Tissue transglutaminase (TTG) is a multifunctional protein that plays a role in several different hematological processes. TTG is a unique member in transglutaminase gene family in that it exhibits multiple enzymatic properties including transglutaminase (TGase) and GTP/ATP hydrolysis activities. The Ca+2-dependent TGase activity catalyzes an isopeptide bond between a specific γ-glutamyl (Q) containing peptide and ε-amine group from a peptide-bound lysine (K) residue that functions to stabilize proteins and plays a role in wound healing, angiogenesis, cell proliferation, and apoptosis. In the presence of Mg+2, TTG hydrolyzes GTP to GDP, and functions as a G protein (Gαh). TTG can also hydrolyze ATP and functions as a kinase that phosphorylates histones and P53. In contrast to GTP, ATP binding does not inhibit TGase activity and involves distinct binding residues. Although normal activation of TTG is required for normal physiological process, aberrant activation of TGase function is reported to play a role in many inflammatory disorders and has been selected as target for therapeutic intervention to control fibrosis, angiogenesis, inflammation and apoptosis. In an effort to screen for specific small chemical inhibitors of TTG, we investigated a structurally diverse Lopac library (Sigma) containing 1280 pharmacologically active compounds that span a broad range of biological areas. This library contains marketed drugs, failed development candidates and “gold standards” that have well-characterized activities. Initial screening was performed using a solution-phase continuous fluorescent TGase assay performed at 6 μg/ml of purified recombinant human TTG at 37°C for 1 hour in the presence of 10 mM Ca+2 in a 96-well microtiter plate designed for high-throughput screening. In the absence of chemicals, the assay had a 3-fold increase in fluorescent intensity (λEx 340nm; λEm 520nm) when BOC-K-EDA-Dansyl (KXD; a K-substrate) was crosslinked to N, N′-dimethylcasein (a Q substrate). Initially, 20 hits were identified based on inhibition of ≥ 90% of TGase activity at 50 μM of chemicals excluding the well-characterized inhibitors cystamine and iodoacetamide, which target the active site Cys-SH of TTG. These hits were more potent than GTP as 50 μM of GTP only inhibited ≤ 25 % of TGase activity. These hits were subjected to a secondary screening using a colorimetric TGase assay that measured the covalent incorporation of biotinylated pentylamine (BP) into NMC coated microtiter plate. A total of six chemicals designated as LL1 (a c-Raf1 kinase inhibitor), LL2 (a JNK-3 kinase inhibitor), LL3 (a EGFR tyrosine kinase inhibitor), LL4 (protein kinase C and Calmodulin Kinase inhibitors), LL5 (cdc25 phosphatase inhibitor) and LL6 (DNA topoisomerase I inhibitor) were validated. All these inhibitors are known to target kinases and phosphatase by functioning as GTP and ATP analogs with guanine, adenine and quinone as backbone structures. The mechanism of inhibition of TGase activity is under investigation and might be similar to that of GTP. The fact that some kinase and phosphatase inhibitors also inhibit TGase activity further consolidating TTG as a member of kinase supergene family, but also raise a challenging task to develop a specific inhibitor to TTG. However, the chemical inhibitors discovered in current study can be used as a warhead to develop more specific inhibitor of TTG.

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