Novel agonists & antagonists for protease-activated receptor 2 and C3a receptor

Abstract

About 30-40% of all drugs currently in the market place target G-protein coupled receptors, however of the 900 plus GPCRs predicted to exist only about 30 are targeted by approved drugs. Two novel human GPCRs were investigated in this thesis, namely protease-activated receptor 2 (PAR2) and C3a receptor (C3aR). These membranebound receptors are implicated in a variety of diseases in which inflammation is a common component, making them attractive targets for drugs that can treat such disorders. This thesis describes the rational design, synthesis and in vitro assay of ligands targeting these two GPCRs and they will become useful probes for investigating the pathology of inflammatory disorders and may lead to possible future treatments.Chapter 1 of this thesis summarises peptidic and non-peptidic ligands for proteaseactivated receptor and C3a receptor reported in the literature to date, together with their known physiological effects.Chapter 2 examines structure-activity relationships for analogues of the only known potent PAR2 antagonist (GB88) discovered at IMB. The chemical features of GB88 were investigated separately by dividing the molecule into fragments, which were independently varied to study the effect of structure on agonist/antagonist function of the ligands. A potent and selective PAR2 agonist (132) was developed with EC 50 of 0.4 µM in calcium release assays on HT29 cells. Agonist 132 has comparable agonist potency and better ligand efficiency to 2f-LIGRLO-NH 2 , which was previously the most potent PAR2 agonist prior to these studies. In addition, a new PAR2 antagonist was developed (167), which was 2-fold more potent than GB88 in the calcium mobilisation assay.Chapter 3 describes the non-peptidic PAR2 agonist (GB110) and SAR studies for the truncation of this compound that led to the development of new PAR2 antagonists (192, 209, 211, 212). These ligands inhibited the activation of PAR2 induced by 1 µM 2f-LIGRLO-NH 2 with IC 50 0.4-0.6 µM in the calcium release assay.Chapter 4 evaluates the anti-inflammatory properties of newly developed PAR2antagonists (133, 159, 167, 192 -from Chapters 2 & 3) in both in vivo and in vitro experiments. The most potent PAR2 antagonists (167 and 192) were stable in rat plasma and rat liver homogenates and were able to inhibit PAR2 activation induced by peptide agonist 2f-LIGRLO-NH 2 as well as the endogenous activator, trypsin. These iv antagonists have been demonstrated to be anti-inflammatory agents, inhibiting proinflammatory cytokine release (IL6 and TNF-α) and were effective in relieving joint inflammation in rat models of arthritis.Chapter 5 explores various aromatic heterocycles that confer a turn conformation to ligands and this mimics the turn conformation observed for the C-terminus of C3a in its crystal structure. The study investigates how the hydrogen bonding capabilities of heteroatoms in each heterocycle affect the binding and functions of the ligands.Increasing the hydrogen bond acceptor properties of the heterocycle improves binding affinity...

Toward Drugs for Protease-Activated Receptor 2 (PAR2)

“…Both the nitrogen and oxygen atoms of the isoxazole are thought to be hydrogen bond acceptors for residues in PAR2. The cyclohexylalanine conferred PAR2 selectivity over PAR1; phenylalanine at this position did not discriminate between PAR1 and PAR2 . The isoleucine was important for binding affinity.…”

“…In addition, an aromatic ring at the C-terminus of the ligand helped to maintain antagonist activity, possibly due to a π-stacking interaction with the receptor. These two PAR2 antagonists ( 32 and 33 ), with enhanced potency over 22 , inhibited PAR2 activation induced by either the PAR2 peptide agonist 2f-LIGRLO-NH 2 (IC 50 0.7 and 0.4 μM, respectively) or trypsin (IC 50 3.6 and 1.9 μM, respectively) in the calcium mobilization assay using HT29 cells and also blocked release of proinflammatory cytokines (IL-6, TNFα) in primary human kidney tubule epithelial cells (HTEC) . These antagonists were stable in rat plasma, with 32 being more orally bioavailable than 33 and more effective in preventing joint inflammation in rats.…”

“…These two PAR2 antagonists (32 and 33), with enhanced potency over 22, inhibited PAR2 activation induced by either the PAR2 peptide agonist 2f-LIGRLO-NH 2 (IC 50 0.7 and 0.4 μM, respectively) or trypsin (IC 50 3.6 and 1.9 μM, respectively) in the calcium mobilization assay using HT29 cells and also blocked release of proinflammatory cytokines (IL-6, TNFα) in primary human kidney tubule epithelial cells (HTEC). 200 These antagonists were stable in rat plasma, with 32 being more orally bioavailable than 33 and more effective in preventing joint inflammation in rats. However, 33 was a more efficient anti-inflammatory agent than 22 and 32 in the rat paw edema model when the compounds were given to rats at 5 mg/kg, sc (WO2013013273).…”

“…The cyclohexylalanine conferred PAR2 selectivity over PAR1; phenylalanine at this position did not discriminate between PAR1 and PAR2. 200 The isoleucine was important for binding affinity. The C-terminal component dictated antagonist versus agonist function.…”

“…However, 33 was a more efficient anti-inflammatory agent than 22 and 32 in the rat paw edema model when the compounds were given to rats at 5 mg/kg, sc (WO2013013273). 200 In summary, compounds 22, 32, and 33 are the most effective small molecule PAR2 antagonists published to date with antiinflammatory properties in vivo. These PAR2 antagonists offer new insights to PAR2 functions in vitro in signaling pathways and in vivo in diverse PAR2-mediated diseases.…”