Development and Evaluation of Small Peptidomimetic Ligands to Protease-Activated Receptor-2 (PAR2) Through the Use of Lipid Tethering
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Development and Evaluation of Small Peptidomimetic Ligands to Protease-Activated Receptor-2 (PAR2) through the Use of Lipid Tethering Scott Boitano1,2*, Justin Hoffman1,2, Dipti V. Tillu3, Marina N. Asiedu3, Zhenyu Zhang2, Cara L. Sherwood1,2, Yan Wang4, Xinzhong Dong5, Theodore J. Price2,3*, Josef Vagner2* 1 Arizona Respiratory Center and Department of Physiology, University of Arizona, Tucson, Arizona, United States of America, 2 The BIO5 Collaborative Research Institute, University of Arizona, Tucson, Arizona, United States of America, 3 Department of Pharmacology, University of Arizona, Tucson, Arizona, United States of America, 4 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China, 5 Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland, United States of America 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 2+ physiological and Ca 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-PEG3-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-PEG3-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-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20–100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260–360 nM) but displayed partial PAR2 activation in both 2+ physiological and Ca signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-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- PEG3-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. Citation: Boitano S, Hoffman J, Tillu DV, Asiedu MN, Zhang Z, et al. (2014) Development and Evaluation of Small Peptidomimetic Ligands to Protease-Activated Receptor-2 (PAR2) through the Use of Lipid Tethering. PLoS ONE 9(6): e99140. doi:10.1371/journal.pone.0099140 Editor: Roland Seifert, Medical School of Hannover, Germany Received March 17, 2014; Accepted May 9, 2014; Published June 13, 2014 Copyright: ß 2014 Boitano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are included within the manuscript. Funding: This manuscript was developed through a multi-principal investigator collaboration between S.B., T.J.P. and J.V. This work was primarily supportedbya multi-principal investigator grant from the National Institutes of Health (NIH)/NINDS, NS073664 (S.B., T.J.P., J.V.). This work was additionally supported by the following grants: NIH/NIEHS Superfund Research Grant ES04940 (S.B.; Raina Maier, P.I.); NIH/NINDS Grant NS065926 (T.J.P.); NIH/NIAID Grant AI083403 (S.B.; Michael O. Daines, P.I.); NIH/NIGMS Grant GM087369 (X.D.); NIH/NINDS Grant NS054791 (X.D.); and the Technology and Research Initiative Fund from Arizona State Proposition 301 (J.V.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: T. P. is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria. * E-mail: [email protected] (SB); [email protected] (JV); [email protected] (TJP) Introduction arthritis, asthma, inflammatory bowel disease, sepsis, and pain disorders [1,2,4]. Stimulation of PAR2 in pain-sensing primary Protease-activated receptors (PARs) are a sub-family of G- sensory neurons (nociceptors) leads to the sensitization of a variety protein coupled receptors (GPCRs) that have a unique mode of of receptors including the noxious heat and capsaicin receptor activation. PARs contain an embedded ligand that is exposed TRPV1 [5,6,7]. This sensitization of sensory neuronal channels following proteolytic cleavage of the extracellular oriented NH2 underlies thermal [7,8,9] or mechanical hypersensitivity [8,10,11] terminus [1]. The different N-termini of the PARs present elicited by activation of PAR2. The involvement of PAR2 in pain substrates for a variety of proteases that create selective activation and other pathologies makes it a prime target for drug discovery. (or inactivation) mechanisms for signal transduction [2,3,4]. The Importantly, PAR2 has been associated with itch based partly on most common, diffusionally limited ‘‘tethered ligand’’ uncovered data obtained using the relatively potent PAR2 signaling peptide, following trypsin-like serine protease activity of PAR [exposing 2 SLIGRL-NH2. It is now clear that this peptide also stimulates an SLIGKV (human) or SLIGRL (rodent)] serves as a potent agonist additional GPCR, Mas1 related G-protein coupled receptor type to the receptor. As an obvious consequence of its activation C11 (MrgprC11), and this receptor is responsible for the pruritic mechanism, PAR is associated with pathologies that have a strong 2 properties of SLIGRL-NH2 [12]. Therefore, assessing the protease release, including inflammatory related diseases such as selectivity of PAR2 ligands against receptors that are selectively PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99140 Full and Partial PAR2 Agonism with Lipid-Tethered Peptidomimetics expressed in sensory ganglia (e.g., MrgprC11; [13,14]) is critical to Novabiochem (San Diego, CA). Aldehyde (4-(4-formyl-3-methox- developing selective probes for PAR2. yphenoxy)butyrylaminomethyl) resin and Rink Amide resins were Small peptides or peptidomimetics that mimic the ligand acquired from Novabiochem (San Diego, CA). N,N’-diisopropyl- binding properties of the tethered ligand exposed by proteolysis carbodiimide (DIC) and diisopropylethylamine (DIEA) were of the N-terminus of the receptor have been used to directly purchased from IRIS Biotech (Marktredwitz, Germany). A Ng- activate PARs [2,15,16,17]. Activating peptides (e.g., SLIGKV- 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl side chain NH2 and SLIGRL-NH2) and peptidomimetics (e.g., 2-furoyl- protecting group was used for Arg. 2-aminothiazole-4-carboxylic LIGRLO-NH2 [18] and 2at-LIGRL-NH2 [19]) have provided acid and 5-isoxazole-carboxylic acid were obtained from Combi- useful tools for establishment of structure-activity relationships Blocks (San Diego, CA); hexadecyl (Hdc-NH2) amine was obtained (SAR) and rational drug design because they limit off-target effects from Sigma-Aldrich. Fmoc-protected version of PEG (1-(9H- that are often a complication of natural protease activation. Early fluoren-9-yl)-3,19-dioxo-2,8,11,14,21-pentaoxa-4,18-diazatrico- SAR studies suggested that the minimal peptide sequence required san-23-oic acid) was obtained from Novabiochem (San Diego, for PAR2 activation is a pentamer (either SLIGR-NH2 or the less CA). Reagent grade solvents, reagents, and acetonitrile for High potent LIGRL-NH2 [17,20]). More recently, heterocycle-dipep- Performance Liquid Chromatography (HPLC) were acquired tide mimetics have been shown to retain PAR2 activity [21]. from VWR (West Chester, PA) or Aldrich-Sigma (Milwaukee, However, full characterization of these shortened compounds has WI), and were used without further purification unless otherwise been hindered by a lack of assays sufficiently sensitive to evaluate noted. Compounds were manually assembled using 5 mL plastic full concentration responses. Commonly used assays require high syringe reactors equipped with a frit, and a Domino manual concentrations (. 50 mM) that potentially limit PAR2-selectivity or synthesizer obtained from Torviq (Niles, MI). The C-18 Sep- TM prevent full solubility for preferred Ca2+ activation studies [21]. It Pak Vac RC cartridges for solid phase extraction were is now evident that a variety of GPCRs, including PAR2, can elicit purchased from Waters (Milford, MA). signaling pathway-specific activation with distinct physiological responses [4,22,23,24,25,26]. A means to establish better evalu- Compound Synthesis ation of the minimal peptidomimetic structure required for full Truncated analogs of 2at-LIGRL-PEG3-Hdc 1–6 and com- PAR2 activation would benefit PAR2 ligand discovery efforts. pounds 11–14 were prepared as previously published by solid- Lipidation of peptide receptor agonists has