Thiol redox state (TRS) is an important parameter to reflect intracellular oxidative stress and is associated with various normal and abnormal biochemical processes. Agents which can be used to increase intracellular TRS will be valuable tools in TRS-related research. Glutathione reductase (GR) is a critical enzyme to the homeostasis of TRS. The enzyme catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) to maintain a high GSH to GSSG ratio. Inhibition of the enzyme can be used to increase TRS. Despite the reporting of various GR inhibitors, N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU), an anticancer drug with an IC(50) value of 647 microM against yeast GR, remains the most commonly used GR inhibitor in the literature. However, the toxicity caused by non-specific interactions as well as inhibition of DNA synthesis complicates the use of BCNU as a GR inhibitor. We would like to report 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthiocarbonylamino)phenylthio-carbamoylsulfanyl]propionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by a one-step synthesis from commercially available reagents. The Ki and kinact values of 2-AAPA against yeast GR were determined to be 56 microM and 0.1 min(-1) respectively. At the concentration which produced over 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. By employing CV-1 cells, 2-AAPA (0.1 mM) produced 97% GR inhibition, a 25% GSH reduction, and a five-fold increase in GSSG in 20 minutes. The compound can be a useful tool in TRS-related research.
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