qHTS Assay for Inhibitors of Mammalian Selenoprotein Thioredoxin Reductase 1 (TrxR1): Counterassay in Glutathione Reductase (GR)
The selenoprotein thioredoxin reductase (TrxR; EC 184.108.40.206) 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 ..
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 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 assay is counterassay using a yeast glutathione reductase (GR) assay. The desired outcome is inactivity in this assay.
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: MH090846
Assay Provider: Elias Arner, Karolinska Institute
Three microliters of reagents (100 microM NADPH and 60 nM hGR or 100 microM NADPH as no-enzyme control) was dispensed into 1,536-well black clear-bottomed plates (Buffer: 0.1 M potassium phosphate, pH 7.4/10 mM EDTA, 0.01 % Tween-20). Compounds (23nl) was transferred via Kalypsys pin tool equipped with 1536-pin array. The plates were incubated for 15 min at room temperature, and 1microl aliquot of 500 microM NADPH was added, immediately followed by a 1 microl aliquot of 15 mM DTNB (3 mM final concentration) to start the reaction. The plate was transferred to a ViewLux high-throughput CCD imager (Perkin-Elmer, Wellesley, MA) where kinetic measurements (one read per 5 min for 6 reads, total time 25 min) of the TNB absorbance was acquired using a 405 excitation filter.
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)