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.