A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase

Abstract

We report the first diselenide-based probe for the selective detection of thioredoxin reductase (TrxR), an enzyme commonly overexpressed in melanomas. The probe design involves conjugation of a seminaphthorhodafluor dye with a diselenide moiety. TrxR reduces the diselenide bond, triggering a fluorescence turn-on response of the probe. Kinetic studies reveal favorable binding of the probe with TrxR with a Michaelis-Menten constant (K_m ) of 15.89 μm. Computational docking simulations predict a greater binding affinity to the TrxR active site in comparison to its disulfide analogue. In vitro imaging studies further confirmed the diselenide probe exhibited improved signaling of TrxR activity compared to the disulfide analogue.

Keywords: diagnostics; melanoma; selenium probes; seminaphthorhodafluor; thioredoxin reductase.

Figure 1.

Docking simulations for: (A) Optimized structure of probe 1a and (B) Probe 1b onto the active site of TrxR1. The distances between the Se atom on the Sec residue (U498a) and the diselenide and disulfide bond atoms are shown. Probe 1a has a binding energy of −11.1 kcal/mol whereas that probe 1b is −9.6 kcal/mol.

Figure 2.

Fluorescence emission vs time for probe 1a (20 μM) in the presence of EDTA (10 mM), BSA (0.2 mg/mL), phosphate buffer (100 mM, pH 7.4) and NADPH (0.24 mM), except as noted for control experiments. TrxR1 activity was monitored via the emission of 2 (λ_ex = 531 nm; λ_em = 580 nm) as well as the depletion of NADPH (λ_abs = 340 nm) upon incubation.

Figure 3.

Reaction rate vs substrate concentration to illustrate the change in the speed of the reaction over time for each substrate concentration. K_m for the reaction was 15.89 μM.

Figure 4.

Fluorescence responses of probe 1a upon 20 min incubation with TrxR1 and other potential biological reducing species (Cys = cysteine, Hcy = homocysteine, GSH = glutathione).

Figure 5:

Live cell imaging of HCC827 cells (i) Untreated controls (ii) Treated with 10 μM of either 1a or 1b and (iii) Treated with 40 μM DNCB and 10 μM of either 1a or 1b. After 1 h,the untreated cells showed no fluorescence whereas those treated with the probes fluoresced. Pretreating the cells with DNCB before introducing the probes resulted in diminished fluorescence. This fluorescence was quantified and was higher for 1a (15805 R.F.U) compared to 1b (9903 R.F.U)

Scheme 1.

Proposed turn-on mechanism of probe 1a in the presence of TrxR1 and NADPH. Nucleophilic attack of the diselenide occurs through the TrxR1 Sec residue. The selenolate formed attacks the carbamate carbonyl, releasing an oxaselenolanone heterocycle and fluorescent 2.

Scheme 2.

Synthesis of diselenide probe 1a and the disulfide probe 1b. Compounds 2 and 7 were prepared as reported previously (ESI^†).