CC Chemokine Receptor 5 Receptor Research Articles

Novel small synthetic HIV-1 V3 crown variants: CCR5 targeting ligands.

The CC chemokine receptor 5 (CCR5) antagonism represents a promising pharmacological strategy for therapeutic intervention as it plays a significant role in reducing the severity and progression of a wide range of pathological conditions. Here we designed and generated peptide ligands targeting the chemokine receptor, CCR5, that were derived from the critical interaction sites of the V3 crown domain of envelope protein glycoprotein gp120 (TRKSIHIGPGRAFYTTGEI) of HIV-1 using computational biology approach and the peptide sequence corresponding to this region was taken as the template peptide, designated as TMP-1. The peptide variants were synthesized by employing Fmoc chemistry using polymer support and were labelled with rhodamine B to study their interaction with the CCR5 receptor expressed on various cells. TMP-1 and TMP-2 were selected as the high-affinity ligands from in vitro receptor-binding assays. Specific receptor-binding experiments in activated peripheral blood mononuclear cells and HOS.CCR5 cells indicated that TMP-1 and TMP-2 had significant CCR5 specificity. Further, the functional analysis of TMP peptides using chemotactic migration assay showed that both peptides did not mediate the migration of responsive cells. Thus, template TMP-1 and TMP-2 represent promising CCR5 targeting peptide candidates.

The CCR5 antagonist maraviroc causes remission of pancreatic cancer liver metastasis in nude rats based on cell cycle inhibition and apoptosis induction.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease, and novel therapeutic strategies are urgently needed. Recently, expression of the C-C chemokine receptor 5 (CCR5) and its ligands has been found to play an important role in cancer progression and metastasis. In this study, we blocked the CCR5 receptor by the FDA approved antagonist maraviroc (MVC) in Suit2-007 and MIA-PaCa-2 human PDAC cells. The treatment significantly inhibited their proliferation and induced apoptosis of exposed cells as evidenced by caspases activation and increased Bax levels. Moreover, MVC inhibited the cell cycle by down regulating the proteins of the complexes of cyclin dependent kinase (CDK) 4/6 - Cyclin D and CDK2 - Cyclin E, as well as by increasing the protein levels of CDK inhibitors p18, p21 and p27. In line with this, MVC caused significant retardation of Suit2-007cells growing in a PDAC liver metastasis xenograft model (p<0.05). These results suggest that maraviroc could be a promising treatment strategy for PDAC patients with liver metastases.

The Abrogation of Phosphorylation Plays a Relevant Role in the CCR5 Signalosome Formation with Natural Antibodies to CCR5.

The exposure to CCR5 (CC chemokine receptor 5) specific natural antibodies in vitro produces a Class B β-arrestin2-dependent CCR5 retention with the aid of ERK1, due to the formation of a CCR5 signalosome, which remains stable for at least 48 h. Considering that β-arrestins and MAPKs are receptive to environmental signals, their signal complexes could be one of the key junction for GPCRs internalization related signal transduction. Here, we demonstrate that, in T cells, the phosphorylation status of either CCR5 receptor or ERK1 protein is necessary to drive the internalized receptor into the early endosomes, forming the CCR5 signalosome. In particular, our data show that β-arrestin2/ERK1 complex is a relevant transducer in the CCR5 signaling pathway. Understanding the mechanism of CCR5 regulation is essential for many inflammatory disorders, tumorigenesis and viral infection such as HIV.

Knockdown of C-C Chemokine Receptor 5 (CCR5) is Protective Against Cerebral Ischemia and Reperfusion Injury.

Stroke is the second leading cause of death and a major cause of disability of adults worldwide. Inflammatory processes are known to contribute to the pathophysiology of cerebral ischemia, especially following reperfusion. Chemokines and their receptors are involved in migration of leukocytes and have been implicated in the pathogenesis of ischemic stroke. In the present study, we investigated the effects of C-C chemokine receptor type 5 (CCR5) deficiency on neurological outcome, brain damage and expression of pro-inflammatory chemokines: chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (CC motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5), and the brain-derived neurotrophic factor (BDNF). Adult male C57BL/6 (wild-type) (WT) and CCR5 deficient mice were subjected to transient cerebral ischemia induced by 25 min of bilateral common carotid artery occlusion (BCCAO) followed by 24 hours of reperfusion. Mice were divided into four groups: WT sham group, which underwent sham operation; WT ischemic group, which was subjected to transient bilateral common carotid artery occlusion, CCR5-/- sham group, which underwent sham operation, and CCR5-/- ischemic group, which was subjected to transient BCCAO. In CCR5 deficiency, we observed a significant improvement in the neurological deficits associated with decreased brain infarcted area as evaluated by triphenyltetrazolium chloride (TTC). Moreover, CCR5 deficiency revealed decreased percentage of necrotic cavities areas and frequency of ischemic neurons by histometric analysis. In addition, CCR5-/- ischemic animals showed lower brain levels of the chemokine CXCL1 and higher levels of BDNF by ELISA, compared with WT BCCAo mice. Taken together, our results suggest a potential neuroprotection in the absence of CCR5 receptor during global brain ischemia and reperfusion injury.

An extended CCR5 ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions.

C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two-dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505.

Structural insight into a novel human CCR5-V130I variant associated with resistance to HIV-1 infection

We report the identification of a novel CC chemokine receptor 5 (CCR5) variant that seems associated with resistance to HIV-1 infection. The V130I mutation of the CCR5 receptor is located in the intracellular loop ICL2 known as DRY box and described in the literature as a nonsynonymous mutation present in nonhuman primates group. Extensive molecular modeling and dynamics simulations were performed to elucidate the mechanism by which the V130I mutation may induce conformational change of the CCR5 folding protein and prevent the interaction with the β-arrestin protein. Our study provides new mechanistic insight into how a specific mutation in the regulatory domain of CCR5 might alter the structural folding of the DRY box and the possible ICL2 loop binding with the β-arrestin protein, as described in our previous computational study. The results from our large-scale simulations complement recent experimental results and clinical features and offer useful insights into the mechanism behind CCR5 protein folding and signal transduction. In order for HIV, the entry of the virus to the cells must fuse with the CCR5 receptor that sits on the surface of T-helper immune cells. The described V130I mutation in the gene encoding the CCR5 protein may results in a defective CCR5-Arrestin binding complex that blocks entry of the virus.

Characterization of the expression of macrophage inflammatory protein‐1α (MIP‐1α) and C‐C chemokine receptor 5 (CCR5) after kainic acid‐induced status epilepticus (SE) in juvenile rats

To identify the potential role of macrophage inflammatory protein-1α (MIP-1α) with its C-C chemokine receptor 5 (CCR5) in epileptogenic brain injury, we examined their expression in juvenile rat hippocampus and explored the potential link between MIP-1α, CCR5 and neuropathological alterations after status epilepticus (SE) induced by intracerebroventricular (i.c.v.) kainic acid (KA) injection. Based on the determination of the development of spontaneous seizures initiated by SE in developing rat brain, we firstly examined hippocampal neurone damage through Nissl and Fluoro-Jade B staining, and evaluated microglial reaction during the early phase following KA-induced SE in 21-day-old rats. MIP-1α and CCR5 protein were quantified by ELISA and Western blot respectively following mRNA by real-time PCR. We also mapped MIP-1α and CCR5 expression in the hippocampus by immunohistochemistry and identified their cellular sources using double-labelling immunofluorescence. In juvenile rats, KA caused characteristic neurone damage in the hippocampal subfields, with accompanying microglial accumulation. In parallel with mRNA expression, MIP-1α protein in hippocampus was transiently increased after KA treatment, and peaked from 16 to 72 h. Double-labelling immunofluorescence revealed that MIP-1α was localized to microglia. Up-regulated CCR5 remained prominent at 24 and 72 h and was mainly localized to activated microglia. Further immunohistochemistry revealed that MIP-1α and CCR5 expression were closely consistent with microglial accumulation in corresponding hippocampal subfields undergoing degenerative changes. Our data indicated that MIP-1α as a regulator, linking with the CCR5 receptor, may be involved within the early stages of the epileptogenic process following SE by i.c.v. KA injection.

Docking and CoMFA study on novel human CCR5 receptor antagonists

The CC-chemokine receptor 5 (CCR5), a membrane protein belonging to the G protein-coupled receptor super-family, has been identified as an essential co-receptor for HIV entry into the cells. Small molecules were used to inhibit HIV entry by targeting CCR5. In this study, comparative molecular field analysis (CoMFA) was carried out on 70 novel phenoxybenzyl derivatives as antagonists of CCR5 HIV co receptor. This CoMFA model yielded satisfactory statistical data with the cross-validated q2 of 0.611 and non-cross-validated r2 of 0.903. Mapping this model according to the topology of the active site of CCR5 end to a better understanding of antagonist-CCR5 interactions. Because of lacking crystallography structure of CCR5, the 3D structure from threading assembly refinement (TASSER) method was used. These antagonists were docked into the binding site of the 3D model of CCR5 using ligandfit, and the probable interaction model between CCR5 and the antagonists were obtained. The correlation of predicted binding affinities between antagonists and CCR5 has good statistical quality.

Using a genetic, observational study as a strategy to estimate the potential cost-effectiveness of pharmacological CCR5 blockade in dialysis patients

Randomized clinical trials are expensive and time consuming. Therefore, strategies are needed to prioritise tracks for drug development. Genetic association studies may provide such a strategy by considering the differences between genotypes as a proxy for a natural, lifelong, randomized at conception, clinical trial. Previously an association with better survival was found in dialysis patients with systemic inflammation carrying a deletion variant of the CC-chemokine receptor 5 (CCR5). We hypothesized that in an analogous manner, pharmacological CCR5 blockade could protect against inflammation-driven mortality and estimated if such a treatment would be cost-effective. A genetic screen and treat strategy was modelled using a decision-analytic Markov model, in which patients were screened for the CCR5 deletion 32 polymorphism and those with the wild type and systemic inflammation were treated with pharmacological CCR5 blockers. Kidney transplantation and mortality rates were calculated using patient level data. Extensive sensitivity analyses were performed. The cost-effectiveness of the genetic screen and treat strategy was &OV0556;18 557 per life year gained and &OV0556;21 896 per quality-adjusted life years gained. Concordance between the genetic association and pharmacological effectiveness was a main driver of cost-effectiveness. Sensitivity analyses showed that even a modest effectiveness of pharmacological CCR5 blockade would result in a treatment strategy that is good value for money. Pharmacological blockade of the CCR5 receptor in inflamed dialysis patients can be incorporated in a potentially cost-effective screen and treat programme. These findings provide formal rationale for clinical studies. This study illustrates the potential of genetic association studies for drug development, as a source of Mendelian randomized evidence from an observational setting.

In Vitro and Clinical Investigation of the Relationship Between CCR5 Receptor Occupancy and Anti‐HIV Activity of Aplaviroc

Aplaviroc (GW873140) binds specifically to human cellular CC chemokine receptor 5 (CCR5) and demonstrates potent anti-human immunodeficiency virus activity in vitro in the subnanomolar range. In vitro studies show that aplaviroc selectively inhibits the binding of a particular monoclonal antibody, 45531, to CCR5. Based on this observation, a flow cytometry-based assay was developed to determine percentage CCR5 receptor occupancy (RO). CCR5 receptor occupancy was aplaviroc concentration-dependent and related to anti-human immunodeficiency virus activity in vitro. In the clinical setting, CCR5 receptor occupancy in peripheral blood was >98% in all subjects within 2 to 3 hours of dosing, which is consistent with the peak plasma concentrations of drug. Longitudinal analysis in the drug washout period revealed the time to 50% CCR5 receptor occupancy averaged >100 hours, in both human immunodeficiency virus-positive and human immunodeficiency virus-negative subjects, substantially longer than the plasma pharmacokinetic half-life of 3 hours. The duration of CCR5 receptor occupancy appeared to be dose-dependent and associated with antiviral activity as measured by plasma human immunodeficiency virus RNA nadir following 10 days of multiple dose administration. These data demonstrate that the analysis of CCR5 receptor occupancy, in addition to conventional plasma-based pharmacokinetic measures, provides an informative tool to assist in evaluating the pharmacodynamic and antiviral effects of cellular CC chemokine receptor antagonists.

A Novel Approach to Regulate Experimental Visceral Leishmaniasis in Murine Macrophages using CCR5 siRNA

Early infection with Leishmania donovani during visceral leishmaniasis (VL) results in the enhanced expression of C-C chemokine receptor 5 (CCR5) in macrophages. CCR5 expression at different time points of infection revealed increased expression at protein and mRNA level from 3 to 24 h and beyond 24 h expression of CCR5 is downregulated. To better understand the functional role of CCR5 during leishmania infection, RNA interference strategy has been used to modulate macrophage function by targeting the expression of CCR5 gene. We found that silencing of CCR5 receptor expression by transfection of CCR5-specific small interfering RNA (siRNA) in murine peritoneal macrophages restricted the parasitic burden up to 70% during early hours of infection. In addition, gene silencing of CCR5 prior to L. donovani infection enhanced the pro-inflammatory response of the host macrophages in comparison with infection alone as shown by high nitric oxide generation and the TNF-alpha:IL-10 ratio. These findings suggest that CCR5 receptor plays a significant role in the entry and establishment of L. donovani in murine macrophages and CCR5 gene silencing would be a potent therapeutic approach to control VL by restricting parasite entry.

CCR5 Receptor

The CC chemokine receptor 5 (CCR5) is a member of CC-chemokine receptor family. CCR5 has the characteristic structure of a seven transmembrane G protein-coupled receptor (GPCR), which regulates trafficking and effector functions of memory/effector Th1 cells, macrophages, NK cells, and immature dendritic cells. CCR5 and its ligands are important molecules in viral pathogenesis. CCR5 represents the co-receptor for macrophage (M) and dual (T cell and M)-tropic immunodeficiency viruses. Recent evidence has also demonstrated the role of CCR5 in a variety of human diseases, ranging from infectious and inflammatory diseases to cancer. In this article, we describe the involvement of CCR5 in two age-related diseases, atherosclerosis and Alzheimer's disease, suggesting a possible role of chemokine system on these diseases' pathophysiology. Finally, we review the data on the probable association between CCR5Delta32 deletion and cardiovascular diseases and Alzheimer's disease.

JM4 is a four-transmembrane protein binding to the CCR5 receptor

The CC chemokine receptor 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV) and CCR5 mutants lacking the carboxy (C)-terminus interfere with HIV infection. Therefore, we analysed the C-terminus of CCR5 and here describe Jena-Muenchen 4 (JM4), a novel CCR5-interacting protein. JM4 is membrane-associated, co-precipitates with CCR5, and is ubiquitously expressed. It shares about 62% sequence similarity with JWA and glutamate transporter-associated protein 3-18 (GTRAP3-18), a regulator of an amino acid transporter. JWA, like JM4, is a four-transmembrane protein, which binds to the CCR5 receptor. Furthermore, JM4, JWA, and GTRAP3-18 co-localise and heterodimerise indicating a functional relationship. JM4 co-localises with calnexin in the endoplasmic reticulum and with the mannose 6-phosphate receptor in the Golgi. JM4 and GTRAP3-18 harbor a Rab-acceptor motif, indicating a function in vesicle formation at the Golgi complex. In conclusion, we describe a CCR5-interacting protein, which is suggested to function in trafficking and membrane localisation of the receptor, possibly also other receptors or amino acid transporters.

CC-chemokine receptor 5 (CCR5) in hepatitis C--at the crossroads of the antiviral immune response?

An effective immune response to hepatitis C virus (HCV) infection requires efficient recruitment and activation of inflammatory cells to the liver, the site of infection. Chemokines are critically involved in this process, since they exert both chemotactic and immunoregulatory actions. In particular, the interaction between chemokines CCL3 (MIP-1alpha), CCL4 (MIP-1beta) and CCL5 (RANTES) and their receptor, CC-chemokine receptor 5 (CCR5), may be critical in regulating T cell functions by mediating recruitment, polarization, activation and differentiation of antiviral type 1 cytokine secreting T helper and cytotoxic T cells. A 32 bp deletion in the encoding region of CCR5 leads to complete loss of the functional CCR5 receptor in subjects homozygous for this mutation and decreased expression in heterozygous patients. This fact provides the unique opportunity to study the role of the CCR5 receptor in chronic hepatitis C infection by comparing immune responses between HCV infected CCR5-Delta32 carriers and CCR5 wild-type patients. This article will summarize and discuss the available data with respect to possibly altered disease susceptibility, clinical course and treatment outcomes associated with the CCR5-Delta32 mutation in hepatitis C.

A synthetic peptide fragment derived from RANTES is a potent inhibitor of HIV-1 infectivity despite a surprising lack of CCR5 receptor affinity

CCR5 (CC-chemokine receptor 5) is a key co-receptor, in concert with CD4, for infectivity of HIV-1 (human immunodeficiency virus type-1) into healthy human cells, and RANTES, an endogenous ligand for CCR5, is a potent inhibitor of HIV-1 infectivity. In this structure-activity relationship (SAR) study, peptide fragments derived from RANTES were designed, synthesized and evaluated for their ability to inhibit HIV-1 infectivity. The goal was to determine the effect of peptide length on anti-HIV activity and to obtain an optimally sized RANTES peptide probe for further SAR studies. The analogue Ac[Ala10,11]RANTES-(1–14)NH2, AA14, was identified as an effective inhibitor of HIV-1 infectivity at 10 nM but despite the functional activity, surprisingly it did not exhibit any notable affinity for the CCR5 chemokine receptor. Further, increasing peptide size enhanced neither the inhibition of HIV-1 infectivity nor CCR5 receptor affinity. As a potent inhibitor of HIV-1 infectivity, the lead analogue most likely utilizes a different (and currently unknown) mechanism than interaction with CCR5 for anti-HIV activity.

Co-receptor usage of HIV-1 primary isolates, viral burden, and CCR5 genotype in mother-to-child HIV-1 transmission

To investigate the relationship between CC chemokine receptor 5 (CCR5) genotype, viral load and co-receptor usage of maternal HIV-1 isolates in perinatal HIV-1 transmission. A total of 181 mothers and infants were studied at the time of delivery. Wild-type (wt) and delta32 CCR5 alleles were determined by means of polymerase chain reaction (PCR). The viral load in maternal plasma samples was determined by a quantitative reverse transcriptase-PCR assay; co-receptor usage of maternal isolates was determined by viral infection in cells stably expressing CCR5 or CXC chemokine receptor 4 (CXCR4) co-receptors. HIV-1 transmission rates in wt/wt and wt/delta32 mothers (14.7 versus 15.8%), and in wt/wt and wt/delta32 infants (14.6 versus 14.3%) were similar. Mothers transmitting infection to wt/delta32 infants had significantly higher HIV-1-RNA levels than those who transmitted infection to wt/wt infants (5.4 versus 4.1 log10 copies/ml, P = 0.03). In wt/wt children there was a positive relationship between transmission rate and maternal viral load over the entire range of HIV-1 values, whereas in wt/delta32 children transmission occurred only at viral loads greater than 4.0 log10 copies/ml. Logistic regression analysis confirmed that the relationship between viral load and transmission varied according to the child's CCR5 genotype (P = 0.035; adjusted for zidovudine prophylaxis and mode of delivery, P = 0.090). Moreover, the majority of wt/wt transmitting mothers had R5-type isolates, whereas none of the wt/delta32 mothers with an R5-type virus transmitted HIV-1 to their wt/delta32 infants. Taken together, these findings suggest that CCR5 delta32 heterozygosity exerts a protective effect against perinatal transmission in children exposed to a low maternal viral burden of an R5-type isolate.