A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists

CXCR4 is a G protein-coupled receptor for stromal-derived factor 1 (SDF-1) that plays a critical role in leukocyte trafficking, metastasis of mammary carcinoma, and human immunodeficiency virus type-1 infection. To elucidate the mechanism for CXCR4 activation, a constitutively active mutant (CAM) was derived by coupling the receptor to the pheromone response pathway in yeast. Conversion of Asn-119 to Ser or Ala, but not Asp or Lys, conferred autonomous CXCR4 signaling in yeast and mammalian cells. SDF-1 induced signaling in variants with substitution of Asn-119 to Ser, Ala, or Asp, but not Lys. These variants had similar cell surface expression and binding affinity for SDF-1. CXCR4-CAMs were constitutively phosphorylated and present in cytosolic inclusions. Analysis of antagonists revealed that exposure to AMD3100 or ALX40-4C induced G protein activation by CXCR4 wild type, which was greater in the CAM, whereas T140 decreased autonomous signaling. The affinity of AMD3100 and ALX40-4C binding to CAMs was less than to wild type, providing evidence of a conformational shift. These results illustrate the importance of transmembrane helix 3 in CXCR4 signaling. Insight into the mechanism for CXCR4 antagonists will allow for the development of a new generation of agents that lack partial agonist activity that may induce toxicities, as observed for AMD3100.     

Determinants for Sensitivity of Human Immunodeficiency Virus Coreceptor CXCR4 to the Bicyclam AMD3100

The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.     

The peptidomimetic CXCR4 antagonist TC14012 recruits beta-arrestin to CXCR7: roles of receptor domains

CXCR7 is an atypical chemokine receptor that signals through β-arrestin in response to agonists without detectable activation of heterotrimeric G-proteins. Its cognate chemokine ligand CXCL12 also binds CXCR4, a chemokine receptor of considerable clinical interest. Here we report that TC14012, a peptidomimetic inverse agonist of CXCR4, is an agonist on CXCR7. The potency of β-arrestin recruitment to CXCR7 by TC14012 is much higher than that of the previously reported CXCR4 antagonist AMD3100 and differs only by one log from that of the natural ligand CXCL12 (EC(50) 350 nM for TC14012, as compared with 30 nM for CXCL12 and 140 μM for AMD3100). Moreover, like CXCL12, TC14012 leads to Erk 1/2 activation in U373 glioma cells that express only CXCR7, but not CXCR4. Given that with TC14012 and AMD3100 two structurally unrelated CXCR4 antagonists turn out to be agonists on CXCR7, this likely reflects differences in the activation mechanism of the arrestin pathway by both receptors. To identify the receptor domain responsible for these opposed effects, we investigated CXCR4 and CXCR7 C terminus-swapping chimeras. Using quantitative bioluminescence resonance energy transfer, we find that the CXCR7 receptor core formed by the seven-transmembrane domains and the connecting loops determines the agonistic activity of both TC14012 and AMD3100. Moreover, we find that the CXCR7 chimera bearing the CXCR4 C-terminal constitutively associates with arrestin in the absence of ligands. Our data suggest that the CXCR4 and CXCR7 cores share ligand-binding surfaces for the binding of the synthetic ligands, indicating that CXCR4 inhibitors should be tested also on CXCR7.     

T134, a Small-Molecule CXCR4 Inhibitor, Has No Cross-Drug Resistance with AMD3100, a CXCR4 Antagonist with a Different Structure

T22, an analog of polyphemusin II (18 amino acid residues), was found to block T-tropic human immunodeficiency virus type 1 (HIV-1) entry into target cells as a CXCR4 inhibitor. We synthesized T134, a small analog (14 amino acid residues) of T22 with reduced positive charges. T134 exhibited highly potent activity and significantly less cytotoxicity in comparison to that of T22. T134 prevents the anti-CXCR4 monoclonal antibody from binding to peripheral blood mononuclear cells but has no effect on the binding of anti-CCR5 monoclonal antibodies. Since T134 inhibits the binding of stromal cell-derived factor-1 (SDF-1) to MT-4 cells, it seems that T134 prevents HIV-1 entry by binding to CXCR4. The bicyclam AMD3100 has also been shown to block HIV-1 entry via CXCR4 but not via CCR5. Both T134 and AMD3100 are CXCR4 antagonists and low-molecular-weight compounds but have different structures. Our results indicate that T134 is active against wild-type T-tropic HIV-1 strains and against AMD3100-resistant strains.     

Identification of allosteric peptide agonists of CXCR4

The chemokine receptor CXCR4 is a co-receptor for T-tropic strains of HIV-1. A number of small molecule antagonists of CXCR4 are in development but all are likely to lead to adverse effects due to the physiological function of CXCR4. To prevent these complications, allosteric agonists may be therapeutically useful as adjuvant therapy in combination with small molecule antagonists. A synthetic cDNA library coding for 160,000 different SDF-based peptides was screened for CXCR4 agonist activity in a yeast strain expressing a functional receptor. Peptides that activated CXCR4 in an autocrine manner induced colony formation. Two peptides, designated RSVM and ASLW, were identified as novel agonists that are insensitive to the CXCR4 antagonist AMD3100. In chemotaxis assays using the acute lymphoblastic leukemia cell line CCRF-CEM, RSVM behaves as a partial agonist and ASLW as a superagonist. The superagonist activity of ASLW may be related to its inability to induce receptor internalization. In CCRF-CEM cells, the two peptides are also not inhibited by another CXCR4 antagonist, T140, or the neutralizing monoclonal antibodies 12G5 and 44717.111. These results suggest that alternative agonist-binding sites are present on CXCR4 that could be screened to develop molecules for therapeutic use.     

Chemokine receptor inhibition by AMD3100 is strictly confined to CXCR4

This study was undertaken to demonstrate the unique specificity of the chemokine receptor CXCR4 antagonist AMD3100. Calcium flux assays with selected chemokine/cell combinations, affording distinct chemokine receptor specificities, revealed no interaction of AMD3100 with any of the chemokine receptors CXCR1 through CXCR3, or CCR1 through CCR9. In contrast, AMD3100 potently inhibited CXCR4-mediated calcium signaling and chemotaxis in a concentration-dependent manner in different cell types. Also, AMD3100 inhibited stromal cell-derived factor (SDF)-1-induced endocytosis of CXCR4, but did not affect phorbol ester-induced receptor internalization. Importantly, AMD3100 by itself was unable to elicit intracellular calcium fluxes, to induce chemotaxis, or to trigger CXCR4 internalization, indicating that the compound does not act as a CXCR4 agonist. Specific small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of human immunodeficiency virus infections and many other pathological processes that are dependent on SDF-1/CXCR4 interactions (e.g. rheumatoid arthritis, atherosclerosis, asthma and breast cancer metastasis).     

Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor

AMD3100 is a symmetric bicyclam, prototype non-peptide antagonist of the CXCR4 chemokine receptor. Mutational substitutions at 16 positions located in TM-III, -IV, -V, -VI, and -VII lining the main ligand-binding pocket of the CXCR4 receptor identified three acid residues: Asp(171) (AspIV:20), Asp(262) (AspVI:23), and Glu(288) (GluVII:06) as the main interaction points for AMD3100. Molecular modeling suggests that one cyclam ring of AMD3100 interacts with Asp(171) in TM-IV, whereas the other ring is sandwiched between the carboxylic acid groups of Asp(262) and Glu(288) from TM-VI and -VII, respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp(262) and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build-up in the rather distinct CXCR3 receptor, for which the compound normally had no effect. Introduction of only a Glu at position VII:06 and the removal of a neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in affinity of AMD3100 to within 10-fold of its affinity in CXCR4. We conclude that AMD3100 binds through interactions with essentially only three acidic anchor-point residues, two of which are located at one end and the third at the opposite end of the main ligand-binding pocket of the CXCR4 receptor. We suggest that non-peptide antagonists with, for example, improved oral bioavailability can be designed to mimic this interaction and thereby efficiently and selectively block the CXCR4 receptor.     

Mapping the ligand-binding site on a G protein-coupled receptor (GPCR) using genetically encoded photocrosslinkers

We developed a general cell-based photocrosslinking approach to investigate the binding interfaces necessary for the formation of G protein-coupled receptor (GPCR) signaling complexes. The two photoactivatable unnatural amino acids p-benzoyl-L-phenylalanine and p-azido-L-phenylalanine were incorporated by amber codon suppression technology into CXC chemokine receptor 4 (CXCR4). We then probed the ligand-binding site for the HIV-1 coreceptor blocker, T140, using a fluorescein-labeled T140 analogue. Among eight amino acid positions tested, we found a unique UV-light-dependent crosslink specifically between residue 189 and T140. These results are evaluated with molecular modeling using the crystal structure of CXCR4 bound to CVX15.     

Glycoprotein 120 binding to CXCR4 causes p38-dependent primary T cell death that is facilitated by, but does not require cell-associated CD4

HIV-1 infection causes the depletion of host CD4 T cells through direct and indirect (bystander) mechanisms. Although HIV Env has been implicated in apoptosis of uninfected CD4 T cells via gp120 binding to either CD4 and/or the chemokine receptor 4 (CXCR4), conflicting data exist concerning the molecular mechanisms involved. Using primary human CD4 T cells, we demonstrate that gp120 binding to CD4 T cells activates proapoptotic p38, but does not activate antiapoptotic Akt. Because ligation of the CD4 receptor alone or the CXCR4 receptor alone causes p38 activation and apoptosis, we used the soluble inhibitors, soluble CD4 (sCD4) or AMD3100, to delineate the role of CD4 and CXCR4 receptors, respectively, in gp120-induced p38 activation and death. sCD4 alone augments gp120-induced death, suggesting that CXCR4 signaling is principally responsible. Supporting that model, AMD3100 reduces death caused by gp120 or by gp120/sCD4. Finally, prevention of gp120-CXCR4 interaction with 12G5 Abs blocks p38 activation and apoptosis, whereas inhibition of CD4-gp120 interaction with Leu-3a has no effect. Consequently, we conclude that gp120 interaction with CXCR4 is required for gp120 apoptotic effects in primary human T cells.     

NeoR6 inhibits HIV-1-CXCR4 interaction without affecting CXCL12 chemotaxis activity

Aminoglycoside-arginine conjugates (AACs) are multi-target HIV-1 inhibitors. The most potent AAC is neomycin hexa-arginine conjugate, NeoR6. We here demonstrate that NeoR6 interacts with CXCR4 without affecting CXCL12-CXCR4 ordinary chemotaxis activity or loss of CXCR4 cell surface expression. Importantly, NeoR6 alone does not affect cell migration, indicating that NeoR6 interacts with CXCR4 at a distinct site that is important for HIV-1 entry and mAb 12G5 binding, but not to CXCL12 binding or signaling sites. This is further supported by our modeling studies, showing that NeoR6 and CXCL12 bind to two distinct sites on CXCR4, in contrast with other CXCR4 inhibitors, e.g. T140 and AMD3100. This complementary utilization of chemical, biology, and computation analysis provides a powerful approach for designing anti-HIV-1 drugs without interfering with the natural function of CXCL12/CXCR4 binding.     

Mutations at the CXCR4 interaction sites for AMD3100 influence anti-CXCR4 antibody binding and HIV-1 entry

The interaction of the CXCR4 antagonist AMD3100 with its target is greatly influenced by specific aspartate residues in the receptor protein, including Asp(171) and Asp(262). We have now found that aspartate-to-asparagine substitutions at these positions differentially affect the binding of four different anti-CXCR4 monoclonal antibodies as well as the infectivity of diverse human immunodeficiency virus type 1 (HIV-1) strains and clinical isolates. Mutation of Asp(262) strongly decreased the coreceptor efficiency of CXCR4 for wild-type but not for AMD3100-resistant HIV-1 NL4.3. Thus, resistance of HIV-1 NL4.3 to AMD3100 is associated with a decreased dependence of the viral gp120 on Asp(262) of CXCR4, pointing to a different mode of interaction of wild-type versus AMD3100-resistant virus with CXCR4.     

Structural Basis of the Interaction between Chemokine Stromal Cell-derived Factor-1/CXCL12 and Its G-protein-coupled Receptor CXCR4

The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 β-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the “fly-casting” mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.     

Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists

Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking, and structural consequences of ubiquitin binding to CXCR4 are unknown. Here, we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle, and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the second transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase, and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In β-arrestin recruitment assays, ubiquitin did not sufficiently recruit β-arrestin2 to CXCR4 (EC₅₀ > 10 μM), whereas the EC₅₀ for CXCL12 was 4.6 nM (95% confidence interval 3.1–6.1 nM). Both agonists induced similar chemical shift changes in the ¹³C-¹H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the ¹³C-¹H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.     

Insights into the mechanism of enhanced mobilization of hematopoietic progenitor cells and release of CXCL12 by a combination of AMD3100 and aminoglycoside-polyarginine conjugates

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow to the peripheral blood is utilized in clinical HSPC transplantation protocols. Retention of HSPCs in the bone marrow is determined by relationships between the chemokine chemokine (C-X-C motif) ligand 12 (CXCL12) and its major receptor C-X-C chemokine receptor type 4 (CXCR4), and disruption of this retention by CXCR4 antagonists such as AMD3100 induces rapid HSPC mobilization. Here, we report that aminoglycoside-polyarginine conjugates (APACs) and N-α-acetyl-nona-D-arginine (r9) induce mobilization of white blood cells and, preferentially, immature hematopoietic progenitor cells (HPCs) in mice, similarly to AMD3100. Remarkably, administration of AMD3100 with each one of the APACs or r9 caused additional HPC mobilization. The mobilizing activity of APACs and r9 was accompanied by a significant elevation in plasma CXCL12 levels. To further understand how APACs, r9 and their combinations with AMD3100 compete with CXCL12 binding to CXCR4, as well with antibody against CXCR4 for CXCR4 binding, we have undertaken an approach combining experimental validation and docking to determine plausible binding modes for these ligands. On the basis of our biological and docking findings, and recently published NMR data, we suggest that combination of pairs of compounds such as APACs (or r9) with AMD3100 induces more efficient disruption of the CXCL12-CXCR4 interaction than AMD3100 alone, resulting in enhanced HPC mobilization.     

Selective Enhancement of Donor Hematopoietic Cell Engraftment by the CXCR4 Antagonist AMD3100 in a Mouse Transplantation Model

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.     

Long-term high-fat diet induces pancreatic injuries via pancreatic microcirculatory disturbances and oxidative stress in rats with hyperlipidemia

Stromal cell-derived factor 1 (CXCL12) is an angiogenic chemokine that is believed to act solely via its cognate receptor CXCR4. Evidence is now provided for the existence of a different CXCL12 binding and signaling receptor on endothelial cells. Bovine aortic endothelial cells (BAECs) strongly expressed CXCR4 and exhibited high binding capacity for fluorescently labeled CXCL12. However, CXCL12 binding was not correlated with the CXCR4 expression level and was virtually unaffected by the specific CXCR4 antagonists AMD3100 or T22. Similar observations were made in endothelial cells of mouse and human origin. Also, AMD3100 failed to block CXCL12 internalization and CXCL12-induced intracellular signal transduction via extracellular signal-regulated kinases 1/2 in BAECs. In contrast, CXCL12 binding and signaling were almost completely inhibited by the CXCR4 antagonist in T-lymphoid SupT1 cells. Together, our data point to the existence of an additional receptor through which CXCL12 exerts its biological effects in endothelial cells.     

Molecular mechanism of action of monocyclam versus bicyclam non-peptide antagonists in the CXCR4 chemokine receptor

AMD3465 is a novel, nonpeptide CXCR4 antagonist and a potent inhibitor of HIV cell entry in that one of the four-nitrogen cyclam rings of the symmetrical, prototype bicyclam antagonist AMD3100 has been replaced by a two-nitrogen N-pyridinylmethylene moiety. This substitution induced an 8-fold higher affinity as determined against (125)I-12G5 monoclonal CXCR4 antibody binding, and a 22-fold higher potency in inhibition of CXCL12-induced signaling through phosphatidylinositol accumulation. Mutational mapping of AMD3465 and a series of analogs of this in a library of 23 mutants covering the main ligand binding pocket of the CXCR4 receptor demonstrated that the single cyclam ring of AMD3465 binds in the pocket around AspIV:20 (Asp(171)), in analogy with AMD3100, whereas the N-pyridinylmethylene moiety mimics the other cyclam ring through interactions with the two acidic anchor-point residues in transmembrane (TM)-VI (AspVI:23/Asp(262)) and TM-VII (GluVII:06/Glu(288)). Importantly, AMD3465 has picked up novel interaction sites, for example, His(281) located at the interface of extracellular loop 3 and TM-VII and HisIII:05 (His(113)) in the middle of the binding pocket. It is concluded that the simple N-pyridinylmethylene moiety of AMD3465 substitutes for one of the complex cyclam moieties of AMD3100 through an improved and in fact expanded interaction pattern mainly with residues located in the extracellular segments of TM-VI and -VII of the CXCR4 receptor. It is suggested that the remaining cyclam ring of AMD3465, which ensures the efficacious blocking of the receptor, in a similar manner can be replaced by chemical moieties allowing for, for example, oral bioavailability.     

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.     

Early Repeated Administration of CXCR4 Antagonist AMD3100 Dose-Dependently Improves Neuropathic Pain in Rats After L5 Spinal Nerve Ligation

AMD3100 is a specific C-X-C chemokine receptor type 4 (CXCR4) antagonist which blocks the interaction between CXCR4 and CXCL12. Multiple lines of evidence suggest that AMD3100 has analgesic effects on many pathological pain states, including peripheral neuropathic pain. However, little is known about the underlying mechanisms. In the current study, we investigated the effect of different doses of AMD3100 on neuropathic pain in rats after L5 spinal nerve ligation. We used naloxone methiodide (NLXM) to further determine whether AMD3100-mediated analgesic effect was opioid-dependent. Behavioral study showed that early repeated administration of AMD3100 (2 and 5 mg/kg, i.p.) dose-dependently alleviates peripheral neuropathic pain. Flow cytometry, immunofluorescence and NLXM experiments showed that AMD3100 alleviates neuropathic pain partially by augmenting leukocyte-derived endogenous opioid secretion. Furthermore, we found that pro-inflammatory cytokines were down-regulated by AMD3100 using Enzyme-linked Immunosorbent Assay. Our data indicate that AMD3100 dose-dependently alleviates neuropathic pain partially by augmenting leukocyte-derived endogenous opioid secretion. This finding suggests that AMD3100 may be a viable pharmacotherapeutic strategy for the treatment of neuropathic pain.     

Consequences of ChemR23 Heteromerization with the Chemokine Receptors CXCR4 and CCR7

Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.