Discovery of an internal alkyne warhead scaffold for irreversible hTG2 inhibition

Human tissue transglutaminase (hTG2) is a multifunctional enzyme with both protein cross-linking and G-protein activity. Dysregulation of these functions has been implicated in diseases such as celiac disease and cancer, prompting the development of hTG2 inhibitors, many of which act covalently <i>via</i> a pendant electrophilic warhead. Most small molecule hTG2 inhibitors to date feature terminal, sterically minimal warheads, based on the assumption that bulkier electrophiles impair binding and reactivity. Here, we report structure-activity relationships (SAR) of a novel internal alkynyl warhead scaffold for irreversible inhibition of hTG2. This series includes one of the most potent non-peptidic hTG2 inhibitors reported to date. We demonstrate that this scaffold not only inhibits transamidase activity but also abolishes GTP binding, while exhibiting excellent isozyme selectivity. In addition, we investigate the tunability and stability of this warhead, providing insights into its broader applicability. Through detailed kinetic analysis, this study establishes a new scaffold for irreversible hTG2 inhibition and expands the design principles for covalent warheads beyond traditional terminal systems.