Identification of a Selective Nonpeptide Antagonist of the Anaphylatoxin C3a Receptor That Demonstrates Antiinflammatory Activity in Animal Models

Abstract

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

Comparative anti‐inflammatory activities of antagonists to C3a and C5a receptors in a rat model of intestinal ischaemia/reperfusion injury

“…This compound, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine, antagonised C3a-induced calcium mobilisation, smooth muscle contraction and attenuated paw oedema in a rat model of adjuvant-induced arthritis (Ames et al, 2001). Given the in vivo anti-inflammatory activities reported, and because to date the effects of this C3aRA have only been reported in one in vitro study (Mollnes et al, 2002), we decided to investigate its pharmacology in more detail and compare its actions with a small molecule C5aRA, which has been well characterised (Strachan et al, 2000;Arumugam et al, 2002;Woodruff et al, 2002;.…”

“…A previous study (Ames et al, 2001) reported the discovery of a nonpeptidic C3aRA with high affinity for the human C3aR. This compound, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine, antagonised C3a-induced calcium mobilisation, smooth muscle contraction and attenuated paw oedema in a rat model of adjuvant-induced arthritis (Ames et al, 2001).…”

“…The C3aRA significantly attenuates paw oedema in a rat model of adjuvant-induced arthritis (Ames et al, 2001). This effect was observed when the C3aRA was administered at the high dose of 30 mg kg À1 i.p.…”

“…This situation has perhaps been, in part, due to the lack of potent and selective C3a receptor agonists and antagonists. Recently, Ames et al (2001) have reported the discovery of a C3a receptor antagonist (C3aRA) that selectively blocked the C3a receptor (C3aR) and C3a activity in vitro and in vivo, but did not inhibit Ca 2 þ mobilisation in response to C5a, leukotriene B 4 , formyl-MetLeu-Pro, platelet activating factor or chemokine (CXCR1 and CXCR2) activation of neutrophils.…”

Efficient chemical synthesis of human complement protein C3a

“…2 Currently, C3a is 20 commercially produced in relatively low yields by biological means (recombinant expression or purification from plasma) 3, 4 resulting in a market value of approximately US$ 5000 per mg of protein. In addition, these approaches often require protein purification tags and additional steps for their removal and suffer 25 from the inherent lability of C3a in biological fluids. C3a is rapidly inactivated within seconds by carboxypeptidase-mediated cleavage of a single C-terminal arginine residue (Arg 77 ).…”