Characterization of a novel dithiocarbamate glutathione reductase inhibitor and its use as a tool to modulate intracellular glutathione.
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 microm 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 microm 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.
Seefeldt
Teresa
T
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007.
Zhao
Yong
Y
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007.
Chen
Wei
W
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007.
Raza
Ashraf S
AS
Covance Bioanalytical Services LLC, Indianapolis, Indiana 46214.
Carlson
Laura
L
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007.
Herman
Jocqueline
J
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007.
Stoebner
Adam
A
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007.
Hanson
Sarah
S
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007.
Foll
Ryan
R
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007.
Guan
Xiangming
X
Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Brookings, South Dakota 57007. Electronic address: Xiangming.Guan@sdstate.edu.
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005978:Glutathione / Q000096:biosynthesis / Q000378:metabolism*; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors*; D009249:NADP / Q000378:metabolism; D013859:Thiocarbamates / Q000494:pharmacology*
C539612:2-acetylamino-3-(4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl)propionic acid; D004791:Enzyme Inhibitors; D013859:Thiocarbamates; D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione; D000111:Acetylcysteine