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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 ):           View Active Data    View All Data
Target
BioActive Compounds: 21
Related Experiments
Show more
AIDNameTypeComment
1452qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase)Confirmatorydepositor-specified cross reference: primary qHTS assay for 12hLO
2162Confirmation qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase)Confirmatorydepositor-specified cross reference
2164Probe Development Summary of Inhibitors of 12-hLO (12-human lipoxygenase)Summarydepositor-specified cross reference: Probe Development Summary of Inhibitors of 12-hLO (12-human lipoxygenase)
2584qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase) - Confirmatory and Counterscreen DataConfirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase) - Confirmatory and Counterscreen Data
493209Activators of 12-hLO (12-human lipoxygenase): 12hLO Cuvette-Based Hit Validation Assay of Followup CompoundsConfirmatorydepositor-specified cross reference
493216Inhibitors of 12-hLO (12-human lipoxygenase): Cuvette-Based Hit Assay for Validation of Followup CompoundsConfirmatorydepositor-specified cross reference
493219Inhibitors of 12-hLO (12-human lipoxygenase): 15hLO-1 Cuvette-Based Activation Counterscreen Assay for 12hLO Followup CompoundsConfirmatorydepositor-specified cross reference
493220Inhibitors of 12-hLO (12-human lipoxygenase): 15hLO-1 Cuvette-Based Inhibition Counterscreen Assay for 12hLO Followup CompoundsConfirmatorydepositor-specified cross reference
504581Inhibitors of 12-hLO: PBS Stability ProfilingOthersame project related to Summary assay
504590Inhibitors of 12-hLO: Aqueous Solubility ProfilingOthersame project related to Summary assay
504605Inhibitors of 12-hLO: Mouse Liver Microsome Stability ProfilingOthersame project related to Summary assay
504610Inhibitors of 12-hLO: Mouse Plasma Stability ProfilingOthersame project related to Summary assay
504611Inhibitors of 12-hLO: Caco-2 Permeability ProfilingOthersame project related to Summary assay
504612Inhibitors of 12-hLO: Efflux Ratio ProfilingOthersame 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

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]
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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