Identification and Characterization of Small Molecule Functional Antagonists of the CCR1 Chemokine Receptor

Abstract

The CC chemokines macrophage inflammatory protein-1␣ (MIP-1␣) and RANTES (regulated on activation normal T cell expressed) have been implicated in rheumatoid arthritis and multiple sclerosis. Since their effects are mediated through the CCR1 chemokine receptor, we set up a small molecule CCR1 antagonist program to search for inhibitors. Through high capacity screening we discovered a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here. Scatchard analysis of the competition binding data revealed that the compounds had K i values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitrite), was further evaluated. The directed migration of select populations of leukocytes from the circulation to sites of inflammation is an integral part of the immune response. The chemokines are a diverse group of proteins that play an important role in this process (1). They are classified into two major groups, CXC and CC, based on the position of the first two of their four invariant cysteines (2). Each of the chemokines recognizes and induces the chemotaxis of a particular subset of leukocytes. For example, the CXC chemokines, like IL-8 1 and melonoma growth stimulatory activity, mainly chemoattract and activate neutrophils, in contrast to the CC chemokines, like RANTES and monocyte chemoattractant protein, which preferentially attract T lymphocytes and monocytes and induce their activation by producing changes in cellular morphology, transient increases in cellular calcium concentration, and the up-regulation of surface adhesion proteins.The chemokines produce their biologic effects by interacting with specific receptors on the cell surface of their target cells (3). To date, 14 different chemokine receptors including eight CC chemokine receptors have been identified by cloning (4, 5). All of these receptors are characterized by a heptahelical structure and belong to a superfamily of serpentine receptors that are coupled to guanine nucleotide-binding proteins (G-proteins) (6).Occasionally the immune system can turn upon its host, giving rise to chronic inflammation and disease. Given their important role in this process, chemokines have been implicated in the pathophysiology of autoimmune diseases like multiple sclerosis and rheumatoid arthritis. For example a recent study by Karpus et al. (7) provides strong in vivo concept validation for a role of MIP-1␣ in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. These investigators were able to show that antibodies to MIP-1␣ 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-1␣ antibody was also able to ameliorate the severity of ongoing clinical disease. These results led the authors to conclude that MIP-1␣ plays an important role in this T-cell me...

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“…BX471 given from day 0-5 had no effect. * P < 0.05. the plasma were determined by HPLC (14). As shown in Figure 3, BX471 reached peak plasma levels of 9 µM by around 30 minutes, and this rapidly declined to approximately 0.4 µM after 2 hours.…”

“…HEK cells transfected with murine CCR1 were incubated with 125 I-MIP-1α/CCL3 in the presence of increasing concentrations of BX471. The binding reactions were terminated by centrifugation of cells as described previously (14). Binding shown represents specific binding from a typical experiment (n = 3).…”

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