ß-Arrestin-biased proteinase-activated receptor-2 antagonist C781 limits allergen-induced airway hyperresponsiveness and inflammation.

BACKGROUND AND PURPOSE: Asthma is a heterogenous disease strongly associated with inflammation that has many different causes and triggers. Current asthma treatments target symptoms such as bronchoconstriction and airway inflammation. Despite recent advances in biological therapies, there remains a need for new classes of therapeutic agents with novel, upstream targets. The proteinase-activated receptor-2 (PAR2) has long been implicated in allergic airway inflammation and asthma and it remains an intriguing target for novel therapies. Here, we describe the actions of C781, a newly developed low MW PAR2 biased antagonist, in vitro and in vivo in the context of acute allergen exposure. EXPERIMENTAL APPROACH: A human bronchial epithelial cell line expressing PAR2 (16HBE14o- cells) was used to evaluate the modulation in vitro, by C781, of physiological responses to PAR2 activation and downstream ß-arrestin/MAPK and Gq/Ca2+ signalling. Acute Alternaria alternata sensitized and challenged mice were used to evaluate C781 as a prophylactically administered modulator of airway hyperresponsiveness, inflammation and mucus overproduction in vivo. KEY RESULTS: C781 reduced in vitro physiological signalling in response to ligand and proteinase activation. C781 effectively antagonized ß-arrestin/MAPK signalling without significant effect on Gq/Ca2+ signalling in vitro. Given prophylactically, C781 modulated airway hyperresponsiveness, airway inflammation and mucus overproduction of the small airways in an acute allergen-challenged mouse model. CONCLUSION AND IMPLICATIONS: Our work demonstrates the first biased PAR2 antagonist for ß-arrestin/MAPK signalling. C781 is efficacious as a prophylactic treatment for allergen-induced airway hyperresponsiveness and inflammation in mice. It exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development.

Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression.

Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR-/-) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiologic responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH2) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR-/- line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2-/- grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.

Proteinase-activated receptor-2 antagonist C391 inhibits Alternaria-induced airway epithelial signalling and asthma indicators in acute exposure mouse models.

BACKGROUND AND PURPOSE: Despite the 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 preclinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 raises intracellular Ca2+ , inducing MAPK and ß-arrestin signalling in the airway, leading to inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signalling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 antagonism by C391 would attenuate allergen-induced acutely expressed asthma indicators in murine models. EXPERIMENTAL APPROACH: We evaluated the ability of C391 to alter Alternaria alternata-induced PAR2 signalling 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 acutely expressed asthma indicators in vivo in two murine strains. KEY RESULTS: C391 blocked A. alternata-induced, PAR2-dependent Ca2+ and MAPK signalling in 16HBE14o- cells, as well as ß-arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute allergen-challenged murine models. CONCLUSIONS AND IMPLICATIONS: To our best 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.

Validation Study for PathogenDx EnviroX-Rv Assay for the Detection of SARS-CoV-2 from Stainless Steel Environmental Surface Swabs Performance Tested MethodSM 122003.

BACKGROUND: The PathogenDx EnviroX-Rv uses endpoint PCR + DNA microarray technology to detect SARS-CoV-2, the causative agent of COVID-19, from stainless-steel environmental sample swabs. OBJECTIVE: To validate the PathogenDx EnviroX-Rv assay as part of the Emergency Response Validation (ERV) Performance Tested Method(s)SM (PTM) program. METHOD: The PathogenDx EnviroX-Rv assay was evaluated for specificity using in silico analysis of ≥41 000 SARS-CoV-2 sequences and over 50 exclusivity organisms (both near neighbors and background organisms). The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the US Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus (2019-nCoV) Real-Time-Polymerase Chain Reaction (RT-PCR) Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). RESULTS: Results of the in silico analysis demonstrated the high specificity of the method in being able to detect target SARS-CoV-2 sequences and discriminate them from near neighbors and environmental background organisms. In the matrix study, the candidate method demonstrated a statistically significant difference when compared to the results of the CDC method utilized in this study, with the candidate method resulting in more positive replicates as it only requires one target to be present for a positive sample. CONCLUSIONS: The EnviroX-Rv assay rapidly and accurately detected SARS-CoV-2 RNA on environmental swabs from stainless-steel surfaces at a concentration of 2000 genomic copies per 2 × 2" test area. HIGHLIGHTS: The EnviroX-Rv assay employs dual PCR and hybridization techniques to provide highly accurate results when detecting SARS-CoV-2 from surfaces.

The novel PAR2 ligand C391 blocks multiple PAR2 signalling pathways in vitro and in vivo.

BACKGROUND AND PURPOSE: Proteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described. EXPERIMENTAL APPROACH: We modified the peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH2 , to create a novel PAR2 peptidomimetic ligand, C391. C391 was evaluated for PAR2 agonist/antagonist activity to PAR2 across Gq signalling pathways using the naturally expressing PAR2 cell line 16HBE14o-. For antagonist studies, a highly potent and specific peptidomimetic agonist (2-aminothiazo-4-yl-LIGRL-NH2 ) and proteinase agonist (trypsin) were used to activate PAR2. C391 was also evaluated in vivo for reduction of thermal hyperalgesia, mediated by mast cell degranulation, in mice. KEY RESULTS: C391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (Gq -dependent Ca2+ , MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75 µg maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation. CONCLUSIONS AND IMPLICATIONS: C391 is the first low MW antagonist to block both PAR2 Ca2+ and MAPK signalling pathways activated by peptidomimetics and/or proteinase activation. C391 represents a new molecular structure for PAR2 antagonism and can serve as a basis for further development for this important therapeutic target.