The C3a receptor antagonist SB 290157 has agonist activity
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 Gi-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 Th2-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 Th2-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 Th2-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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