Dynamically driven cellular redox networks power a broad range of physiological cellular processes, and additionally are often dysregulated in various pathologies including cancer and inflammatory diseases. Therefore it is vital to be able to image and to respond to the turnover of the key players in redox homeostasis, to understand their physiological dynamics and to target pathological conditions. However, selective modular probes for assessing specific redox enzyme activities in cells are lacking. Here we report the development of cargo-releasing chemical probes that target the mammalian selenoprotein thioredoxin reductase (TrxR) while being fully resistant to thiol reductants in cells, such as the monothiol glutathione (GSH). We used a rationally oriented cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger that matches the chemistry of the unique TrxR active site, and integrated this reducible trigger into modular probes that release arbitrary cargos upon reduction. The probes' redox biochemistry was evaluated over a panel of thiol-type oxidoreductases, particularly showing remarkable, selenocysteine-dependent sensitivity of the "RX1" probe design to cytosolic TrxR1, with little response to mitochondrial TrxR2. The probe was cross-validated in cells by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and use of TrxR-selective chemical inhibitors, showing excellent TrxR1-dependent cellular performance. The RX1 design is therefore a robust, cellularly-validated, modular probe system for mammalian TrxR1. This sets the stage for in vivo imaging of TrxR1 activity in health and disease; and the thermodynamic and kinetic considerations behind its selectivity mechanism represent a significant advance towards rationally-designed probes for other key players in redox biology.

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Cellular redox networks power a multitude of cellular processes, and are often dysregulated in pathologies including cancer and inflammatory diseases. Quantifying the turnover of the key players in redox homeostasis is crucial for understanding their physiological dynamics and for targeting them in pathologies. However, suitably selective probes for assessing specific redox enzyme activities in cells are lacking. We rationally developed the first chemical probes targeting the mammalian selenoprotein thioredoxin reductase (TrxR) while fully resisting other cellular thiols and oxidoreductases. We used a cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger, oriented to harness the chemistry of TrxR's unique selenolthiol active site, and integrated it into modular probes releasing arbitrary cargos upon reduction. The probes showed remarkable selenocysteine-dependent sensitivity to cytosolic TrxR1, against a panel of oxidoreductases. Lead probe RX1 also had excellent TrxR1-selective performance in cells, as cross-validated by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and TrxRselective chemical inhibitors. Its background-free fluorogenicity enabled us to perform the first quantitative high-throughput live cell screen for TrxR1 inhibitors. This indicated that tempered SNAr electrophiles may be a more favorable drug class than classically-used electrophiles. The RX1 design is thus a robust, cellularly validated, high-performance modular system for mammalian TrxR1. This sets the stage for in vivo imaging TrxR1 activity in health and disease, and can also drive and reorient TrxR1-inhibitor drug design. The thermodynamic and kinetic considerations behind RX1's selectivity also outline paths towards rationally-designed probes for other key players in redox biology.