Mechanistic studies of gamma-glutamyl transpeptidase inhibition: A novel approach to modulating serum levels of cysteine

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Gamma-glutamyl transpeptidase (GGT) is essential for cysteine homeostasis in vivo. There are no potent, specific non-toxic inhibitors of GGT that can be used as molecular probes in studies of the disruption of cysteine homeostasis. Such studies are needed to increase our understanding of many disease processes including cancer. Tumors have an enhanced requirement for cysteine. The long term goal of this research is to study the mechanisms by which cysteine availability impacts tumor progression and response to therapy. The central hypothesis in this proposal is that potent, non-toxic, highly specific inhibitors of GGT1 administered systemically will deplete the serum of cysteine. The objectives of this application are to investigate the catalytic mechanism of GGT, using insights from kinetic and structural studies to develop two mechanistically distinct classes of GGT inhibitors and provide in vivo proof-of-biology that small molecule inhibitors of GGT1 can be used to modulate serum cysteine levels. These objective will be met by studies outlined in three specific aims. In Aim I structural studies of inhibitor-bound hGGT1 and molecular modeling will be used to design new sets of analogs of a high potency competitive inhibitor of hGGT1. The new compounds will be synthesized and analyzed for potency and specificity as inhibitors of hGGT1 in kinetic assays and cell culture model systems. The most potent inhibitors will be co-crystallized with hGGT1 and the structures solved. We will use an iterative strategy to optimize the potency and specificity of the analogs. Using the strategy outlined in Aim I, in Aim II we will undertake development of a novel class of uncompetitive inhibitors that we have identified of hGGT1. Based on our preliminary data, we hypothesize that studying the interaction of these compounds with the enzyme will provide additional knowledge of the catalytic activity of GGT. This knowledge will be applied to the studies in both Aims I and II. Aim III will focus on proof-of-biology studies in mice. The mice will be treated with the most potent and specific compounds from each of the two aims. Cysteine concentrations in the serum will be monitored following administration of the compound. These experiments will be compare the efficacy of competitive and uncompetitive inhibitors in vivo. These studies will be undertaken by an established team of investigators with expertise in structural biology, molecular modeling, medicinal chemistry, enzyme kinetics, and use of inhibitors as probes in in vitro and in vivo studies. We are well positioned for these studies as the first and only group to crystallize and solve the structure of any eukaryotic GGT. Successful completion of the proposed studies will provide valuable molecular probes for future mechanistic studies of the effect of GGT inhibition and modulation of serum cysteine on tumors.