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BioAssay: AID 1020

Counter Screen for Glucose-6-Phosphate Dehydrogenase-based Primary Assay

This functional assay was developed for detection of compounds inhibiting glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides. These compounds would be observed as false positives of assays employing G6PDH, e.g. cytochrome P450 enzyme assays where G6PDH is utilized in the NADPH-regenerating system. The knowledge of compounds behavior in the GAPDH assay is required for characterization of their effect in CYP450 systems. ..more
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 Tested Compounds
 Tested Compounds
All(195564)
 
 
Active(236)
 
 
Inactive(195328)
 
 
 Tested Substances
 Tested Substances
All(195634)
 
 
Active(236)
 
 
Inactive(195398)
 
 
AID: 1020
Data Source: Burnham Center for Chemical Genomics (SDCCG-A042-G6PDH-Primary)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-01-08
Modify Date: 2010-09-24

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 236
Related Experiments
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AIDNameTypeProbeComment
777CYP2C9 AssayScreening depositor-specified cross reference
778CYP2C19 AssayScreening depositor-specified cross reference
1545Summary - Compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay.Summary2 depositor-specified cross reference
1209HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay.Confirmatory same project related to Summary assay
1217uHTS Identification of Diaphorase Inhibitors and Chemcical Oxidizers: Counter Screen for Diaphorase-based Primary AssaysScreening same project related to Summary assay
1220HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphateConfirmatory same project related to Summary assay
1535Confirmation of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay.Confirmatory same project related to Summary assay
1536Confirmation of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphate.Confirmatory same project related to Summary assay
1553Screening for Phosphomannose Isomerase inhibitors in cellular based assay using Hela cells.Other same project related to Summary assay
1620Toxicity Screening of PMI Inhibitors in Hela cellsOther same project related to Summary assay
1655Counter screen SAR assay for PMM2 inhibitors via a fluorescence intensity assayConfirmatory same project related to Summary assay
1666SAR assay for compounds that inhibit PHOSPHO1Confirmatory same project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)

This functional assay was developed for detection of compounds inhibiting glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides. These compounds would be observed as false positives of assays employing G6PDH, e.g. cytochrome P450 enzyme assays where G6PDH is utilized in the NADPH-regenerating system. The knowledge of compounds behavior in the GAPDH assay is required for characterization of their effect in CYP450 systems.
Protocol
Materials:
1) Glucose-6-phosphate dehydrogenase (cat # G5885 Sigma-Aldrich)
2) Beta-Nicotinamide adenine dinucleotide phosphate (Amresco)
3) Glucose-6-phosphate (Sigma-Aldrich)
4) Diaphorase (Sigma-Aldrich)
5) Resazurin (Sigma-Aldrich)
6) Potassium phosphate (Sigma-Aldrich)

Assay protocol:
Assay was performed in 8-uL volume using 1536-well black plates (Corning, 3724). First, 4-uL of a solution containing G6PDH, diaphorase, and resazurin was added to each well of the assay plate followed by the addition of 20-nL of test compounds in DMSO using a V & P Scientific Pintool. The reaction was initiated by the addition of 4-uL of a solution containing NADP, MgCl2, and G6P. Assay was allowed to proceeed at room temperature for 45 minutes before being read on a PerkinElmer ViewLux.

Final concentrations of components in the assay were as follows:
1) 0.025 U/mL glucose-6-phosphate dehydrogenase
2) 1.5 mM nicotinamide adenine dinucleotide phosphate
3) 3.3 mM glucose-6-phosphate
4) 3.3 mM magnesium chloride
5) 1.0 U/mL diaphorase
6) 1.0 mM resazurin
7) 5.0 uM compounds (columns 5-48)
8) 0.025 M potassium phosphate buffer, pH 7.4
9) 0.25% DMSO (columns 1-48)
10) Above components, minus G6PDH, as a positive control (columns 1-2)
Comment
Compounds with inhibition > 50% are defined as 'actives' in the outcome column.

Activity Scoring
Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics were utilized in this screening. Details of the Scoring System will be published elsewhere.

Briefly, the outline of the scoring system utilized for the G6PDH assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data-the score is correlated with % displacement in the assay demonstrated by a compound at 5 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4

2) Second tier (41-80 range) is reserved for dose-response confirmation data and therefore is not applicable in this assay.

3) Third tier (81-100 range) is reserved for resynthesized confirmed positives and their analogues and is not applicable in this assay.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Inhibition at 5 uM (5μM**)% inhibition of G6PDH activity relative to the controlsFloat
2Mean HighMean luminescence signal of negative controls in the corresponding plateFloatcps
3STD Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatcps
4Mean LowMean luminescence signal of positive controls in the corresponding plateFloatcps
5STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatcps

** Test Concentration.
Additional Information
Grant Number: R03 MH082386-01

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