Development of an ¹⁸F-Labeled Irreversible Inhibitor of Transglutaminase 2 as Radiometric Tool for Quantitative Expression Profiling in Cells and Tissues

Abstract

The transamidase activity of transglutaminase 2 (TGase 2) is considered to be important for several pathophysiological processes including fibrotic and neoplastic tissue growth, whereas in healthy cells this enzymatic function is predominantly latent. Methods that enable the highly sensitive detection of TGase 2, such as application of radiolabeled activity-based probes, will support the exploration of the enzyme’s function in various diseases. In this context, the radiosynthesis and detailed in vitro radiopharmacological evaluation of an 18F-labeled N ε-acryloyllysine piperazide are reported. Robust and facile detection of the radiotracer-TGase 2 complex by autoradiography of thin layer plates and polyacrylamide gels after chromatographic and electrophoretic separation owing to irreversible covalent bond formation was demonstrated for the isolated protein, cell lysates, and living cells. By use of this radiotracer, quantitative data on the expression profile of activatable TGase 2 in mouse organs and selected tumors were obtained for the first time by autoradiography of tissue sections.

Structure–activity relationships of hydrophobic alkyl acrylamides as tissue transglutaminase inhibitors

“…We expected that inhibitors bearing the longest linkers would show the greatest affinity since we had observed this trend among many series of peptidomimetic TG2 inhibitors. 22,26,27,38 However, it is clear from the data shown in Table 1 that the shortest linker apparently positions the reactive acrylamide at the optimal distance from the active site cysteine, providing an efficiency ( k inact / K I ratio) for compound 24a of 27 500 M −1 min −1 , whereas the longer chain derivatives 24b–e were roughly an order of magnitude less efficient.…”

“…The scaffolds of many inhibitors have been designed to allow binding in the D-site, while presenting an electrophilic group that can react with the catalytic cysteine in the active site tunnel. These warhead groups include acrylamides, 21,22,26,27 epoxides, 28 6-diazo-5-oxo-norleucine (DON), 10 and other α,βunsaturated carbonyl groups. 24,29 Very recently, peptidomimetic inhibitors designed to inhibit intestinal TG2 have progressed through phase II clinical trials for the treatment of celiac disease.…”

Development and Preclinical Evaluation of Radiolabeled Covalent G12C-Specific Inhibitors for Direct Imaging of the Oncogenic KRAS Mutant

“…The protein binding assay was executed as previously described. , The probes ([ 131 I]­I-ARS-1620 and [ 18 F]­F-ARS-1620) were dissolved in a buffer containing 10% DMSO, 5 mM MgCl 2 , and 5 mM nucleotide (GDP or GMPPNP) with an activity of 37 MBq/mL. KRAS protein (G12C, WT as indicated, and BSA for control) with a final concentration of 2 μmol/L was incubated with 3.7 MBq of probes in a buffer containing 20 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 1 mmol/L MgCl 2 , and 1 mmol/L DTT for 1 h. This process was quenched by adding formic acid to 0.2%.…”

“Clickable” Albumin Binders for Modulating the Tumor Uptake of Targeted Radiopharmaceuticals

“…Determination of log D 7.4 of (S)-1f and (S)-2b by HPLC. The log D 7.4HPLC value was determined as previously reported by us 105,106 utilizing a modified HPLC method originally described by Donovan and Pescatore. 107 Radiolabeling of DOTA/NODAGA-Bearing Peptides.…”