DISCUSSION
In this study we demonstrate that the potent PAR2 antagonist, C391, can effectively inhibit allergen (Alternaria alternata )-induced canonical PAR2 signaling (Ca2+ and β-arrestin/MAPK) in human cell lines and significantly diminish A. alternata -induced asthma indicators in pre-clinical murine models. Immortalized human bronchial epithelial cells, such as 16HBE14o-, are widely used in pharmacological in vitro studies centered around pulmonary physiology (Forbes, Shah, Martin & Lansley, 2003). They are ideal for drug discovery in the space of PAR-2-directed asthma therapeutics because they naturally express PAR2 and retain many features of differentiated bronchial epithelial cells including tight junction formation, barrier function and directional ion transport (Cozens et al., 1994). C391 was the first PAR-2 antagonist we identified, using 16HBE14o- for initial screening and subsequent assessment of Ca2+ and MAPK activation in response to trypsin and high-affinity peptidomimetic PAR2 agonists (Boitano et al., 2015; Flynn et al., 2011). Here, we have expanded the in vitro analyses of C391 to verify that it can effectively inhibit natural asthma-associated allergen (A. alternata )-induced, PAR2-dependent Ca2+ and MAPK activation in airway epithelial cells. Since PAR-2 can promote MAPK activation through both G-protein and β-arrestin pathways (Ge, Ly, Hollenberg & DeFea, 2003), and β-arrestin-dependent signaling is crucial for the inflammatory effects of A. alternata -induced PAR2 activation in the airway (Nichols et al., 2012; Yee et al., 2018), we have also confirmed that C391 inhibits β-arrestin recruitment, using BRET in transfected HEK293 cells.
We show that this PAR2 antagonist effectively reduces asthma indicators (including inflammation, eosinophil infiltration, mucus cell hyperplasia/mucus overproduction and airway hyperresponsiveness) in twoAlternaria alternata -induced murine asthma models. Asthma-associated allergen exposure in mouse models normally triggers a cascade of Th2 cytokine producing processes that result in eosinophil and leukocyte recruitment and residence in the airway (Wardlaw, 1999). While we cannot conclude as to where in the inflammatory pathway C391 is altering recruitment (e.g., airway epithelial- or other cell-dependent cytokine/chemokine release, or in the inflammatory cell response to increased proteinase), it is clear that concurrent or prophylactic treatment of C391 is effective in limiting A. alternata -induced inflammatory cell recruitment, mucus overproduction and airway hyperresponsiveness. More work is needed to clarify if C391 antagonism of A. alternata-induced signaling is sufficient to reduce asthma indicators (i.e., act as a therapeutic) or, if C391 is additionally antagonizing downstream, endogenous protease activation of PAR2 on epithelial cells or other cells in the airway.
Serine proteinases that activate PAR2 are a recurring theme among asthma-associated allergens (Adams et al., 2011), including those fromAlternaria and other molds (Boitano et al., 2011; Chiu, Perng, Yu, Su & Chow, 2007; Yee et al., 2018), house dust mite (Stewart, Thompson & Simpson, 1989; Sun, Stacey, Schmidt, Mori & Mattoli, 2001) and cockroach (Ock, Kim, Kim & Byun, 2005; Polley et al., 2017; Sudha, Arora, Gaur, Pasha & Singh, 2008). PAR2-/- murine models have been used to demonstrate a key role for PAR2 in the inflammation induced by these allergens (Davidson et al., 2013; Day, Zhou, Ledford & Page, 2010; Page, Ledford, Zhou, Dienger & Wills-Karp, 2010; Yee et al., 2018). Additionally, mast cell tryptase and neutrophil elastase represent endogenous PAR2-activating proteases that contribute to the asthma phenotype (Andersson, Tufvesson, Diamant & Bjermer, 2016; Kabashima et al., 2018; Ogawa et al., 2018). PAR2-neutralizing antibodies were recently shown to reduce asthma symptoms in acute and chronic allergic asthma mouse models (Asaduzzaman, Davidson, Nahirney, Fiteih, Puttagunta & Vliagoftis, 2018; Asaduzzaman et al., 2015), and over-expression of PAR2 or intranasal administration of PAR-2 activating peptides (SLIGRL or 2-furoyl-LIGRLO-NH2) promotes cellular inflammation and airway hyperresponsiveness(Ebeling, Forsythe, Ng, Gordon, Hollenberg & Vliagoftis, 2005; Nichols et al., 2012). Paradoxically, PAR2 activation also results in a beneficial Ca2+-dependent PGE2 secretion from airway epithelial cells, allowing for smooth muscle relaxation (Chow, Moffatt & Cocks, 2000; Cocks et al., 1999; Henry, 2006; Jairaman, Yamashita, Schleimer & Prakriya, 2015; Nichols et al., 2012) and resolution of neutrophilia (Rayees et al., 2019). We have demonstrated that the protective and inflammatory responses are mediated by separate G-protein and β-arrestin-2 signaling pathways, respectively (Nichols et al., 2012; Yee et al., 2018). While these studies might suggest that a biased antagonist that effectively prevents PAR2-dependent β-arrestin signaling, C391 mitigates multiple asthma indicators, including airway hyperresponsiveness, despite blocking both pathways.
There are only a handful of PAR2 antagonists that have been developed [reviewed in (Yau, Lim, Liu & Fairlie, 2016)]. These include K-14585 (Goh, Ng, Nilsson, Kanke & Plevin, 2009; Kanke et al., 2005), GB88 (Hollenberg et al., 2014; Suen et al., 2012; Suen et al., 2014), two small molecules isolated from a large screening library (AC-55541 and AC-264613; (Gardell et al., 2008)), two PAR2 allosteric antagonists AZ8838 and AZ3451 from Astra Zeneca (Cheng et al., 2017; Kennedy et al., 2020), a group of small molecules: I-343, I-191 and I-287, from Vertex (Avet et al., 2020; Jiang et al., 2018; Jimenez-Vargas et al., 2018), and C391 (Boitano et al., 2015). C391 is distinct from these other antagonists in that it requires minimal pre-incubation for inhibition of multiple PAR2 signaling pathways. Although we observed differences in calculated IC50 values for C391 between the in vitro Ca2+ and MAPK signaling (µM) and β-arrestin recruitment (nM), these experiments were in different cell types that include naturally expressed PAR2 (16HBE14o- cells used for Ca2+ and MAPK) vs transfected PAR2 (HEK 293 cells for β-arrestin), and we cannot conclude that C391 is a biased antagonist. Other small molecule PAR2 antagonists also show differences in potency for different signaling pathways. For example, AZ3451 was reported to have a lower IC50 for Ca2+mobilization (nM) than for β-arrestin-2 recruitment (µM) and AZ8588 was reported to have a lower IC50 for β-arrestin-2 recruitment than Ca2+ mobilization. Both compounds inhibited inflammation to a similar extent in a paw edema model (Kennedy et al., 2020). The PAR2 antagonists from Vertex all inhibited PAR2-induced G-protein but not β-arrestin signaling and show partial efficacy in mouse models of pain and subcutaneous inflammation (Avet et al., 2020; Jiang et al., 2018; Jimenez-Vargas et al., 2018). While these inhibitors show efficacy in peripheral inflammation and pain models, the ability to inhibit β-arrestin signaling appears to be important for reduction of asthma indicators, making C391 a more promising therapeutic. It is possible C391 could be further modified to be more selective for β-arrestin-2 and this might increase its efficacy as an asthma therapeutic.
Asthma can be triggered by environmental allergens such asAlternaria , pollens, insect droppings and infection. Due to the heterogeneity of its pathology, it remains difficult to manage in some patients, particularly those with moderate to severe disease. Many of the commonly used treatments for asthma address symptoms without addressing underlying triggers for the disease. Traditional treatments include corticosteroids and mast cell stabilizers for inflammation control, short and long acting β2-agonists to relax bronchial smooth muscle and increase airflow; anticholinergics as supplemental broncho-relaxants and, leukotriene disruptors (receptor antagonists or leukotriene synthesis inhibitors) used as an anti-inflammatory therapy (Martinez & Vercelli, 2013; Pera & Penn, 2016). Monoclonal antibody treatments that target immunoglobulin E, IL-13, IL-5 or IL-4-Receptor show some potential for patients with moderate to severe asthma who remain poorly controlled with steroids, although clinical trials have had mixed results. Some patients experienced improved lung function; however, in many cases overall eosinophilic inflammation was not significantly reduced and clinical endpoint reduction was limited (Chung, 2018; Hanania et al., 2016; Stephenson et al., 2016). Despite these varied approaches, asthma remains uncontrolled in more than half the patients receiving standard asthma medication (Peters, Jones, Haselkorn, Mink, Valacer & Weiss, 2007). While a portion of this population includes non-compliant patients, a large proportion of patients with refractory asthma would stand to benefit from improved therapeutics that ameliorate allergic asthma symptoms. Given that PAR2 is upregulated in both human respiratory epithelium and peripheral blood monocytes in asthmatics (Knight et al., 2001; Shrestha Palikhe et al., 2015), PAR2 signaling is of great interest in asthma development and severity.
A primary advance in this work is the first demonstration of pharmacological inhibition of allergen-induced asthma indicators (inflammation, mucus secretion and airway hyperresponsiveness) in pre-clinical animal models. That C391 reduces airway mucus production is also particularly exciting because of its potential for reducing asthma mortality. It has been observed that airway mucus plugging due to goblet cell hyperplasia coupled with peripheral airway mucus accumulation appears to be more prevalent in patients who eventually succumb to a severe asthma attack (Aikawa, Shimura, Sasaki, Ebina & Takishima, 1992). These same patients appear to be hypo-responsive to bronchodilators and anti-inflammatory medications. Providing a therapy that could control mucus production would allow for control of disease through minimizing airway obstruction caused by mucus plugging and possibly provide improved patient outcomes. C391 also reduced the development of airway hyperresponsiveness (AHR) in our pre-clinical models. AHR closely resembles a bronchoconstriction event or asthma attack in sensitized individuals and represents a measure that is commonly used in diagnostic procedures in the clinic. This suggests inhibition of PAR2 would be useful for prophylaxis in vulnerable individuals and as a potential treatment for airway hyperresponsiveness in allergic asthmatics.
We acknowledge that the studies here represent a prophylactic treatment and it is unknown if application of PAR2 pharmacophores could be effective in a therapeutic setting, e.g., after the establishment of asthma. However, these data, and the recent report that PAR2-specific antibodies can be effective in both preventing and controlling asthma indicators in pre-clinical exposure models (Asaduzzaman, Davidson, Nahirney, Fiteih, Puttagunta & Vliagoftis, 2018; Davidson et al., 2013) support PAR2 as a druggable target for addressing allergen-induced asthma. Furthermore, C391 specifically presents as a novel lead compound in the pursuit of novel asthma drugs.