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

Abstract

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.

Cyclic 5-Membered Disulfides Are Not Selective Substrates of Thioredoxin Reductase, but Are Opened Nonspecifically

“…By identifying and avoiding problematic and nonselective substrate types such as 1,2-dithiolane, chemical development may instead be oriented towards selective and robust redox chemotypes for bioreductive probe and prodrug research. Indeed, novel reducible motifs with chemotype-based selectivity, as in the recent Trx-selective SS60-PQ 9 and the TrxR-reporting probe RX1, 66 are just now emerging. The modular reductiontriggered phenolic carbamate system we developed for this work already ensures valuable performance in biology, through its zero signal background, excellent hydrolytic robustness, and retained cell-marking signal, which combine to offer high-spatial-resolution imaging and cell-resolved statistics (Fig 4).…”

“…We then tested the TrxR-specificity of cellular activation of both 1,2-dithiolane probes, using the TrxR-independent linear disulfide SS00-PQ 9 and the strongly TrxR-dependent selenenylsulfidecontaining RX1 66 as references to indicate the expected outcomes of selectivity-testing cellular experiments.…”