Potential of a γ-glutamyl-transpeptidase-stable glutathione analogue against amyloid-β toxicity

Abstract

The antioxidant properties of glutathione (GSH) and their relevance to oxidative stress induced pathological states such as Alzheimer's disease is well-established. The utility of GSH itself as a pharmacotherapeutic agent for such disorders is limited because of the former's lability to breakdown through amide cleavage by the ubiquitous enzyme γ-glutamyl transpeptidase (γ-GT). In the present study, a GSH analogue, Ψ-GSH, where the γ-glutamylcysteine amide linkage is replaced with a ureide linkage, was synthesized. Ψ-GSH was found to be stable toward γ-GT mediated breakdown. Ψ-GSH fulfilled four cardinal properties of GSH, namely, traversing across the blood brain barrier (BBB) via the GSH active uptake machinery, replacing GSH in the glyoxalase-I mediated detoxification of methylglyoxal, protecting cells against chemical oxidative insult, and finally lowering the cytotoxicity of amyloid-β peptide. These results validate Ψ-GSH as a viable metabolically stable replacement for GSH and establish it as a potential preclinical candidate for treatment of oxidative stress mediated pathology.

Keywords: Alzheimer’s disease; Glutathione; advanced glycation end products; glyoxalase I; methylglyoxal; β-amyloid peptide.

Figure 1

Structure of Ψ-GSH.

Scheme 1. Synthesis of Ψ-GSH

Figure 2

Glyoxalase I enzyme kinetics assay. GSH or ΨGSH at various concentrations was incubated with MG at 30 °C in phosphate buffer (0.05 M, pH 6.6) for 6 min to allow formation of the hemimercaptal 2, followed by the addition of yeast Glx-I. The enzyme reaction was monitored for 180 s by measuring the increase in absorption at 240 nm. Rates of enzymatic reaction were plotted against substrate concentrations. Data shown here is representative of three independent experiments.

Scheme 2. Glx-I Mediated Formation of 2

Figure 3

Effect of GSH and Ψ-GSH on intracellular levels of methylglyoxal. SH-SY-5Y cells were treated with glucose (50 mM) for 72 h in the presence and absence of GSH or Ψ-GSH (500 μM). Intracellular concentrations of MG were quantified by HPLC after derivatization with 1,2-diaminobenzene as described in Methods. The results of this experiment demonstrated that GSH and Ψ-GSH caused reduction in MG levels to similar extents.

Figure 4

Protection against Aβ1-42 cytotoxicity by GSH and Ψ-GSH. The percent cell death caused in SH-SY-5Y cells by 24 h exposure to Aβ1-42 (20 μM) exposure was determined by the standard MTT assay as described in Methods. The decrease in cytotoxicity of Aβ1-42 was observed by preincubation of cells with (A) GSH or ΨGSH and (B) GSBB (1 mM) for 24 h and was dose-dependent with respect to their concentrations. Data are expressed as the (mean ± SEM) of three independent experiments (a, significantly higher than βA1–42 only group, p < 0.0001; b, significantly higher than corresponding GSH treatment group, p < 0.05).

Figure 5

Reduction in the cytotoxicity of H₂O₂ in the presence of GSH and Ψ-GSH. Pretreatment of SH-SY-5Y cells with GSH or Ψ-GSH (1 mM) for 24 h prior to H₂O₂ (50 μM) exposure for 30 min showed a significant protection against peroxide toxicity. The protection observed due to GSH (white bars) and Ψ-GSH (gray bars) was comparable and dose-dependent with respect to their concentrations. The data are expressed as the (mean ± SEM) of three independent experiments (** p < 0.001; *** p < 0.0001).

Figure 6

Measurement of ROS using DCFH-DA. Oxidative stress was induced in SH-SY-5Y cells by exposure to (A) H₂O₂ (500 μM) for 90 min or (B) MG (1 mM) for 180 min at 37 °C in the presence or absence of GSH or Ψ-GSH (250 μM). Increase in fluorescence of DCF was regarded as an indicator of oxidative stress as described in Methods. Both GSH and Ψ-GSH were efficient at reducing the ROS generated by peroxide and MG. The data are represented as the (mean ± SEM) of two independent experiments (p < 0.0001).

Figure 7

(A) Uptake kinetics of [³H]GSH in SH-SY-5Y cells. SH-SY-5Y cells were exposed to [³H]GSH (100 nM) at 37 °C for various time intervals and intracellular radioactivity was measured using scintillation counter. The net uptake was normalized to protein content in each well as described in Methods. The data are represented as the (mean ± SEM) of three independent experiments. (B) Inhibition of [³H]GSH uptake by Ψ-GSH. Uptake of [³H]GSH (1000 nM) in the presence and absence of GSH and Ψ-GSH (10, 100, and 1000 nM) was determined in SH-SY-5Y cells as described in Methods. The inhibition of [³H]GSH uptake by Ψ-GSH was comparable to that by cold GSH. The data are represented as the (mean ± SEM) of three independent experiments (p < 0.0001).