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BioAssay: AID 488772

qHTS Assay for Substrates of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1)

The selenoprotein thioredoxin reductase (TrxR; EC 1.8.1.9) is a FAD containing homodimeric pyridine nucleotide-disulfide oxidoreductase with many cellular roles. Together with NADPH and its prime substrate thioredoxin (Trx), the enzyme forms the core of the Trx system. The mammalian Trx system exerts a wide spectrum of functions including redox regulation, antioxidant defense, regulation of more ..
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 Tested Compounds
 Tested Compounds
All(1266)
 
 
Active(2)
 
 
Inactive(1192)
 
 
Inconclusive(73)
 
 
 Tested Substances
 Tested Substances
All(1280)
 
 
Active(2)
 
 
Inactive(1205)
 
 
Inconclusive(73)
 
 
AID: 488772
Data Source: NCGC (TRXR188)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeComment
587qHTS Assay for Spectroscopic Profiling in Texas Red Spectral RegionOtherdepositor-specified cross reference: qHTS Assay for Spectroscopic Profiling in Texas Red Spectral Region.
488771Probe Development Summary for Modulators of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1)Summarydepositor-specified cross reference
488773qHTS Assay for Inhibitors of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1)Confirmatorydepositor-specified cross reference
588453qHTS Assay for Inhibitors of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1): qHTSConfirmatorysame project related to Summary assay
588456qHTS Assay for Substrates of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1): qHTSConfirmatorysame project related to Summary assay
652020qHTS Assay for Inhibitors of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1): Counterassay in Glutathione Reductase (GR)Confirmatorysame project related to Summary assay
652023qHTS Assay for Inhibitors of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1): Hit Validation in Primary ScreenConfirmatorysame project related to Summary assay
652037qHTS Assay for Inhibitors of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1): Insulin MOA AssayOthersame project related to Summary assay
652256qHTS Assay for Inhibitors of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1): DTNB AssayOthersame project related to Summary assay
Description:
The selenoprotein thioredoxin reductase (TrxR; EC 1.8.1.9) is a FAD containing homodimeric pyridine nucleotide-disulfide oxidoreductase with many cellular roles. Together with NADPH and its prime substrate thioredoxin (Trx), the enzyme forms the core of the Trx system. The mammalian Trx system exerts a wide spectrum of functions including redox regulation, antioxidant defense, regulation of transcription factors as well as support of cell growth and replication. Many of these functions involve the reduction of Trx, which may subsequently reduce a number of different substrates including ribonucleotide reductase, peroxiredoxins or NFkB. Mammalian TrxR itself also has a broad substrate specificity, reducing both protein and non-protein substrates, including low molecular weight compounds such as dehydroascorbate, lipoic acid, ubiquinone, and juglone. In addition, several drugs in clinical use for anticancer treatment are indeed known to target TrxR1.

This BioAssay is a validation of an NADPH fluorescence-based, dual-purpose qHTS rTrxR1 assay designed to find potential inhibitors and substrates of rTrxR1. For this particular BioAssay, actives are potential substrates. See related BioAssay for inhibitors data deposition.
Protocol
Assay protocol: 2 uL of reagents (buffer in column 4 as negative control and 90 nM rTrxR1 in columns 1-3 and 5-48) were dispensed into Greiner black solid-bottom 1,536-well assay plates, followed by 1 uL of NADPH (400 uM final concentration) to each well. The plates were centrifuged at 1000 rpm for 15 seconds and subsequently incubated for 5 min at room temperature (~22 deg C) to allow for rTrxR1 reduction. Compounds (23 nL) were then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). In addition, a duplicate 2-fold serial dilution of the control compounds auranofin, a known gold-based TrxR1 inhibitor, and juglone (5-hydroxy-1,4-naphthoquinone), a natural TrxR1 substrate, were pin-transferred to columns 2 and 3, respectively. After incubation for 15 min at room temperature (~22 deg C), 1 uL of selenite (400 uM final concentration) were dispensed to each well. The plate was immediately transferred to a ViewLux high-throughput CCD imager (PerkinElmer), wherein kinetic measurements of NADPH fluorescence (Ex 340 nm, Em 450 nm) were acquired (8 minute kinetic read, see Table 1). Read 1 was utilized to assess the capacity of a compound to serve as an rTrxR1 substrate, i.e. a decrease in NADPH fluorescence compared to the no-compound background is an indication of a substrate behavior for that particular compound. For inhibitory activity of a compound, delta values, computed as the difference in fluorescence intensity between the first and last reads of an 8-minute time kinetic window, were used. All reagents were diluted in an assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 0.01% Tween-20.

Throughout the screen, reagent bottle and all liquid lines were made light-tight to minimize reagent degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration, and a 'double-pinning' step of the highest concentration was required to access higher concentrations of compounds. Vehicle-only plates, with DMSO being pin-transferred to the columns 5-48, were inserted uniformly at the beginning and the end of each library in order to monitor and record any shifts in the background, which can be affected by reagent dispensers or loss in enzyme activity over time. Screening data were corrected, normalized, and concentration-effect relationships were derived by using publicly-available curve fitting algorithms developed in-house (http://ncgc.nih.gov/pub/openhts).
Comment
Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.

2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.00366 uM (0.00366μM**)% Activity at given concentration.Float%
15Activity at 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
16Activity at 0.091 uM (0.0914μM**)% Activity at given concentration.Float%
17Activity at 0.457 uM (0.457μM**)% Activity at given concentration.Float%
18Activity at 2.290 uM (2.29μM**)% Activity at given concentration.Float%
19Activity at 11.40 uM (11.4μM**)% Activity at given concentration.Float%
20Activity at 57.10 uM (57.1μM**)% Activity at given concentration.Float%
21Compound QCSource of compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH090846-01

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