A Profiling Platform for the Characterization of Transglutaminase 2 (TG2) Inhibitors

Abstract

Huntington's disease (HD) is associated with increased expression levels and activity of tissue transglutaminase (TG2), an enzyme primarily known for its cross-linking of proteins. To validate TG2 as a therapeutic target for HD in transgenic models and for eventual clinical development, a selective and brain-permeable inhibitor is required. Here, a comprehensive profiling platform of biochemical and cellular assays is presented which has been established to evaluate the potency, cellular efficacy, subtype selectivity and the mechanism-of-action of known and novel TG2 inhibitors. Several classes of inhibitors have been characterized including: the commonly used pseudo-substrate inhibitors, cystamine and putrescine (which are generally nonspecific for TG2 and therefore not practical for drug development), the various peptidic inhibitors that target the active site cysteine residue (which display excellent selectivity but in general have poor cellular activity), and the allosteric reversible small-molecule hydrazides (which show poor selectivity and a lack of cellular activity and could not be improved despite considerable medicinal chemistry efforts). In addition, a set of inhibitors identified from a collection of pharmacologically active compounds was found to be unselective for TG2. Moreover, inhibition at the guanosine triphosphate binding site has been examined, but apart from guanine nucleotides, no such inhibitors have been identified. In addition, the promising pharmacological profile of a TG2 inhibitor is presented which is currently in lead optimization to be developed as a tool compound. (Journal of Biomolecular Screening 2010:478-487)

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells

“…4D). It is important to note that despite these TG2 inhibitors being cell-permeable, inhibition of cellular TG2 is only achieved at concentrations significantly above their IC50 value versus purified enzyme (Schaertl et al, 2010;Freund et al, 1994). Overall, these data indicate that TG2 is the TG isoform activated by the PAC1 receptor.…”

“…To confirm that TG2 is responsible for PAC1 receptor-mediated transglutaminase activity in differentiating N2a cells, two structurally different cell permeable TG2 specific inhibitors were tested; R283 (a small molecule; Freund et al, 1994) and Z-DON (peptide-based; Schaertl et al, 2010). N2a cells were pre-treated for 1 h with Z-DON (150 µM) or R283 (200 µM) prior to stimulation with PACAP-27 for 10 min.…”

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival

“…To confirm that TG2 is the isoform linked to A1 adenosine receptor transglutaminase activity in H9c2 cardiomyocytes, two structurally different cell permeable TG2 specific inhibitors were tested; R283 (a small molecule; [34]) and Z-DON (peptide-based; [35]). activity similar inhibitory trends were observed but these were not always statistically significant.…”

Regulation of the activities of the mammalian transglutaminase family of enzymes

“…35,37 The binding site of these inhibitors has not yet been verified, and some controversy exists whether it is the GTP binding pocket itself, or a site that is coupled to the GTP binding site. 35,38 The acylidene oxindoles such as CK-IV-38 [ Fig. 3(B)] comprise another class of allosteric inhibitors based on their noncompetitive inhibition characteristics.…”