Justin Hoffman

Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR2

Allergens are diverse proteins from mammals, birds, arthropods, plants, and fungi. Allergens associated with asthma (asthmagens) share a common protease activity that may directly impact respiratory epithelial biology and lead to symptoms of asthma. Alternaria alternata is a strong asthmagen in semiarid regions. We examined the impact of proteases from A. alternata on lung inflammation in vivo and on cleaving protease-activated receptor-2 (PAR(2)) in vitro. A. alternata filtrate applied to the airway in nonsensitized Balb/c mice induced a protease-dependent lung inflammation. Moreover, A. alternata filtrate applied to human bronchial epithelial cells (16HBE14o-) induced changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), consistent with PAR(2) activation. These effects were blocked by heat inactivation or by serine protease inhibition of A. alternata filtrates, and mimicked by PAR(2) specific ligands SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2), but not the PAR(1)-specific ligand TFLLR-NH(2). Desensitization of PAR(2) in 16HBE14o- cells with 2-furoyl-LIGRLO-NH(2) or trypsin prevented A. alternata-induced [Ca(2+)](i) changes while desensitization of PAR(1), PAR(3), and PAR(4) with thrombin had no effect on A. alternata-induced Ca(2+) responses. Furthermore, the Ca(2+) response to A. alternata filtrates was dependent on PAR(2) expression in stably transfected HeLa cell models. These data demonstrate that A. alternata proteases act through PAR(2) to induce rapid increases in human airway epithelial [Ca(2+)](i) in vitro and cell recruitment in vivo. These responses are likely critical early steps in the development of allergic asthma.

The Protease-activated Receptor-2-specific Agonists 2-Aminothiazol-4-yl-LIGRL-NH2 and 6-Aminonicotinyl-LIGRL-NH2 Stimulate Multiple Signaling Pathways to Induce Physiological Responses in Vitro and in Vivo

Protease-activated receptor-2 (PAR2) is one of four protease-activated G-protein-coupled receptors. PAR2 is expressed on multiple cell types where it contributes to cellular responses to endogenous and exogenous proteases. Proteolytic cleavage of PAR2 reveals a tethered ligand that activates PAR2 and two major downstream signaling pathways: mitogen-activated protein kinase (MAPK) and intracellular Ca2+ signaling. Peptides or peptidomimetics can mimic binding of the tethered ligand to stimulate signaling without the nonspecific effects of proteases. The most commonly used peptide activators of PAR2 (e.g. SLIGRL-NH2 and SLIGKV-NH2) lack potency at the receptor. However, although the potency of 2-furoyl-LIGRLO-NH2 (2-f-LIGRLO-NH2) underscores the use of peptidomimetic PAR2 ligands as a mechanism to enhance pharmacological action at PAR2, 2-f-LIGRLO-NH2 has not been thoroughly evaluated. We evaluated the known agonist 2-f-LIGRLO-NH2 and two recently described pentapeptidomimetic PAR2-specific agonists, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at-LIGRL-NH2) and 6-aminonicotinyl-LIGRL-NH2 (6-an-LIGRL-NH2). All peptidomimetic agonists stimulated PAR2-dependent in vitro physiological responses, MAPK signaling, and Ca2+ signaling with an overall rank order of potency of 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2 ≫ SLIGRL-NH2. Because PAR2 plays a major role in pathological pain conditions and to test potency of the peptidomimetic agonists in vivo, we evaluated these agonists in models relevant to nociception. All three agonists activated Ca2+ signaling in nociceptors in vitro, and both 2-at-LIGRL-NH2 and 2-f-LIGRLO-NH2 stimulated PAR2-dependent thermal hyperalgesia in vivo. We have characterized three high potency ligands that can be used to explore the physiological role of PAR2 in a variety of systems and pathologies.

Development of highly potent protease-activated receptor 2 agonists via synthetic lipid tethering

Protease‐activated receptor‐2 (PAR2) is a G‐protein coupled receptor (GPCR) associated with a variety of pathologies. However, the therapeutic potential of PAR2 is limited by a lack of potent and specific ligands. Following proteolytic cleavage, PAR2 is activated through a tethered ligand. Hence, we reasoned that lipidation of peptidomimetic ligands could promote membrane targeting and thus significantly improve potency and constructed a series of synthetic tethered ligands (STLs). STLs contained a peptidomimetic PAR2 agonist (2‐aminothiazol‐4‐yl‐LIGRL‐NH2) bound to a palmitoyl group (Pam) via polyethylene glycol (PEG) linkers. In a high‐throughput physiological assay, these STL agonists displayed EC50 values as low as 1.47 nM, representing a ~200 fold improvement over the untethered parent ligand. Similarly, these STL agonists were potent activators of signaling pathways associated with PAR2: EC50 for Ca2+ response as low as 3.95 nM; EC50 for MAPK response as low as 9.49 nM. Moreover, STLs demonstrated significant improvement in potency in vivo, evoking mechanical allodynia with an EC50 of 14.4 pmol. STLs failed to elicit responses in PAR2 cells at agonist concentrations of >300‐fold their EC50 values. Our results demonstrate that the STL approach is a powerful tool for increasing ligand potency at PAR2 and represent opportunities for drug development at other protease activated receptors and across GPCRs.—Flynn, A. N., Hoffman, J., Tillu, D. V., Sherwood, C. L., Zhang, Z., Patek, R., Asiedu, M. N. K., Vagner, J., Price, T. J., Boitano, S. Development of highly potent protease‐activated receptor 2 agonists via synthetic lipid tethering. FASEB J. 27, 1498–1510 (2013). www.fasebj.org

Potent agonists of the protease activated receptor 2 (PAR2).

Novel peptidomimetic pharmacophores to PAR(2) were designed based on the known activating peptide SLIGRL-NH(2). A set of 15 analogues was evaluated with a model cell line (16HBE14o-) that highly expresses PAR(2). Cells exposed to the PAR(2) activating peptide with N-terminal 2-furoyl modification (2-furoyl-LIGRLO-NH(2)) initiated increases in intracellular calcium concentration ([Ca(2+)](i) EC(50) = 0.84 μM) and in vitro physiological responses as measured by the xCELLigence real time cell analyzer (RTCA EC(50) = 138 nM). We discovered two selective PAR(2) agonists with comparable potency: compound 1 (2-aminothiazol-4-yl; Ca(2+) EC(50) = 1.77 μM, RTCA EC(50) = 142 nM) and compound 2 (6-aminonicotinyl; Ca(2+) EC(50) = 2.60 μM, RTCA EC(50) = 311 nM). Unlike the previously described agonist, these novel agonists are devoid of the metabolically unstable 2-furoyl modification and thus provide potential advantages for PAR(2) peptide design for in vitro and in vivo studies. The novel compounds described herein also serve as a starting point for structure-activity relationship (SAR) design and are, for the first time, evaluated via a unique high throughput in vitro physiological assay. Together these will lead to discovery of more potent agonists and antagonists of PAR(2).

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

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.

Lanthanide labeling of a potent protease activated receptor-2 agonist for time-resolved fluorescence analysis.

Protease activated receptor-2 (PAR(2)) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR(2) can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR(2) to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR(2) peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH(2) (2-f-LIGRLO-dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR(2) agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH(2), 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH(2), 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH(2), 6-an-LIGRL) and PAR(2). A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH(2), 3-ia-LIGRL) that does not activate PAR(2) signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR(2) binding. In summary, we have developed a europium-containing PAR(2) agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR(2) ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR(2) ligands.

Development and Evaluation of Small Peptidomimetic Ligands to Protease-Activated Receptor-2 (PAR2) through the Use of Lipid Tethering

Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG 3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2–2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG 3-Hdc retained potency (EC50 = 2.1 nM; 1.3–3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG 3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20–100 nM). 2at-LI-PEG 3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260–360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG 3-Hdc and 2at-PEG 3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG 3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.