentrations depending on potency.
5; No change in absorbance ensured.
6; Enzyme concentration to achieve reaction rate between 0.02 and 0.04 AU/s.
7; Enzymatic reaction visualized until completion.
Comment
Keywords: NIH Roadmap, MLSCN, MLPCN, MLI, MLSMR, qHTS, NCGC, human
lipoxygenase, 12hLO, 12-hLO

Compound Ranking:

For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given based on -Log[Potency*10^-6]
Result Definitions
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, IC50. Extrapolated IC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM

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

Data Table (Concise)
Classification
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