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BioAssay: AID 899

qHTS Assay for Inhibitors and Substrates of Cytochrome P450 2C19

The P450 gene superfamily is involved in the metabolism and clearance of xenobiotics. This assay used human CYP2C19 to measure the hydroxylation of ethylene glycol ester of 6' deoxyluciferin (Luciferin-H EGE; luciferin detection buffer) to luciferin. The luciferin is then measured by luminescence after the addition of a luciferase detection reagent. Luciferin-H EGE concentration in the assay was equal to its KM for CYP2C19. Inhibitors and some substrates limit the production of luciferin, and decrease measured luminescence. ..more
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 Tested Compounds
 Tested Compounds
All(9385)
 
 
Active(1901)
 
 
Inactive(6443)
 
 
Inconclusive(1279)
 
 
 Tested Substances
 Tested Substances
All(10320)
 
 
Active(1964)
 
 
Inactive(7043)
 
 
Inconclusive(1313)
 
 
 Related BioAssays
 Related BioAssays
AID: 899
Data Source: NCGC (2C19490)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-18
Modify Date: 2010-07-06

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1901
Related Experiments
AIDNameTypeComment
410p450-cyp1a2Confirmatorydepositor-specified cross reference
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: None

NCGC Assay Overview:

The P450 gene superfamily is involved in the metabolism and clearance of xenobiotics. This assay used human CYP2C19 to measure the hydroxylation of ethylene glycol ester of 6' deoxyluciferin (Luciferin-H EGE; luciferin detection buffer) to luciferin. The luciferin is then measured by luminescence after the addition of a luciferase detection reagent. Luciferin-H EGE concentration in the assay was equal to its KM for CYP2C19. Inhibitors and some substrates limit the production of luciferin, and decrease measured luminescence.
Protocol
NCGC Assay Protocol Summary:
Two uL of enzyme and substrate mix (final concentrations: 5nM CYP2C19, 10uM Luciferin-H EGE, 50mM KPO4) were dispensed into a white, solid bottom 1536-well microtiter plate. 23nL of compound was transferred by a pin tool. The plate was incubated at 37C for 10 min. Two uL of an NADPH regeneration solution (Promega supplied reagent) was dispensed and the reaction was allowed to continue for 60 min at 37C. Then the reaction was stopped by reconstituted luciferin detection buffer. After another 20 min of incubation the plate was read on the ViewLux (Perkin Elmer) at 60 sec exposure and 2x2 binning. This protocol was carried out with Promega-Glo reagents and further information can be found at http://www.promega.com/tbs/tb325/tb325.pdf. Data were normalized to the to AC100 inhibition (57uM ketoconazole).
Keywords: P450, CYP2C19, Luciferase, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
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. 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: ADME
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
Classification
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