Interdicting Protease-Activated Receptor-2-Driven Inflammation with Cell-Penetrating Pepducins

Interdicting protease-activated receptor-2-driven inﬂammation with cell-penetrating pepducins

Leila M. Sevignya,b, Ping Zhanga, Andrew Bohmc, Katherine Lazaridesa, George Peridesd, Lidija Covica,c,e, and Athan Kuliopulosa,b,c,e,1

aMolecular Oncology Research Institute and Departments of dSurgery and eMedicine, Tufts Medical Center, and Departments of bGenetics and cBiochemistry, Tufts University School of Medicine, Boston, MA 02111

Edited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved April 12, 2011 (received for review November 12, 2010) Protease-activated receptor-2 (PAR2), a cell surface receptor for mice, which correlated with a decline in inflammatory mediators trypsin-like proteases, plays a key role in a number of acute and (13). Conversely, overstimulation of PAR2 can lead to severe chronic inflammatory diseases of the joints, lungs, brain, gastroin- edema, granulocyte infiltration, increased tissue permeability, tis- testinal tract, and vascular systems. Despite considerable effort sue damage, and hypotension (1, 12). Agonists of PAR2, including by the pharmaceutical industry, PAR2 has proven recalcitrant to trypsin and the synthetic SLIGRL peptide, also trigger the release targeting by small molecule inhibitors, which have been unable to of calcitonin and substance P from sensory neurons causing neu- effectively prevent the interaction of the protease-generated trophil infiltration, edema, hyperalgesia, and cancer pain (3, 14). tethered ligand with the body of the receptor. Here, we report the PAR2 has been linked to arthritis as evidenced by significant de- development of first-in-class cell-penetrating lipopeptide “pepducin” creases in joint inflammation in PAR2-deficient mice (15) and up- antagonists of PAR2. The design of the third intracellular (i3) loop regulated expression of the receptor in osteoarthritis and rheu- pepducins were based on a structural model of a PAR2 dimer and by matoid arthritis synovial tissues (16). mutating key pharmacophores in the receptor intracellular loops and Tryptase, a major proinflammatory serine protease, can also analogous pepducins. Individual pharmacophores were identified, cleave and activate PAR2 (17). Local or systemic release of high which controlled constitutive, agonist, and antagonist activities. This levels of mast cell-derived tryptase can have life-threatening con- fi approach culminated in the identi cation of the P2pal-18S pepducin sequences including acute asthma, systemic mastocytosis, and which completely suppressed trypsin and mast cell tryptase signaling anaphylaxis (18). A specific and effective pharmacological in- PHARMACOLOGY through PAR2 in neutrophils and colon cancer cells. The PAR2 pepdu- hibitor of PAR2 therefore has the potential to provide beneficial fi fl cin was highly ef cacious in blocking PAR2-dependent in ammatory anti-inflammatory effects and reduce the detrimental activity of responses in mouse models. These effects were lost in PAR2-deficient – fi fi mast cells, neutrophils, and other PAR2-expressing leukocytes and mast-cell de cient mice, thereby validating the speci city of the that contribute to tissue damage. To date, it has been challenging pepducin in vivo. These data provide proof of concept that PAR2 to identify an effective PAR2 antagonist that lacks agonist ac- pepducin antagonists may afford effective treatments of potentially tivity or is efficacious at submillimolar levels (4, 19, 20). debilitating inflammatory diseases and serve as a blueprint for de- We have developed a unique way to inhibit G protein-coupled veloping highly potent and specific i3-loop–based pepducins for receptors on the inside surface of the lipid bilayer with cell- other G protein-coupled receptors (GPCRs). penetrating pepducins. Pepducins are derived from the intracellular loops of their target receptor and comprise a lipid tether protease-activated receptor-1 | protease-activated receptor-4 conjugated to the peptidic portion of the loop (20). These lipidated peptides have the ability to rapidly flip across the mem- rotease-activated receptor-2 (PAR2) mediates a number of brane and interfere with receptor-G protein signaling in a highly P(patho)physiological pathways involved in acute and chronic specific manner (20–24). Here, we report the rational design and fl fl in ammation, arthritis, allergic reactions, sepsis, in ammatory development of a potent and specific PAR2 antagonist pepducin – pain, as well as cancer cell invasion and metastasis (1 6). PAR2 is based on the third intracellular loop, which effectively inhibits a member of the G protein-coupled protease-activated receptor protease-PAR2 signaling induced by trypsin and tryptase. In subfamily, which includes the related PAR1, PAR3, and PAR4. mouse models of acute inflammation, we demonstrate that the Proteases such as trypsin (7), thrombin, and MMP-1 (8, 9) cleave PAR2 pepducin provides salutary effects in reducing inflam- the N-terminal extracellular domain of individual PAR members, mation and edema, which are lost in PAR2-deficient mice. These thereby unmasking a tethered ligand that binds to the outer sur- data indicate that targeting PAR2 may prove to be beneficial in face of the receptor to activate transmembrane signaling to intra- inflammatory conditions and other diseases that involve trypsin, cellular G proteins. The pleiotropic downstream pathways tryptase, and other protease agonists of PAR2. activated by PAR2 include calcium mobilization, phospholipase C-β–dependent production of inositol phosphates and diacylgly- Results cerol, Rho and Rac activation, MAPK signaling, and gene tran- Identification of Critical Pharmacophores in the Intracellular i3 Loop scription (10). PAR2 itself is strongly up-regulated by diverse of PAR2. As has been shown with many GPCRs, there is con- fl α α in ammatory mediators such as TNF- , IL-1 , bacterial endo- siderable evidence that PARs can interact with each other and toxin, and autoimmunity-inducing antigens produced during the form homo- and heterodimers, and pepducins are postulated to tissue response to inflammation (11). As a cell surface sensor of proteases, PAR2 endows the cell with

the ability to respond or overrespond to the rapidly changing pro- Author contributions: L.M.S., A.B., L.C., and A.K. designed research; L.M.S., P.Z., A.B., K.L., teolytic microenvironment that occurs during inflammation. PAR2 and L.C. performed research; A.B. and G.P. contributed new reagents/analytic tools; is widely expressed in inflammatory cells, stroma, endothelium, and L.M.S., L.C., and A.K. analyzed data; and L.M.S., A.B., and A.K. wrote the paper. intestinal epithelium (10). PAR2-deficient mice exhibit reduced The authors declare no conflict of interest. granulocytic infiltration and tissue damage and suppression of This article is a PNAS Direct Submission. inflammatory cytokines in models of intestinal inflammation, au- 1To whom correspondence should be addressed. E-mail: [email protected]. toimmunity, and encephalomyelitis (11, 12). Reduced cardiac is- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. chemia/reperfusion injury was also observed in PAR2-deficient 1073/pnas.1017091108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1017091108 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 mimic these dimeric interactions on the intracellular surface of terface with the eighth helix region from an adjacent i4 domain. the lipid bilayer (25). To initiate the design of PAR2 pepducin Mutation of M274 to alanine ablated the constitutive signal, antagonists, we constructed a molecular model of a PAR2 dimer. whereas mutation of K287 to alanine had no effect (Fig. 1C). The model of the PAR2 monomer was based on the structure of Strikingly, mutation of K287 to phenylalanine gave a twofold in- rhodopsin (26), which shares 45% identity with PAR2. Residues crease in the constitutive signal. Despite losing its constitutive 51–397 of PAR2 were substituted for residues 1–346 of bovine activity, M274A was able to fully signal to agonist (Fig. 1D). rhodopsin. Stereochemically reasonable positions were used for Likewise, K287A and K287F were also able to fully signal to ag- side chains that were later subjected to molecular dynamics and onist (Fig. 1E). Conversely, the R284S mutant exhibited a loss of energy minimization. A series of twofold symmetric PAR2 dimer constitutive signal and was unable to be activated by even high models were then manually constructed with the aim of maxi- concentrations of peptide ligand. Similarly, the PAR2ΔH8 mu- mizing the surface area between the adjacent receptors. Among tant, which lacks the eighth helix in the i4 domain, was completely these, we selected the model that was most consistent with signaling dead to ligand (Fig. 1D). Together, these data indicate available data regarding interactions between the receptor pair that the juxtamembrane residues of the i3 loop of PAR2 play and the G protein (SI Materials and Methods). critical roles in both constitutive and ligand-dependent activity. To provide functional evidence that one PAR2 receptor can interact with an adjacent PAR2 receptor, we constructed a sig- Molecular Engineering of PAR2 Antagonist Pepducins. Having iden- naling dead PAR2-RQ mutant by transposing residues at Q172 tified residues important for the signaling of PAR2, we next syn- and R173 located in the critical “DRY” TM3 motif (Fig. 1A). The thesized a series of i3 loop-based pepducins with mutations of the PAR2-RQ mutant has an intact protease cleavage site and teth- critical M274, R284, or K287 pharmacophores (Fig. 2 A and B). ered ligand but cannot signal to G proteins. A noncleavable An initial screen of PAR2 pepducin agonist and antagonist activity PAR2-R36A mutant was also constructed, which retains the was performed using the PAR2-expressing human colorectal ad- ability to fully signal to SLIGRL ligand, but is not able to be enocarcinoma cell line SW620. The wild-type full-length i3 loop proteolyzed and directly activated by trypsin (Fig. S1 A–C). Cells pepducin, P2pal-21, gave a weak agonist signal and lacked an- expressing PAR2-R36A or PAR2-RQ alone were unable to mi- tagonist activity against SLIGRL as assessed by calcium flux (Fig. 2 grate toward gradients of trypsin or to activate Gq-PLC-β signal- A and C). Consistent with the gain-of-constitutive activity ob- ing. However, when the two mutant receptors were cotransfected, served in the PAR2-K287F mutant, the analogous P2pal-21F chemotactic migration and signaling were restored (Fig. 1B and pepducin (20) gave full agonist activity but no antagonist activity in Fig. S1 B and D), consistent with a mechanism whereby PAR2-RQ the SW620 cells (Fig. 2 A and C). Deletion of the first three res- can donate its tethered ligand to transactivate PAR2-R36A. To idues to make P2pal-18F gave a slight decrease in agonist activity, provide direct evidence that PAR2 can form homodimers or but still was devoid of antagonist activity. oligomers, we showed that myc-tagged PAR2 can stably associate The P2pal-18S pepducin, which replaces the critical R284 with T7-tagged PAR2 by coimmunoprecipitation (Fig. S2). These pharmacophore with serine, was a full antagonist of PAR2 and had data indicate that PAR2 has the ability to associate with itself no detectable agonist activity in the calcium flux assay (Fig. 2 A and within a homodimer or oligomeric complex. C). Substitution of the C-terminal K287 of P2pal-18S with phe- Interestingly, we also found that wild-type (WT) PAR2 has nylalanine to make P2pal-18SF restored agonist activity. The re- constitutive activity in the absence of ligand (Fig. 1C). Constitutive placement of R284 with glutamine to make P2pal-18Q had signaling has been observed in GPCRs and is often dependent on nearly no agonist activity but had partial antagonist activity. The critical residues located in the C-terminal juxtamembrane region N-terminally truncated P2pal-14GF had no agonist nor antag- of the i3 loop (27). The PAR2 homodimer model shown in Fig. 1A, onist activity; however, substitution of R284 with glutamine to predicts that the juxtamembrane i3 loop residues M274, R284, create P2pal-14GQ gained 53% antagonist activity (Fig. 2 A and and K287 could potentially interact across the PAR2 dimer in- C). From this initial calcium flux screen, we chose to further

Trypsin, Tryptase K287 R284 A Ligand PAR2 WT: 30-NRSSKGR-SLIGKV DGTSHVTGK-51 R36A: 30-NRSSKGA-SLIGKV DGTSHVTGK-51 TM3 i2 Fig. 1. Constitutive and ligand-dependent ac- PAR2 WT: 170-SVQRYWV IVNPMGHSRKKANI-190 QR RQ: 170-SVRQYWV IVNPMGHSRKKANI-190 tivity of PAR2 is regulated by juxtamembrane 274 284 287 residues located in the third intracellular (i3) TM5 i3 TM6 PAR2 WT: 266-IR MLRSSAMDENSEKKRK RAIK-287 loop. (A) Location of PAR2 mutants and model of M274A: 266-IR MLRSSAADENSEKKRK RAIK-287 R284S: 266-IR MLRSSAADENSEKKRK SAIK-288 the receptor dimer used in this study. The PAR2 K287A: 266-IR MLRSSAMDENSEKKRKRAIA -287 model depicting the i3 loop–eighth helix dimer K287F: 266-IR MLRSSAMDENSEKKRKRAIF -287 TM7 H8 interface was constructed using X-ray structures PAR2 WT: 346-F VSHDFRDHAKNALLCRSVRT-366 M274 of rhodopsin as described in Materials and PAR2ΔH8: 346-F VS…………AAA…..LCRSVRT-366 i3 loop 8th helix Methods.(B) Migration of HEK cells transiently B C D E transfected with PAR2 (wild type, WT), PAR2- 4 R36A, PAR2-RQ, or PAR2-R36A plus PAR2-RQ DMEM 2.2 * pcDEF3 toward chemotactic gradients of 30 nM trypsin Trypsin WT PAR2 M274A 100 R284S 100 WT PAR2 or DMEM media alone for 18 h in a transwell ld) # * PAR2ΔH8 K287A

o 1.9 K287F μ 3 apparatus (8- m pore), n = 2, repeated three ) )

(f 80 80 independent times. (C–E) Constitutive and %

n * 1.6 60 60 SLIGRL-activated signaling of PAR2 mutants to β β 3 nsP ( InsP (fold) PLC- . PLC- activity was measured by [ H]-InsP InsP (% * * I

2 l 40 1.3 40 gratio formation over 30 min (39) and was typically i

M 20 20 stimulated four- to ﬁvefold above basal with 30– Basa 1.0 100 μM SLIGRL. Mean PAR2 mutant surface ex- 1 0 0 pression was assessed by FACS and was 1.0 ± 0.3- 0.7 -6 -5 -4 -6 -5 -4 ± A 01010 10 0 10 10 10 fold relative to wild type in COS7 cells and 1.00 T A Q Q 74A 87 87F R 2 284S 2 2 SLIGRL (M) W R vector-PAR2M R K K SLIGRL (M) – R36 A+ 0.18 in HEK cells (Fig. S1D). Data (n =28) rep- 36 WT # R resent the mean ± SD. *P < 0.05 and P = 0.07.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1017091108 Sevigny et al. Downloaded by guest on September 25, 2021 A

BC PHARMACOLOGY

Fig. 2. Design and screening of agonist and antagonist PAR2 i3-loop pepducins. (A) Agonist and antagonist activity of third intracellular (i3) loop PAR2 pepducins using calcium flux assays with SW620 colon adenocarcinoma cells that endogenously express PAR2. Each column of three calcium flux traces corresponds to the i3 pepducin sequence shown above. The Top row is the agonist activity of 3–4 μM pepducin and the Middle row is the agonist activity of 14–15 μM pepducin. The Bottom row depicts the calcium signal of 100 μM SLIGRL (open arrowheads) following 1-min pretreatment with 6 μM pepducin (closed arrowheads). Final concentration of DMSO vehicle was 0.2%. (B) Model of the WT PAR2 i3 pepducin P2pal-21 bound to the intracellular surface of PAR2. The location of the i3 pepducin was derived by substituting the coordinates of the i3 loop on the intact receptor with the i3 pepducin using the PAR2 dimer model of Fig. 1A. Key pharmacophores M274 (brown), R284 and K287 (yellow), and palmitate (green) are shown. (C) Agonist activity for each pepducin from A is reported as initial velocity of calcium flux at 3–4 μM pepducin (†), or at 14–15 μM pepducin (‡). Antagonist activity of 6 μM pepducin against 100 μM SLIGRL was measured by area under the curve of calcium flux from the Bottom row of A. Experiments were repeated at least two to three times each and gave highly similar results.

analyze the properties of the P2pal-18S antagonist pepducin for 18S did not inhibit PAR1 or PAR4 by calcium flux assays in SW620 its ability to block other PAR2 functions. cells or in inositol phosphate signaling in COS7 cells, despite providing effective inhibition to the PAR2 ligand SLIGRL (Fig. S3 P2pal-18S Is a Specific Antagonist of PAR2 Activity in Neutrophils. We A and B). The P2pal-14GQ pepducin was also selective for PAR2 first tested the ability of P2pal-18S to antagonize PAR2-dependent and not PAR1, but was not as efficacious in suppressing migration activity of human neutrophils. Neutrophils were isolated from the of SW620 cells to SLIGRL compared with P2pal-18S (Fig. S3 peripheral blood of healthy volunteers and found to express high A–C). P2pal-18S had no effect on PAR1-dependent platelet ag- levels of surface PAR2 and PAR4 and lower apparent levels of gregation (Fig. S4A). Neither P2pal-18S nor P2pal-14GQ caused PAR1 by flow cytometry (Fig. 3A). Neutrophils robustly migrated membrane disruption or apoptosis as assessed by propidium io- toward gradients of the PAR2 agonists trypsin and SLIGRL, dide uptake in SW620 cells with up to 30 μM concentrations of which was completely blocked by P2pal-18S with IC50 values of pepducin (Fig. S4B). 0.14–0.2 μM (Fig. 3B). Likewise, 0.3 μM P2pal-18S completely We also found that P2pal-18S blocked transactivation of PAR2 blocked chemotactic migration of human neutrophils to 100 nM homodimers as shown by complete suppression of chemotactic tryptase (Fig. 3C). P2pal-18S also completely inhibited migration migration of HEK cells coexpressing PAR2-R36A and PAR2-RQ of mouse neutrophils toward 30 nM trypsin (Fig. 3D). This cross- mutants (Fig. S3D). Furthermore, to provide evidence that the species inhibition was predicted as human PAR2 shares 85% P2pal-18S peptide directly interacts with PAR2, we showed that identity with mouse PAR2, and the mouse i3 loop retains all of the PAR2 had enhanced binding to avidin beads coupled with the critical pharmacophores identified in the human PAR2 i3 loop. PAR2 i3 loop 18S peptide compared with beads alone (Fig. S5). Specificity of P2pal-18S for PAR2 was evident as it had no an- Additionally, we tested whether P2pal-18S inhibited proteolytic tagonist activity to the closely related PAR1, PAR4, or CXCR1/2 cleavage of PAR2 or endocytosis (28–30). P2pal-18S had no effect IL-8 receptors in neutrophil chemotaxis assays (Fig. 3E). P2pal- on trypsin cleavage of PAR2 (Fig. S6) and did not inhibit ligand-

Sevigny et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 A Human Neutrophils following λ-carrageenan/kaolin injection, which was nearly identical to the protective effect observed in WT mice treated with 100 100 100 −/− 80 80 80 P2pal-18S. Notably, treatment of PAR2 mice with P2pal-18S

x ο ο 60 2ο 60 2 60 2 fi Ma Max did not further reduce swelling, con rming that the anti- f f o 40 o 40 40 fl % % %ofMax Counts Counts Counts in ammatory effects of the PAR2 pepducin required the presence 20 20 20 of its cognate receptor. 0 0 0 0102 103 104 105 102 103 104 105 0102 103 104 105 Histologic analysis of the inflamed footpads harvested 7 h post PAR1 FluorescenceI C-A PAR2 FluorescenceFITC-A PAR4 FluorescenceFITC-A λ-carrageenan/kaolin injection revealed that P2pal-18S provided fi < 1.8 RPMI Tryptase signi cant 60% protection (P 0.005) against the leukocytic B 30nM Trypsin C 1.6 infiltrates in the dermis of the footpads, which was identical to the 10uM SLIGRL − − 1.6 protection observed in PAR2 / relative to WT mice (Fig. 4C and 1.4 Fig. S8A). The λ-carrageenan/kaolin challenge caused a twofold c Index

1.4 i

t * *

c increase in myeloperoxidase activity in WT mice, which was 1.2 1.2 blocked by P2pal-18S (Fig. S8B). Together, these data demonstrate that the PAR2 pepducin P2pal-18S affords signiﬁcant 1.0 1.0 Chemota Chemotactic Index protection against acute leukocytic inﬂammation and edema and

0.8 these protective effects are dependent on the presence of PAR2. -9 -8 -7 -6 -5 0.8 01010 10 10 10 P2pal-18S (μM): 0, 0.3, 3 0, 0.3, 3 [P2pal18S] (M) P2pal-18S Protects Against Mast Cell Tryptase-Induced Inﬂammation. Previous studies have established that mast cell tryptase cleaves Mouse Neutrophils D E 2.2 RPMI IL-8 TFLLRN AYPGKF and activates PAR2 signaling in human endothelium and kerati- 1.6 2.0 nocytes and in mouse models of arthritis (4, 17, 33). To determine ex nd

I 1.8 whether mast cells and mast cell tryptase were contributing to the c

1.4 * i t observed PAR2-dependent effects in the mouse models of paw

c 1.6 a inflammation, we stimulated mast cells with the degranulating ot 1.4 λ 1.2 agent 48/80, or -carrageenan/kaolin and collected conditioned em 1.2 media. As shown in Fig. 5A, the stimulated mast cells secreted Ch Chemotactic Index 1.0 tryptase, which was then used as a chemoattractant source in 1.0 0.8 neutrophil chemotaxis assays. The conditioned media from the Trypsin (30 nM): - + + + P2pal-18S (μM):0 0,0.3,3 0,0.3,3 0,0.3,3 P2pal-18S (μM): - - 0.3 3 stimulated mast cells gave comparable chemotactic migration as 100 nM tryptase (Fig. 5B). Treatment of human neutrophils with Fig. 3. The P2pal-18S pepducin is a full antagonist of PAR2-dependent tryptase inhibitor, APC-366, or the PAR2 pepducin P2pal-18S, neutrophil chemotaxis. (A) Human neutrophils (n = 4 normal volunteers) completely inhibited chemotactic migration toward the tryptase- were analyzed for surface expression of human PAR1, PAR2, and PAR4 by flow cytometry with PAR-specific antibodies. (B) P2pal-18S inhibits human containing mast cell media (Fig. 5B). To provide in vivo evidence neutrophil chemotaxis to gradients of trypsin (30 nM) and SLIGRL (10 μM) that mast cells and mast cell tryptase were activating PAR2 in the

with IC50 values of 0.14–0.2 μM. Chemotaxis index is the ratio of directed paw edema model, we depleted mice of mast cells by pretreatment versus random migration over 30 min through a 5-μm pore filter. (C) P2pal- with compound 48/80 (34). A decrease in λ-carrageenan/kaolin- 18S completely inhibits human neutrophil chemotaxis to 100 nM tryptase. induced edema in the 48/80-depleted animals was observed, (D) P2pal-18S completely inhibits mouse neutrophil (n = 6) chemotaxis to 30 compared with nontreated controls (Fig. S9A). Similarly, mice nM trypsin. (E) P2pal-18S does not affect human neutrophil chemotaxis to treated with tryptase inhibitor APC-366, gave a significant 40% gradients of 100 nM IL-8 (CXCR1/CXCR2), 10 μM TFLLRN (PAR1), or 100 μM λ – C – ± < protection in -carrageenan/kaolin induced paw edema (Fig. 5 ). AYPGKF (PAR4). n =4 6, mean SEM. *P 0.05. An intraplantar injection of the tryptase-containing mast cell media resulted in a similar peak increase in paw edema (Fig. 5D), dependent endocytosis of PAR2 or PAR1 (Fig. S7). Therefore, the as induced by the selective PAR2 agonist SLIGRL (Fig. 4B). Systemic treatment with P2pal-18S gave a 50% decrease in peak P2pal-18S i3 loop pepducin can inhibit PAR2-dependent calcium development of edema at 4 h and afforded complete protection at signaling, PLC-β inositol phosphate formation, and cell migration, 8 h and thereafter (Fig. 5D). To provide further support for the but not proteolytic cleavage or endocytosis. notion that mast cell-derived tryptase mediates the observed fl fi fl PAR2-dependent in ammatory responses, we challenged mast Ef cacy of P2pal-18S in Mouse Models of In ammatory Paw Edema. fi λ To evaluate the in vivo efficacy and specificity of P2pal-18S, we cell-de cient mice with -carrageenan/kaolin and observed that these mice had an identical reduced paw edema response as P2pal- tested the ability of the pepducin to protect against inflammatory 18S–treated littermate control mice that have intact mast cells hindlimb paw edema in WT and PAR2-deficient mouse strains fl (Fig. S9B). Furthermore, the observed paw edema in the mast cell- (31). Acute in ammatory edema was induced by an intraplantar fi λ de cient mice could not be further reduced by treatment with injection of -carrageenan and kaolin, irritants that cause a mas- P2pal-18S (Fig. S9B). Together, these data suggest that the P2pal- sive leukocytosis and hyperemic response, which leads to local- 18S pepducin provides significant protection against inflammatory ized swelling (32). We also directly assessed the PAR2-dependent edema triggered by mast cell-derived tryptase. activity of P2pal-18S by quantifying its inhibitory effects against the PAR2-specific agonist SLIGRL when injected into the hind Discussion fl footpad of WT C57BL/6 mice. Acute in ammation induced by In this paper, we report the development of first-in-class lip- λ-carrageenan/kaolin resulted in a nearly twofold increase in paw opeptide pepducin antagonists of PAR2. Pepducins are an edema with vehicle-treated WT mice, peaking 8 h after injection emerging new technology to target recalcitrant transmembrane (Fig. 4A). The PAR2 agonist peptide, SLIGRL, also induced an receptors such as PAR2. These highly stable lipidated peptides increase in edema of WT mice, peaking 4 h after injection (Fig. are targeted to the intracellular surface of their cognate GPCR 4B). Systemic administration of P2pal-18S caused a significant and stabilize the receptor in either an active or inactive confor- 50% decrease in λ-carrageenan/kaolin–induced edema and an mation, resulting in modulation of signal transduction (20, 21, 85% decrease in SLIGRL-induced edema (Fig. 4 A and B). PAR2 24). Pepducins typically comprise two components: a short pep- deficiency conferred a 50% protective effect relative to WT mice tide sequence, derived from an i1–i4 intracellular loop of the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1017091108 Sevigny et al. Downloaded by guest on September 25, 2021 Fig. 4. The PAR2 antagonist A B C pepducin P2pal-18S signifi- Inflam cells P value cantly reduces mouse paw 1.16 (# per field) edema and inflammation in 2.0 WT, Vehicle Vehicle WT, P2pal-18S WT + _ wild-type but not PAR2- PAR2-/-, Vehicle 108 15 deficient mice. (A) λ-carra- 1.8 1.12 PAR2-/-, P2pal-18S geenan/kaolin or (B)the PAR2-specific agonist SLIGRL 1.6 * 1.08 was administered by intra- P2pal-18S ** WT 42+ 3 <0.005 plantar injection to the left 1.4 hindpaw of C57BL/6 wild- 1.04 −/− * type or PAR2 mice that Edema (fold) * * 1.2 were treated with s.c. injec- ** * 1.00 tion of 10 mg/kg of P2pal-18S Vehicle or vehicle. Paw area was mea- 1.0 PAR2-/- + 0102030 48 40 3 <0.005 sured and reported as fold 0102030 48 Hours after λλ-carr/k injection Hours after SLIGRL injection increase relative to baseline paw area. (C) Histology of representative H&E-stained footpads 7 h after λ-carrageenan/kaolin injection and quantification of the infiltrating inflammatory cells at 40× magnification. Data (n =4–6 per group) mean ± SEM. *P < 0.05 and **P < 0.005.

target GPCR, and an acyl-chain fatty acid (e.g., palmitate) or PAR2 has been observed to be beneficial in the mechanism of other lipid conjugated to the peptide. The rational design of the clearance of Pseudomonas aeruginosa pathogens from infected − − − − i3 loop agonist and antagonist pepducins was based on a struc- lungs using PAR2 / mice (37). In this model, PAR2 / mice had tural model of a PAR2 dimer and by manipulating key residues in the receptor loops and analogous pepducins. We identified individual pharmacophores that controlled constitutive, agonist, and antagonist activites. The most potent pepducin antagonist, 2.0 P2pal-18S, fully ablated PAR2 signaling but did not inhibit the ABHMC-1 media PHARMACOLOGY closely related PAR1 or PAR4 receptors or other tested GPCRs. 48/80: - + - 1.8 λ The PAR2 pepducin antagonist had significant in vivo efficacy -carr/k: - - + fi 40 1.6 in suppressing leukocytic in ltration and edema induced by * λ-carrageenan/kaolin or a PAR2-selective agonist in mouse paw 33 1.4 inflammation models. The anti-inflammatory effect of the P2pal- WB: mast cell tryptase *** 18S pepducin was lost in PAR2-deficient mice, demonstrating 1.2 # *

that the pepducin was highly specific for PAR2 in vivo. Moreover, Chemotactic Index 1.0 the anti-inflammatory effect observed in the PAR2-deficient mice relative to wild type was nearly identical to that observed in wild- 0.8 type mice treated with P2pal-18S. Together, these data indicate Tryptase (100 nM): - + + ------Buffer: that P2pal-18S affords effective pharmacologic blockade of PAR2 HMC-1 - - - + ------in models of acute inflammation and that these effects require the CM 48/80: - - - - + + + - - - λ-carr/k: ------+ ++ presence of PAR2. APC 366 (10 μM): - - + - - - + - - + Many studies have implicated PAR2 as playing critical roles P2pal-18S (1 μM): - - - - - + - - + - in a wide range of diseases including asthma (2), arthritis (16), hyperalgesia (3), neurogenic and cancer pain (14), and cancer C D 1.20 1.8 invasion (5). We provided several lines of evidence that the in- Vehicle Vehicle flammatory response observed in the mouse footpad model was APC 366 1.15 P2pal-18S largely dependent on mast cells and mast cell-derived tryptase, an 1.6 important agonist of PAR2-driven inflammation. We found that 1.10 * 1.4 * the PAR2 pepducin could completely suppress tryptase signaling * * through PAR2. Moreover, mast cell-deficient mice had an iden- 1.2 1.05 * Edema (fold) * tical reduced paw edema response as P2pal-18S treated littermate * ** controls which had intact mast cells. Furthermore, the paw edema 1.0 1.00 fi in the mast cell-de cient mice could not be further reduced by 0 8 16 24 32 40 48 0 8 16 24 32 40 48 treatment with P2pal-18S, providing further support for the notion Hours after λ-carr/k injection Hours after HMC-1 CM injection that mast cell-derived tryptase mediates the PAR2-dependent fi fl fl Fig. 5. P2pal-18S signi cantly attenuates mast cell tryptase-dependent neu- in ammatory responses in this acute in ammation model. It is trophil migration and paw edema in mice. (A) Human mast cells (HMC-1) were possible that the PAR2 pepducin may inhibit signaling induced by treated with 2 mg/mL of mast cell degranulating agent 48/80 or 2% λ-carra- other PAR2 protease agonists present in the inflammatory milieu geenan/4% kaolin. Additionally, conditioned media (CM) was harvested at in addition to tryptase. Indeed, PAR2 has been shown to be ac- 24 h and a Western blot of mast cell tryptase shows release of tryptase. (B) tivated by other proteases, including the TF-FXa-FVIIa complex P2pal-18S inhibits human neutrophil chemotaxis (n = 6) to mast cell media. and membrane-type serine protease 1 (MT-SP1) matriptase (35). Human neutrophils were incubated with 1 μM P2pal-18S, or 10 μMmastcell Several studies indicate that it may not always be advantageous tryptase inhibitor APC-366 and allowed to migrate 30 min toward CM from to inhibit PAR2 (21, 36, 37). For example, PAR2 agonists can mast cells. (C) C57BL/6 mice (n = 5) were pretreated with the tryptase inhibitor APC-366 (5 mg/kg, s.c.) or vehicle (20% DMSO) and then challenged with provide physiologic protective responses against bronchocon- intraplantar injection of λ-carrageenan/kaolin. (D) Mast cell-conditioned media striction in rat models (36), and PAR2 has been shown to be es- (30 μLofλ-carrageenan/kaolin–stimulated media) was injected into the hind- sential for the late protective effects of PAR1 agonism in mouse paws of C57BL/6 mice treated with 10 mg/kg P2pal-18S or vehicle (n =5).Data models of cecal ligation and puncture sepsis (21). Additionally, represent mean ± SEM. #P =0.07,*P < 0.05, and **P < 0.005.

Sevigny et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 severe defects in their neutrophil and macrophage phagocytic ef- which will aid in the delineation of complex mechanisms of ficiency, suggesting that PAR2 may also confer protective effects GPCR signaling and pathophysiology and may lead to novel in the host response to bacterial pathogens (37). pharmacological agents in a potentially wide range of diseases. Two other groups have disclosed PAR2 antagonists on the basis of the tethered peptide ligand (4, 38). A PAR2 small mol- Materials and Methods ecule inhibitor, ENMD-1068, was tested in a model of joint in- Detailed methods, including PAR2 structural modeling, reagents, cell culture, fl ammation (4). ENMD-1068 requires millimolar concentrations PAR surface expression analysis, transwell migration, mouse paw edema to observe its protective effects in vitro and considerably higher models, neutrophil chemotaxis, and statistical analyses, appear in SI Materials doses in vivo. The peptide antagonist K-14585 was shown to in- and Methods. All animal experiments were performed in accordance with κ hibit PAR2-dependent IL-8 production, NF- B phosphorylation, the National Institutes of Health guidelines and approved by Tufts University and p38 signaling (19). However, the K-14585 compound has Institutional Animal Care and Use Committee. Neutrophils were obtained partial agonist activity (19, 38), as also observed with the wild- from the peripheral blood of healthy volunteers using informed consent type PAR2 pepducin P2pal-21 (20). In this regard, we discovered procedures approved by the institutional review board of Tufts Medical that wild-type PAR2 has constitutive activity, indicating that Center. Statistical significance was defined as *P < 0.05 or **P < 0.005. certain extracellular or intracellular PAR2 ligands might stablize the latent on state. The realization that constitutive activity could ACKNOWLEDGMENTS. We thank Dr. J. H. Butterfield for providing the HMC- be ablated or enhanced by mutation of critical i3 loop pharma- 1 cells; Dr. Martin Beinborn for the COS7 cells; the Rosenblatt laboratory for cophores in the intact receptor led us to rationally design PAR2 the HEK cells; George Koukos for technical assistance; and Katie O’Calla- ghan, Anika Agarwal, Caitlin Foley, and Nga Nguyen for their expert advice. pepducin antagonists that lost residual agonist activity. The ability This work was funded in part by Grants R01 HL-57905, R01 HL-64701, and to design pepducin antagonists against difficult GPCR targets R01 CA-122992 from the National Institutes of Health (to A.K.) and R01 CA- such as PAR2 is a valuable research and therapeutic approach, 104406 (to L.C.).

1. Vergnolle N, Hollenberg MD, Sharkey KA, Wallace JL (1999) Characterization of the 21. Kaneider NC, et al. (2007) ‘Role reversal’ for the receptor PAR1 in sepsis-induced inflammatory response to proteinase-activated receptor-2 (PAR2)-activating peptides vascular damage. Nat Immunol 8:1303–1312. in the rat paw. Br J Pharmacol 127:1083–1090. 22. Covic L, Tchernychev B, Jacques S, Kuliopulos A (2007) Handbook of Cell-Penetrating 2. Schmidlin F, et al. (2002) Protease-activated receptor 2 mediates eosinophil infiltration Peptides, ed Langel U (Taylor and Francis, Boca Raton), pp 245–257. and hyperreactivity in allergic inflammation of the airway. J Immunol 169:5315–5321. 23. Wielders SJ, Bennaghmouch A, Reutelingsperger CP, Bevers EM, Lindhout T (2007) 3. Vergnolle N, et al. (2001) Proteinase-activated receptor-2 and hyperalgesia: A novel Anticoagulant and antithrombotic properties of intracellular protease-activated pain pathway. Nat Med 7:821–826. receptor antagonists. J Thromb Haemost 5:571–576. 4. Kelso EB, et al. (2006) Therapeutic promise of proteinase-activated receptor-2 24. Tressel SL, et al. (2011) Pharmacology, biodistribution, and efficacy of GPCR-based antagonism in joint inflammation. J Pharmacol Exp Ther 316:1017–1024. pepducins in disease models. Methods Mol Biol 683:259–275. 5. Shi X, Gangadharan B, Brass LF, Ruf W, Mueller BM (2004) Protease-activated receptors 25. Leger AJ, et al. (2006) Blocking the protease-activated receptor 1-4 heterodimer in – (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol Cancer Res 2: platelet-mediated thrombosis. Circulation 113:1244 1254. 395–402. 26. Teller DC, Okada T, Behnke CA, Palczewski K, Stenkamp RE (2001) Advances in 6. Kaneider NC, Agarwal A, Leger AJ, Kuliopulos A (2005) Reversing systemic inflammatory determination of a high-resolution three-dimensional structure of rhodopsin, – response syndrome with chemokine receptor pepducins. Nat Med 11:661–665. a model of G-protein-coupled receptors (GPCRs). Biochemistry 40:7761 7772. 7. Nystedt S, Emilsson K, Wahlestedt C, Sundelin J (1994) Molecular cloning of a potential 27. Kjelsberg MA, Cotecchia S, Ostrowski J, Caron MG, Lefkowitz RJ (1992) Constitutive proteinase activated receptor. Proc Natl Acad Sci USA 91:9208–9212. activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single 8. Boire A, et al. (2005) PAR1 is a matrix metalloprotease-1 receptor that promotes site. Evidence for a region which constrains receptor activation. JBiolChem267: 1430–1433. invasion and tumorigenesis of breast cancer cells. Cell 120:303–313. 28. DeFea KA, et al. (2000) beta-arrestin-dependent endocytosis of proteinase-activated 9. Trivedi V, et al. (2009) Platelet matrix metalloprotease-1 mediates thrombogenesis by receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol 148: activating PAR1 at a cryptic ligand site. Cell 137:332–343. 1267–1281. 10. Ossovskaya VS, Bunnett NW (2004) Protease-activated receptors: Contribution to 29. Roosterman D, Schmidlin F, Bunnett NW (2003) Rab5a and rab11a mediate agonist- physiology and disease. Physiol Rev 84:579–621. induced trafficking of protease-activated receptor 2. Am J Physiol Cell Physiol 284: 11. Noorbakhsh F, et al. (2006) Proteinase-activated receptor2 modulates neuroinflammation C1319–C1329. in experimental autoimmune encephalomyelitis and multiple sclerosis. JExpMed203: 30. Stalheim L, et al. (2005) Multiple independent functions of arrestins in the regulation 425–435. of protease-activated receptor-2 signaling and trafficking. Mol Pharmacol 67:78–87. 12. Cenac N, et al. (2002) Induction of intestinal inflammation in mouse by activation of 31. Damiano BP, et al. (1999) Cardiovascular responses mediated by protease-activated proteinase-activated receptor-2. Am J Pathol 161:1903–1915. receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient 13. Antoniak S, et al. (2010) Protease-activated receptor 2 deficiency reduces cardiac in PAR-2 or PAR-1. J Pharmacol Exp Ther 288:671–678. ischemia/reperfusion injury. Arterioscler Thromb Vasc Biol 30:2136–2142. 32. Day SM, Lockhart JC, Ferrell WR, McLean JS (2004) Divergent roles of nitrergic and 14. Lam DK, Schmidt BL (2010) Serine proteases and protease-activated receptor 2- prostanoid pathways in chronic joint inflammation. Ann Rheum Dis 63:1564–1570. – dependent allodynia: A novel cancer pain pathway. Pain 149:263 272. 33. Palmer HS, et al. (2007) Protease-activated receptor 2 mediates the proinflammatory 15. Ferrell WR, et al. (2003) Essential role for proteinase-activated receptor-2 in arthritis. effects of synovial mast cells. Arthritis Rheum 56:3532–3540. – J Clin Invest 111:35 41. 34. Carvalho M, Benjamim C, Santos F, Ferreira S, Cunha F (2005) Effect of mast cells 16. Ferrell WR, Kelso EB, Lockhart JC, Plevin R, McInnes IB (2010) Protease-activated depletion on the failure of neutrophil migration during sepsis. Eur J Pharmacol 525: receptor 2: A novel pathogenic pathway in a murine model of osteoarthritis. Ann 161–169. – Rheum Dis 69:2051 2054. 35. Takeuchi T, et al. (2000) Cellular localization of membrane-type serine protease 1 and 17. Molino M, et al. (1997) Interactions of mast cell tryptase with thrombin receptors and identification of protease-activated receptor-2 and single-chain urokinase-type PAR-2. J Biol Chem 272:4043–4049. plasminogen activator as substrates. J Biol Chem 275:26333–26342. 18. Caughey GH (2007) Mast cell tryptases and chymases in inflammation and host 36. Cocks TM, et al. (1999) A protective role for protease-activated receptors in the defense. Immunol Rev 217:141–154. airways. Nature 398:156–160. 19. Goh FG, Ng PY, Nilsson M, Kanke T, Plevin R (2009) Dual effect of the novel peptide 37. Moraes TJ, et al. (2008) Role of PAR2 in murine pulmonary pseudomonal infection. antagonist K-14585 on proteinase-activated receptor-2-mediated signalling. Br J Am J Physiol Lung Cell Mol Physiol 294:L368–L377. Pharmacol 158:1695–1704. 38. Kanke T, et al. (2009) Novel antagonists for proteinase-activated receptor 2: Inhibition 20. Covic L, Gresser AL, Talavera J, Swift S, Kuliopulos A (2002) Activation and inhibition of cellular and vascular responses in vitro and in vivo. Br J Pharmacol 158:361–371. of G protein-coupled receptors by cell-penetrating membrane-tethered peptides. 39. Swift S, et al. (2010) A novel protease-activated receptor-1 interactor, Bicaudal D1, Proc Natl Acad Sci USA 99:643–648. regulates G protein signaling and internalization. J Biol Chem 285:11402–11410.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1017091108 Sevigny et al. Downloaded by guest on September 25, 2021