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

Abstract

Background and Purpose: Despite availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in pre-clinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 induces intracellular Ca2+, mitogen activated protein kinase (MAPK) and -arrestin signaling the airway, leading to both inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signaling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 reduction with C391 would attenuate allergen-induced asthma indicators in murine models. Experimental Approach: We evaluated the ability for C391 to alter Alternaria alternata-induced PAR2 signaling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata-induced asthma indicators in vivo in two murine strains. Key Results: C391 blocked A. alternata-induced, PAR2-dependent Ca2+ and MAPK signaling in 16HBE14o- cells, as well as -arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute asthma murine models. Conclusions and Implications: To our knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs.

β‐Arrestin‐biased proteinase‐activated receptor‐2 antagonist C781 limits allergen‐induced airway hyperresponsiveness and inflammation

“…Cells were grown in media consisting of minimum essential media with Earle's salts (Life Technologies Cat# 11095–080), supplemented with 10% fetal bovine serum (FBS), 1% Pen/Strep, and 1% glutamax (MEM). Cells were grown on matrix‐treated culture ware, with the matrix consisting of collagen, fibronectin, and bovine serum albumin as described previously (Rivas et al, 2021).…”

“…ICW studies were adapted from methods described earlier (Flynn et al, 2011; Rivas et al, 2021). 16HBE14o‐ cells were plated onto matrix‐coated, clear bottom, black‐walled 96‐well plates at a density of 2 × 10 4 cells per well.…”

“…Male mice were used due to their enhanced response to allergens that allowed for statistical analysis with a reduced amount of animals. At 8 weeks, mice were randomly divided into four treatment groups of six mice each and subjected to allergen sensitization and challenge using A. alternata filtrate as described (Rivas et al, 2021). Mice were first anaesthetised by placing them in a chamber supplied with vaporized isoflurane and oxygen.…”

“…In vivo mouse models using cockroach frass, house dust mite extracts or potent and selective PAR2 activating peptides have all shown that PAR2 plays critical roles in allergic asthma sensitization (Arizmendi et al, 2011; Davidson et al, 2013; Page et al, 2010) and inflammation (Day et al, 2010; Ebeling et al, 2005; Nichols et al, 2012). We have shown that Alternaria alternata filtrates similarly cause proteinase‐dependent asthma associated responses—airway hyperresponsiveness (AHR), inflammation and mucus overproduction—in mouse models of acute allergen exposure (Boitano et al, 2011; Yee et al, 2018) that can be moderated with a PAR2 antagonist (Rivas et al, 2021). Complementary studies that neutralize PAR2 with antibodies reduced asthma indicators in both acute and chronic allergen exposure mouse models of asthma (Asaduzzaman et al, 2015, 2018).…”

“…Complementary studies that neutralize PAR2 with antibodies reduced asthma indicators in both acute and chronic allergen exposure mouse models of asthma (Asaduzzaman et al, 2015, 2018). Using genetically modified mouse models, we have further demonstrated that acute exposure to A. alternata induces physiological indicators of asthma, such as AHR, pulmonary inflammation, mucus cell hyperplasia and mucus hypersecretion, after an 8 day sensitization and challenge protocol, in a manner that requires both PAR2 and β‐arrestin‐2 expression (Rivas et al, 2021; Yee et al, 2018). Interestingly, proteinase activated, PAR2‐dependent Gq/Ca 2+ signalling at the airway epithelium is associated with broncho‐relaxation (Cocks et al, 1999; Henry, 2006; Nichols et al, 2012; Yee et al, 2018), a clearly beneficial outcome for asthmatics.…”