Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype

Abstract

Transglutaminase 2 (TG2) is a multifunctional enzyme primarily responsible for crosslinking proteins. Ubiquitously expressed in humans, TG2 can act either as a transamidase by crosslinking two substrates through formation of an Nε(ɣ-glutaminyl)lysine bond or as an intracellular G-protein. These discrete roles are tightly regulated by both allosteric and environmental stimuli and are associated with dramatic changes in the conformation of the enzyme. The pleiotropic nature of TG2 and multi-faceted activities have resulted in TG2 being implicated in numerous disease pathologies including celiac disease, fibrosis, and cancer. Targeted TG2 therapies have not been selective for subcellular localization, such that currently no tools exist to selectively target extracellular over intracellular TG2. Herein, we have designed novel TG2-selective inhibitors that are not only highly potent and irreversible, but also cell impermeable, targeting only extracellular TG2. We have also further derivatized the scaffold to develop probes that are intrinsically fluorescent or bear an alkyne handle, which target both intra- and extracellular TG2, in order to facilitate cellular labelling and pull-down assays. The fluorescent probes were internalized and imaged in cellulo, and provide the first implicit experimental evidence that by comparison with their cell-impermeable analogues, it is specifically intracellular TG2, and presumably its G-protein activity, that contributes to transglutaminase-associated cancer progression.

The Role of Transglutaminase 2 in Cancer: An Update

“…The pleiotropic nature of TG2 makes it an essential player in numerous cellular processes; it participates in cell growth and differentiation, adhesion and migration, autophagy, inflammation, tissue repair, fibrosis, extracellular matrix formation, proteostasis, apoptosis, angiogenesis, the EMT transition, and epigenetic modification [ 10 , 21 , 23 , 28 , 33 , 34 ]. The impairment of such a crucial enzyme that participates in several processes leads to different hallmarks of cancer, such as cancer progression, metastasis spread, cancer cell survival and invasion, and drug resistance.…”

“…Moreover, making the study of this enzyme complicated, TG2 activity is diverse depending on its subcellular localization. Therefore, TG2 is involved in different cellular processes like cell death, growth, adhesion, and differentiation, as well as inflammation, tissue remodeling, wound healing, and extracellular matrix (ECM) organization [ 6 , 20 , 21 ].…”

Preclinical evaluation of an 18F-labeled Nε-acryloyllysine piperazide for covalent targeting of transglutaminase 2

“…by using glutamine and lysine residues to intra- and intermolecularly cross-link the proteins (Folk 1980 ; Griffin et al 2002 ; Keillor et al 2014 ). Its involvement in various pathophysiological processes including fibrosis (Elli et al 2009 ; Olsen et al 2011 ; Fell et al 2021 ), celiac disease (Rauhavirta et al 2019 ; Schuppan et al 2021 ), and cancer (Eckert 2019 ; Tabolacci et al 2019 ) stimulated the development of selective and potent inhibitors, especially targeted covalent inhibitors (Badarau et al 2015 ; Büchold et al 2022 ; Rangaswamy et al 2022 ; McNeil et al 2022 ; Mader et al 2023 ; Cundy et al 2023 ; Gates et al 2023 ). Recently, we selected compound 7b (numbering according to our previous publications), which is equipped with an acrylamide group as electrophilic warhead for reaction with the active site cysteine residue of TGase 2, for labeling with fluorine-18 due to its favorable parameters with regards to inhibitory activity, selectivity and physicochemical properties (Wodtke et al 2018 ).…”