Dihydroisoxazole analogs for labeling and visualization of catalytically active transglutaminase 2

Abstract

We report the synthesis and preliminary characterization of "clickable" inhibitors of human transglutaminase 2 (TG2). These inhibitors possess the 3-halo-4,5-dihydroisoxazole warhead along with bioorthogonal groups such as azide or alkyne moieties that enable subsequent covalent modification with fluorophores. Their mechanism for inhibition of TG2 is based on halide displacement, resulting in the formation of a stable imino thioether. Inhibition assays against recombinant human TG2 revealed that some of the clickable inhibitors prepared in this study have comparable specificity as benchmark dihydroisoxazole inhibitors reported earlier. At low micromolar concentrations they completely inhibited transiently activated TG2 in a WI-38 fibroblast scratch assay and could subsequently be used to visualize the active enzyme in situ. The potential use of these inhibitors to probe the role of TG2 in celiac sprue as well as other diseases is discussed.

Figure 1

Labeling and visualization of catalytically active (but not catalytically inactive) transglutaminase 2 with alkynyl-DHI inhibitors. Similar labeling strategy can be employed using azido-DHI inhibitors.

Figure 2

General synthesis of azido- and alkynyl-DHI inhibitors. Supported by Figure S1.

Figure 3

Titration of 11 against active TG2 in a fibroblast scratch assay. TG2 activity was visualized in situ after scratching a confluent WI-38 monolayer with a small pipette tip. Significant TG2 activity was detected around the wound in the presence of 300 μM 5BP and vehicle (DMSO) (a-c). Clear inhibition of active TG2 was observed in the presence of 6.25 μM 11 (g-i), and complete inhibition was observed at or above 12.5 μM 11 (j-o). In this assay, active TG2 was detected by exposing fixed cultures to streptavidin Alexa fluor 555 (a, d, g, j, m). The scratch geometry is conveniently visualized by co-staining with polyclonal anti-fibronectin antibody, followed by a secondary antibody conjugated Alexa fluor 488 (b, e, h, k, n). Overlays of the left and middle images are in the right column (c, f, i, l, o). Scale bar represents 200 μm and applies to all panels.

Figure 4

Visualization of recombinant TG2 with alkynyl-DHI inhibitor 12. Recombinant TG2 was inhibited with 12 in the presence of CaCl₂ (5 mM) or GTP (500 μM) and MgCl₂ (1 mM). The resulting mixture was reacted with biotin-azide, resolved by SDS-PAGE, and detected by Western blotting with neutravidin-HRP. Lane 1:TG2 (0.2μg) + Ca²⁺ + 12, lane 2:TG2 (0.4μg) + Ca²⁺ + 12, lane 3:TG2 (1μg) + Ca²⁺ + 12, lane 4:TG2 (4μg) + Ca²⁺ + 12, lane 5:TG2 (0.4μg) + GTP/MgCl₂ + 12, lane 6:TG2 (1μg) + GTP/MgCl₂ + 12, lane 7: TG2 (1μg) + GTP/MgCl₂ + 12, lane 8: TG2 (4μg)+GTP/MgCl₂ + 12.

Figure 5

Visualization of catalytically active TG2 in a scratched WI-38 fibroblast culture. TG2 activity was visualized after scratching confluent WI-38 monolayers in the presence of 50 μM 11 (a-f) or 17 (g-i) for 1 h. Cells were then fixed, permeabilized, reacted with biotin azide followed by strepatavidin-Alexa fluor 555 conjugates (red), and imaged by epifluorescence microscopy through a 10x objective. (a, d, g) Rabbit anti-fibronectin antibody, followed by a secondary antibody labeled with Alexa fluor 488, were used to visualize fibronectin on the WI-38 monolayers. (b, e, h) Catalytically active TG2 was visualized by coupling the alkynyl-DHI inhibitor to biotin azide. (c, f, i) Overlays of the left and middle image pairs are shown in the right column. Scale bar represents 200 μm and applies to all panels. Supported by Figure S4 and S5.