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) by 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 through the use of knockout, selenium starvation, knockin, 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 S_NAr 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.

量化关键细胞氧化还原参与者的活动对于理解生理稳态和针对其在病理学（包括癌症和炎症性疾病）中的扰动状态至关重要。然而，非常缺乏用于氧化还原酶转换的细胞选择性探针。我们合理地开发了第一个选择性靶向哺乳动物硒蛋白硫氧还蛋白还原酶 (TrxR) 的探针，方法是使用环状硒烯硫化物，利用 TrxR 独特的硒硫醇化学，同时抵抗细胞单硫醇背景。引线探头RX1在细胞中具有出色的 TrxR1 选择性性能，通过使用敲除、硒饥饿、敲入和化学抑制剂进行交叉验证。它的无背景荧光性使我们能够对 TrxR1 抑制剂进行首次定量高通量活细胞筛选，这表明与迄今为止使用的经典亲电试剂相比，经过调和的 S N Ar 亲电子试剂可能是更具选择性的 TrxR 药物_。因此， RX1设计为这种关键氧化还原酶在健康和疾病中的活性体内成像奠定了基础，并且还可以推动 TrxR1 抑制剂药物设计。