Identification and profiling of CXCR3–CXCR4 chemokine receptor heteromer complexes

Background and Purpose

The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers.

Experimental Approach

CXCR3–CXCR4 heteromers were identified in HEK293T cells using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer, saturation BRET and the GPCR-heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity.

Key Results

We provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co-expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross-inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR-HIT approach, we have evidence for specific β-arrestin2 recruitment to CXCR3-CXCR4 heteromers in response to agonist stimulation.

Conclusions and Implications

This study indicates that heteromeric CXCR3–CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities.     

The chemokine receptor antagonist,TAK-779, decreased experimental autoimmune encephalomyelitis by reducing inflammatory cell migration into the central nervous system,without affecting T cell function

Background and purpose:

The C–C chemokine receptor CCR5, and the C–X–C chemokine receptor CXCR3 are involved in the regulation of T cell-mediated immune responses, and in the migration and activation of these cells. To determine whether blockade of these chemokine receptors modulated inflammatory responses in the central nervous sytem (CNS), we investigated the effect of a non-peptide chemokine receptor antagonist, TAK-779, in mice with experimental autoimmune encephalomyelitis (EAE).

Experimental approach:

EAE was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35–55. TAK-779 was injected s.c. once a day after immunization. Disease incidence and severity (over 3 weeks) were monitored by histopathological evaluation and FACS assay of inflammatory cells infiltrating into the spinal cord, polymerase chain reaction quantification of mRNA expression, assay of T cell proliferation, by [³H]-thymidine incorporation and cytokine production by enzyme-linked immunosorbent assay.

Key results:

Treatment with TAK-779 reduced incidence and severity of EAE. It strongly inhibited migration of CXCR3/CCR5 bearing CD4⁺, CD8⁺ and CD11b⁺ leukocytes to the CNS. TAK-779 did not reduce proliferation of anti-MOG T cells, the production of IFN-γ by T cells or CXCR3 expression on T cells. In addition, TAK-779 did not affect production of IL-12 by antigen-presenting cells, CCR5 induction on T cells and the potential of MOG-specific T cells to transfer EAE.

Conclusions and implications:

TAK-779 restricted the development of MOG-induced EAE. This effect involved reduced migration of inflammatory cells into the CNS without affecting responses of anti-MOG T cells or the ability of MOG-specific T cells to transfer EAE.     

HIV-1协同受体及其抑制剂研究进展

协同受体CCR5和CXCR4分别是嗜巨噬细胞性HIV-1和嗜T细胞性TIV-1侵入靶细胞的主要受体。CCR5抑制剂如TAK-779、SCH—C等，和CXCR4抑制剂如AMD3100、T22等，能分别与CCR5和CXCR4结合，从而阻断HIV-1侵入靶细胞。本文综述了HIV-1与CCR5和CXCR4的结合机制及其抑制剂的研究进展。     

The development of an europium-GTP assay to quantitate chemokine antagonist interactions for CXCR4 and CCR5

Chemokine receptors have been implicated in several disease processes such as acute and chronic inflammation, cancer, and allograft rejection and are therefore targets for drug development. The chemokine receptors CCR5 and CXCR4 are of particular interest as they serve as entry cofactors for human immunodeficiency virus. These receptors are members of the G protein-coupled receptor (GPCR) family. In this respect, assessing GPCR activation by GTP binding is an important tool to study the early stage of signal transduction. The assay normally utilizes the non-hydrolysable GTP analogue guanosine 5'-gamma-[35S]thiotriphosphate. In order to avoid the problems involved in working with radioactivity, a new non-radioactive version of the assay was developed using a europium-labeled GTP analogue in which europium-GTP binding can be assayed using time-resolved fluorescence. The assay was optimized for CXCR4 and CCR5 and validated for screening of chemokine antagonists using the small molecule CXCR4 antagonist AMD3100 and CCR5 antagonists.     

The molecular pharmacology of AMD11070: an orally bioavailable CXCR4 HIV entry inhibitor

In order to enter and infect human cells HIV must bind to CD4 in addition to either the CXCR4 or the CCR5 chemokine receptor. AMD11070 was the first orally available small molecule antagonist of CXCR4 to enter the clinic. Herein we report the molecular pharmacology of AMD11070 which is a potent inhibitor of X4 HIV-1 replication and the gp120/CXCR4 interaction. Using the CCRF-CEM T cell line that endogenously expresses CXCR4 we have demonstrated that AMD11070 is an antagonist of SDF-1α ligand binding (IC50 = 12.5 ± 1.3 nM), inhibits SDF-1 mediated calcium flux (IC50 = 9.0 ± 2.0 nM) and SDF-1α mediated activation of the CXCR4 receptor as measured by a Eu-GTP binding assay (IC50 =39.8 ± 2.5 nM) or a [(35)S]-GTPγS binding assay (IC50 =19.0 ± 4.1 nM), and inhibits SDF-1α stimulated chemotaxis (IC50 =19.0 ± 4.0 nM). AMD11070 does not inhibit calcium flux of cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, or ligand binding to CXCR7 and BLT1, demonstrating selectivity for CXCR4. In addition AMD11070 is able to inhibit the SDF-1β isoform interactions with CXCR4; and N-terminal truncated variants of CXCR4 with equal potency to wild type receptor. Further mechanistic studies indicate that AMD11070 is an allosteric inhibitor of CXCR4.     

Shikonin, a component of antiinflammatory Chinese herbal medicine, selectively blocks chemokine binding to CC chemokine receptor-1

Shikonin is a chemically characterized component of traditional Chinese herbal medicine and has been shown to possess antiinflammatory activities. We ascertained that shikonin blocked radiolabelled Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) and macrophage inflammatory protein-1 (MIP-1alpha) binding to human monocytes with IC50 values of 3.58 x 10(-6) and 2.57 x 10(-6) M, respectively. In contrast, up to 1.7 x 10(-5) M of shikonin failed to inhibit stromal cell-derived factor-1 (SDF-1alpha) binding to the cells. Additionally, shikonin blocked RANTES and MIP-1alpha binding to stable CC chemokine receptor-1 (CCR1) transfected human embryonic kidney (HEK)/293 cells with IC50 values of 2.63 x 10(-6) and 2.57 x 10(-6) M, respectively. However, shikonin inhibited neither RANTES nor MIP-1alpha binding to CCR5 transfected HEK/293 cells. Shikonin also did not inhibit monocyte chemoattractant protein-1 (MCP-1) binding to CCR2 cells, eotaxin binding to CCR3 cells, interferon-inducible T cell alpha-chemoattractant (I-TAC) binding to CXCR3 cells and SDF-1alpha binding to CXCR4 cells. Additionally, shikonin inhibited RANTES-induced CCR1 cell migration, but did not inhibit CCR1 cell migration induced by epidermal growth factor (EGF). Our study suggests shikonin may be a target for the future design of more potent, highly selective therapeutics that could be useful antiinflammatory agents for selectively blocking the binding of CCR1 ligands.     

Therapeutic potential of chemokine receptor antagonists for liver disease

Chemokines are a family of small heparin-binding proteins that orchestrate the infiltration of immune cells into the liver, but also directly influence the biology of resident liver cells. A nonredundant role of different chemokines and their receptors has been identified within the last years in animal models of acute and chronic liver diseases. Chemokine receptors that have been shown to play an import pathophysiological role in the liver are CCR1, CCR2, CCR5, D6, CXCR2 and CXCR3. Interestingly, most of these receptors have already been targeted by specific antagonists in early human trials and a CCR5 antagonist is already licensed for use in HIV infection. Most of these trials have been performed in autoimmune and infectious human diseases, but no controlled clinical trials have yet been performed in patients with liver diseases. Nevertheless, in light of growing evidence that chemokines are important mediators of liver damage, these trials seem to be on the clinical horizon.     

Therapeutic potential of chemokine receptor antagonists for liver disease

Chemokines are a family of small heparin-binding proteins that orchestrate the infiltration of immune cells into the liver, but also directly influence the biology of resident liver cells. A nonredundant role of different chemokines and their receptors has been identified within the last years in animal models of acute and chronic liver diseases. Chemokine receptors that have been shown to play an import pathophysiological role in the liver are CCR1, CCR2, CCR5, D6, CXCR2 and CXCR3. Interestingly, most of these receptors have already been targeted by specific antagonists in early human trials and a CCR5 antagonist is already licensed for use in HIV infection. Most of these trials have been performed in autoimmune and infectious human diseases, but no controlled clinical trials have yet been performed in patients with liver diseases. Nevertheless, in light of growing evidence that chemokines are important mediators of liver damage, these trials seem to be on the clinical horizon.     

Regulation by allergens of chemokine receptor expression on in vitro-generated dendritic cells

Immature dendritic cells (DCs) derived from CD34+ progenitor cells or peripheral monocytes, are used as in vitro sensitization models in many chemical allergen treatment studies. During the sensitization, DCs follow maturation process and gain the capacity to migrate to lymph nodes where they stimulate T cells. Chemokine receptor allows DCs to migrate along chemotactic gradients. In this work, we used immature DCs from peripheral monocytes to evaluate the influence of allergens on chemokine receptor and surface-marker expression. We tested the sensitizers dinitrochlorobenzene, Bandrowski's base, and coumarin, as well as the tolerogen dichloronitrobenzene, the irritant sodium dodecyl sulfate and the solvent dimethyl sulfoxide. All skin sensitizers up-regulated the co-stimulatory molecule CD86 and increased the CD83+ cell population. No expression of the chemokine receptors CCR2, CCR3, CCR6, or CXCR5 was observed on DCs exposed to the tested chemicals. The strong allergen dinitrochlorobenzene slightly increased CCR7 expression on DCs but down-regulated CCR1 surface expression. CCR1 down-regulation was not mediated by a classical maturation pathway, as it was unaffected by the corticosteroid dexamethasone.     

Orally bioavailable competitive CCR5 antagonists

The chemokine receptor CCR5 plays an important role in inflammatory and autoimmune disorders as well as in transplant rejection by affecting the trafficking of effector T cells and monocytes to diseased tissues. Antagonists of CCR5 are believed to be of potential therapeutic value for the disorders mentioned above and HIV infection. Here we report on the structure-activity relationship of a new series of highly potent and selective competitive CCR5 antagonists. While all compounds tested were inactive on rodent CCR5, this series includes compounds that cross-react with the cynomolgus monkey (cyno) receptor. One of these compounds, i.e., 26n, has good PK properties in cynos, and its overall favorable profile makes it a promising candidate for in vivo profiling in transplantation and other disease models.     

Cytokine and chemokine based control of HIV infection and replication

HIV infects and propagates into CD4+ T lymphocytes and macrophages, although many other cell types play an important role in virus spreading and pathogenesis. In addition to regulatory viral proteins, the cytokine network has early been implicated as a major controller of the plastic capacity of HIV to spread productively or rather remain silently integrated in the chromosomes of infected cells. The recent discovery of CCR5 and CXCR4 as essential entry co-receptors together with CD4 has highlighted a novel and potentially important step in the pharmacological hunt for more effective antiviral agents. In addition to regulate HIV expression and replication, several cytokines have demonstrated the capacity of up- or down-modulating chemokine receptors including CCR5 and CXCR4 with the consequence of influencing the susceptibility of T cells and macrophages to HIV infection. Pharmacological agents such as pertussis toxin B-oligomer have demonstrated HIV suppressive effects via non competitive binding of CCR5, whereas interferons or interleukin-16 (IL-16) can prevent post-entry steps in HIV expression. At the clinical level, several cytokines or their receptors are useful markers for monitoring disease progression and its consequence on the immune system. Cytokine-based therapy represents a realistic complementary approach to traditional antiretroviral therapy potentially capable of restoring important adaptive or innate immune functions ultimately curtailing HIV spreading and its consequences on the immune system, as exemplified by the experimental clinical use of IL-2.     

Characterization of the molecular pharmacology of AMD3100: a specific antagonist of the G-protein coupled chemokine receptor, CXCR4

The chemokine receptor CXCR4 is widely expressed on different cell types, is involved in leukocyte chemotaxis, and is a co-receptor for HIV. AMD3100 has been shown to be a CXCR4 receptor antagonist, and to block HIV infection of T-tropic, X4-using, virus in vitro and in vivo. AMD3100 is an effective mobilizer of hematopoietic stem cells and is being investigated in clinical trials in multiple myeloma and non-Hodgkins lymphoma patients. Using the CCRF-CEM T-cell line that constitutively expresses CXCR4 we confirmed that AMD3100 was an antagonist of SDF-1/CXCL12 ligand binding (IC50=651+/-37 nM). We have also shown that AMD3100 inhibits SDF-1 mediated GTP-binding (IC50=27+/-2.2 nM), SDF-1 mediated calcium flux (IC50=572+/-190 nM), and SDF-1 stimulated chemotaxis (IC50=51+/-17 nM). AMD3100 did not inhibit calcium flux against cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7 when stimulated with their cognate ligands, nor did it inhibit receptor binding of LTB4. AMD3100 did not, on its own, induce a calcium flux in the CCRF-CEM cells, which express multiple GPCRs including CXCR4, CCR4 and CCR7. Furthermore, AMD3100 neither stimulated GTP-binding, an assay for GPCR activation, in CEM cell membranes; nor chemotaxis of CCRF-CEM cells. These data therefore demonstrate that AMD3100 is a specific antagonist of CXCR4, is not cross-reactive with other chemokine receptors, and is not an agonist of CXCR4.     

Altered sensitivity of an R5X4 HIV-1 strain 89.6 to coreceptor inhibitors by a single amino acid substitution in the V3 region of gp120

The replication of several R5X4 strains is blocked by single CXCR4 inhibitors such as AMD3100 or T140 although the target cells express both CXCR4 and CCR5 in vitro. To identify which region(s) of the Env are involved in the increased sensitivity to CXCR4 inhibitors, we isolated a T140-escape mutant using R5X4 HIV-1 strain 89.6. An isolated mutant harbored a single amino acid substitution in the V3 region of the Env (arginine 308 to serine R308S). Luciferase-reporter HIV-1 pseudotyped with the mutant Env showed that the substitution conferred total resistance to CXCR4 antagonists but increased sensitivity to a CCR5 antagonist TAK-779 in the infection of the cells expressing both CCR5 and CXCR4. Analyses using the cells expressing a single coreceptor showed that the mutant Env predominantly and efficiently utilized CCR5 rather than CXCR4 while retaining R5X4 phenotype. These results indicated that the sensitivities of the R5X4 strain to coreceptor inhibitors were altered by a single amino acid substitution in the V3 region of gp120.     

Adhesion molecules and kinases involved in gammadelta T cells migratory pathways: implications for viral and autoimmune diseases

gammadelta T lymphocytes are involved in the defence from viral and mycobacterial infections; however they are also responsible for autoimmune reactions. Herein, we discuss the characteristics of these cells, focusing on the mechanism(s) underlying extravasation and tissue localization. We show that Vdelta1 and Vdelta2 gammadeltaT cells display differential expression of adhesion molecules and chemokine receptors, the former being preferentially PECAM-1(+)CXCR4(+), the latter expressing NKRP1A and CXCR3. The two cell populations transmigrate across endothelial cells by activation of distinct kinase pathways and in response to interferon-gamma-inducing protein-10 (IP-10/CXCL10) or stromal-derived factor-1 (SDF-1/CXCL12) according to the expression of the specific receptors CXCR3 and CXCR4. IP-10/CXCL10 and SDF-1/CXCL12-induced transmigration are phosphoinositide-3 kinase (PI-3K) and Akt/PKB-dependent. In addition, occupancy of CXCR3, but not of CXCR4, leads to CAMKII activation; blocking of CAMKII decreases IP-10/CXCL10 and 6Ckine/SLC/CCL21-driven transmigration. We report that HIV-1-infected patients have an increased number of circulating Vdelta1 T cells possibly due to the interference of Tat protein on the function of chemokine receptors. In turn, patients with relapsing-remitting multiple sclerosis (MS), display an increase in peripheral Vdelta2 gammadelta T cells and this is related to interleukin-12-mediated upregulation of NKRP1A. Finally, the possible role of gammadelta T lymphocytes in post-transplantation immune reconstitution is discussed.     

Conformational properties of HIV-1 gp120/V3 immunogenic domains

Infection of target host cells by the human immunodeficiency virus-1 (HIV-1) is a multi-step process involving a series of conformational changes in the viral gp120 and gp41 proteins. Gp120 binding to the main cell receptor, CD4, on the surface of cells expressing this molecule, and interaction with the cell chemokine receptors CCR5 and CXCR4, are among the key events for HIV-1 infection. These steps are crucial for the virus and offer potential therapeutic targets. For this reason, understanding the structure and the physicochemical characteristics of the gp120 in relation to these interactions has drawn much attention. This review article focuses on the biologically important V3 region of the gp120 and summarizes the functional role, the sequence variation and the conformational features of V3 peptides, which are important for co-receptor selectivity, specificity and interaction. Synthetic V3 peptides have been extensively studied by NMR spectroscopy and X-ray crystallography, in solution or in solid state, in their free or bound form, and valuable information was generated with the aim to be exploited in the design of new, effective inhibitors of HIV-1 infection. The features of the potential gp120 interacting sites on the two chemokine co-receptors, CCR5 and CXCR4, are also discussed, and co-receptor blocking molecules under clinical trial are also reported.     

The therapeutic potential of CXCR4 antagonists in the treatment of HIV

Since the identification of the chemokine receptors CXCR4 and CCR5 as co-receptors for HIV-1 entry, several antagonists against these receptors have been synthesised. A highly selective CXCR4 antagonist, T22, and its downsized analogues T140 and TC14012, which inhibit X4-HIV-1 infection through their specific binding to CXCR4, have been identified. Besides T22 analogues, several other CXCR4 antagonists have been reported, such as AMD3100, ALX40-4C, KRH-1120 and AMD8664. Discovery of entry inhibitors, such as chemokine antagonists, may lead to the development of a new generation of antiHIV agents, since these inhibitors are thought to be useful for the clinical treatment of HIV-1-infected patients, especially at the late stage of treatment for AIDS patients developing multi-drug-resistant strains. In this review, recent research into CXCR4 antagonists in comparison with development of other antagonists is summarised.     

Chemokine receptor-directed agents as novel anti-HIV-1 therapies

Historically, therapeutic benefit in the treatment of human immunodeficiency virus infection (HIV-1) infection has been best achieved by targeting viral proteins like HIV protease involved in viral replication rather than host cell proteins, like CD4, which facilitate the process of viral infection. Two discoveries in 1996 presented a novel opportunity to redress this issue: 1) the understanding that heptahelical G-protein coupled chemokine receptors on the surface of T cells and macrophages functioned together with CD4 to mediate viral entry, and 2) the observation that CD4 positive T cells from individuals homozygous for the CCR5 delta 32 null allele were resistant to infection by macrophage-tropic strains of the virus in vitro and in vivo. Since that time, data demonstrating that selective blockade of two chemokine receptors, CCR5 and CXCR4, by small molecule chemokine receptor antagonists or receptor-directed biologics could robustly inhibit the infection of human peripheral blood mononuclear cells (PBMCs) by macrophage-tropic and T-cell line tropic strains respectively in vitro has validated this potential approach to therapy. Early clinical trial data now also confirms that these types of agents will have anti-viral activity in some HIV-1 infected individuals; however to date, dose limiting off-target activities have prohibited a full test of their potential clinical value. It also remains to be seen how these types of agents will fare in synergy with existing HIV-1 targeted antivirals, or those currently in development.     

Chemokines in allergic airway disease

Expression of chemokine receptors on T helper 2 cells and eosinophils has been postulated to be the mechanism by which these cells are selectively recruited to the lung during allergic inflammatory reactions. Mouse models have provided evidence to show that blocking the ligands for these receptors is successful in abrogating the pathophysiological effects of allergen challenge. However, recent studies describing the effect of genetic deletions of these chemokine receptors have not confirmed the results obtained with ligand knockouts or neutralising antibodies. Coupled with the realisation that, because of a lack of species cross-reactivity, it is not possible to test small molecule antagonists against human receptors in the original in vivo animal models, the future of chemokine receptor therapeutics is in question. However, recent advances have been made regarding the therapeutic potential of blocking the chemokine receptors CCR3, CCR4 and CCR8 in allergic airway disease.