Identification of Fungal Metabolite Gliotoxin as a Potent Inhibitor Against Bacterial O-Acetylserine Sulfhydrylase CysK and CysM

Cysteine is an essential amino acid for sustaining life, including protein synthesis, and serves as a precursor for antioxidant glutathione. Pathogenic bacteria synthesize cysteine via a two-step enzymatic process using serine as the starting material. The first step is catalyzed by serine acetyltransferase, also known as CysE, and the second by <i>O</i>-acetylserine sulfhydrylase (OASS), referred to as CysK or CysM. This cysteine biosynthetic pathway in bacteria differs significantly from that in mammals, making it an attractive target for the development of novel antibacterial agents. In this study, we aimed to identify OASS inhibitors. To achieve this, a high-throughput screening system was developed to analyze compounds capable of inhibiting CysK/CysM activity. Screening 168,640 compounds from a chemical library revealed that gliotoxin, a fungal metabolite, strongly inhibits both CysK and CysM. Furthermore, gliotoxin significantly suppressed the growth of <i>Salmonella enterica</i> serovar Typhimurium, a Gram-negative bacterium, under cystine-deficient conditions. Gliotoxin possesses a unique disulfide structure classified as epipolythiodioxopiperazine. To date, no studies have reported OASS inhibition by compounds with this structural motif, highlighting its potential for future structural optimization. The screening system developed in this study is expected to accelerate the discovery of functional CysK/CysM inhibitors, providing a foundation for novel antibacterial strategies.