Abstract
Small molecule probes are indispensable tools to explore diverse cellular events. However, finding a specific probe of a target remains a high challenge. Here we report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. Mechanistic studies reveal that the fluorescence signal is switched on by a simple reduction of the disulfide bond within the probe, which is in stark contrast to the sensing mechanism of published probes. The favorable properties of Fast-TRFS enable development of a high-throughput screening assay to discover inhibitors of thioredoxin reductase by using crude tissue extracts as a source of the enzyme.
Highlights
Small molecule probes are indispensable tools to explore diverse cellular events
The reaction process of TRFS-green and TCEP was monitored by highperformance liquid chromatography (HPLC) with a mass detector or photodiode array (PDA) detector (Fig. 2a, b), and our results showed clearly that the cyclization of the intermediate contributes to the sluggish response of the probe
We disclosed the structural factors determining the rate of such process by rational design and evaluation of a series of thioredoxin reductase (TrxR) probes (TRFS1-8)
Summary
Small molecule probes are indispensable tools to explore diverse cellular events. finding a specific probe of a target remains a high challenge. We report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. TRFS-red was reported with improved properties, such as a faster response rate (~1.5 h) and higher elevation of the fluorescence signal (~90-fold)[11]. Both probes showed good selectivity to TrxR over other related biological species, such as glutathione (GSH) and protein thiols. Further mechanistic studies reveal that switching on the fluorescence of Fast-TRFS is achieved by the reduction of the disulfide bond only (Fig. 1c), which is different
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