Highly Selective Off–On Fluorescent Probe for Imaging Thioredoxin Reductase in Living Cells

Abstract

The first fluorescent probe for mammalian thioredoxin reductase (TrxR), TRFS-green, was designed, synthesized, and fully evaluated. The probe features a 1,2-dithiolane scaffold with a quenched naphthalimide fluorophore. TRFS-green displays a green fluorescence off-on change induced by the TrxR-mediated disulfide cleavage and subsequent intramolecular cyclization to liberate the masked naphthalimide fluorophore. It was demonstrated in vitro that TRFS-green manifests high selectivity toward TrxR over other related enzymes and various small molecule thiols as well as biological reducing molecules. HPLC analyses indicated that TRFS-green was exclusively converted to naphthalimide catalyzed by TrxR. The ability in triggering on the fluorescence signal by cellular protein extracts correlates well with the endogenous TrxR activity in different cells. Furthermore, inhibition of TrxR by 2,4-dinitrochlorobenzene or depletion of TrxR by immunoprecipitation remarkably decreases the reduction of TRFS-green by cellular protein extracts. Finally, TRFS-green was successfully applied in imaging TrxR activity in living cells. The fluorescence signal of TRFS-green in living cells was inhibited by pretreating the cells with TrxR inhibitor in a dose-dependent manner, potentiating the development of living cell-based screening assay for identifying TrxR inhibitors. We expect the novel fluorescent probe TRFS-green would facilitate the discovery of TrxR-targeting small molecules for potential therapeutic agents and provide significant advances in understanding the physiological/pathophysiological functions of TrxR in vivo.

Synthesis of Piperlongumine Analogues and Discovery of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Activators as Potential Neuroprotective Agents

“…However, the high cytotoxicity of PL hinders its application as a cytoprotective agent. 21 As our continued interests in discovering and developing novel redox active small molecules as potential therapeutic or diagnostic agents, 30,32,34,[44][45][46][47] we described the synthesis of PL and its analogues, and the discovery of two potent compounds (4 and 5) as potential neuroprotective agents to rescue the H 2 O 2 -or 6-OHDA-induced PC12 cell damage. Compared to the cytotoxicity data of 4 (a compound lacking the M2 moiety), PL shows greater toxic to both L02 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 13 cells and PC12 cells.…”

Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging

“…Probe 18 enables selective and rapid imaging of TrxR activity in live cells. 48 The selectivity arises from the five-membered cyclic disulfide attached to a naphthalimide fluorophore via a carbamate linker. Reduction by TxrR generates a thiolate that cleaves the carbamate linker to form a stable cyclic carbonothioate, releasing the naphthalimide fluorophore.…”

Targeting Thioredoxin System with an Organosulfur Compound, Diallyl Trisulfide (DATS), Attenuates Progression and Metastasis of Triple-Negative Breast Cancer (TNBC)

“…All cells were treated polysulfide for 21 h, then TrxR probe was added for 4 h. PBS was used to wash the unloaded probe 2-3 times. The fluorescence intensity was detected in fluorescent microplate reader at 438/538nm wavelength [31,32].…”