Protease-activated receptor-2 ligands reveal orthosteric and allosteric mechanisms of receptor inhibition

Abstract

Protease-activated receptor-2 (PAR2) has been implicated in multiple pathophysiologies but drug discovery is challenging due to low small molecule tractability and a complex activation mechanism. Here we report the pharmacological profiling of a potent new agonist, suggested by molecular modelling to bind in the putative orthosteric site, and two novel PAR2 antagonists with distinctly different mechanisms of inhibition. We identify coupling between different PAR2 binding sites. One antagonist is a competitive inhibitor that binds to the orthosteric site, while a second antagonist is a negative allosteric modulator that binds at a remote site. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. Importantly, both antagonists are active in vivo, inhibiting PAR2 agonist-induced acute paw inflammation in rats and preventing activation of mast cells and neutrophils. These results highlight two distinct mechanisms of inhibition that potentially could be targeted for future development of drugs that modulate PAR2.

Novel proteinase-activated receptor-2 (PAR2) antagonist C391 blocks Alternaria-induced human airway epithelial signaling and asthma indicators in murine models

“…For example, AZ3451 was reported to have a lower IC 50 for Ca 2+ mobilization (nM) than for β-arrestin-2 recruitment (μM) and AZ8588 was reported to have a lower IC 50 for β-arrestin-2 recruitment than Ca 2+ mobilization. Both compounds inhibited inflammation to a similar extent in a paw edema model (Kennedy et al, 2020). The PAR2 antagonists from Vertex all inhibited PAR2-induced Gprotein but not β-arrestin signaling and show partial efficacy in mouse models of pain and subcutaneous inflammation (Avet et al, 2020;Jiang et al, 2018;Jimenez-Vargas et al, 2018).…”

“…There are only a handful of PAR2 antagonists that have been developed [reviewed in (Yau, Lim, Liu & Fairlie, 2016)]. These include K-14585 (Goh, Ng, Nilsson, Kanke & Plevin, 2009;Kanke et al, 2005), GB88 (Hollenberg et al, 2014;Suen et al, 2012;Suen et al, 2014), two small molecules isolated from a large screening library (AC-55541 and AC-264613; (Gardell et al, 2008)), two PAR2 allosteric antagonists AZ8838 and AZ3451 from Astra Zeneca (Cheng et al, 2017;Kennedy et al, 2020), a group of small molecules: I-343, I-191 and I-287, from Vertex (Avet et al, 2020;Jiang et al, 2018;Jimenez-Vargas et al, 2018), and C391 (Boitano et al, 2015). C391 is distinct from these other antagonists in that it requires minimal pre-incubation for inhibition of multiple PAR2 signaling pathways.…”

Proteinase‐activated receptor‐2 antagonist C391 inhibits Alternaria‐induced airway epithelial signalling and asthma indicators in acute exposure mouse models

“…There are only a handful of PAR2 antagonists that have been developed (reviewed in Yau et al, 2016). These include K‐14585 (Goh et al, 2009; Kanke et al, 2005); GB88 (Hollenberg et al, 2014; Suen et al, 2012); two PAR2 allosteric antagonists AZ8838 and AZ3451 from AstraZeneca (Cheng et al, 2017; Kennedy et al, 2020); a group of small molecules (I‐343, I‐191 and I‐287) from Vertex (Avet et al, 2020; Jiang et al, 2018; Jimenez‐Vargas et al, 2018); cell permeant PAR2 pepducin, 18S, that disrupts PAR2–G‐protein coupling (Sevigny et al, 2011); a monoclonal antibody, PAR650097 (Kopruszinski et al, 2020); and C391 (Boitano et al, 2015). We show herein that C391 effectively inhibits multiple signalling pathways associated with PAR2 signalling.…”

Mechanism of activation and the rewired network: New drug design concepts

“…Proteins act through structural changes and drugs aim to block their action. A competitive drug binding mechanism is powerful since it directly blocks ligand docking 125,157–166 . A noncompetitive drug binding can be powerful by altering the active site shape, leading to the same outcome 167–177 .…”

“…The modulation of the active site structure prompted by the allosteric drug restored effective binding to a competitive inhibitor 12 . Protease‐activated receptor‐2 (PAR2) provides another potential example 125 as does B‐Raf 126 . (iv) The drug can bind non‐covalently, which is the case most of the time, or especially in the absence of sufficiently deep pockets, it can be covalent 121,127–137 .…”