Identification and monitoring of gamma‐glutamylcysteine ligase inhibition in cells

Glutathione (GSH), an intracellular reductant, acts as a detoxicant in cells in both mammalian and bacterial systems. Previous research has shown that GSH is important for survival of organisms across the animal and microbial kingdom. Therefore, studies related to GSH production may be informative for therapeutics. One approach to alter GSH production is through inhibition of gamma‐glutamylcysteine ligase (gamma‐GCL), the enzyme that catalyzes the rate‐limiting step in GSH biosynthesis. Four molecules (D‐Ethionine, D‐Cysteine, L‐2,4‐diaminobutyric acid and L‐2,3‐diaminopropionic acid) were found to inhibit purified E. coli gamma‐GCL with K i values of 8 μM, 15 μM, 11.5 μM and 3.8 μM, respectively. In order to determine the impact of these compounds in vivo, a liquid chromatography‐mass spectrometry (LC‐MS) method for GSH detection with C 13 N 15 GSH as an internal standard was optimized. This method will be used to detect GSH levels in J774A mouse macrophage cells that have been subjected to the gamma‐GCL inhibitors. Funding for this project has been provided by Merck Institute for Science Education and Gustavus Adolphus College.