The C3a receptor antagonist SB 290157 has agonist activity

doi:10.1016/j.imlet.2005.03.003

Immunology Letters

Volume 100, Issue 2, 15 September 2005, Pages 139-145

https://doi.org/10.1016/j.imlet.2005.03.003 Get rights and content

Abstract

The anaphylatoxin C3a is an important immune regulator with a number of distinct functions in both innate and adaptive immunity. Many of these roles have been ascribed to C3a based on studies in mice genetically modified to lack its precursor, C3, or its receptor, C3aR. However, other presumed functions of C3a are based on results obtained with a recently described small molecule ligand of C3aR, SB 290157. Although this compound was originally described as an antagonist and appears to act as such in some systems, it has recently been shown to have effects that cannot be explained by simple antagonism of C3aR. In the current study, SB 290157 is shown to have full agonist activity on C3aR in a variety of cell systems, including a calcium mobilization assay in transfected RBL cells, a β-lactamase assay in CHO-NFAT-bla-Gα₁₆ cells and an enzyme-release assay in differentiated U-937 cells. On the other hand, the compound lacks agonist activity in guinea pig platelets, cells known to express C3aR at very low levels. SB 290157 agonism of C3aR is consistent with recent discrepant data obtained using this molecule. These results caution against attributing novel roles to C3a based on data obtained with SB 290157 and highlight a continuing need for the identification of true small molecule C3aR antagonists.

Introduction

Complement factor 3a (C3a) is a 77-amino acid peptide produced following activation of the complement cascade. Specifically, it is derived from C3 upon cleavage of this protein by C3 convertase, the central mediator of the complement cascade [1]. Some of the cellular and physiological events associated with C3a include contraction of smooth muscle, chemotaxis and activation of leukocytes, primarily mast cells and eosinophils, and an increase in vascular permeability [1], [2]. The functional receptor for C3a, C3aR, is a predominantly G_i-coupled GPCR, the binding kinetics and downstream mechanisms of which have been studied in a number of cellular systems [3], [4], [5], [6], [7].

Although it has been described as having broad pro-inflammatory effects, the main role of C3a appears to be in T_h2-type inflammatory reactions [2], [8], [9], [10], [11]. Consistent with this, a large body of evidence has emerged over the last few years implicating C3a in allergic asthma. These data include: (i) a complete abrogation of airway hyper-reactivity upon allergen-challenge in mice lacking C3 or C3aR [8], [9], [10], [12], (ii) an upregulation of C3 and C3aR mRNA [13], as well as C3aR protein [14] in ovalbumin-sensitized and -challenged mice, (iii) increased C3a levels in bronchoalveolar lavage fluid and serum of asthmatics upon allergen challenge [10], [15] or during exacerbations [16], (iv) elevated C3aR levels in patients with fatal asthma (Fregonese et al., 99th American Thoracic Society International Conference, 2003) and (v) genetic linkage of the C3 and C3aR genes with asthma [17], [18].

One of the difficulties in evaluating the role of C3a in animal models of asthma has been the lack of a potent, orally available small molecule C3aR antagonist. Recently, Ames et al. [19] described just such an antagonist, SB 290157. The arginine-like molecule was shown to compete with C3a binding to C3aR, expressed in RBL cells, as well as inhibit C3a-induced calcium mobilization in these cells and in human neutrophils (with IC₅₀ values ranging from 28 to 200 nM for the human enzyme). The molecule also inhibits chemotaxis of HMC-1 cells and ATP-release from guinea pig platelets. In addition, SB 290157 was found to be active in two animal models of inflammation, a guinea pig LPS-induced lung neutrophilia model, and a rat adjuvant-induced arthritis model. SB 290157 has since been used in a number of in vitro and in vivo studies [20], [21], [22], [23], [24] with sometimes unexpected results [20], [23]. We wished to further characterize SB 290157 with the goal of evaluating its effects in T_h2-driven in vivo models. Surprisingly, we found that SB 290157 has full agonist activity in a variety of cell systems, particularly those with a relatively high receptor density. These results reconcile the discordant results obtained in vivo with SB 290157 and serve to caution against use of this compound as a C3aR antagonist.

Section snippets

DNA constructs and cell lines

The genes for human C3aR (hC3aR; GenBank Accession Number: NM_004054) and mouse C3aR (mC3aR; GenBank Accession Number: NM_009779) were amplified by polymerase chain reaction from Marathon-ready cDNA libraries (BD Biosciences, San Jose, CA, USA) or full-length cDNA clones (Invitrogen, Carlsbad, CA, USA). The gene for the cynomolgus monkey C3aR (cC3aR; GenBank Accession Number: AY426336) was amplified using primers corresponding to the human sequence flanking the coding region, and from a

Results

The compound SB 290157 has been described as a potent small molecule antagonist of C3aR suitable for use in animal models of inflammation [19]. We synthesized SB 290157 and characterized its binding affinity to human C3aR on membranes of rat basophil leukemia (RBL) cells transfected with hC3aR (RBL-hC3aR), a similar system to that used by Ames et al [19]. In saturation binding analyses (not shown), maximal specific binding of [¹²⁵I]-C3a to membranes of these cells was determined to be 101 ± 86

Discussion

Although studies on C3- and C3aR-null mice have provided strong evidence of a role of C3a in T_h2-driven diseases, use of a small molecule antagonist of C3aR would allow for demonstration of such a role in the absence of developmentally derived issues that can arise with genetically modified mice. The publication of SB 290157 [19] provided the tool necessary for such investigations. However, Proctor et al. [23] and Baelder et al. [20] recently observed in vivo effects of SB 290157 that are

Acknowledgments

We thank Drs. Christopher M. Tan, Marlene A. Jacobson (Merck Research Laboratories) and André deLéan (University of Montreal) for helpful discussions, as well as Rino Stocco and Ken McDonald (Merck Research Laboratories) for experimental contributions.

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The C3a receptor antagonist SB 290157 has agonist activity

Abstract

Introduction

Section snippets

DNA constructs and cell lines

Results

Discussion

Acknowledgments

Curr Opin Immunol

Mol Immunol

J Biol Chem

Biochim Biophys Acta

J Allergy Clin Immunol

Blood

Blood

Trends Pharmacol Sci

Immunopharmacology

Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from differentiated U-937 cells

Eur J Immunol

Complement fragment C3a stimulates Ca2+ influx in neutrophils via a pertussis-toxin-sensitive G protein

Eur J Biochem

Complement c3a and c5a induce different signal transduction cascades in endothelial cells

J Immunol

Absence of the complement anaphylatoxin C3a receptor suppresses Th2 effector functions in a murine model of pulmonary allergy

J Immunol

Cutting edge: the absence of C3 demonstrates a role for complement in Th2 effector functions in a murine model of pulmonary allergy

J Immunol

A role for the C3a anaphylatoxin receptor in the effector phase of asthma

Nature

Lack of C3 affects Th2 response development and the sequelae of chemotherapy in schistosomiasis

J Immunol