In vitro activity: AC-55541 is an agonist of protease-activated receptor (PAR) 2. AC-55541 activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca(2+) mobilization assays, with potencies ranging from 200 to 1000 nM. AC-55541 was well absorbed when administered intraperitoneally to rats, reaching micromolar peak plasma concentrations. AC-55541 was stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 h. 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.

Kinase Assay: Phosphatidylinositol (PI) hydrolysis assays were performed as follows. For PAR2 WT, HEK 293T cells were seeded at 10,000 cells/well in DMEM (Invitrogen) supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) in a 37°C humidified atmosphere containing 5% CO2. Eighteen hours later, the cells were transfected as described above with the indicated plasmid DNAs (30 ng/well of a 96-well plate). Approximately 20 to 24 h after transfection, the cells were washed and labeled overnight with DMEM culture medium containing 0.2 μCi of NET1114 (37 MBq/ml; PerkinElmer Life and Analytical Sciences, Waltham, MA) per well (0.1 ml).

Cell Assay: In brief, cells were plated 1 day before transfection using 7 × 103 cells in 0.1 ml of media per well of a 96-well plate. Cells were transiently transfected with 10 ng of receptor DNA and 30 ng of pSI-β-galactosidase (Promega) per well of a 96-well plate using Polyfect according to the manufacturer's instructions. One day after transfection, medium was changed, and cells were combined with ligands in DMEM supplemented with 25% Ultraculture synthetic supplement instead of calf serum to a final volume of 200 μl/well. After 5 days in culture, β-galactosidase levels were measured essentially as described. Cells were rinsed with phosphate-buffered saline (PBS), pH 7.4, before the addition of 200 μl of PBS supplemented with 3.5 mM O-nitrophenyl-β-d-galactopyranoside and 0.5% Nonidet P-40 (both Sigma-Aldrich). After incubation (2–4 h), the plates were read at 420 nm on a plate reader.