Novel antagonists for proteinase-activated receptor 2: inhibition of cellular and vascular responses in vitro and in vivo

Abstract

Background and purpose: Proteinase-activated receptor 2 (PAR(2)) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR(2) antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR(2) antagonists and demonstrate inhibitory effects on PAR(2)-mediated intracellular signalling pathways and vascular responses.

Experimental approach: The peptide-mimetic compound library based on the structures of PAR(2) agonist peptides were screened for inhibition of PAR(2)-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFkappaB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models.

Key results: Two compounds, K-12940 and K-14585, significantly reduced SLIGKV-induced Ca(2+) mobilisation in primary human keratinocytes. Both K-12940 and K-14585 exhibited competitive inhibition for the binding of a high-affinity radiolabelled PAR(2)-ligand, [(3)H]-2-furoyl-LIGRL-NH(2), to human PAR(2) with K(i) values of 1.94 and 0.627 microM respectively. NFkappaB reporter activity and IL-8 production were also significantly reduced. Furthermore, relaxation of rat-isolated aorta induced by SLIGRL-NH(2) was inhibited competitively by K-14585. K-14585 also significantly lowered plasma extravasation in the dorsal skin of guinea pigs and reduced salivation in mice.

Conclusions and implications: K-12940 and K-14585 antagonized PAR(2) competitively, resulting in inhibition of PAR(2)-mediated signalling and physiological responses both in vitro and in vivo. These peptide-mimetic PAR(2) antagonists could be useful in evaluating PAR(2)-mediated biological events and might lead to a new generation of therapeutically useful antagonists.

Figure 1

Chemical structures of peptide-mimetic proteinase-activated receptor 2 antagonists K-12940 and K-14585.

Figure 2

Representative trace for inhibitory effect of K-14585 on SLIGKV-induced Ca²⁺ mobilization in normal human epidermal keratinocytes. Cells were stimulated with SLIGKV (100 µM), and intracellular Ca²⁺ mobilization was measured using a fluorescence method as described in the Methods section. K-14585 (10 µM) was incubated with cells 15 min prior to addition of agonist peptide. Fluorescence was measured over 180 s.

Figure 3

Displacement of [³H]2-furoyl-LIGRL-NH₂ binding in NCTC2544-PAR₂ cells by K-12940 and K-14585. Cells were incubated with [³H]2-furoyl-LIGRL-NH₂ (9.2 nM, 1 µCi·mL⁻¹) in either absence or presence of increasing concentrations of unlabelled agonist/antagonist under conditions as outlined in the Methods section. Binding competition curves for unlabelled compounds are presented as % specific binding. Results are shown as the mean ± SEM (n= 3).

Figure 4

Concentration-dependent inhibitory effects of K-12940 and K-14585 on SLIGKV- or trypsin-mediated NFκB-luciferase activation in NCTC2544 cells. Cells were stimulated with SLIGKV (30 µM) (Panel A) or trypsin (10 nM) (Panel B) in the presence of increasing concentrations of either K-12940 or K-14585. NFκB reporter activity was assayed as outlined in the Methods section. Data represent % of the control activity stimulated with agonist alone (n= 6). *P < 0.05, compared with control stimulation either peptide or trypsin alone.

Figure 5

Effect of K-12940 and K-14585 on IL-8 production in NCTC2544-PAR₂ cells. NCTC2544-PAR₂ cells were stimulated with vehicle (Control), SLIGKV (100 µM) or tumuor necrosis factorα (TNFα) (10 ng·mL⁻¹) in the absence (DMSO) or presence of antagonists (5 µM) for 24 h. IL-8 levels in the culture medium were determined by ELISA. Data is expressed as the mean ± SEM of three experiments. *P < 0.05 compared with SLIGKV stimulated control.

Figure 6

Effect of K-14585 on relaxation of rat aortic rings induced by the proteinase-activated receptor 2 (PAR₂) agonist, Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH₂). In A, SLIGRL-NH₂ was applied cumulatively to the aortic ring preparation pre-contracted with 1 µM phenylephrine (PE). K-14585 (10 µM) was added to the bath 2 min prior to the addition of SLIGRL-NH₂. Summary data (in B) are the mean ± SEM of nine control experiments and 16 with K-14585. The response curve was significantly shifted to the right by K-14585; P < 0.001 compared with the control curve (Bonferoni's test). In C, results from experiments using histamine instead of PAR₂ activating peptide and data shown are the mean ± SEM of eight experiments. n.s., not significant.

Figure 8

The effect of K-14585 on saliva secretion in anaesthetized mice. K-14585 at 10 µmol·kg⁻¹ dissolved in DMSO was administered i.p. in a volume of 10 µL per 10 g body weight, 5 min before i.p. Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH₂) at 1.5 or 2.5 µM, in combination with amastatin at 2.5 µM. Saliva secretion was measured at 5 min intervals for 15 min [basal levels at time 0 were below detection (1 mg)]. Control animals received only the same volume of vehicle (DMSO) before the agonist peptide. Summary results from the whole 15 min collection period are shown in histogram form (in B) and each value represents the mean ± SEM. *P < 0.001 compared with control; numbers of animals are shown in parentheses.

Figure 7

Effect of K-14585 on the plasma exudation caused by the proteinase-activated receptor 2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH₂), in guinea pig dorsal skin. Evans blue dye (50 mg·kg⁻¹) was injected intravenously in male Hartley guinea pigs. Immediately after the dye injection, vehicle (10% DMSO), SLIGRL- NH₂ (300 nM) or a mixture of SLIGRL-NH₂ (300 n^M) and K-14585 (300 µg) was injected intradermally on dorsal skin in a volume of 100 µL per site. Dye extravasation (in A) was quantified (in B) spectrophotometrically, as outlined in the Methods section. Each value represents the mean ± SEM from at least four experiments. *P < 0.01 compared with test control (SLIGRL-NH₂).