Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2.

BACKGROUND AND PURPOSE: Diverse proteases cleave protease-activated receptor-2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N-terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin-S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists. EXPERIMENTAL APPROACH: We investigated whether the PAR2 antagonist GB88 inhibits protease-evoked activation of nociceptors and protease-stimulated oedema and hyperalgesia in rodents. KEY RESULTS: Intraplantar injection of trypsin, cathespsin-S or elastase stimulated mechanical and thermal hyperalgesia and oedema in mice. Oral GB88 or par2 deletion inhibited the algesic and proinflammatory actions of all three proteases, but did not affect basal responses. GB88 also prevented pronociceptive and proinflammatory effects of the PAR2-selective agonists 2-furoyl-LIGRLO-NH2 and AC264613. GB88 did not affect capsaicin-evoked hyperalgesia or inflammation. Trypsin, cathepsin-S and elastase increased [Ca(2+) ]i in rat nociceptors, which expressed PAR2. GB88 inhibited this activation of nociceptors by all three proteases, but did not affect capsaicin-evoked activation of nociceptors or inhibit the catalytic activity of the three proteases. CONCLUSIONS AND IMPLICATIONS: GB88 inhibits the capacity of canonical and biased protease agonists of PAR2 to cause nociception and inflammation.

Demonstration of elevated levels of active cathepsin S in dextran sulfate sodium colitis using a new activatable probe.

BACKGROUND: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. METHODS: We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. KEY RESULTS: NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). CONCLUSIONS & INFERENCES: Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.