Working towards proteinase activated receptor-2 antagonists

Proteinase activated receptor-2 (PAR2) is a G-protein coupled receptor, that is activated by means of proteolytic cleavage by serine proteases (Macfarlane et al, 2001). PAR2 has been implicated in several inflammatory diseases including those of the cardiovascular system, the airways, and the gastrointestinal tract. We have previously identified PAR2 to play a key role in mediating chronic joint inflammation (Ferrell et al, 2003; McIntosh et al, 2007). PAR2 represents an excellent therapeutic target however target validation and translation to clinical application has been hampered by the lack of potent and selective antagonists. In 2012, progress was made when a novel non-peptidic PAR2 antagonist called GB88 was developed (Suen et al, 2012). However subsequent pharmacological analysis of this compound resulted in its reclassification as a biased antagonist in 2014. In these studies GB88 was found to selectively inhibit Gαq/11 aspects of PAR2 signalling, such as calcium mobilisation and PKC phosphorylation, while activating components of Gi/o and G12 signalling (Suen et al, 2014).

Characterisation of GB88 was carried out by examining its role in a number of PAR2 mediated signalling pathways, utilising both over-expression and endogenous cell systems. Receptor internalisation was investigated in NCTC2544 cells expressing PAR2-YFP. Phosphorylation of ERK and calcium mobilisation examined in HEK293s by Western blotting and a fluorescent based assay respectively. NFκB activity was measured using PAR2 expressing NCTC2544 cells (Clone G) containing a NFκB-luciferase reporter plasmid. Inhibition of Gαq/11 was achieved using the novel Gαq/11 inhibitor YM-254890 (YM).

Initial experiments demonstrated GB88 mediated a concentration-dependent increase in NFκB activity with an EC50 of 0.9μM (885nM) in comparison to 2FLIGRLO-NH2 with an EC50 of 0.3μM (304nM). Pre-treatment with GB88 failed to inhibit 2FLIGRLO-NH2 mediated NFκB activity. Surprisingly, treatment with 3μM GB88 mediated a time-dependent internalisation of PAR2-YFP in NCTC2544 cells, which was comparable in kinetics to internalisation driven by 2FLIGRLO-NH2. Further investigation examining the signalling mechanisms of GB88 in a cell system endogenously expressing PAR2 (HEK293 cells) suggest it may function as a partial agonist with demonstrable calcium and ERK signalling, albeit less potent than 2FLIGRLO-NH2 responses (2FLIGRLO-NH2 EC50 0.3μM, vs. GB88 EC50 3μM). GB88 appears however to be non-competitive against trypsin, failing to inhibit trypsin mediated PAR2 signalling in any system tested. Treatment with the novel Gαq/11 inhibitor YM254890 (300nM) effectively inhibited GB88-mediated calcium and ERK responses thus demonstrating the Gαq/11-dependency of these signalling events.

In light of this data, the potential therapeutic benefits of GB88 as a PAR2 antagonist require re-evaluation. Considering GB88 internalises the receptor after 15 min, pre-treatment experiments could be misleading and any results originally interpreted as inhibition could instead be reduced receptor responsiveness due to receptor desensitization.