Synthèse des O-Aryl glycosides ainsi que le design et la génération de nouvelles classes de groupes partants

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Activation and Inhibition of Transglutaminase 2 in Mice

Transglutaminase 2 (TG2) is an allosterically regulated enzyme with transamidating, deamidating and cell signaling activities. It is thought to catalyze sequence-specific deamidation of dietary gluten peptides in the small intestines of celiac disease patients. Because this modification has profound consequences for disease pathogenesis, there is considerable interest in the design of small molecule TG2 inhibitors. Although many classes of TG2 inhibitors have been reported, thus far an animal model for screening them to identify promising celiac drug candidates has remained elusive. Using intraperitoneal administration of the toll-like receptor 3 (TLR3) ligand, polyinosinic-polycytidylic acid (poly(I∶C)), we induced rapid TG2 activation in the mouse small intestine. Dose dependence was observed in the activation of TG2 as well as the associated villous atrophy, gross clinical response, and rise in serum concentration of the IL-15/IL-15R complex. TG2 activity was most pronounced in the upper small intestine. No evidence of TG2 activation was observed in the lung mucosa, nor were TLR7/8 ligands able to elicit an analogous response. Introduction of ERW1041E, a small molecule TG2 inhibitor, in this mouse model resulted in TG2 inhibition in the small intestine. TG2 inhibition had no effect on villous atrophy, suggesting that activation of this enzyme is a consequence, rather than a cause, of poly(I∶C) induced enteropathy. Consistent with this finding, administration of poly(I∶C) to TG2 knockout mice also induced villous atrophy. Our findings pave the way for pharmacological evaluation of small molecule TG2 inhibitors as drug candidates for celiac disease

Treatment of C57BL/6J mice with poly(I∶C) results in villous atrophy and activation of TG2 at the villus tips.

<p>Single intraperitoneal injections of 30 (a–d), 20 (e–h), 15 (i–l), 5 (m–p), or 0 (q–t) mg/kg poly(I∶C) were administered. Mice were sacrificed after 9 h, and small intestines collected and frozen in OCT. Sections of 20 µm thickness were cut, fixed, washed, permeabilized, and exposed first to a rabbit anti-TG2 antibody (ab421) followed by a secondary Alexa fluor 488-congugated goat anti-rabbit IgG (c, g, k, o, s). Total TG2 protein (active and non active) is shown. TG2 activity after poly(I∶C) injury, measured using 5BP crosslinking, was visualized by co-staining with Alexa fluor 555-conjugated streptavidin (b, f, j, n, r). Nuclei were stained with DAPI (a, e, i, m, q). Overlays of the three stains are shown in (d, h, l, p, t). At least 3 animals were tested at each poly(I∶C) dose shown; one representative duodenal section is shown.</p

Inhibition of TG2 activity by ERW1041E in the small intestine of poly(I∶C)-treated mice.

<p>Mice were treated with 20 mg/kg (i–p), or 15 mg/kg (q–x) poly(I∶C), followed by two intraperitoneal injections of vehicle (i–l; q–t) or 50 mg/kg ERW1041E (m–p; u-bb) at 6 h and 9 h. Control cohorts were dosed with no pharmacological agent (a–d), only 5BP (e–h) or ERW1041E and 5BP (y-bb). TG2 activity was detected by exposing duodenal cross-sections to Alexa fluor 555-conjugated streptavidin (b, f, j, n, r, v, z). TG2 protein was visualized by co-staining with polyclonal anti-TG2 antibody, followed by a secondary antibody conjugated to Alexa fluor 488 (c, g, k, o, s, w, aa). Nuclei were visualized by staining with DAPI (a, e, i, m, q, u, y). The right column contains overlay images (d, h, l, p, t, x, bb). At least 3 animals were tested in each cohort; one representative duodenal section is shown.</p

Effects of poly(I∶C) treatment on TG2 knockout mice.

<p>Mice were treated with 0 mg/kg (a–d) or 20 mg/kg (e–l) poly(I∶C), followed by two intraperitoneal injections of PBS (a–d), or 100 mg/kg (e–l) 5BP at 6 h and 9 h. Poly(I∶C)-treated mice were subsequently administered 50 mg/kg ERW1041E. TG2 activity was detected by exposing duodenal cross-sections to Alexa fluor 555-conjugated streptavidin (b, f, j). TG2 protein was visualized by co-staining with polyclonal anti-TG2 antibody, followed by a secondary antibody conjugated with Alexa fluor 488 (c, g, k). Nuclei were stained with DAPI (a, e, i). Overlays are shown in the right column (d, h, l). At least 3 animals were tested in each cohort; one representative duodenal section is shown. Unlike wild-type mice, significant lethality (2/7) was observed in poly(I∶C)-treated mice. The structure of ERW1041E (<b>1</b>) is shown.</p

Localization of TG2 activity in the small intestine of poly(I∶C)-treated mice.

<p>Mice were treated with 30 mg/kg poly(I∶C), and samples were collected from the duodenum (a–d), jejunum (e–h), and ileum (i–l). Active TG2 was detected by exposing cross-sections to Alexa fluor 555-conjugated (b, f, j). The total amount of TG2 protein in these sections was visualized by co-staining with polyclonal anti-TG2 antibody, followed by an Alexa fluor 488-conjugated secondary antibody (c, g, k). Nuclei were stained with DAPI (a, e, i). Overlays are shown in the right column (d, h, l). Scale bar represents 300 µm and applies to all panels. At least 4 animals were tested; one representative section is shown for each intestinal segment.</p

H&E staining of duodenal cross sections from mice treated with poly(I∶C) at 30 mg/kg (A), 15 mg/kg (B), or 0 mg/kg poly(I∶C) (C).

<p>Animals were sacrificed 9 h after poly(I∶C) administration, and their small intestines were harvested.</p