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

Amanda J Kennedy,Dean G Brown,Rongfeng Chen,Linda Sundström,Rink-Jan Lohman,Maria Fridén-Saxin,Yuhong Jiang,Andrew Madin,Wenzhen Yang,Niek Dekker,Eunice K Y Poon,Junxian Lim,Anneli Nordqvist,David P Fairlie,Rob Cooke,Stefan Geschwindner,Shawn Johnstone,Qingqi Liu

doi:10.1038/s42003-020-01504-0

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.

Highlights

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
Protease-activated receptor-2 (PAR2) is predominately activated by serine proteases, such as trypsin and tryptase, which cleave between R35–S36 thereby exposing a new N-terminal sequence SLIGKV, that acts as a tethered agonist for intramolecular PAR2 activation[3,4,5]
In 2017, we reported the crystal structures of PAR2 bound to AZ8838 and AZ345124

Summary

Introduction

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. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. 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. The wide range of effects of PAR2 underscores the importance of this receptor in human physiology and disease As a consequence, this target is highly sought after in drug discovery and for a number of years has been a focus of major pharmaceutical endeavours[19]. AZ8838 was derived from an initial highthroughput screen hit[24] and AZ3451 was obtained from a DNA-

Methods

Results

Conclusion