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

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

Communications biology (2020-12-19)
Amanda J Kennedy, Linda Sundström, Stefan Geschwindner, Eunice K Y Poon, Yuhong Jiang, Rongfeng Chen, Rob Cooke, Shawn Johnstone, Andrew Madin, Junxian Lim, Qingqi Liu, Rink-Jan Lohman, Anneli Nordqvist, Maria Fridén-Saxin, Wenzhen Yang, Dean G Brown, David P Fairlie, Niek Dekker
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
PAR-2 (1-6) amide (mouse, rat) trifluoroacetate salt, ≥97% (HPLC)
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Ser-Leu-Ile-Gly-Lys-Val-amide, ≥95% (HPLC), solid
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Fluo-3, suitable for fluorescence, ~70%
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Toluidine Blue, 8.74% (ZN (THEORY)), for microscopy (Hist., Vit.)
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p-Xylene-bis(N-pyridinium bromide), ≥95% (TLC)
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Nitrilotriacetic acid disodium salt, Sigma Grade, ≥99%
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Anti-Histamine antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
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AZ3451, ≥98% (HPLC)