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 membrane bound 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 protease activated 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 EC50 of 0.4 μM in calcium release assays on HT29 cells. Agonist 132 has comparable agonist potency and better ligand efficiency to 2f-LIGRLO-NH2, 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-NH2 with IC50 0.4–0.6 μM in the calcium release assay.
Chapter 4 evaluates the anti-inflammatory properties of newly developed PAR2 antagonists (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-NH2 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 for C3aR as demonstrated by imidazole and thiazole containing a heterocycle nitrogen. A potent and selective C3aR agonist (242, containing an imidazole ring) was generated with agonist potency comparable to the native peptide C3a. On the other hand, replacing the heterocycle with a thiophene ring, containing the non-hydrogen bond accepting sulfur, resulted in a potent C3aR antagonist (233) with IC50 80 nM (calcium mobilisation assay in HMDM).
Chapter 6 investigates various guanidine mimetics and carboxylic acid bioisosteres to replace the basic arginine residue, in order to develop more drug-like agonists and antagonists for C3aR. Of all the guanidine mimetics investigated, the non-basic cyanoguanidine group retained most potency although the binding affinity was lowered by 8-fold. Furthermore the phenylsulfonamide moiety, which is known to be a good carboxylic acid bioisostere, was successfully utilised without significant loss in the potency. These arginine modified compounds, which conform to the reliable predictors of bioavailability such as “Lipinski’s rule of five”, are likely to show improved oral activity, however future studies are required to demonstrate this.
Chapter 7 summarises all the key findings reported in Chapters 2–6 and indicates possible future directions in PAR2 and C3aR research.
These studies have generated some potent agonists and antagonists for PAR2 and C3aR, that are now under patent examination, and have provided us with greater understanding of the structure-activity relationships of these ligands. These compounds will be valuable for interrogating the functions and physiological roles of these receptors in inflammatory diseases as well as other medical disorders.