Waging Warheads: Investigation of Electrophilic Warheads for Targeted Covalent Inhibition of Transglutaminase 2 and Beyond

Abstract

Covalent inhibition has re-emerged as a powerful therapeutic strategy, with attenuated electrophilic warheads enabling selective and sustained target engagement. Despite their central role, warheads are often treated primarily as elements for tuning electrophilicity, and their broader structural contributions to pharmacodynamic and pharmacokinetic behaviour remain underexplored. This thesis investigates how warhead structure influences both reactivity and binding in covalent inhibitors, in the context of target engagement and metabolism. In Chapter 2, a library of electrophilic warheads was evaluated on a common scaffold targeting human tissue transglutaminase 2 (hTG2), demonstrating that warhead structure significantly impacts not only reactivity, but also binding affinity and isozyme selectivity across the transglutaminase family. In Chapter 3, a novel, fully non-peptidic internal alkynyl warhead scaffold was developed, revealing that sterically complex warheads can effectively inhibit hTG2 and providing a foundation for the development of new covalent inhibitor scaffolds. In Chapter 4, a library of warheads was evaluated as substrates for glutathione S-transferase (GST)-mediated metabolism using quantitative assays, demonstrating that metabolic susceptibility is a structurally tunable property that does not correlate with intrinsic reactivity. These results identify structural features that contribute to GST liability and provide strategies for modulating GST-mediated metabolism of covalent inhibitors. Collectively, this work establishes a more integrated view of electrophilic warheads as tunable structural elements governing both reactivity and affinity-driven interactions and provides insight into how warhead design can be leveraged to optimize both target engagement and metabolic stability in covalent inhibitors.