Protocol for assaying irreversible inhibitors of thioredoxin reductase 1

Selenoprotein thioredoxin reductase 1 (TXNRD1) is a promising therapeutic target, with several inhibitors reported to inhibit TXNRD1 activity. These inhibitors have the potential for applications such as anti-tumor medications. Here, we present a protocol for assessing irreversible inhibitors of TXNRD1. We describe four assays covering cellular TXNRD activity measurement, recombinant enzyme-based activity determination, differential scanning fluorimetry (DSF), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This protocol will facilitate the screening and development of potential small-molecule inhibitors of TXNRD1.