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BioAssay: AID 1217

uHTS Identification of Diaphorase Inhibitors and Chemcical Oxidizers: Counter Screen for Diaphorase-based Primary Assays

Diaphorase is an enzyme which reversibly catalyzes the reaction of converting NAD(P)+ to NAD(P)H and transfers its electrons to a variety of Redox dyes, e.g. resazurin also known as Alamar Blue. The enzyme from Clostridium kluyveri could utilize both NAD(H) and NADP(H) with similar efficiency; thus is an attractive coupling enzyme for detection of oxidoreductase- and dehydrogenase-based more ..
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 Tested Compounds
 Tested Compounds
All(194156)
 
 
Active(1341)
 
 
Inactive(192815)
 
 
 Tested Substances
 Tested Substances
All(194226)
 
 
Active(1342)
 
 
Inactive(192884)
 
 
AID: 1217
Data Source: Burnham Center for Chemical Genomics (SDCCG-A064-Diaphorase-Inhibitor-FluoAssay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-03-10
Modify Date: 2010-10-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1341
Related Experiments
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AIDNameTypeProbeComment
1209HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay.Confirmatory depositor-specified cross reference
1545Summary - Compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay.Summary2 depositor-specified cross reference
1020Counter Screen for Glucose-6-Phosphate Dehydrogenase-based Primary AssayScreening 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)
Grant Number: R03 MH082386-01
Assay Provider: Dr. Hudson H. Freeze, Sanford-Burnham Medical Research Institute, San Diego, CA

Diaphorase is an enzyme which reversibly catalyzes the reaction of converting NAD(P)+ to NAD(P)H and transfers its electrons to a variety of Redox dyes, e.g. resazurin also known as Alamar Blue. The enzyme from Clostridium kluyveri could utilize both NAD(H) and NADP(H) with similar efficiency; thus is an attractive coupling enzyme for detection of oxidoreductase- and dehydrogenase-based reactions. For example, diaphorase from Clostridium kluyveri was utilized in the screening of phosphomannose isomerase (PMI) paired with resazurin (AID 1209). In this fluorogenic assay the NADPH produced in the reaction reduces resazurin to form resorufin with a concomitant increase in red fluorescence.

The purpose of the current assay assay is to identify inhibitors of diaphorase. It is expected that strong inhibitors of diaphorase would appear as inhibitors in the PMI assay. Thus, diaphorase is a counter-screen for the PMI and other diaphorase-based assays allowing identification of the artifacts of the coupled assay detection system. The concentration of NADH substrate of diaphorase was kept at its Km value to resemble the conditions of the coupled reactions.

In addition, compounds that have strong oxidative properties are also expected to suppress the resorufin formation and appear as inhibitors of diaphorase based reactions. Thus, the current assay could be utilized for library characterization in respect to redox-active compounds.
Protocol
Assay materials:
1) Substrate working solution: 50 mM HEPES, pH 7.4, 0.228 mM NADH, 9.084 mM MgCl2, 0.01% Tween 20
2) Enzyme working solution: 50 mM HEPES, pH 7.4, 0.14 U/ml Diaphorase, 0.2mM Resazurin.
HTS protocol:
1) 2 uL of Substrate working solution was added to columns 3-48 of a Costar 1536-well black plate (cat #3724) using a Thermo Multidrop Combi dispenser
2) 2 ul of Substrate working solution without NADH was to added to columns 1 and 2 (positive control) of a Costar 1536-well black plate (cat #3724) using a Thermo Multidrop Combi dispenser
3) 40 nL of 100% DMSO was added to columns 1-4 using a HighRes biosolutions pintool and V&P Scientific pins
3) 40 nL of 2 mM compounds in 100% DMSO were dispensed in columns 5-48 using a HighRes biosolutions pintool and V&P Scientific pins
4) 2 uL of Enzyme working solution was added to the whole plate using a Thermo Multidrop Combi dispenser.
5) Plates were incubated at room temperature for 16 min.
6) After 16 minutes the plates were read on a ViewLux plate reader (Perkin Elmer), Ex544, Em590.
7) The screening was performed using a HighRes biosolution fully integrated HTS POD-based system
8) Data analysis was performed using CBIS software (ChemInnovation, Inc).
Comment
Diaphorase enzyme used in this assay is from Clostridium kluyveri and its protein/nucleotide sequence is not available in GeneBank. Sequence of human counterpart of this enzyme is cited in the Target Data reference.
Compounds with greater than 50% inhibition at 20 uM concentration are defined as actives of the primary screening. The primary screening actives proceed to the dose-response confirmation stage.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the diaphorase assay is described below.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the diaphorase 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 20 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
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Result Definitions
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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
2IC50IC50 value determined using sigmoidal dose response equationFloatμM
3Std.Err(IC50)Standard Error of IC50 valueFloatμM
4nHHill coefficient determined using sigmoidal dose response equationFloat
5%Inhibition at 20 uM (20μM**)% inhibition of Diaphorase in primary screeningFloat
6Mean HighMean fluorescent signal of negative controls in the corresponding plateFloatRFU
7STD Deviation HighStandard deviation (n=64) of negative controls in the corresponding plateFloatRFU
8Mean LowMean fluorescent signal of positive controls in the corresponding plateFloatRFU
9STD Deviation LowStandard deviation (n=64) of positive controls in the corresponding plateFloatRFU

** 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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