Introduction:
Asthma is a common and growing lung disease afflicting the world population (Martinez & Vercelli, 2013; To et al., 2012). While there is no cure, many therapeutic options have been successfully used to control mild-to-moderate asthma symptoms for the past 60 years. The most commonly-prescribed asthma medications include bronchodilators composed of aerosolized β-adrenergic agonists or cholinergic antagonists, and anti-inflammatory medicines such as corticosteroids, anti-leukotrienes, and monoclonal antibodies (Stephenson et al., 2016). Despite the seeming plethora of treatment options to control asthma, approximately 50% of asthmatics with moderate to severe disease do not have adequate control of their symptoms (Martinez & Vercelli, 2013).
Allergic asthma can be triggered by a variety of environmental factors including exposure to the ubiquitous fungus Alternaria alternata .A. alternata is a prominent fungal allergen that has been associated with increased asthma-related hospitalization in children, and homes with higher levels of A. Alternata reported a higher incidence of moderate to severe asthma (Batra et al., 2021; Bush & Prochnau, 2004; Halonen, Stern, Wright, Taussig & Martinez, 1997; Lombardi, Savi, Ridolo, Passalacqua & Canonica, 2017; Mendy, Wilkerson, Salo, Zeldin & Thorne, 2020; Pulimood, Corden, Bryden, Sharples & Nasser, 2007). When found in semi-arid regions, A .alternata contains a proteinase, Alternaria alkaline Serine Proteinase (AASP), that acts through the G-protein-coupled receptor (GPCR), proteinase-activated-receptor-2 (PAR2), to induce airway inflammation, mucus production and bronchoconstriction (Boitano et al., 2011; Snelgrove et al., 2014; Yee et al., 2018). Trypsin-like serine proteinases associated with other allergy-inducing pathogens such as cockroach and house dust mite also activate PAR2 to promote airway inflammation [e.g., (Adam et al., 2006; Page, Ledford, Zhou, Dienger & Wills-Karp, 2010; Polley et al., 2017)]. Studies using PAR2 knockout (PAR2-/-) animals have demonstrated that PAR2 expression is critical for allergen-induced asthma indicators (Arizmendi et al., 2011; Davidson et al., 2013; Day, Zhou, Ledford & Page, 2010; Page, Ledford, Zhou, Dienger & Wills-Karp, 2010; Yee et al., 2018). Further, studies using PAR2-neutralizing antibodies have demonstrated a reduction in allergen-induced asthma indicators in both acute and chronic allergen-exposed animal models (Asaduzzaman, Davidson, Nahirney, Fiteih, Puttagunta & Vliagoftis, 2018; Asaduzzaman et al., 2015). Collectively, PAR2 has emerged as a viable drug target for the treatment of asthma (Ramachandran, Noorbakhsh, Defea & Hollenberg, 2012).
A common approach to developing novel GPCR drugs is via screening of large libraries, however, this approach has had limited success with PAR2 ligands (Cheng et al., 2017; Gardell et al., 2008). An alternative approach to developing PAR2 ligands is the use of small peptides [e.g., SLIGRL-NH2 (Hollenberg, Saifeddine & al-Ani, 1996)] or peptidomimetics [e.g., 2-furoyl-LIGRLO-NH2; (McGuire, Saifeddine, Triggle, Sun & Hollenberg, 2004)] that mimic the activating peptide sequence following proteolytic cleavage of the receptor (Ramachandran & Hollenberg, 2008; Yau, Liu & Fairlie, 2013). A more recent advance from our laboratory is the modification of the peptide sequence of the high potency peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH2, to form a novel PAR2 antagonist pharmacophore, compound 391 [C391; (Boitano et al., 2015)]. C391 is one of a handful of recently proposed PAR2 antagonists (Avet et al., 2020; Cheng et al., 2017; Goh, Ng, Nilsson, Kanke & Plevin, 2009; Hollenberg et al., 2014; Huang, Ni, Xi, Chu, Zhang & You, 2019; Kanke et al., 2005; Suen et al., 2012; Suen et al., 2014; Yau, Lim, Liu & Fairlie, 2016). It is distinct from other reported antagonists in that it requires minimal pre-incubation for inhibition of peptide activation of PAR2-dependent Ca2+ signaling and it acts as an antagonist for mitogen activating protein kinase (MAPK) signaling pathways. Further, C391 is one of the only known antagonists shown to be effective in blocking both natural proteinase and peptidomimetic activation of PAR2 in vitro and to be effective for blocking pain response in vivo (Boitano et al., 2015).
In this study we sought to evaluate the ability of C391 to inhibit allergen-induced, PAR2-dependent signaling in airway epithelial cells, and asthma indicators in murine models. We hypothesized that C391 would be effective at protecting against the development of cellular inflammation and broncho-constriction in pre-clinical asthma models. To this endeavor we first used established cellular models to demonstrate that C391 reduced or eliminated A. alternata -induced Ca2+ mobilization, β-arrestin recruitment and MAPK activation. Finally, we demonstrated that C391 was able to reduce or eliminate A. alternata -induced asthma indicators (inflammation, mucus production and airway hyperresponsiveness) in acutely-exposed murine models. These experiments demonstrate the ability of C391 to alter multiple allergen-induced and PAR2-dependent signaling pathwaysin vitro and demonstrate the first successful pharmacological reduction of asthma indicators in a pre-clinical model. Taken together, these data further support PAR2 as a druggable target to control allergen-induced asthma.