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BioAssay: AID 1227

GAPDH Dose Response Colorimetric Assay

This glyceraldehydes-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) dose-response assay is developed and performed at the Sanford-Burnham Center for Chemical Genomics for characterization of the hits of biochemical assays. ..more
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 Tested Compounds
 Tested Compounds
All(744)
 
 
Active(33)
 
 
Inactive(563)
 
 
Inconclusive(148)
 
 
 Tested Substances
 Tested Substances
All(744)
 
 
Active(33)
 
 
Inactive(563)
 
 
Inconclusive(148)
 
 
AID: 1227
Data Source: Burnham Center for Chemical Genomics (SDCCG-A056-GAPDH-Colorimetric-Dose-Response)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-03-10
Modify Date: 2010-10-28

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 33
Depositor Specified Assays
AIDNameTypeComment
1548Summary assay for compounds activating TNAP performed in a luminescent assaysummary
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)
Grant Number: None

This glyceraldehydes-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) dose-response assay is developed and performed at the Sanford-Burnham Center for Chemical Genomics for characterization of the hits of biochemical assays.

GAPDH is found in all mammalian tissues, and is considered a "housekeeping enzyme" unaffected by most physiological, hormonal, and metabolic changes. Therefore, compounds that inhibit GAPDH are expected to have pronounced cytotoxic effect and would be unfavorable as chemical probes. In addition, GAPDH has a labile cysteine in its active site that is crucial for the catalysis, resembling several other enzyme groups, such as cysteine-based proteases and phosphatases. As a result, the compounds capable of cysteine oxidation or modification are likely to appear as screening hits for numerous targets from those groups. This makes GAPDH a valuable counter-screen for identification of non-specific and reactive compounds.
Protocol
GAPDH assay materials:
1)rabbit GAPDH, NAD+ and glyceraldehydes-3-phosphate were purchased from Sigma-Aldrich
2)Assay Buffer: 62.5 mM glycine, 62.5 mM phosphate, pH 8.9, containing 2.5 mM EDTA, 2.5 mM NAD+, and 2.5 mM DTT.
3)GAPDH working solution: 12 nM GAPDH in the assay buffer
4)G3P working solution: 0.735 mM glyceraldehyde-3-phosphate solution in water.

GAPDH dose response assay protocol:
1) Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4 uL compounds in 10% DMSO were transferred into columns 3-22 of Greiner 384-well clear plates (781101). Columns 1-2 and 23-24 were added with 4 uL of 0.15% H2O2 and 10% DMSO.
2) 8 uL of GAPDH working solution was added to columns 1-24 using WellMate bulk dispenser (Matrix).
3) 8 uL of G3P working solution was added to columns 1-24 using WellMate bulk dispenser (Matrix).
4) 8 uL of G3P working solution was added to columns 1-24 using WellMate bulk dispenser (Matrix).
5) Absorbance at 340 nm was measured using M5 plate reader (Molecular Devices, Inc) in kinetic mode for 4 min.
6) The slopes of progress curves were determined using SoftMax Pro software (Molecular Devices, Inc).
7) Data analysis was performed using sigmoidal dose-response equation through non-linear regression using CBIS software (ChemInnovations, Inc). Compound concentrations that were determined to have the compound precipitated were not included in the analysis.
Comment
Bioassay activity outcome is defined as follows: 1) active compound demonstrates >50% inhibition below 100 uM, 2) inactive compound shows less than 50% inhibition at 100 uM or above, 3) inconclusive compound shows less than 50% inhibition at the highest concentration (Cmax) used for data analysis which is <100 uM.

Activity scoring rules developed at Sanford-Burnham Center for Chemical Genomics were devised to take into consideration compound efficacy, the screening stage of the data and apparent compound behavior in the assay. Details of the Scoring System will be published elsewhere.

Briefly, the outline of the scoring system utilized for this assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data and therefore is not applicable in this assay.

2) Second tier (41-80 range) is reserved for dose-response confirmation data of the primary hits.
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. Active compounds have pIC50 = -log(IC50), where IC50 value expressed in mole/L concentration units.
c. Inconclusive compounds have pIC50 = -log(Cmax) - 1.5, where Cmax value expressed in mole/L concentration units.
d. The score is linearly correlated with a compound#s potency and calculated using the following equation:
Score=44+6*(pIC50-3),
This equation results in the Score values above 50 for compounds with IC50 below 10 uM.

3) Third tier (81-100 range) is reserved for dry-powder compounds that represent purchased and resynthesized positives and their analogues and utilized for SAR studies.
a. Inactive compounds of the SAR stage are assigned a score value equal 81.
b. Active compounds have pIC50 = -log(IC50), where IC50 value expressed in mole/L concentration units.
c. Inconclusive compounds have pIC50 = -log(Cmax) - 1.5, where Cmax value expressed in mole/L concentration units.
d. The score is linearly correlated with a compound#s potency and calculated using the following equation:
Score = 82+3*(pIC50-4)
This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior in the assays.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column; if qualifier is ">", IC50 result is greater than that value. String
2IC50*IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat

* Activity Concentration.

Data Table (Concise)
Classification
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