Selective cellular probes for mammalian thioredoxin reductase TrxR1: Rational design of RX1, a modular 1,2-thiaselenane redox probe

Quantifying the activity of key cellular redox players is crucial for understanding physiological homeostasis, and for targeting their perturbed states in pathologies including cancer and inflammatory diseases. However, cellularly-selective probes for oxidoreductase turnover are sorely lacking. We rationally developed the first probes that selectively target the mammalian selenoprotein thioredoxin reductase (TrxR), using a cyclic selenenylsulfide oriented to harness TrxR's unique selenolthiol chemistry while resisting the cellular monothiol background. Lead probe RX1 had excellent TrxR1-selective performance in cells, cross-validated by knockout, selenium starvation, knock-in, and chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors, which indicated that tempered SNAr electrophiles may be more selective TrxR drugs than the classical electrophiles used hitherto. The RX1 design thus sets the stage for in vivo imaging of the activity of this key oxidoreductase in health and disease, and can also drive TrxR1-inhibitor drug design.