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.