Abstract
We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide], each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca2+ mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH2 had similar potency, whereas SLIGRL-NH2 was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.
Footnotes
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L.R.G. and J.M. contributed equally to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.142570.
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ABBREVIATIONS: PAR, protease-activated receptor; CGRP, calcitonin gene-related peptide; TRPV, transient receptor potential vanilloid; AC-55541, N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide; AC-264613, 2-oxo-4-phenyl-pyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney; R-SAT, receptor selection and amplification technology; PBS, phosphate-buffered saline; PI, phosphatidylinositol; WT, wild type; NET1114, [myo-2-3H]inositol; IP, [3H]inositol phosphate; DMSO, dimethyl sulfoxide; LC, liquid chromatography; NK, neurokinin; PG, prostaglandin; L732,138, (S)-2-acetylamino-3-(1H-indol-3-yl)propionic acid 3,5-trifluoromethyl-benzyl ester.
- Received June 23, 2008.
- Accepted August 28, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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