uHTS Identification of Diaphorase Activators and Chemical Reducers: 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, such as resazurin also known as Alamar Blue. The enzyme purified from Clostridium kluyveri can utilize both NAD(H) and NADP(H) with similar efficiency, and, therefore, is an attractive coupling enzyme for detection of oxidoreductase- and more ..
BioActive Compounds: 11
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, such as resazurin also known as Alamar Blue. The enzyme purified from Clostridium kluyveri can utilize both NAD(H) and NADP(H) with similar efficiency, and, therefore, 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 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 is to identify activators of diaphorase. More importantly, compounds that have strong reductive properties are also expected to increase the resorufin formation and would appear as activators in the diaphorase and diaphorase-based reactions. 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. In addition, the current assay could be utilized for library characterization in respect to redox-active compounds.
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.
3) Negative control (NC) solution - 50 mM HEPES, pH 7.4, 9.084 mM MgCl2, 0.01% Tween 20
1) 2 ul of negative control solution was added to columns 1 and 2 of a Costar 1536-well black plate (cat #3724) using a Thermo Multidrop Combi dispenser
2) 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
3) 40 nL of 100% DMSO was added to columns 1-4 using a HighRes biosolutions pintool and V&P Scientific pins. Columns 3-4 are positive controls (PC).
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) Final reagent concentrations are:
a) 50 mM HEPES, pH 7.4
b) 0.114 mM NADH (except columns 1-2)
c)4.542 mM MgCl2
d) 0.22 mM NADP+
e) 0.005% Tween 20
f) 0.1 mM Resazurin
g) 0.07 U/ml Diaphorase
h) 1% DMSO (all columns)
i) 20 uM compounds (columns 3-48)
6) Plates were incubated at room temperature for 16 min.
7) After 16 minutes the plates were read on a ViewLux plate reader (Perkin Elmer), Ex544, Em590.
8) The screening was performed using a HighRes biosolution fully integrated HTS POD-based system
9) Data analysis was performed using CBIS software (ChemInnovation, Inc).
Diaphorase enzyme used in this assay is from Clostridium kluyveri and its protein/nucleotide sequence is not available in GeneBank. The sequence of the human counterpart of this enzyme is cited in the Target Data reference.
Diaphorase activation was calculated using the following formula:
Activation Factor (AF) = (Signal_Well - Mean_NC)/(Mean_PC - Mean_NC),
where Signal_Well corresponds to luminescence signal in the well with a compound, Mean_NC and Mean_PC correspond to mean values of corresponding controls in the plate.
Compounds with greater than or equal to 2-fold activation (AF >= 2) of diaphorase at 20 uM concentration are defined as actives of the primary screening.
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 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 and the score is correlated with the activation factor demonstrated by a compound at 20 uM concentration:
a. If AF<1, then the assigned score is 0
b. For all other AF values, Score = 40 - 40/AF
This formula results in a score that is equal 20 for AF=2 and asymptotically approaches 40 with increasing AF values.
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.
** Test Concentration.
Data Table (Concise)