Identification of Juglone, a "first-in-class" inhibitor of the human glutathione degrading enzyme, ChaC1, using yeast-based high throughput screens

Abstract

The cytosolic glutathione-degrading enzyme, ChaC1, is highly upregulated in several cancers, with the upregulation correlating to poor prognosis. The ability to inhibit ChaC1 thus becomes important in pathophysiological situations where elevated glutathione levels would be beneficial. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. We also evaluated synthetic substrate analogs, but these too were not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 {micro}M. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to their active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds represent the first-in-class inhibitors of the ChaC enzymes discovered using a robust yeast screen.