Development of a fluorescent molecular probe for transglutaminase 2 activity in mammals

Celiac disease is an autoimmune disorder in genetically predisposed individuals where ingestion of gluten drives an inflammatory response that presents as enteropathy of the small intestine. A necessary but not sufficient genetic predisposition in individuals involves harboring the HLA-DQ2 or HLA-DQ8 gene cluster, with HLA-DQ2 being far more common. The disease is estimated to affect approximately 1 in 100 individuals worldwide, with an estimated 6 million patients between the U.S. and Europe and an estimated 2.5 million undiagnosed and at-risk Americans. There have also been studies showing that late diagnosis of celiac disease increases the risk of further autoimmune disorders. Clinically, the primary symptoms include crypt hyperplasia, a massive increase in intraepithelial lymphocytes (IELs), and villous atrophy. The only diagnostic method for the disease involves a small intestinal biopsy through upper gastrointestinal (GI) endoscopy, although elevated levels of anti-TG2 antibodies aid in the diagnosis. Currently, the only treatment for celiac disease is a lifelong avoidance of gluten as efforts to develop pharmacological treatments for celiac disease have been hindered by fundamental gaps in our understanding of its molecular pathogenesis, particularly involving the role of transglutaminase 2 (TG2). The work in this thesis helps address these issues. First, we highlight the structure-based design of a new TG2 probe named RZ-5 using available crystal structure data and develop a novel synthesis route towards developing 6-diazo-5-oxo-L-norleucine (DON) based peptides and probes. We highlight the challenges involved with DON synthesis prior to this work and present a solution to efficiently produce RZ-5 and similar analogs such as DP3-3 to validate the versatility of this method. After producing a novel probe, we explored the in vitro characteristics of RZ-5 and DP3-3 to validate RZ-5 as a potent TG2 inhibitor and interaction partner through various kinetic assays. We also present a novel assay to survey strong irreversible binding partners to TG2 and present kinetic parameters for RZ-5. Additionally, we studied RZ-5 in tissue and cell-based assays to validate its efficacy as an inhibitor and sensor. Starting with studies on frozen thin sections, we then moved towards higher-order biological systems. We present data validating RZ-5's binding and probing efficacies towards TG2 in mouse embryonic fibroblasts (MEF) systems, and show promising preliminary data of its potential as a compound capable of labeling TG2 in live animal systems either through direct delivery or through enteric formulation as an orally available drug. Collectively, these works serve as a guideline to highlight our work towards a next-generation probe for TG2 that has the potential to label the active enzyme in live animals. Future usage of this probe can strongly advance our molecular understanding of the roles of TG2 activity in celiac disease pathogenesis and its flexibility can lead to understanding of TG2 in other systems

Keywords: Chemical Engineering.
Advisor: Khosla Chaitan
Bogyo Matthew
Sattely Elizabeth