Characterization of a Novel Dithiocarbamate Glutathione Reductase Inhibitor and Its Use as a Tool to Modulate Intracellular Glutathione

Abstract

Thiol redox state (TRS) is an important parameter to reflect intracellular oxidative stress and is associated with various normal and abnormal biochemical processes. Agents that can be used to increase intracellular TRS will be valuable tools in TRS-related research. Glutathione reductase (GR) is a critical enzyme in the homeostasis of TRS. The enzyme catalyzes the reduction of GSSG to GSH to maintain a high GSH:GSSG ratio. Inhibition of the enzyme can be used to increase TRS. Despite the reports of various GR inhibitors, N,N-bis(2-chloroethyl)-N-nitrosourea, an anticancer drug with IC 50 ‫؍‬ 647 M against yeast GR, remains the most commonly used GR inhibitor in the literature. However, the toxicity caused by nonspecific interactions, as well as inhibition of DNA synthesis, complicates the use of N,N-bis(2-chloroethyl)-N-nitrosourea as a GR inhibitor. We report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The K i and k inact of 2-AAPA against yeast GR were determined to be 56 M and 0.1 min ؊1 , respectively. At the concentration that produced >80% yeast GR inhibition, 2-AAPA showed no inhibition against glutamylcysteine synthetase, glutathione synthetase, catalase, and superoxide dismutase, but minimal inhibition against glutathione S-transferase and glutathione peroxidase. In CV-1 cells, 2-AAPA (0.1 mM) produced 97% GR inhibition, 25% GSH reduction, and a 5-fold increase in GSSG in 20 min. The compound can be a useful tool in TRS-related research.

The Potent and Novel Thiosemicarbazone Chelators Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone and 2-Benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone Affect Crucial Thiol Systems Required for Ribonucleotide Reductase Activity

“…The glutathione reductase (GR) activity was assayed by following the rate of decrease in NADPH absorbance at 340 nm as a result of the reduction of GSSG (Seefeldt et al, 2009). After a 24-h/37°C incubation with the chelators, cells were washed with ice-cold PBS and resuspended in sample buffer (50 mM potassium phosphate, pH 7.5, containing 1 mM EDTA) and then lysed by three freeze-thaw cycles.…”

“…Thus, the ability of thiosemicarbazones to reduce the GSH/GSSG ratio could be a result of compromised GR activity, and this was then examined. As a positive control, a known GR inhibitor, bis-chloronitrosourea (BCNU; 100 M), was included (Seefeldt et al, 2009) (Fig. 3A).…”

Evaluation of a dithiocarbamate derivative as an inhibitor of human glutaredoxin-1

“…Previously, this group reported 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) (Figure 2) as an irreversible inhibitor of GR with a K i of 56 μM and a k inact of 0.143 min −1 against yeast GR. (16) 2-AAPA was also shown to inhibit GR, increase GSSG, and produce increased glutathionylation in CV-1 (monkey kidney) cells. (16, 17) In this study, the potential for human GRX-1 inhibition by 2-AAPA was evaluated.…”

“…(16) 2-AAPA was also shown to inhibit GR, increase GSSG, and produce increased glutathionylation in CV-1 (monkey kidney) cells. (16, 17) In this study, the potential for human GRX-1 inhibition by 2-AAPA was evaluated.…”

Dithiocarbamate Toxicity - An Appraisal

“…Similarly, disulfiram also induces oxidative stress that changes mitochondrial permeability leading to mitochondrial injury (Balakirev and Zimmer, 2001). A number of enzymes are inhibited by DTC which include cyclooxygenase, , heme oxygenase (Kushida et al, 2002), cytochrome P450, superoxide dismutase, glutathione reductase, and caspase (Dalvi et al, 2002;Cvek & Dvorak, 2007;Seefeldt et al, 2009). The superoxide dismutase inhibitory activity of thiram and disulfiram is implicated in their anti-angiogenic effects (Marikovsky et al, 2002;Shian et al, 2003).…”