Abstract
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.
Highlights
Protease-activated receptors (PARs) are a sub-family of Gprotein coupled receptors (GPCRs) that have a unique mode of activation
Determination of a minimal peptidomimetic structure needed for Protease-activated receptor-2 (PAR2) activation using synthetic tethered ligands (STLs)
Agonist activity was similar between compound 1, the full length STL (2atLIGRL-PEG3-Hdc; previously published as Compound 12 in Flynn, et al [28]), and an STL missing the C-terminal Leu6, compound 2 (2at-LIGR-PEG3-Hdc), in their respective physiological responses (Figure 2) and in their calculated EC50s
Summary
Protease-activated receptors (PARs) are a sub-family of Gprotein coupled receptors (GPCRs) that have a unique mode of activation. As an obvious consequence of its activation mechanism, PAR2 is associated with pathologies that have a strong protease release, including inflammatory related diseases such as arthritis, asthma, inflammatory bowel disease, sepsis, and pain disorders [1,2,4]. Stimulation of PAR2 in pain-sensing primary sensory neurons (nociceptors) leads to the sensitization of a variety of receptors including the noxious heat and capsaicin receptor TRPV1 [5,6,7]. This sensitization of sensory neuronal channels underlies thermal [7,8,9] or mechanical hypersensitivity [8,10,11] elicited by activation of PAR2. Assessing the selectivity of PAR2 ligands against receptors that are selectively expressed in sensory ganglia (e.g., MrgprC11; [13,14]) is critical to developing selective probes for PAR2
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