Discovery and development of inhibitors of transglutaminase 2 as chemical tools and medicinal leads [electronic resource]
Keywords: Chemistry.

The family of transglutaminases (TG) encompasses nine members in humans, eight of which are catalytically competent (TG1-7 and Factor XIIIa), whereas one (band 4.2) is not. As their name suggests, the enzymes catalyze the posttranslational modification of specific glutamine residues in their protein or peptide substrates. In the transamidating activity, these acyl donor substrates are coupled to biogenic small molecule amines (such as serotonin) or lysine sidechains on proteinogenic acyl acceptor substrates through a newly formed [gamma]-glutaminyl isopeptide bond. Under suitable conditions, transglutaminases are also capable of deamidating the parent glutamine to glutamic acid. TGs exert a broad range of functions in physiology. TG1 and TG3 are, for example, activated during keratinocyte maturation and confer mechanical stability to the skin barrier. Factor XIIIa is part of the blood clotting cascade and its crosslinking is important for stabilizing fibrin clots. TG2 is ubiquitously expressed and has been implicated with various functions that depend on the precise location and physiological context. In virtually all cases, the activity of TGs is subject to an elaborate regulation at the transcriptional and posttranslational level. The posttranslational regulatory cues for TG1-3 and FXIIIa are reviewed in Chapter 1 and include the concentration of cofactors such as Ca2+ ion or GTP nucleotides or the proteolytic processing of TG zymogens. While it was previously known that oxidation of a pair of vicinal cysteines inactivates TG2, the discovery that the reducing cofactor thioredoxin can specifically re-activate the oxidized enzyme in the extracellular environment (Appendix B) positioned the redox state as another layer of regulatory cues for TG2. Aberrant activity of TGs has been implicated in a variety of human ailments. For TG1 and FXIIIa, loss-of-function mutations give rise to skin barrier dysfunction and bleeding disorders, respectively. In contrast, ablation of TG2 activity appears benign, but its (aberrant) activation appears pathogenic in a number of diseases, such as celiac sprue, septic shock, ischemic reperfusion injury, renal fibrosis, certain cancers and Huntington's disease. Among these disorders, the role of TG2 is arguably best characterized in celiac sprue and Chapter 2 reviews the arguments supporting its pathogenic role in this disease. Given the mild phenotype of its ablation and its potentially pathogenic activity, TG2 appears as an attractive target for the treatment of these diseases and this observation has motivated the discovery and development of TG2 inhibitors reported in the main body of this dissertation. Specifically, Chapter 3 describes the discovery as well as the structure-activity relationship of 3-acylidene-2-oxoindoles as inhibitors of TG2. These compounds are slow-tight binding inhibitors of the enzyme and appear to bind to an allosteric regulatory site. Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have previously been developed and two members of this series, KCC009 and ERW1041E have been extensively used as tool compounds to study the function of TG2 in biology and in disease. The work described in Chapter 4 sought to develop this promising class of inhibitors towards medicinally useful lead compounds. In particular, the previous compounds had only modest potency toward TG2 and a largely unknown selectivity profile against other pertinent TG homologs (TG1, TG3 and FXIIIa). Observing significant reactivity towards TG1 in the initial assessment, structure-activity and -selectivity analyses led to the discovery of several improved analogues. After profiling the pharmacokinetic and --dynamic properties of these inhibitors, two lead compounds emerged; ZH147A as an orally bioavailable lead that might be suitable for the treatment of e.g. celiac sprue and a potent and selective derivative, CK996 that might be useful for the treatment of systemic inflammatory disorders. Specifically, CK996 was efficacious in reducing the proinflammatory cytokine load in murine endotoxemia at doses as low as 1.5 mg/kg. The work in Chapter 5 illustrates how the DHI inhibitors were useful to study the role of TG2 in hypoxia-induced pulmonary hypertension. While it is not yet clear whether TG2 is a target for treating the disease, it might be a biomarker for diagnosis and management of the disease and activity-based probes for TG2 might be useful imaging probes in positron-emission tomography (PET). The chapter describes DHI-based TG2 inhibitors that could be adapted as probes or provide avenues for future probe-development.