The cytosolic glutathione-degrading enzyme, ChaC1, is highly up-regulated in several cancers, with the up-regulation correlating to poor prognosis. The ability to inhibit ChaC1 is therefore important in different pathophysiological situations, but is challenging owing to the high substrate Km of the enzyme. 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. Synthetic substrate analogs were also 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 µM. 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 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 are the first inhibitor leads against the ChaC enzymes using a robust yeast screen.

中文翻译：

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通过酵母中的高通量筛选鉴定人 ChaC1 的抑制剂，ChaC1 是一种细胞质谷胱甘肽降解酶。


胞质谷胱甘肽降解酶 ChaC1 在多种癌症中高度上调，上调与不良预后相关。因此，抑制 ChaC1 的能力在不同的病理生理情况下很重要，但由于酶的高底物 Km 而具有挑战性。由于尚无已知的 ChaC1 抑制剂，因此本研究将重点放在这一目标上。我们最初采用一种计算方法，其中执行了基于系统结构的虚拟筛选。然而，没有一个预测的命中被证明是有效的抑制剂。合成底物类似物也不具有抑制作用。由于这两种方法都针对活性位点，因此我们转向开发两种高通量、稳健、基于酵母的检测方法，这些检测方法不依赖于活性位点。使用这些筛选的自动化液体处理系统筛选小分子化合物库。使用体外测定进一步分析命中。其中，天然存在的萘醌 juglone 完全抑制 ChaC1 活性，IC50 为 8.7 μM。它对 ChaC2 酶也有效。动力学研究表明，抑制作用与底物无关。已知 Juglone 与谷胱甘肽形成加合物，并且还已知通过与活性位点半胱氨酸残基共价结合来选择性抑制酶。然而，juglone 继续抑制不含半胱氨酸的 ChaC1 变体，表明它通过一种新的机制起作用。我们评估了不同的抑制机制，以及 juglone 的类似物，发现 plumbagin 作为抑制剂有效。这些化合物是使用强大的酵母筛选的针对 ChaC 酶的第一个抑制剂先导物。