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

**Background:** Celiac disease (CD) affects about 1% of the Western population, and a strict lifelong gluten-free diet remains the only available therapy. Transglutaminase 2 (TG2) is the central autoantigen in CD; it deamidates gliadin epitopes, increasing their immunogenicity and triggering the Th1-mediated immune response. TG2 also participates in endocytotic recycling and transcytosis of gliadin peptides. Inhibition of TG2 is a promising non-dietary therapeutic strategy. This study compared the oxidative inhibitor PX-12 with the active-site directed competitive inhibitor ERW1041 in multiple in vitro and ex vivo models.

**Methods:** TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) was quantified by fluorometry using a 96-well plate coated with 10 nM TG2. Cellular TG2 activity was measured in Caco-2 cell lysates using the substrate 5BP. Ex vivo TG2 activity was assessed in cryosections (400 nm) of duodenal biopsies from 6 CD patients (Marsh 3a-3b, anti-TG2-IgA ≥134 IU/mL) by immunofluorescence, with activity normalized to TG2 expression. Cell viability was tested using a resazurin-based assay (PrestoBlue HS) after 24 h exposure. Extracellular TG2 activity on intact Caco-2 monolayers was measured after 48 h IFN-γ stimulation (1000 IU/mL). Transepithelial transport of fluorochrome-conjugated gliadin peptides P31-43 and P56-88 was quantified by fluorometry of basal supernatants after 3 h, and cellular uptake was assessed by confocal microscopy after 1 h.

**Key Results:** PX-12 inhibited TG2-mediated cross-linking of PTG more effectively than ERW1041 at both 10 µM (15 ± 3% vs. 48 ± 8%, p < 0.001) and 100 µM (5 ± 1% vs. 11 ± 2%, p < 0.001). In Caco-2 cell lysates, PX-12 also showed greater inhibition (12 ± 7% vs. 45 ± 19%, p < 0.05). In duodenal biopsies, both inhibitors significantly reduced TG2 activity in the lamina propria at 100 µM (ERW1041: 0.94 ± 0.26 vs. 0.31 ± 0.17, p < 0.001; PX-12: 0.91 ± 0.28 vs. 0.44 ± 0.21, p < 0.05), with no significant difference between them. Neither inhibitor affected Caco-2 cell viability up to 100 µM. On intact Caco-2 monolayers, ERW1041 reduced extracellular TG2 activity in a dose-dependent manner (32% reduction at 10 µM, p < 0.01; 70% reduction at 100 µM, p < 0.0001), whereas PX-12 showed no significant inhibition at any concentration tested (10 nM–100 µM). ERW1041 significantly inhibited transepithelial passage of P56-88 at 1 µM (33 ± 22%, p < 0.05) and 100 µM (24 ± 16%, p < 0.01), while PX-12 had no effect. Transport of P31-43 was unaffected by either inhibitor. Confocal microscopy confirmed dose-dependent reduction of P56-88 uptake by ERW1041, with nearly absent uptake at 100 µM.

**Clinical Implications:** This study demonstrates that irreversible inhibition of TG2 by ERW1041 reduces both TG2 activity and the transepithelial transport of immunogenic gliadin peptides, suggesting a dual mechanism of action that may be beneficial in celiac disease. PX-12, despite superior efficacy in cell-free and lysate assays, failed to inhibit TG2 on intact cells or reduce peptide transport, likely due to rapid degradation or reduction by living cells. These findings support the continued development of specific, stable TG2 inhibitors as non-dietary therapies for CD, though further research is needed to evaluate their effects on gliadin-induced inflammation and T-cell activation.