qHTS Assay for Inhibitors of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1)
The selenoprotein thioredoxin reductase (TrxR; EC 220.127.116.11) 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 ..
BioActive Compounds: 65
The selenoprotein thioredoxin reductase (TrxR; EC 18.104.22.168) 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 inhibitors. See related BioAssay for substrate data deposition.
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).
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)