Fig 2: I-287 inhibits PAR2-mediated activation of DAG/Ca2+/PKC and RhoA/SRF-RE, as well as FAK and ERK1/2 signaling pathways.a, b Impact of increasing concentrations of I-287 (15 min) on DAG production (a) and PKC activation (b) induced after 1 (DAG) or 5 (PKC) min stimulation with an EC80 concentration of hTrypsin or SLIGKV-NH2 in HEK293 cells co-expressing hPAR2 and the indicated unimolecular BRET2-based biosensors. Results are expressed as ΔBRET in % of the response induced by EC80 of respective agonists in the absence of I-287 (mean ± SEM; n = 4–5). c Impact of increasing concentrations of I-287 (30 min) on intracellular Ca2+ mobilization induced by an EC80 concentration of hTrypsin or SLIGKV-NH2 in HEK293 cells endogenously expressing hPAR2. Results are expressed as % of the response induced by respective agonists in the absence of I-287 (mean ± SEM; n = 3-4). d Impact of I-287 (10 µM, 30 min) on hPAR2-promoted SRF-RE reporter gene activation induced after 6 h stimulation with hTrypsin (10 U/mL) or SLIGKV-NH2 (100 µM) in HEK293 cells expressing hPAR2. FBS (10%) was used as control. Results are expressed as % of the response induced by respective agonists in the absence of I-287 (mean ± SEM; n = 3–5; unpaired t-test: *p < 0.05 and **p < 0.01 compared to respective control cells, ns: nonsignificant). e, f Kinetics of FAK and ERK1/2 phosphorylation in HEK293 cells expressing hPAR2 and pretreated with DMSO or I-287 (10 µM, 30 min) before stimulation with hTrypsin (1 U/mL) or SLIGKV-NH2 (100 µM) at the indicated times. Representative immunoblots of FAK and ERK1/2 phosphorylation are shown. Western blots were quantified and expressed as the ratio of phosphorylated protein level (P-FAK or P-ERK1/2) normalized over total protein (t-FAK or t-ERK1/2; mean ± SEM; n = 3–5; two-way ANOVA followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, and ***p < 0.001 compared to DMSO-treated cells at the respective time).

Fig 3: I-287 inhibits PAR2-induced secretion of IL-8 cytokine in vitro and reduces CFA-induced inflammation in mice.a, b Impact of I-287 (10 µM, 30 min) on hPAR2-promoted IL-8 cytokine release induced after 6 h stimulation with vehicle, hTrypsin (1 U/mL) or SLIGKV-NH2 (100 µM) in culture medium of HCT 116 (a) and A549 (b) cells expressing hPAR2. Data are expressed as IL-8 concentration in pg/mL (mean ± SEM; n = 3–5; two-way ANOVA followed by Tukey’s post hoc test: ***p < 0.001 compared to control cells with vehicle; ##p < 0.01 and ###p < 0.001 compared to control cells with respective agonist). c Impact of I-287 on complete Freund’s adjuvant (CFA)-induced inflammation in mice. One hour after CFA injection, mice were given I-287 (50 mg/kg) or vehicle (95% TPGS – 5% NMP) by gavage. A group of animals received Ibuprofen (140 mg/kg) as a reference drug. The volume of the hindpaw was measured every hour to evaluate swelling/inflammation using a plethysmometer (mean ± SEM; n = 6 for vehicle and I-287 groups and n = 8 for Ibuprofen group; two-way repeated-measures ANOVA followed by Dunnett’s post hoc test: *p < 0.05 for I-287 vs. vehicle and ##p < 0.01, ###p < 0001 for Ibuprofen vs. vehicle).

Fig 4: Identification of I-287 as a negative PAR2 allosteric modulator.a Chemical structure of compound I-287. b Impact of I-287 pretreatment (30 min) on the Ca2+ responses evoked by increasing concentrations of hTrypsin (left panel) or SLIGKV-NH2 (right panel) in HEK293 cells endogenously expressing hPAR2. Results are expressed as % of the maximal induced-response in the absence of I-287 (% activity; mean ± SEM; n = 3–6). c, d Impact of I-287 pretreatment (15 min) on the Gαq (c) and Gαi2 (d) proteins activation induced after 1 min stimulation with increasing concentrations of hTrypsin (left panel) or SLIGKV-NH2 (right panel) in HEK293 cells co-expressing hPAR2 and the human BRET2-based biosensors Gαq-RlucII or Gαi2-RlucII and GFP10-Gγ1. Results are expressed as BRET2 ratio of absolute values (mean ± SEM; n = 3).

Fig 5: Effect of I-287 on intracellular signaling pathways induced by the two human PAR2 agonists, Trypsin, and SLIGKV-NH2.The pathways inhibited by I-287 are in black, whereas the unaffected pathways are in gray.