Fluorescent Probes for Mammalian Thioredoxin Reductase: Mechanistic Analysis, Construction Strategies, and Future Perspectives

Abstract

The modulation of numerous signaling pathways is orchestrated by redox regulation of cellular environments. Maintaining dynamic redox homeostasis is of utmost importance for human health, given the common occurrence of altered redox status in various pathological conditions. The cardinal component of the thioredoxin system, mammalian thioredoxin reductase (TrxR) plays a vital role in supporting various physiological functions; however, its malfunction, disrupting redox balance, is intimately associated with the pathogenesis of multiple diseases. Accordingly, the dynamic monitoring of TrxR of live organisms represents a powerful direction to facilitate the comprehensive understanding and exploration of the profound significance of redox biology in cellular processes. A number of classic assays have been developed for the determination of TrxR activity in biological samples, yet their application is constrained when exploring the real-time dynamics of TrxR activity in live organisms. Fluorescent probes offer several advantages for in situ imaging and the quantification of biological targets, such as non-destructiveness, real-time analysis, and high spatiotemporal resolution. These benefits facilitate the transition from a poise to a flux understanding of cellular targets, further advancing scientific studies in related fields. This review aims to introduce the progress in the development and application of TrxR fluorescent probes in the past years, and it mainly focuses on analyzing their reaction mechanisms, construction strategies, and potential drawbacks. Finally, this study discusses the critical challenges and issues encountered during the development of selective TrxR probes and proposes future directions for their advancement. We anticipate the comprehensive analysis of the present TrxR probes will offer some glitters of enlightenment, and we also expect that this review may shed light on the design and development of novel TrxR probes.

Keywords: 1,2-dithiolane; diselenide; disulfide; fluorescent probe; redox chemistry; selenenylsulfide; thioredoxin; thioredoxin reductase.

Figure 1

The principal functions of Trx system.

Figure 2

(a) The dimeric structure of human TrxR1 (PDB accession number: 3QFA, with two mutant sites, i.e., Cys497-to-Ser497 and Sec498-to-Cys498) [24]. One subunit is shown in cyan and the other is in green. The two subunits are arranged in a head-to-tail style. Molecule in yellow that binds to each monomer is FAD receiving electrons from NADPH. Residues in red are Cys59 and Cys64 in N-terminal active site –CVNVGC–, whereas residues in magenta are Ser497 and Cys498 in C-terminal active site –GSCG. (b) Cartoon representation of electrons flow in TrxR, which is indicated by the dashed boxes. The designations A-D correspond to different states of TrxR during the process of electron transfer. Arrows indicate the transformation of biological molecules from one status to other status.

Figure 3

The principle of DTNB reduction assay and DTNB reduction assay in combination with TrxR inhibitors. Arrows indicate the transformation of biological molecules or reagents from one status to other status.

Figure 4

The principle of Trx-mediated insulin reduction assay. Path 1, also called endpoint insulin reduction assay, uses DTNB to quantify the newly generated sulfhydryl groups in insulin, and determination of TNB absorbance at λ = 412 nm indicates TrxR activity. Path 2, the dashed box in blue, shows an alternative approach to determinate TrxR activity, only by monitoring the NADPH decay at λ = 340 nm.

Figure 5

The principle of SC-TR assay. Selenocystine acts as the substrate of TrxR, and monitoring the decay of NADPH indicates the TrxR activity.

Figure 6

1,2-Dithiolane-based TrxR probes. Probes 1–4 employ carbamate motif as the linker. Probes 5 and 6 use urea and carbonate motif as the linkers, respectively. The model probe 7 uses a stable phenolic carbamate motif as the linker. Following a two-step process, the free fluorophores are liberated in probes 1–4 and 6–7. Probe 5 is triggered only by cleavage of the disulfide bond in the recognition part. ①–⑤ indicate the influences of changing atoms in the linker unit and recognition part on the properties of probe 5. ①–⑤ also show the development process of probe 5. The cyan and yellow balls having sulfydryl on them shown in the working process of probe 7 indicate various biological thiols. Protein TrxR is shown in pink. The colors covering the chemical structures show their fluorescence colors after TrxR activation, and the gray indicates the fluorescence of the probes has been quenched.

Figure 7

1,2-Thiaselenane-based TrxR1 probe RX1. Probe RX1 interacts with the desired TrxR1 to liberate the fluorescent PQ-OH, during which process the key intermediates 8-1 and 8-3 are generated. Probe RX1 can also react with high concentrations of monothiols to give intermediate 8-2; however, this intermediate encounters significant kinetic and thermodynamic barriers to yield key intermediate 8-3. Thus, this unwanted parallel pathway induced by monothiols does not trigger the RX1. The bidirectional arrows indicate corresponding transformations are reversible. Protein TrxR1 is shown in pink and various biological thiols are shown in blue. Dashed ellipse in red shows the tunable side chain of probe 8, and the corresponding words in red explain the function of the side chain. Dashed cycle in green shows the potential reactive thiol in intermediate 8-1, and the corresponding words in green show the name of thiol appearing in the main text. The colors covering the chemical structures show their fluorescence colors after TrxR activation, and the gray indicates the fluorescence of the probes has been quenched.

Figure 8

Linear diselenide-based TrxR probe. Probes 9 and 10 are diselenide- and disulfide-based TrxR probes. Experimental evidence indicates probe 9 is better than probe 10 in recognizing TrxR. During the activation process of probe 9 by TrxR, free selenolate is generated, which leads to the production of ROS. The dashed box in red shows the process of generation of ROS through free selenolate byproduct, and the arrows indicate the transformation of the substances. The curved arrows in black show the generated side products after activation by TrxR. Protein TrxR is shown in pink. The colors covering the chemical structures show their fluorescence colors after TrxR activation, and the gray indicates the fluorescence of the probes has been quenched.

Figure 9

α,β-Unsaturated ketone-based TrxR probes. Probe 12, TR-green, is constructed from a specific TrxR inhibitor 2a (compound 11) and fluorescent coumarin. TR-green undergoes the Michael addition with the nucleophilic TrxR’s Sec residue. TR-green can be used in gel-imaging of TrxR and live-cell imaging of TrxR. Probe 13, TPP2a, is built by the integration of compound 2a with TPP. TPP2a can be used as a labelling agent for TrxR2, or a theranostic agent for cancers overexpressing TrxR2. The dashed box in red shows the structure of fluorophore coumarin, and the dashed ellipse in red show the structural determinant of compound 2a for inhibition of TrxR activity. Protein TrxR is shown in pink. The colors covering the chemical structures show their fluorescence colors after TrxR activation, and the gray indicates the fluorescence of the probes has been quenched.

Figure 10

Linear disulfide-based TrxR probes. Probes 14 and 15 are based on carbon dots, and both have linear disulfide as their recognition parts for TrxR. Biotin-CD-Naph is a ratiometric probe that uses biotin to selectively target cancer cells. When the disulfide bond in Biotin-CD-Naph is cleaved by TrxR, the FRET process is blocked, and carbon dots emit fluorescence. fCDs-Cu²⁺ is quenched because of the chelation of Cu²⁺ within this probe. When the disulfide bond in fCDs-Cu²⁺ is cleaved by TrxR, Cu²⁺ is removed from the surface of carbon dots, and the fluorescence generates. Both probes 14 and 15 are able to image cancer cells that overexpress TrxR. The curved arrows in red indicate the FRET process, which leads to the turning-on of naphthalimide fluorophore (shown in yellow) and quench of carbon dots (shown in grey). The red × in the upper panel indicates the blockage of FRET process, which results in turning-on of carbon dots (shown in blue) and quench of naphthalimide fluorophore (shown in grey). The red × in the bottom panel indicates the quench process by chelation of Cu²⁺. Protein TrxR is shown in pink.

Figure 11

Possible activation pathways and mechanisms of linear disulfide/diselenide and 1,2-dithianes by monothiols and dithiol/selenolthiol enzymes. (A). The bidirectional arrows indicate the transformation is reversible. The blue arrows indicate the forward processes, while the green arrows show the reverse processes. The pink icons having dithiol/selenolthiol on them represent enzymes in biological systems. (B). The one-way arrows in black show the irreversible activation processes of the probe.