The search for an inhibitor of ChaC1, a glutathione degrading enzyme

Abstract

ChaC1 is an enzyme involved in the degradation of glutathione. It is found in higher eukaryotes, including humans. ChaC1 is highly upregulated in several cancers, with the upregulation correlating with poor prognosis. The ability to inhibit ChaC1 could therefore be potentially important in cancer and situations where elevated glutathione levels could be beneficial. However, no inhibitors of ChaC1 have been discovered. In this thesis, therefore, the focus has been to identify a “first-in-class” inhibitor of the human ChaC1 enzyme. We have taken both computational and wet lab approaches towards this goal. In the absence of a crystal structure for ChaC1, a 3-D structure of human ChaC1 was predicted using homology modeling. Its active site map was predicted through substrate docking and MD simulations, which were further validated using mutagenesis followed by functional evaluation. A systemic pharmacophore-based virtual screening was performed, where approximately 6.6 lakh compounds from two different natural compound libraries were screened. The top hits were evaluated for their inhibition against the human ChaC1 enzyme using an in vitro enzymatic assay. None of these compounds, along with a few analogues of GSH, were able to inhibit ChaC1 activity at 100 µM concentration. In a second approach, we developed two different high-throughput, robust, yeast-based assays for identifying human ChaC1 inhibitors. A small molecule compound library was screened using an automated liquid handling system. The hits were further analyzed using the in vitro enzymatic assay. Juglone, a naturally occurring naphthoquinone, completely inhibited human ChaC1 activity at 50 µM concentration. The half-maximal inhibitory concentration (IC50) was determined to be 8.7 µM. 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 inhibited ChaC1 in a cysteine-independent manner. Molecular docking and MD simulations of Juglone with the human ChaC1 model show that it enters the active site pocket of the enzyme and forms strong hydrogen bonds with the Tyr38 and Tyr121 residues. Several other quinone derivatives were evaluated, and while some were able to inhibit ChaC1, others were unable to do so. Among them, none appeared to be significantly superior in their ability to inhibit ChaC1 compared to Juglone.