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High activity of tissue transglutaminase (TGase 2) in various tumors is associated with both their increased metastatic and invasive potential and their resistance towards chemotherapy and radiation. This renders TGase 2 an attractive target for the development of agents that are capable of targeting the tumor-associated TGase 2 for both imaging and therapeutic approaches [1]. TGase 2 exists in two different conformations, with the closed one being the major intracellular form. It functions as a GTP-binding protein (Gh protein) at low Ca2+ levels, whereas transition to the open conformation (initiated by an increase in Ca2+ concentration) allows for the transamidase activity leading to protein-protein crosslinking [2]. Characterization of the GTP-binding activity of TGase 2 was done by means of a new fluorescence anisotropy assay using the literature known BODIPY FL-GTPγS [3]. This compound, internally quenched by an intramolecular stacking of the BODIPY and the guanosin moieties, is commonly used in fluorescence-based assays that make use of the unfolding and thereby dequenching of the fluorophore upon binding, leading to an increase in fluorescence. [3] Such behavior, however, interferes with the analysis of fluorescence anisotropy as the measured data have to be corrected [4]. To optimize the new assay, we investigated a small series of newly developed GTP- and GDP-analogues labeled with fluorescein for TGase affinity and change in fluorescence upon protein binding. All compounds show a significantly smaller increase in fluorescence intensity compared to the BODIPY FL-labeled nucleotide and – in some cases – an up to ten-fold superior binding affinity towards TGase 2. The fluorescence anisotropy assay was then validated for inhibition studies by investigation of GTP and GTPγS, which both show IC50 values (64 nM and 109 nM, respectively) that are in agreement with literature data [5,6]. Further investigations included titration of the GTP/TGase 2 interaction with CaCl2 to determine the Ca2+ concentration needed to shift TGase 2 to the extended conformation and inhibition studies with GDP and ATP as well as compounds targeting the acyltransferase domain of TGase 2. References: [1] Pietsch, M. et al. Bioorg. Med. Chem. Lett. 2013, 23, 6528. [2] Kerr, C. et al. Oncogene 2016, doi: 10.1038/onc.2016.452. [3] McEwen, D. P. et al. Methods Enzymol. 2002, 344, 403. [4] Jameson, D. M. & Mocz, G. Methods Mol. Biol. 2005, 305, 301-322. [5] Datta, S. et al. Biochemistry 2007, 46, 14819. [6] Schaertl, S. et al. J. Biomol. Screen. 2010, 15, 478. |
