Development and Testing of Pharmacological Tools to Evaluate the Role of Protease-Activated Receptor-2 in Allergic Asthma

Abstract

Despite the variety of asthma pharmacotherapy options that exist, a large subset of asthma patients present with poorly controlled disease. Extensive studies supporting a central role for Protease-Activated Receptor-2 (PAR2) in allergic asthma pathophysiology have promoted drug development efforts towards this molecular target. I report here that PAR2 antagonist C391 blocks signaling and symptoms caused by the asthma causing allergen Alternaria alternata. I show C391 effectively inhibits A. alternata-induced PAR2-MAPK and -Ca2+ signaling in vitro and attenuates asthma symptom development (e.g. airway hyperresponsiveness, excessive mucus production, and inflammation) in a murine model of asthma. Use of in vitro studies enable the cell-specific examination of PAR2 contribution to A. alternata-induced airway inflammation. I show PAR2 signaling plays an important role in the initiation of asthma through the finding that PAR2 signaling is required for full cytokine and chemokine release from A. alternata-exposed epithelial cells when I compare release from parental cells and CRISPR-produced PAR2 knockout cells. C391 inhibition of PAR2 signaling in A. alternata-induced lymphocyte migration reveal PAR2 signaling is required for effective chemotaxis. Finally, I show development and screening of three novel PAR2 ligands, C732, C781 and C782. Cell impedance screening and PAR2 binding assessment highlight C781 as a lead compound for asthma therapy. Further PAR2 signaling characterization reveals C781 to be a PAR2-specific, biased MAPK antagonist. My studies demonstrate for the first time that a PAR2-specific antagonist provides pharmacological control of asthma in a pre-clinical model and presents novel lead compounds for improved drug development.