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
Target
BioActive Compounds: 236
Depositor Specified Assays
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.
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)
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.
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
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | %Inhibition at 5 uM (5μM**) | % inhibition of G6PDH activity relative to the controls | | Float | ||
| 2 | Mean High | Mean luminescence signal of negative controls in the corresponding plate | | Float | cps | |
| 3 | STD Deviation High | Standard deviation (n=64) of negative controls in the corresponding plate | | Float | cps | |
| 4 | Mean Low | Mean luminescence signal of positive controls in the corresponding plate | | Float | cps | |
| 5 | STD Deviation Low | Standard deviation (n=64) of positive controls in the corresponding plate | | Float | cps |
** Test Concentration.
Additional Information
Grant Number: R03 MH082386-01
Data Table (Concise)
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