International Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors.

Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145-176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.

Cell-autonomous regulation of neutrophil migration by the D6 chemokine decoy receptor.

Chemokines, acting on their cognate receptors on infiltrating leukocytes, drive the inflammatory response. We have been interested in determining roles and potential mechanisms for the atypical chemokine-scavenging receptor D6 in the regulation of inflammation. In this study, we show that a psoriasis-like pathology that arises in inflamed skins of D6-deficient mice is characterized by a massive and aberrant localization of neutrophils to the dermal/epidermal junction, which is associated with development of the pathology. Such misplacement of neutrophils is also seen with D6-deficient mice in other inflammatory models, suggesting a role for D6 in the spatial positioning of neutrophils within inflamed sites. We further show that D6 functions cell autonomously in this context and that D6, expressed by neutrophils, limits their migrational responses to CCR1 ligands such as CCL3. Our data therefore indicate that D6 is able to play a cell-autonomous role as a migratory rheostat restricting migration of D6-expressing cells such as neutrophils toward ligands for coexpressed inflammatory chemokine receptors. These data have important implications for our understanding of the roles for D6 in regulating inflammation and for our understanding of the control of spatial positioning of leukocytes at inflamed sites.

The chemokine, CCL3, and its receptor, CCR1, mediate thoracic radiation-induced pulmonary fibrosis.

Patients receiving thoracic radiation often develop pulmonary injury and fibrosis. Currently, there are no effective measures to prevent or treat these conditions. We tested whether blockade of the chemokine, CC chemokine ligand (CCL) 3, and its receptors, CC chemokine receptor (CCR) 1 and CCR5, can prevent radiation-induced lung inflammation and fibrosis. C57BL/6J mice received thoracic radiation, and the interaction of CCL3 with CCR1 or CCR5 was blocked using genetic techniques, or by pharmacologic intervention. Lung inflammation was assessed by histochemical staining of lung tissue and by flow cytometry. Fibrosis was measured by hydroxyproline assays and collagen staining, and lung function was studied by invasive procedures. Irradiated mice lacking CCL3 or its receptor, CCR1, did not develop the lung inflammation, fibrosis, and decline in lung function seen in irradiated wild-type mice. Pharmacologic treatment of wild-type mice with a small molecule inhibitor of CCR1 also prevented lung inflammation and fibrosis. By contrast, mice lacking CCR5 were not protected from radiation-induced injury and fibrosis. The selective interaction of CCL3 with its receptor, CCR1, is critical for radiation-induced lung inflammation and fibrosis, and these conditions can be largely prevented by a small molecule inhibitor of CCR1.

Chemokine receptor CCR1 regulates inflammatory cell infiltration after renal ischemia-reperfusion injury.

Neutrophils and macrophages rapidly infiltrate the kidney after renal ischemia-reperfusion injury, however specific molecular recruitment mechanisms have not been fully delineated for these cell types. Here we provide genetic and pharmacologic evidence supporting a positive role for the chemokine receptor CCR1 in macrophage and neutrophil infiltration in a 7 day mouse model of renal ischemia-reperfusion injury. By day 7, injured kidneys from mice lacking CCR1 contained 35% fewer neutrophils and 45% fewer macrophages than injured kidneys from wild-type control mice. Pretreatment of wild-type mice with the specific CCR1 antagonist BX471 also suppressed neutrophil and macrophage infiltration in the model. Injured kidneys from mice lacking CCR1 also had reduced content of the CCR1 ligands CCL3 (MIP-1alpha) and CCL5 (RANTES) compared with injured kidneys from wild-type controls, suggesting a leukocyte source for these inflammatory chemokines and existence of a CCR1-dependent positive feedback loop for leukocyte infiltration in the model. Local leukocyte proliferation and apoptosis were detected after injury, but were not dependent on CCR1. Also, the extent of necrotic and fibrotic damage and decline in renal function in injured kidneys was similar in wild-type and CCR1-deficient mice. Thus, CCR1 appears to regulate trafficking of macrophages and neutrophils to kidney in a mouse model of renal ischemia-reperfusion injury, however this activity does not appear to affect tissue injury.

CCR5 deficiency aggravates crescentic glomerulonephritis in mice.

The chemokine receptor CCR5 is predominantly expressed on monocytes and Th1-polarized T cells, and plays an important role in T cell and monocyte recruitment in inflammatory diseases. To investigate the functional role of CCR5 in renal inflammation, we induced a T cell-dependent model of glomerulonephritis (nephrotoxic serum nephritis) in CCR5(-/-) mice. Induction of nephritis in wild-type mice resulted in up-regulation of renal mRNA expression of the three CCR5 chemokine ligands, CCL5 (15-fold), CCL3 (4.9-fold), and CCL4 (3.4-fold), in the autologous phase of the disease at day 10. The up-regulated chemokine expression was paralleled by infiltration of monocytes and T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Nephritic CCR5(-/-) mice showed a 3- to 4-fold increased renal expression of CCL5 (61.6-fold vs controls) and CCL3 (14.1-fold vs controls), but not of CCL4, in comparison with nephritic wild-type mice, which was accompanied by augmented renal T cell and monocyte recruitment and increased lethality due to uremia. Furthermore, CCR5(-/-) mice showed an increased renal Th1 response, whereas their systemic humoral and cellular immune responses were unaltered. Because the CCR5 ligands CCL5 and CCL3 also act via CCR1, we investigated the effects of the pharmacological CCR1 antagonist BX471. CCR1 blockade in CCR5(-/-) mice significantly reduced renal chemokine expression, T cell infiltration, and glomerular crescent formation, indicating that increased renal leukocyte recruitment and consecutive tissue damage in nephritic CCR5(-/-) mice depended on functional CCR1. In conclusion, this study shows that CCR5 deficiency aggravates glomerulonephritis via enhanced CCL3/CCL5-CCR1-driven renal T cell recruitment.

Elucidation of binding sites of dual antagonists in the human chemokine receptors CCR2 and CCR5.

Design of dual antagonists for the chemokine receptors CCR2 and CCR5 will be greatly facilitated by knowledge of the structural differences of their binding sites. Thus, we computationally predicted the binding site of the dual CCR2/CCR5 antagonist N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzohepten-8-yl] carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium (TAK-779), and a CCR2-specific antagonist N-(carbamoylmethyl)-3-trifluoromethyl benzamido-parachlorobenzyl 3-aminopyrrolidine (Teijin compound 1) in an ensemble of predicted structures of human CCR2 and CCR5. Based on our predictions of the protein-ligand interactions, we examined the activity of the antagonists for cells expressing thirteen mutants of CCR2 and five mutants of CCR5. The results show that residues Trp98(2.60) and Thr292(7.40) contribute significantly to the efficacy of both TAK-779 and Teijin compound 1, whereas His121(3.33) and Ile263(6.55) contribute significantly only to the antagonistic effect of Teijin compound 1 at CCR2. Mutation of residues Trp86(2.60) and Tyr108(3.32) adversely affected the efficacy of TAK-779 in antagonizing CCR5-mediated chemotaxis. Y49A(1.39) and E291A(7.39) mutants of CCR2 showed a complete loss of CCL2 binding and chemotaxis, despite robust cell surface expression, suggesting that these residues are critical in maintaining the correct receptor architecture. Modeling studies support the hypothesis that the residues Tyr49(1.39), Trp98(2.60), Tyr120(3.32), and Glu291(7.39) of CCR2 form a tight network of aromatic cluster and polar contacts between transmembrane helices 1, 2, 3, and 7.

Promiscuous drugs as therapeutics for chemokine receptors.

Chemokine receptor antagonists that held much promise for the treatment of autoimmune and inflammatory diseases have recently performed poorly in clinical trials, resulting in disappointment for both pharmaceutical companies and patients. This review focuses on the redundancy of the molecular target as one potential reason for the failure of some of these antagonists to fulfil their initial promise, and discusses the use of drugs that are capable of interacting with more than one drug target - so-called promiscuous drugs - as possible approaches to overcome this difficulty. Several clinically approved promiscuous drugs, such as aspirin and olanzapine, are already used successfully. This review discusses examples of promiscuous drugs for G-protein-coupled receptors, including progress in developing dual-specific chemokine receptor antagonists, and considers evidence for the possible therapeutic utility of such drugs.

Expression, purification and in vitro functional reconstitution of the chemokine receptor CCR1.

Chemokine receptors are a specific class of G-protein-coupled receptors (GPCRs) that control cell migration associated with routine immune surveillance, inflammation and development. In addition to their roles in normal physiology, these receptors and their ligands are involved in a large number of inflammatory diseases, cancer and AIDS, making them prime therapeutic targets in the pharmaceutical industry. Like other GPCRs, a significant obstacle in determining structures and characterizing mechanisms of activation has been the difficulty in obtaining high levels of pure, functional receptor. Here we describe a systematic effort to express the chemokine receptor CCR1 in mammalian cells, and to purify and reconstitute it in functional form. The highest expression levels were obtained using an inducible HEK293 system. The receptor was purified using a combination of N- (StrepII or Hemagglutinin) and C-terminal (His8) affinity tags. Function was assessed by ligand binding using a novel fluorescence polarization assay with fluorescein-labeled chemokine. A strict dependence of function on the detergent composition was observed, as solubilization of CCR1 in n-dodecyl-beta-D-maltopyranoside/cholesteryl hemisuccinate yielded functional receptor with a K(d) of 21 nM for the chemokine CCL14, whereas it was non-functional in phosphocholine detergents. Differences in function were observed despite the fact that both these detergent types maintained the receptor in a state characterized by monomers and small oligomers, but not large aggregates. While optimization is still warranted, yields of approximately 0.1-0.2mg of pure functional receptor per 10(9) cells will permit biophysical studies of this medically important receptor.

A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation.

The expression of chemokines and their receptors is thought to contribute to leukocyte infiltration and progressive renal fibrosis after unilateral ureter obstruction (UUO). We hypothesized that blocking the chemokine receptor CCR1 using the nonpeptide antagonist BX471 could reduce leukocyte infiltration and renal fibrosis after UUO. UUO kidneys from BX471-treated mice (day 0-10 and day 6-10) revealed a 40-60% reduction of interstitial macrophage and lymphocyte infiltrate compared with controls. Treated mice also showed a marked reduction of CCR1 and CCR5 mRNA levels, and FACS analysis showed a comparable reduction of CD8+/CCR5+ T cells. Markers of renal fibrosis, such as interstitial fibroblasts, interstitial volume, mRNA and protein expression for collagen I, were all significantly reduced by BX471-treatment compared with vehicle controls. By contrast treatment was ineffective when the drug was supplied only from days 0 to 5. In summary, blockade of CCR1 substantially reduces cell accumulation and renal fibrosis after UUO. Most interestingly, late onset of treatment is also effective. We therefore conclude that CCR1 blockade may represent a new therapeutic strategy for reducing cellular infiltration and renal fibrosis as major factors in the progression to end-stage renal failure.

Chemokines.

Chemokines are a family of polypeptides that direct the migration of leukocytes toward a site of infection. They play a major role in autoimmune disease and chemokine receptors have recently been found to mediate HIV-1 fusion. In this short review we examine the role of chemokines in host defence and in the pathophysiology of autoimmune diseases. We conclude by discussing various therapeutic approaches that target chemokine receptors and that could be beneficial in disease.

I want a new drug: G-protein-coupled receptors in drug development.

Huey Lewis and the News summed it up nicely in their 1980s hit record: 'I want a new drug, one that won't make me sick, one that won't make me crash my car, or make me feel three feet thick'. The song could be an anthem for drug discovery in the pharmaceutical industry. We all want new and better drugs with fewer side effects, which are effective for combating the major diseases of our time: cancer, heart disease, obesity and autoimmune diseases. How do we get these new drugs? There are currently some new ideas in drug discovery, centered on that staple diet of the pharmaceutical industry, the G-protein-coupled receptor (GPCR) superfamily. In silico methods, employing receptor-based modeling, offer a more rational approach in the design of drugs targeting GPCRs. These approaches can be used to understand receptor selectivity and species specificity of drugs that interact with GPCRs. In addition, there are various novel approaches, such as the design and potential utility of drugs that target more than one GPCR ('dual specificity' drugs).

Role of CCR1 and CCR5 in homing and growth of multiple myeloma and in the development of osteolytic lesions: a study in the 5TMM model.

Multiple myeloma (MM) is a plasma cell malignancy, characterized by the localization of the MM cells in the bone marrow (BM), where they proliferate and induce osteolysis. The MM cells first need to home or migrate to the BM to receive necessary survival signals. In this work, we studied the role of CCR1 and CCR5, two known chemokine receptors, in both chemotaxis and osteolysis in the experimental 5TMM mouse model. A CCR1-specific (BX471) and a CCR5-specific (TAK779) antagonist were used to identify the function of both receptors. We could detect by RT-PCR and flow cytometric analyses the expression of both CCR1 and CCR5 on the cells and their major ligand, macrophage inflammatory protein 1alpha (MIP1alpha) could be detected by ELISA. In vitro migration assays showed that MIP1alpha induced a 2-fold increase in migration of 5TMM cells, which could only be blocked by TAK779. In vivo homing kinetics showed a 30% inhibition in BM homing when 5TMM cells were pre-treated with TAK779. We found, in vitro, that both inhibitors were able to reduce osteoclastogenesis and osteoclastic resorption. In vivo end-term treatment of 5T2MM mice with BX471 resulted in a reduction of the osteolytic lesions by 40%; while TAK779 treatment led to a 20% decrease in lesions. Furthermore, assessment of the microvessel density demonstrated a role for both receptors in MM induced angiogenesis. These data demonstrate the differential role of CCR1 and CCR5 in MM chemotaxis and MM associated osteolysis and angiogenesis.

The clinical potential of chemokine receptor antagonists.

Chemokines belong to a family of chemotactic cytokines that direct the migration of immune cells towards sites of inflammation. They mediate their biological effects by binding to cell surface receptors, which belong to the G protein-coupled receptor superfamily. Since chemokines and their receptors have been implicated in the pathophysiology of a number of autoinflammatory diseases, chemokine receptor antagonists could prove to be useful therapeutics to target these diseases. Here, we review the role of chemokines in autoimmunity, concentrating mainly on the chemokine receptors CCR1 and CCR5, and discuss the potential utility of antagonists that target these 2 receptors as they progress through the clinic.

MIP-1alpha utilizes both CCR1 and CCR5 to induce osteoclast formation and increase adhesion of myeloma cells to marrow stromal cells.

OBJECTIVES: Macrophage inflammatory protein-1alpha (MIP-1alpha), an osteoclast (OCL) stimulatory factor produced by primary multiple myeloma (MM) cells, increases bone destruction and tumor burden in murine models of MM. Several chemokine receptors (CCR1, CCR5, and CCR9) mediate the effects of MIP-1alpha. In this study, we determined which of these mediates the effects of MIP-1alpha on human OCL formation and myeloma cells. METHODS: We employed RT-PCR analysis, neutralizing antibodies to CCR1 and CCR5 as well as a CCR1-specific antagonist and OCL formation assays to identify the MIP-1alpha receptors involved in MIP-1alpha's effects on myeloma cells and OCL formation. RESULTS: RT-PCR analysis demonstrated that both CCR1 and CCR5 were expressed by highly purified human OCL precursors, myeloma cell lines, and purified marrow plasma cells from MM patients. Neutralizing antibodies to CCR1 or CCR5 inhibited MIP-1alpha-induced OCL formation. Furthermore, monocyte chemotactic protein-3 (MCP-3), which binds CCR1 but not CCR5 and the CCR1-specific antagonist, BX471, markedly inhibited OCL formation stimulated with MIP-1alpha. Anti-CCR1, anti-CCR5, or BX471 also inhibited the upregulation of beta1 integrin mRNA in myeloma cells induced by MIP-1alpha, as well as the adherence of myeloma cells to stromal cells and IL-6 production by stromal cells in response to myeloma cells. CONCLUSION: These data demonstrate that MIP-1alpha utilizes either CCR1 or CCR5 for its effects on OCL formation and myeloma cells, and that blocking either CCR1 or CCR5 inhibits OCL formation and myeloma cell adhesion to stromal cells.

Chemokines, chemokine receptors and small-molecule antagonists: recent developments.

The physiological roles of chemokine receptors have expanded beyond host defense and now represent important targets for intervention in several disease indications. Chemokine receptors have joined the ranks of other members of the G-protein-coupled receptor (GPCR) family in therapeutic potential as small-molecule chemokine receptor antagonists move from discovery to the clinic. Chemokine receptors belong to the rhodopsin family of GPCRs and, as such, are expected to be closely related in structure to other Class A members. In this review, we summarize information that is pertinent to chemokine receptors as therapeutic targets, the status of low molecular weight antagonists in clinical development, molecular modeling of receptor-small-molecule interactions, and the challenges that face drug discovery and development programs.

Differential inhibition of inducible T cell cytokine secretion by potent iron chelators.

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.

BX471: a CCR1 antagonist with anti-inflammatory activity in man.

Chemokines belong to a large family of chemoattractant molecules involved in the directed migration of immune cells. They achieve their cellular effects by direct interaction with cell surface receptors. The chemokine receptor CCR1 appears to be involved in a variety of proinflammatory and autoimmune diseases and this makes it a very attractive therapeutic target. This review discusses the identification, chemistry, biology and therapeutic potential of BX 471 a potent CCR1 antagonist that is currently in the clinic for a variety of indications.

CXCR3 antagonist VUF10085 binds to an intrahelical site distinct from that of the broad spectrum antagonist TAK-779.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 is implicated in a variety of clinically important diseases, notably rheumatoid arthritis and atherosclerosis. Consequently, antagonists of CXCR3 are of therapeutic interest. In this study, we set out to characterize binding sites of the specific low MW CXCR3 antagonist VUF10085 and the broad spectrum antagonist TAK-779 which blocks CXCR3 along with CCR2 and CCR5. EXPERIMENTAL APPROACH: Molecular modelling of CXCR3, followed by virtual ligand docking, highlighted several CXCR3 residues likely to contact either antagonist, notably a conserved aspartate in helix 2 (Asp-112(2:63) ), which was postulated to interact with the quaternary nitrogen of TAK-779. Validation of modelling was carried out by site-directed mutagenesis of CXCR3, followed by assays of cell surface expression, ligand binding and receptor activation. KEY RESULTS: Mutation of Asn-132(3.33) , Phe-207 and Tyr-271(6.51) within CXCR3 severely impaired both ligand binding and chemotactic responses, suggesting that these residues are critical for maintenance of a functional CXCR3 conformation. Contrary to our hypothesis, mutation of Asp-112(2:63) had no observable effects on TAK-779 activity, but clearly decreased the antagonist potency of VUF 10085. Likewise, mutations of Phe-131(3.32) , Ile-279(6.59) and Tyr-308(7.43) were well tolerated and were critical for the antagonist activity of VUF 10085 but not for that of TAK-779. CONCLUSIONS AND IMPLICATIONS: This more detailed definition of a binding pocket within CXCR3 for low MW antagonists should facilitate the rational design of newer CXCR3 antagonists, with obvious clinical potential.

Chemokines in transplant rejection.

Recent studies in transplant biology have demonstrated a role for chemokines in the migration of leukocytes into transplanted tissue. There is further evidence that modulation of the action of chemokines at their receptors can improve the outcome of acute rejection as well as long-term graft survival. A number of clinical therapies, based on both small molecules and biologicals, are currently under investigation with the potential for selective immunosupression. This article discusses the chemokines and receptors identified as the best characterized molecular targets, as well as the therapies under investigation for these targets.

Recent progress in the development of antagonists to the chemokine receptors CCR3 and CCR4.

INTRODUCTION: The chemokine receptors CCR3 and CCR4 have been shown to be important therapeutic targets for the treatment of a variety of diseases. Although only two chemokine receptor inhibitors have been approved so far, there are numerous compounds that are in various stages of development. AREAS COVERED: In this review article, the authors provide an update on the progress made in the identification of antagonists against the chemokine receptors CCR3 and CCR4 from 2009 to the present. The rationale of writing this review article is to cover the most important approaches to identifying antagonists to these two receptors, which could prove to be useful therapeutics in treating proinflammatory diseases. EXPERT OPINION: Pharmaceutical companies have expended a considerable amount of money and effort to identify potent inhibitors of CCR3 and CCR4 for the treatment of asthma and atopic diseases. Although a variety of compounds have been described and several have progressed into the clinic, none have so far made it as approved drugs. There are, however, novel approaches such as mogamulizumab, a monoclonal antibody to CCR4 currently is in clinical trials for cancer and ASM8, an antisense nucleotide to CCR3, which is in Phase II clinical trials for asthma that might still prove to be successful new therapeutics.