pubs.acs.org/jmc Published on Web 09/28/2010 r 2010 American Chemical Society 7428 J. Med. Chem. 2010, 53, 7428–7440 DOI: 10.1021/jm100984y Novel Agonists and Antagonists for Human Protease Activated Receptor 2 Grant D. Barry, † Jacky Y. Suen, † Giang T. Le, Adam Cotterell, Robert C. Reid, and David P. Fairlie* Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane Qld 4072, Australia. † Joint first authors. Received July 31, 2010 Human protease activated receptor 2 (PAR2) is a G protein-coupled receptor that is associated with inflammatory diseases and cancers. PAR2 is activated by serine proteases that cleave its N-terminus and by synthetic peptides corresponding to the new N-terminus. Peptide agonists are widely used to characterize physiological roles for PAR2 but typically have low potency (e.g., SLIGKV-NH 2 , SLIGRL-NH 2 ), uncertain target selectivity, and poor bioavailability, limiting their usefulness for specifically interrogating PAR2 in vivo. Structure-activity relationships were used to derive new PAR2 agonists and antagonists containing nonpeptidic moieties. Agonist GB110 (19, EC 50 0.28 μM) selectively induced PAR2-, but not PAR1-, mediated intracellular Ca 2þ release in HT29 human colorectal carcinoma cells. Antagonist GB83 (36, IC 50 2 μM) is the first compound at micromolar concentrations to reversibly inhibit PAR2 activation by both proteases and other PAR2 agonists (e.g., trypsin, 2f-furoyl-LIGRLO-NH 2 , 19). The new compounds are selective for PAR2 over PAR1, serum stable, and suitable for modulating PAR2 in disease models. Introduction Among the most unusual of almost 1000 known human G protein-coupled receptors (GPCRs a ) 1,2 are four protease acti- vated receptors (PARs), 3,4 which have no known endogenous extracellular ligands. Instead, serine proteases indirectly activate at least four PAR subtypes by cleaving their N-terminus, exposing a new extracellular N-terminus that folds back onto and intramolecularly self-activates PAR. 5 Research on PARs has focused on PAR1 because it is a receptor for the serine protease thrombin, and thus a potential therapeutic target in cardiovascular disease. 3 PAR2 is not activated by thrombin, but it is activated by other serine proteases (e.g., trypsin, tryptase, cathepsin G) and has been linked to inflammatory and prolif- erative disorders. 3-7 Most PAR2 research has attempted to define physiological and pathophysiological roles for this receptor. Distributed widely throughout the body, PAR2 signals through intracellular G proteins 3,8 associated with release of intracellular Ca 2þ via phospholipase C activation and inositol triphosphate (GR q ), 8 p38 MAP kinase and ERK phosphorylation, as well as adenylyl cyclase inhibition (GR i ), 9-11 and Rho-dependent phagocytosis (GR 12/13 ). 12 PAR2 activation has been linked to proliferation, metastasis, and angiogenesis in many cancers especially of the stomach, 13 colon, 11,14 breast, and pancreas. 11,15-17 PAR2 has been implicated as a pro-inflammatory mediator in arthritis, 18 inflammatory bowel disease, 19,20 pancreatitis, 21-27 and cardio- vascular disease, 28 while it has also been reported as anti- inflammatory and protective in other conditions such as gastric ulcer, 29 colitis, 30 asthma, 27,31-33 and liver fibrosis. 30,34-36 Clearly, these roles are not yet well understood. 22,24-26,37 PAR2 -/- deficient mice show delayed appearance and decreased development of mammary adenocarcinoma 38 and abolished revascularization in hypoxia-induced angiogenesis. 39 In inflammation, PAR2 knockouts had reduced dendritic cell trafficking and T cell activation, 40 and higher survival against antiphospholipid syndrome by lowered neutrophil activa- tion. 41 PAR2 deficient mice also had impaired production of IgE and IL-4, 42 reduced contact sensitivity in airway inflammation, 43 resistance to adjuvant-induced arthritis, 18 or delayed onset of inflammation. 44 Roles for PAR2 in inflam- mation were further supported by studies with small interfering RNA (siRNA) for PAR2, which repeatedly showed suppres- sion of innate immunity markers, such as IL8, 40,45 CXCL5, and CCL20. 46 By contrast, PAR2 activation attenuated pancreatitis-related hyperalgesia, 47 and thus, the role of PAR2 could be cell type-specific. This is supported by a *To whom correspondence should be addressed. Telephone: þ61- 733462989. Fax: þ61 733462990. E-mail: d.fairlie@imb.uq.edu.au. a Abbreviations: ACN, acetonitrile; Boc, tert-butoxycarbonyl; BOP, (benzotriazol-1-yloxy)-tris(dimethylamino) phosphonium hexafluoro- phosphate; Cha, cyclohexylalanine; DCM, dichloromethane; DIC, 1,3- diisopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; DTPM, 1,3-dimethyl- 5-[(dimethylamino)methylene]-2,4,6-(1H,3H,5H)-trioxopyrimidine; EC 50 , molar concentration that produces 50% of the maximum response of an agonist; ERK, extracellular signal-regulated kinase; ESMS, electrospray mass spectroscopy; FCS, fetal calf serum; Fmoc, 9H-fluoren-9-ylmethoxy- carbonyl; GI, gastrointestinal, G protein, guanosine monophosphate- protein; GPCR, G protein-coupled receptor; HBSS, Hank’s balanced salt solution; HEK293, human embryonic kidney 293 cells; HBTU, O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro- phosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); HOBt, 1-hydroxybenzotriazole; HT29, human colon adenocar- cinoma grade II cells; HRMS, high resolution mass spectroscopy; IC 50 , molar concentration of an antagonist that inhibits 50% of a known concentration of agonist activity; IgE, immunoglobulin E; IL, interleukin; IMDM, Iscove’s modified Dulbucco’s medium; iCa 2þ , intracellular Ca 2þ ; MAP kinase, mitogen-activated protein kinase; MBHA, methylbenzhy- drylamine; PAR, protease activated receptor; rpHPLC, reversed phase high performance liquid chromatography; SAR, structure-activity relation- ships; siRNA, small interfering ribonucleic acid; SEM, standard error of mean; SPPS, solid phase peptide synthesis; TFA, trifluoroacetic acid; TIPS, triisopropylsilane; TCP, tritylchloride-polystyrene.