The Selenazal Drug Ebselen Potently Inhibits Indoleamine 2,3-Dioxygenase by Targeting Enzyme Cysteine Residues

摘要

The heme enzyme indoleamine 2,3-dioxygenase (IDO) plays an important immune regulatory rolenby catalyzing the oxidative degradation of L-tryptophan. Here we show that the selenezal drug ebselen is anpotent IDO inhibitor. Exposure of human macrophages to ebselen inhibited IDO activity in a mannernindependent of changes in protein expression. Ebselen inhibited the activity of recombinant human IDOn(rIDO) with an apparent inhibition constant of 94 ( 17 nM. Optical and resonance Raman spectroscopynshowed that ebselen altered the active site heme of rIDO by inducing a transition of the ferric heme iron fromnthe predominantly high- to low-spin form and by lowering the vibrational frequency of the Fe-CO stretch ofnthe CO complex, indicating an opening of the distal heme pocket. Substrate binding studies showed thatnebselen enhanced nonproductive L-tryptophan binding, while circular dichroism indicated that the drugnreduced the helical content and protein stability of rIDO. Thiol labeling and mass spectrometry revealed thatnebselen reacted withmultiple cysteine residues of IDO. Removal of cysteine-bound ebselen with dithiothreitolnreversed the effects of the drug on the heme environment and significantly restored enzyme activity. Thesenfindings indicate that ebselen inhibits IDO activity by reacting with the enzyme’s cysteine residues that resultnin changes to protein conformation and active site heme, leading to an increase in the level of nonproductivensubstrate binding. This study highlights thatmodification of cysteine residues is a novel and effectivemeans ofninhibiting IDO activity. It also suggests that IDO is under redox control and that the enzyme represents anpreviously unrecognized in vivo target of ebselen.