Species selectivity of a small molecule antagonist for the CCR1 chemokine receptor

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Abstract

The species specificity of a small molecule antagonist for the human CCR1 chemokine receptor, 2-2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile (CCR1 antagonist 1), has been examined using cloned CCR1 receptors from various species. The compound was able to bind to rabbit, marmoset, and human CCR1, and was able to block the functional activation of these receptors. However, it failed to significantly displace radiolabeled macrophage inflammatory protein-1α (MIP-1α) binding to mouse CCR1 at concentrations up to 10 μM. These data suggested that the antagonist binding site is well-conserved in rabbit, marmoset and human CCR1, but not in mouse CCR1. The functional selectivity and mechanism of action for CCR1 antagonist 1 were further characterized. CCR1 antagonist 1 blocked the increase in intracellular Ca2+ stimulated by CCR1 agonists, but had no effect on N-formyl-Met–Leu–Phe (FMLP), monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor 1α (SDF1α)-induced Ca2+ mobilization, demonstrating functional selectivity for CCR1. Since CCR1 antagonist 1 is a functional antagonist of marmoset and rabbit CCR1 receptors, it should be possible to test its efficacy in animal models of disease.

Introduction

The chemokines are a group of small proteins that play an important role in leukocyte-trafficking during immune response Schall, 1994, Baggiolini, 1998. They can be classified into four groups, CC, CXC, C, and CX3C, according to the positions of the cysteines at the N-terminus of the molecule (Schall, 1994). The chemokines produce their biological effects by activating specific receptor proteins on their target cells (Horuk, 1994). To date, a total of 16 chemokine receptors, including nine CC chemokine receptors and five CXC chemokine receptors, have been identified Mackay, 1996, Luster, 1998. All of these receptors belong to a G-protein-coupled receptor superfamily (Dohlman et al., 1991).

Chemokines have been implicated in the pathogenesis of chronic inflammatory diseases, such as multiple sclerosis and rheumatoid arthritis. For example, expression of RANTES and macrophage inflammatory protein-1α (MIP-1α) correlated with the disease onset in a mouse experimental autoimmune encephalomyelitis model of multiple sclerosis (Godiska et al., 1995). A recent study by Karpus et al. (1995) demonstrated that antibodies to MIP-1α prevented the development of both acute and relapsing paralytic disease as well as infiltration of mononuclear cells into the central nervous system. Treatment with MIP-1α antibody was also able to ameliorate the severity of ongoing clinical disease. These results led the authors to conclude that MIP-1α plays an important role in this T-cell-mediated disease.

A number of studies have also suggested a role for RANTES in rheumatoid arthritis. Both RANTES mRNA and protein appear to be upregulated in rheumatoid arthritis Rathanaswami et al., 1993, Snowden et al., 1994. Antibodies against RANTES significantly decreased the severity of ongoing clinical disease in a rat adjuvant-induced rheumatoid arthritis model (Barnes et al., 1998).

These studies provided strong evidence for an important role of the chemokines, RANTES, and MIP-1α in chronic inflammatory diseases. Since MIP-1α and RANTES are ligands for CCR1, antagonists for this receptor may prove to be useful in treatment of these diseases. In our previous report, we identified a novel series of small molecule antagonists for human CCR1 receptor. In this study, we further characterize the pharmacological properties of one of these compounds, 2-2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile (CCR1 antagonist 1), report the cloning of CCR1 receptor from marmoset and rabbit, and discuss the selectivity of CCR1 antagonist 1 for CCR1 receptors from a number of animal species. The study will provide useful information for testing the efficacy of CCR1 antagonists in animal disease models in vivo.

Section snippets

Materials

Unlabeled chemokines were from Peprotech (Rocky Hill, NJ). 125I-labeled chemokines were obtained from New England Nuclear (Boston, MA).

Synthesis of compounds

CCR1 antagonist 1 was synthesized as previously described (Hesselgesser et al., 1998).

Marmoset CCR1 gene cloning

Previous studies had shown that the mouse and human CCR1 genes are highly conserved and contain no introns Gao et al., 1993, Neote et al., 1993. A number of polymerase chain reaction (PCR) primers were synthesized based on the 5′ and 3′ ends of the human coding region. In

Cloning of CCR1 receptors from rabbit and marmoset

We have previously identified and described a novel CCR1 antagonist, (2-2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile), designated CCR1 antagonist 1. In order to examine the species selectivity of this antagonist, CCR1 receptors were cloned from rabbit and marmoset. As expected, these receptors were highly homologous (Table 1 and Fig. 1). However, as shown in Fig. 1, the rabbit receptor is missing 12 nucleotides corresponding to four amino acids in the cytoplasmic tail region of the

Discussion

CCR1 could be a therapeutic target for autoimmune diseases like multiple sclerosis and rheumatoid arthritis since its ligands, MIP-1α and RANTES, have been implicated in these diseases Karpus and Kennedy, 1997, Karpus et al., 1995, Plater-Zyberk et al., 1997, Barnes et al., 1998. Thus, a potent and selective CCR1 antagonist could be useful as a potential therapy. We have previously identified and described a novel CCR1 antagonist, CCR1 antagonist 1 (Hesselgesser et al., 1998). This compound was

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    Current address: Coulter Pharmaceutical, South San Francisco, CA 94080, USA.

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