Design, Synthesis, and Evaluation of Chemical Tools for the Study of Transglutaminase 2 and Factor XIII

Abstract

The mammalian transglutaminases (TGases) are a family of eight active and one catalytically inactive calcium dependent enzymes, primarily responsible for crosslinking proteins. Two isozymes within this family with strong therapeutic interest in humans are transglutaminase 2 (TG2) and factor XIII (FXIII). TG2, or tissue transglutaminase, is a multifunctional enzyme ubiquitously expressed and capable of traditional transamidation activity as well as G-protein activity. TG2 is also present both in intracellular and extracellular localizations resulting it being implicated in numerous physiological diseases including Celiac disease, fibrosis, and cancer. Plasma soluble FXIII is a key regulator in the blood coagulation cascade responsible for crosslinking fibrin to form a rigid, insoluble, clot network and is thus associated with hemophilia when activity is low, or venous thrombosis if overactive. Cellular localized FXIII has further been implicated in the activity of macrophages, chondrocytes, and osteocytes. The content of this thesis develops a broad range of tools to study these two critical enzymes. In our study of FXIII we developed novel peptidic inhibitors and tailored the scaffold to produce a highly reactive fluorescent probe. The probe, KM93, effectively labelled FXIIIa in cellulo and enabled fundamental localization studies of this enzyme in bone marrow macrophages. Our work with TG2 developed isozyme selective inhibitors that specifically targeted extracellular TG2, with the lead now referred to as NCEG2. We then further modified this scaffold to produce a cell permeant fluorescent probe (aka NCEG-RHB), a propargylated inhibitor, and several active site directed proteolysis targeting chimeras (PROTACs). Through the use of both cell impermeable inhibitors as well as permeant versions we were able to conclude that intracellular TG2 is responsible for the proliferation and migration of cancer cells. The final tool disclosed within this thesis is a novel fluorogenic activity assay substrate, APH7, which is the highest affinity synthetic TG2 substrate published to date. APH7 aided in us uncovering an unreported enzymatic hysteresis with TG2’s catalytic activity, while also being an extremely reliable, sensitive, and reactive synthetic activity substrate. All of the tools presented within this thesis have already been applied to fundamental studies of TGases and are continuing to aid the field in their respective TGase investigations.