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Inhibition of Transglutaminase 2 as a Therapeutic Strategy in Celiac Disease-In Vitro Studies in Intestinal Cells and Duodenal Biopsies

Sebastian Stricker, Jan de Laffolie, Klaus-Peter Zimmer, Silvia Rudloff

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES(2023)

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Abstract
Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 mu M, 15 +/- 3 vs. 48 +/- 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 mu M; 12 +/- 7% vs. 45 +/- 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 mu M, 25 +/- 13% vs. 22 +/- 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 mu M. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD.
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Key words
celiac disease,transglutaminase 2,TG2 inhibitor,PX-12,gliadin transport
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