A fast and specific fluorescent probe for thioredoxin reductase that works via disulphide bond cleavage

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.

Fig. 1

Reaction mechanism of TRFS-green and summary of current work. a Stepwise activation of TRFS-green. b Strategy for modification of TRFS-green. c One-step activation of Fast-TRFS

Fig. 2

Stepwise activation of TRFS-green by TCEP. a TRFS-green (20 μM) was incubated with TCEP (1 mM) in TE buffer (50 mM Tris-HCl, 1mM EDTA, pH 7.4) at 37 °C for 24 h. The reaction mixture was analyzed by HPLC, and the quantification of intermediate and ANA was shown in (b). c Time-dependent fluorescence changes of TRFS-green (10 μM) in the presence of TCEP (1 mM) in TE buffer at 37 °C. The inset shows the time-dependent changes of emission at 538 nm (λ_ex = 438 nm). Source data are provided as a Source Data file. Source Data file

Fig. 3

Chemical Structures of TRFS series probes. The chemical structures of TRFS1-8 were presented

Fig. 4

Reaction details of TRFS3 and TCEP. a TRFS3 (20 μM) was incubated with TCEP (1 mM) in TE buffer at 37 °C for 4 h. The reaction mixture was analyzed by HPLC-MS. b Proposed mechanism for the reduction of TRFS3 by TCEP. Source data are provided as a Source Data file.

Fig. 5

Structures of TRFS9-13 and their response to TCEP. a Structures of TRFS9-13.The probes (10 μM) were incubated with TCEP (1 mM) at 37 °C in TE buffer. The time-dependent emission spectra were recorded. b λ_ex = 345 nm; c λ_ex = 490 nm; d λ_ex = 324 nm. The insets in b–d showed the time-dependent changes of emission at 460 nm, 510 nm, and 460 nm, respectively. Source data are provided as a Source Data file

Fig. 6

Reduction of Fast-TRFS (TRFS9) by TCEP and Selective activation of Fast-TRFS by TrxR. a Reduction of Fast-TRFS by TCEP. Fast-TRFS (100 μM) was incubated with TCEP (1 mM) for 0 min (a0), 1 min (a1), 30 min (a30) and 240 min (a240), and the mixture was analyzed by HPLC-MS. b Time-dependent emission spectra of Fast-TRFS toward TrxR. Fast-TRFS was incubated with TrxR/NADPH (50 nM/200 μM), and the emission spectra were recorded every 1 min for 15 min. c Time course of the fluorescence increase of Fast-TRFS with TrxR/NADPH and U498C TrxR/NADPH. d Response of Fast-TRFS to various relevant biological species. The fluorescence increase at 460 nm was determined after they were incubated with Fast-TRFS for 15 min. The Sec (10 μM) was generated in situ by mixing Cys (1 mM) and selenocystine (5 μM). e Inhibition of the cell lysate-mediated Fast-TRFS reduction by TrxR inhibitor AF. The NADPH-pretreated HeLa cell lysate (0.5 mg mL⁻¹) was treated with AF for 30 min, and further incubated with Fast-TRFS and NADPH (200 μM) for additional 20 min. The fluorescence increase was determined. f Reduction of Fast-TRFS by lysates (0.5 mg mL⁻¹) from the genetically manipulated HeLa cells in the presence of NADPH (200 μM). g Time course of the fluorescence increase of Fast-TRFS, TRFS-red and TRFS-green with TrxR/NADPH (50 nM/200 μM). All reactions were performed in TE buffer at 37 °C. The excitation/emission wavelengths for Fast-TRFS, TRFS-green and TRFS-red are 345/460 nm, 438/538 nm and 615/661 nm, respectively. The concentration of the probes in (b–g) was 10 μM. Source data are provided as a Source Data file

Fig. 7

Activation of Fast-TRFS in live cells. a HeLa cells were treated with Fast-TRFS (10 μM) for the indicated times, and bright field and fluorescence images were acquired. b HeLa cells were pretreated with different concentrations of TrxR inhibitor AF for 2 h, and then were further stained with Fast-TRFS (10 μM) for 15 min. Bright field and fluorescence images were shown. c HeLa-shTrxR1 cells and HeLa-shNT cells were treated with Fast-TRFS (10 μM) for the indicated times, and fluorescence images were shown. d Quantification of the fluorescence intensity from (a) and (b) by a fluorescence plate reader (Tecan Infinite M200). e Quantification of the fluorescence intensity from (c) by a fluorescence plate reader. Scale bars: 25 μm. Source data are provided as a Source Data file