Adam J. Cotterell

Modulating human proteinase activated receptor 2 with a novel antagonist (GB88) and agonist (GB110)

BACKGROUND AND PURPOSE Many cells express proteinase activated receptor 2 (PAR2) on their plasma membrane. PAR2 is activated by proteolytic enzymes, such as trypsin and tryptase that cleave the receptor N‐terminus, inititating signalling to intracellular G proteins. Studies on PAR2 have relied heavily upon activating effects of proteases and peptide agonists that lack stability and bioavailability in vivo.

Novel agonists and antagonists for human protease activated receptor 2.

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(2+) 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.

An antagonist of human protease activated receptor-2 attenuates PAR2 signaling, macrophage activation, mast cell degranulation, and collagen-induced arthritis in rats

Multiple serine proteases exert proinflammatory actions by signaling through protease‐activated receptor‐2 (PAR2) on the cell surface. Although inhibitors of individual proteases are anti‐inflammatory, we sought to discover whether the first potent antagonist of their common target PAR2 might be beneficial in treating chronic arthritis‐like inflammatory disease. Using a fluorescence assay, a novel compound, GB88, was shown to antagonize PAR2‐induced intracellular Ca2+ release in human monocyte‐derived macrophages, being 1000 times more potent than a control compound, ENMD‐1068 (IC50 1.6±0.5 μM vs. 1.2±0.4 mM, respectively). In Wistar rats, GB88 was orally bioavailable (F=55%, Tmax 4 h, Cmax 1.7 μM, 10 mg/kg). GB88 inhibited the acute paw edema induced in Wistar rats by intraplantar λ‐carrageenan or PAR2 agonists 2‐furoyl‐LIGRLO‐NH2 or mast cell β‐tryptase, without inhibiting proteolytic activity of tryptase in vitro. In the chronic collagen‐induced model of arthritis in rats, GB88 (10 mg/kg) was disease modifying and ameliorated pathological and histopathological changes (edema, pannus formation, synovial hyperplasia, collagen degradation, macrophage invasion, mast cell degranulation) compared to untreated arthritic controls. The results suggest that an orally active PAR2 antagonist is effective in treating chronic arthritis in rats through inhibiting macrophage infiltration, mast cell degranulation, and β‐tryptase‐PAR2 signaling in joint inflammation.—Lohman, R.‐J., Cotterell, A. J., Barry, G. D., Liu, L., Suen, J. Y., Vesey, D. A., Fairlie, D. P. An antagonist of human protease activated receptor‐2 attenuates PAR2 signaling, macrophage activation, mast cell degranulation, and collagen‐induced arthritis in rats. FASEB J. 26, 2877–2887 (2012). www.fasebj.org

Antagonism of protease-activated receptor 2 protects against experimental colitis.

Many trypsin-like serine proteases such as β-tryptase are involved in the pathogenesis of colitis and inflammatory bowel diseases. Inhibitors of individual proteases show limited efficacy in treating such conditions, but also probably disrupt digestive and defensive functions of proteases. Here, we investigate whether masking their common target, protease-activated receptor 2 (PAR2), is an effective therapeutic strategy for treating acute and chronic experimental colitis in rats. A novel PAR2 antagonist (5-isoxazoyl-Cha-Ile-spiro[indene-1,4′-piperidine]; GB88) was evaluated for the blockade of intracellular calcium release in colonocytes and anti-inflammatory activity in acute (PAR2 agonist-induced) versus chronic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] models of colitis in Wistar rats. Disease progression (disease activity index, weight loss, and mortality) and postmortem colonic histopathology (inflammation, bowel wall thickness, and myeloperoxidase) were measured. PAR2 and tryptase colocalization were investigated by using immunohistochemistry. GB88 was a more potent antagonist of PAR2 activation in colonocytes than another reported compound, N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD-1068) (IC50 8 μM versus 5 mM). Acute colonic inflammation induced in rats by the PAR2 agonist SLIGRL-NH2 was inhibited by oral administration of GB88 (10 mg/kg) with markedly reduced edema, mucin depletion, PAR2 receptor internalization, and mastocytosis. Chronic TNBS-induced colitis in rats was ameliorated by GB88 (10 mg/kg/day p.o.), which reduced mortality and pathology (including colon obstruction, ulceration, wall thickness, and myeloperoxidase release) more effectively than the clinically used drug sulfasalazine (100 mg/kg/day p.o.). These disease-modifying properties for the PAR2 antagonist in both acute and chronic experimental colitis strongly support a pathogenic role for PAR2 and PAR2-activating proteases and therapeutic potential for PAR2 antagonism in inflammatory diseases of the colon.

Pathway-selective antagonism of proteinase activated receptor 2

Proteinase activated receptor 2 (PAR2) is a GPCR associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signalling associated with disease without inhibiting PAR2 activation in normal physiology could be provided by studies of biased signalling.

Differential anti-inflammatory activity of HDAC inhibitors in human macrophages and rat arthritis

Vorinostat and other inhibitors of different histone deacetylase (HDAC) enzymes are currently being sought to modulate a variety of human conditions, including chronic inflammatory diseases. Some HDAC inhibitors are anti-inflammatory in rodent models of arthritis and colitis, usually at cytotoxic doses that may cause side effects. Here, we investigate the dose-dependent pro- and anti-inflammatory efficacy of two known inhibitors of multiple HDACs, vorinostat and BML281, in human macrophages and in a rat model of collagen-induced arthritis by monitoring effects on disease progression, histopathology, and immunohistochemistry. Both HDAC inhibitors differentially modulated lipopolysaccharide (LPS)-induced cytokine release from human macrophages, suppressing release of some inflammatory mediators (IL12p40, IL6) at low concentrations (<3 µM) but amplifying production of others (TNF, IL1β) at higher concentration (>3 μΜ). This trend translated in vivo to rat arthritis, with anti-inflammatory activity inversely correlating with dose. Both compounds were efficacious only at a low dose (1 mg⋅kg−1⋅day−1 s.c.), whereas a higher dose (5 mg⋅kg−1⋅day−1 s.c.) showed no positive effects on reducing pathology, even showing signs of exacerbating disease. These striking effects suggest a smaller therapeutic window than previously reported for HDAC inhibition in experimental arthritis. The findings support new investigations into repurposing HDAC inhibitors for anti-inflammatory therapeutic applications. However, HDAC inhibitors should be reinvestigated at lower, rather than higher, doses for enhanced efficacy in chronic diseases that require long-term treatment, with careful management of efficacy and long-term safety.

Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1–3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents.

Benzylamide antagonists of protease activated receptor 2 with anti-inflammatory activity

Activation of protease activated receptor 2 (PAR2) has been implicated in inflammatory and metabolic disorders and its inhibition may yield novel therapeutics. Here, we report a series of PAR2 antagonists based on C-terminal capping of 5-isoxazolyl-L-cyclohexylalanine-L-isoleucine, with benzylamine analogues being effective new PAR2 antagonists. 5-Isoxazolyl-L-cyclohexylalanine-L-isoleucine-2-methoxybenzylamine (10) inhibited PAR2-, but not PAR1-, induced release of Ca(2+) (IC50 0.5 μM) in human colon cells, IL-6 and TNFα secretion (IC50 1-5 μM) from human kidney cells, and was anti-inflammatory in acute rat paw inflammation (ED50 5 mg/kg sc). These findings show that new benzylamide antagonists of PAR2 have anti-inflammatory activity.

Helixconstraints and amino acid substitution in GLP-1 increase cAMP and insulin secretion but not beta-arrestin 2 signaling

Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a β-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1-8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9-18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19-28) over GLP-1(7-37)-NH2. The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion.

Mapping transmembrane residues of proteinase activated recpetor 2 (PAR2) that influence ligand-modulated calcium signaling

Graphical abstract Figure. No Caption available. Abstract Proteinase‐activated receptor 2 (PAR2) is a G protein‐coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N‐terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR2, while small organic molecules show promising PAR2 antagonism. Developing PAR2 ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR2. Guided by PAR2 homology modeling and ligand docking based on bovine rhodopsin, followed by cross‐checking with newer PAR2 models based on ORL‐1 and PAR1, site‐directed mutagenesis of PAR2 was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin‐exposed tethered ligand) and one antagonist for modulation of PAR2 signaling. Effects of 28 PAR2 mutations were examined for PAR2‐mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty‐eight PAR2 mutations reduced the potency of at least one ligand by >10‐fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR2, differentially influence intracellular Ca2+ induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR2 TM regions for receptor activation by synthetic PAR2 agonists and antagonists. The trypsin‐cleaved N‐terminus that activates PAR2 was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR2 physiology.