Inhibition of thioredoxin reductase 1 by porphyrins and other small molecules identified by a high-throughput screening assay.
The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K(i)=2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k(inact)) of (0.73 ± 0.07) × 10⁻³ μM⁻¹ min⁻¹. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Stefanie
S
Prast-Nielsen
Thomas S
TS
Dexheimer
Lena
L
Schultz
William C
WC
Stafford
Qing
Q
Cheng
Jianqiang
J
Xu
Ajit
A
Jadhav
Elias S J
ES
Arnér
Anton
A
Simeonov
D000098:Acetophenones / Q000494:pharmacology*; D001578:Benzopyrans / Q000494:pharmacology*; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D005453:Fluorescence; D006427:Hemin / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000188:drug therapy; D058990:Molecular Targeted Therapy; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011524:Protoporphyrins / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000032:analysis; D018038:Sodium Selenite / Q000378:metabolism; D054481:Thioredoxin Reductase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000098:Acetophenones; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D011524:Protoporphyrins; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D009249:NADP; D006427:Hemin; C028025:protoporphyrin IX; C085746:rottlerin; D054481:Thioredoxin Reductase 1; D018038:Sodium Selenite
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