Chemokine Analogs Research Articles

Chemical Synthesis of Modularly Modified Analogs of E2/NS1 Peptides on a Novel Chemically and Mechanically Stable Terpolymer of 1,6-Hexanediol Diacrylate, N-Vinylpyrrolidone on Styrene Support

Analysis of the chemokine analogs synthesised on a terpolymer-polystyrene, N-vinylpyrrolidone, 1,6-hexanediol diacrylate crosslinked resin (PS-NVP-HDDA) showed it can be used a good solid support for the chemical synthesis of polypeptides. The polymer with a good swelling character and chemical resistance was synthesised by the radical aqueous suspension polymerisation technique. HPLC analysis of the crude peptides synthesised on PS-NVP-HDDA resin showed that it is a better substitute for most of the currently available polymer support for peptide synthesis. HIGHLIGHTS This article includes the synthesis of modularly modified analogs of E2/NS1 peptides The synthesized peptide is IIe-Phe-Thr-Tyr-His-Phe-Asn-Ser-Ser-Ser-Gly-Cys(Acm)-Pro-Glu-Ala-Asp-Leu-Asp- Cys(Acm)-Arg-Ser-Lys-Leu-Arg The peptide is synthesized on a chemically and mechanically stable terpolymer of 1,6-hexanediol diacrylate, N-vinylpyrrolidone on styrene support The polymer exhibited a good swelling character and chemical resistance The peptide was synthesized with great purity GRAPHICAL ABSTRACT

Purinergic Receptors Crosstalk with CCR5 to Amplify Ca2+ Signaling

Many G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C–C chemokine receptor type 5 (CCR5), which serves as a co-receptor to facilitate cellular entry of human immunodeficiency virus 1 (HIV-1), normally signals through the heterotrimeric G protein, Gi. However, CCR5 also exhibits G protein signaling bias and certain chemokine analogs can cause a switch to Gq pathways to induce Ca2+ signaling. We want to understand how much of the Ca2+ signaling from Gi-coupled receptors is due to G protein promiscuity and how much is due to transactivation and crosstalk with other receptors. We propose a possible mechanism underlying the apparent switching between different G protein signaling pathways. We show that chemokine-mediated Ca2+ flux in HEK293T cells expressing CCR5 can be primed and enhanced by ATP pretreatment. In addition, agonist-dependent lysosomal exocytosis results in the release of ATP to the extracellular milieu, which amplifies cellular signaling networks. ATP is quickly degraded via ADP and AMP to adenosine. ATP, ADP and adenosine activate different cell surface purinergic receptors. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signaling and allow for Gi- and Gq-coupled receptor signaling pathways to converge. Associated secretory release of GPCR ligands, such as chemokines, opioids, and monoamines, should also lead to concomitant release of ATP with a synergistic effect on Ca2+ signaling. Our results suggest that crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs is an important cooperative mechanism to amplify the intracellular Ca2+ signaling response.

CCR5 tyrosine sulfation heterogeneity generates cell surface receptor subpopulations with different ligand binding properties

BackgroundChemokine receptor tyrosine sulfation plays a key role in the binding of chemokines. It has been suggested that receptor sulfation is heterogeneous, but no experimental evidence has been provided so far. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. MethodsWe investigated the CCR5 sulfation heterogeneity and the sensitivity of CCR5 ligands to receptor sulfation by performing ELISA assays on synthetic N-terminal sulfopeptides and by performing binding assays on CCR5-expressing cells under conditions that modulate CCR5 sulfation levels. ResultsTwo commonly used anti-CCR5 monoclonal antibodies with epitopes in the sulfated N-terminal domain of CCR5 show contrasting binding profiles on CCR5 sulfopeptides, incomplete competition with each other for cell surface CCR5, and opposing sensitivities to cellular treatments that affect CCR5 sulfation levels. 5P12-RANTES is less sensitive than native CCL5 to conditions that affect cellular CCR5 sulfation. ConclusionsCCR5 sulfation is heterogeneous and this affects the binding properties of both native chemokines and antibodies. Enhanced capacity to bind to CCR5 is a component of the inhibitory mechanism of 5P12-RANTES. General significanceWe provide the first experimental evidence for sulfation heterogeneity of chemokine receptors and its impact on ligand binding, a phenomenon that is important both for the understanding of chemokine cell biology and for the development of drugs that target chemokine receptors.

Arrestin Recruitment to C-C Chemokine Receptor 5: Potent C-C Chemokine Ligand 5 Analogs Reveal Differences in Dependence on Receptor Phosphorylation and Isoform-Specific Recruitment Bias.

C-C chemokine receptor 5 (CCR5) is a chemokine receptor belonging to the G protein-coupled receptor (GPCR) superfamily. An established anti-human immunodeficiency virus drug target, CCR5 is attracting significant additional interest in both cancer and neuroinflammation. Several N-terminally engineered analogs of C-C chemokine ligand 5 (CCL5), a natural ligand of CCR5, are highly potent CCR5 inhibitors. The inhibitory mechanisms of certain analogs relate to modulation of receptor desensitization, but the cellular and molecular mechanisms have not been fully elucidated. Here we made use of a collection of CCR5 phosphorylation mutants and arrestin variants to investigate how CCL5 analogs differ from CCL5 in their capacity to elicit both CCR5 phosphorylation and arrestin recruitment, with reference to the current "core" and "tail" interaction model for arrestin-GPCR interaction. We showed that CCL5 recruits both arrestin 2 and arrestin 3 to CCR5 with recruitment, particularly of arrestin 2, strongly dependent on the arrestin tail interaction. 5P12-RANTES does not elicit receptor phosphorylation or arrestin recruitment. In contrast, PSC-RANTES induces CCR5 hyperphosphorylation, driving enhanced arrestin recruitment with lower dependence on the arrestin tail interaction. 5P14-RANTES induces comparable levels of receptor phosphorylation to CCL5, but arrestin recruitment is absolutely dependent on the arrestin tail interaction, and in one of the cellular backgrounds used, recruitment showed isoform bias toward arrestin 3 versus arrestin 2. No evidence for ligand-specific differences in receptor phosphorylation patterns across the four implicated serine residues was observed. Our results improve understanding of the molecular pharmacology of CCR5 and help further elucidate the inhibitory mechanisms of a group of potent inhibitors. SIGNIFICANCE STATEMENT: C-C chemokine receptor 5 (CCR5) is a key drug target for human immunodeficiency virus, cancer, and inflammation. Highly potent chemokine analog inhibitors act via the modulation of receptor desensitization, a process initiated by the recruitment of arrestin proteins. This study shows that potent C-C chemokine ligand 5 analogs differ from each other and from the parent chemokine in the extent and quality of CCR5-arrestin association that they elicit, providing valuable insights into CCR5 pharmacology and cell biology that will facilitate the development of new medicines targeting this important receptor.

High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Coreceptor CCR5

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

Development and pharmacokinetics of a combination vaginal ring for sustained release of dapivirine and the protein microbicide 5P12-RANTES

The past fifteen years have witnessed a resurgence of interest in vaginal ring technologies for drug delivery applications, mostly driven by the impetus for development of vaginally-administered antiretroviral microbicides to help reduce the high acquisition rates for human immunodeficiency virus (HIV) among Sub-Saharan African women. Currently, the lead candidate microbicide is a 28-day silicone elastomer vaginal ring releasing dapivirine (Ring-004), an experimental non-nucleoside reverse transcriptase inhibitor. The ring was tested in two pivotal Phase III clinical studies in 2016 and is currently undergoing review by the European Medicines Agency. Recently, we described a new type of silicone elastomer vaginal ring offering sustained release of the protein molecule 5P12-RANTES, a potent experimental chemokine analogue that potently blocks the HIV CCR5 coreceptor. Building on our previous work, here we report the preclinical development of a new combination vaginal ring that offers sustained release of both 5P12-RANTES and dapivirine, in which the 5P12-RANTES is incorporated into an exposed core within the ring body and the dapivirine in the sheath. In this way, in vitro release of dapivirine matches closely that for Ring-004. Also, we report the pharmacokinetic testing of this combination ring formulation in sheep, where vaginal concentrations of both drugs are maintained over 28 days at levels potentially useful for preventing HIV infection in women.

Fine-Tuning of GPCR-Chemokine Interactions. Design and Identification of Chemokine Analogues as Receptor Agonists, Biased Agonists, and Antagonists.

Chemokines play important roles in immune defense by directing migration of leukocytes and serve as key promoters of tumorigenesis and metastasis. This study explores the molecular mechanisms of recognition and activation of two homologous chemokine receptors, CXCR1 and CXCR2, using CXCL8 analogues with residue substitutions in the conserved Glu4Leu5Arg6 (ELR) triad. Analysis of the binding of CXCL8 analogues to CXCR1 is consistent with the two-site model for signal recognition of CXCR1, whereas analysis of the binding of CXCL8 analogues to CXCR2 supported a single-site model for signal recognition of CXCR2. The CXCL8-Arg6His analogue stimulated calcium release, phosphorylation of ERK1/2, and chemotaxis in cells expressing CXCR1. However, CXCL8-Arg6His failed to stimulate calcium release and chemotaxis in cells expressing CXCR2, although it stimulated phosphorylation of ERK1/2, indicating that CXCL8-Arg6His operated as a classical CXCR2 biased agonist. The CXCL8-Glu4AlaLeu5AlaArg6His analogue was inactive in cells expressing CXCR1 and CXCR2. These findings suggest that the Glu4Leu5 motif in CXCL8 is essential for activation of CXCR1 and CXCR2. Importantly, CXCL8-Glu4AlaLeu5AlaArg6His blocked specifically the calcium release and chemotaxis of cells expressing CXCR1 but not of cells expressing CXCR2. CXCL8-Glu4AlaLeu5AlaArg6His was identified as the first specific CXCR1 antagonist. The binding of CXCL8-ELR6H to CXCR1 created a Zn2+ coordination site at the receptor activation domain responsible for calcium release, as ZnCl2 specifically blocked CXCL8-Arg6His-induced calcium release without affecting CXCL8-induced calcium release. This work provides the basis for further exploration of the activation mechanisms of chemokine receptors and will assist in the design of the next generation of modulators of CXCR1 and CXCR2.

Vaginal rings with exposed cores for sustained delivery of the HIV CCR5 inhibitor 5P12-RANTES

Antiretroviral-releasing vaginal rings are at the forefront of ongoing efforts to develop microbicide-based strategies for prevention of heterosexual transmission of the human immunodeficiency virus (HIV). However, traditional ring designs are generally only useful for vaginal administration of relatively potent, lipophilic, and small molecular weight drug molecules that have sufficient permeability in the non-biodegradable silicone elastomer or thermoplastic polymers. Here, we report a novel, easy-to-manufacture ‘exposed-core’ vaginal ring that provides sustained release of the protein microbicide candidate 5P12-RANTES, an experimental chemokine analogue that potently blocks the HIV CCR5 coreceptor. In vitro release, mechanical, and stability testing demonstrated the utility and practicality of this novel ring design. In a sheep pharmacokinetic model, a ring containing two ¼-length excipient-modified silicone elastomer cores – each containing lyophilised 5P12-RANTES and exposed to the external environment by two large windows – provided sustained concentrations of 5P12-RANTES in vaginal fluid and vaginal tissue between 10 and 10,000 ng/g over 28days, at least 50 and up to 50,000 times the reported in vitro IC50 value.

Rapid and low-cost multiplex synthesis of chemokine analogs

Peptides represent a promising source of new medicines, but improved technologies are needed to facilitate discovery and optimization campaigns. In particular, longer peptides with multiple disulfide bridges are challenging to produce, and producing large numbers of structurally related variants is dissuasively costly and time-consuming. The principal cost and time drivers are the multiple column chromatography purification steps that are used during the multistep chemical synthesis procedure, which involves both ligation and oxidative refolding steps. In this study, we developed a method for multiplex parallel synthesis of complex peptide analogs in which the structurally variant region of the molecule is produced as a small peptide on a 384-well synthesizer with subsequent ligation to the longer, structurally invariant region and oxidative refolding carried out in-well without any column purification steps. To test the method, we used a panel of 96 analogs of the chemokine RANTES (regulated on activation normal T cell expressed and secreted)/CCL5 (69 residues, two disulfide bridges), which had been synthesized using standard approaches and characterized pharmacologically in an earlier study. Although, as expected, the multiplex method generated chemokine analogs of lower purity than those produced in the original study, it was nonetheless possible to closely match the pharmacological attributes (anti-HIV potency, capacity to elicit G protein signaling, and capacity to elicit intracellular receptor sequestration) of each chemokine analog to reference data from the earlier study. This rapid, low-cost approach has the potential to support discovery and optimization campaigns based on analogs of other chemokines as well as those of other complex peptide and small protein targets of a similar size.

G protein subtype-specific signaling bias in a series of CCR5 chemokine analogs.

Chemokines and some chemical analogs of chemokines prevent cellular HIV-1 entry when bound to the HIV-1 coreceptors C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4), which are G protein-coupled receptors (GPCRs). The ideal HIV-1 entry blocker targeting the coreceptors would display ligand bias and avoid activating G protein-mediated pathways that lead to inflammation. We compared CCR5-dependent activation of second messenger pathways in a single cell line. We studied two endogenous chemokines [RANTES (also known as CCL5) and MIP-1α (also known as CCL3)] and four chemokine analogs of RANTES (5P12-, 5P14-, 6P4-, and PSC-RANTES). We found that CCR5 signaled through both Gi/o and Gq/11 IP1 accumulation and Ca2+ flux arose from Gq/11 activation, rather than from Gβγ subunit release after Gi/o activation as had been previously proposed. The 6P4- and PSC-RANTES analogs were superagonists for Gq/11 activation, whereas the 5P12- and 5P14-RANTES analogs displayed a signaling bias for Gi/o These results demonstrate that RANTES analogs elicit G protein subtype-specific signaling bias and can cause CCR5 to couple preferentially to Gq/11 rather than to Gi/o signaling pathways. We propose that G protein subtype-specific signaling bias may be a general feature of GPCRs that can couple to more than one G protein family.

Evaluating Protein - Protein Interactions in Chemokine - Inhibitor Complexes using MD Simulation

Chemokines are signaling proteins that play many roles, including the recruitment of immune system cells to infections. Pathogens have evolved chemokine inhibitors that show promise as anti-inflammatory drugs. The poxvirus family produces a viral CC chemokine inhibitor (vCCI) which suppresses immune response by sequestering CC chemokines and thus prevents signaling to immune cells. The herpesvirus family produces a CC chemokine analog, viral macrophage inflammatory protein (vMIP-II), to compete with other CC chemokines at receptors to prevent immune response. In this study, we have used computer modeling to study the binding vCCI to three chemokines, human macrophage inflammatory protein-1β (MIP-1β), vMIP-II, and the human CC chemokine Eotaxin to compare the structural basis for their binding. Molecular Dynamics simulations allow for atomistic level detail of potential interactions between the chemokines and vCCI. Solvent accessible surface area analysis, as well as hydrogen bond count and persistence throughout the length of a simulation, provide a means of comparing the strength and types of interactions between vCCI and each of the chemokines. In addition, evaluating these interactions is key to determining which are critical to binding chemokines. vMIP-II, which has experimentally been shown to bind vCCI more strongly, has more overall hydrogen bonds with vCCI and maintains these hydrogen bonds through more of the simulation. We have also studied the effects of vCCI mutations on chemokine binding. Experimental studies of vCCI have shown that the Y80A mutation, predicted to increase binding strength to chemokines, instead, disables protein function. Simulations of vCCI Y80A reveal that the mutation on the microsecond time scale allows for the flexible loop of vCCI to collapse on the beta sheet docking site for chemokines, blocking key interaction sites for chemokine binding.

Pharmacokinetics of the Protein Microbicide 5P12-RANTES in Sheep following Single-Dose Vaginal Gel Administration.

5P12-RANTES, a chemokine analogue that potently blocks the HIV CCR5 coreceptor, is being developed as both a vaginal and rectal microbicide for prevention of sexual transmission of HIV. Here, we report the first pharmacokinetic data for 5P12-RANTES following single-dose vaginal gel administration in sheep. Aqueous gel formulations containing low (1.24-mg/ml), intermediate (6.18-mg/ml), and high (32.0-mg/ml; suspension-type gel) concentrations of 5P12-RANTES were assessed via rheology, syringeability, and in vitro release testing. Following vaginal gel administration to sheep, 5P12-RANTES concentrations were measured in vaginal fluid, vaginal tissue, and serum over a 96-h period. All gels showed non-Newtonian pseudoplastic behavior, with the high-concentration gels exhibiting a greater viscosity and cohesive structure than the intermediate- and low-concentration gels. In in vitro release testing, >90% 5P12-RANTES was released from the low- and intermediate-concentration gels after 72 h. For the high-concentration gel, ∼50% 5P12-RANTES was detected, attributed to protein denaturation during lyophilization and/or subsequent solvation of the protein within the gel matrix. In sheep, 5P12-RANTES concentrations in vaginal fluid, vaginal tissue, and serum increased in a dose-dependent manner. The highest concentrations were measured in vaginal fluid (105 to 107 ng/ml), followed by vaginal tissue (104 to 106 ng/ml). Both of these concentration ranges are several orders of magnitude above the reported half-maximal inhibitory concentrations. The lowest concentration was measured in serum (<102 ng/ml). The 5P12-RANTES pharmacokinetic data are similar to those reported previously for other candidate microbicides. These data, coupled with 5P12-RANTES's potency at picomolar concentrations, its strong barrier to resistance, and the full protection that it was observed to provide in a rhesus macaque vaginal challenge model, support the continued development of 5P12-RANTES as a microbicide.

Biophysical and Computational Studies of the vCCI:vMIP-II Complex.

Certain viruses have the ability to subvert the mammalian immune response, including interference in the chemokine system. Poxviruses produce the chemokine binding protein vCCI (viral CC chemokine inhibitor; also called 35K), which tightly binds to CC chemokines. To facilitate the study of vCCI, we first provide a protocol to produce folded vCCI from Escherichia coli (E. coli.) It is shown here that vCCI binds with unusually high affinity to viral Macrophage Inflammatory Protein-II (vMIP-II), a chemokine analog produced by the virus, human herpesvirus 8 (HHV-8). Fluorescence anisotropy was used to investigate the vCCI:vMIP-II complex and shows that vCCI binds to vMIP-II with a higher affinity than most other chemokines, having a Kd of 0.06 ± 0.006 nM. Nuclear magnetic resonance (NMR) chemical shift perturbation experiments indicate that key amino acids used for binding in the complex are similar to those found in previous work. Molecular dynamics were then used to compare the vCCI:vMIP-II complex with the known vCCI:Macrophage Inflammatory Protein-1β/CC-Chemokine Ligand 4 (MIP-1β/CCL4) complex. The simulations show key interactions, such as those between E143 and D75 in vCCI/35K and R18 in vMIP-II. Further, in a comparison of 1 μs molecular dynamics (MD) trajectories, vMIP-II shows more overall surface binding to vCCI than does the chemokine MIP-1β. vMIP-II maintains unique contacts at its N-terminus to vCCI that are not made by MIP-1β, and vMIP-II also makes more contacts with the vCCI flexible acidic loop (located between the second and third beta strands) than does MIP-1β. These studies provide evidence for the basis of the tight vCCI:vMIP-II interaction while elucidating the vCCI:MIP-1β interaction, and allow insight into the structure of proteins that are capable of broadly subverting the mammalian immune system.

Generating Chemokine Analogs with Enhanced Pharmacological Properties Using Phage Display.

Phage display technology, which allows extremely rare ligands to be selected from libraries of variants according to user-defined selection criteria, has made a huge impact on the life sciences. In this chapter, we describe phage display methods for the discovery of chemokine analogs with enhanced pharmacological properties. We discuss strategies for chemokine library design and provide a recommended technique for library construction. We also describe cell-based library selection approaches that we have used to discover chemokine analogs, not only receptor antagonists but also variants with unusual effects on receptor signaling and trafficking. By providing a survey of the different phage chemokine projects that we have undertaken, we comment on the parameters most likely to affect success. Finally, we discuss how phage display-derived chemokine analogs with altered pharmacological activity represent valuable tools to better understand chemokine biology, and why certain among them have the potential to be developed as new medicines.

CCR5 susceptibility to ligand-mediated down-modulation differs between human T lymphocytes and myeloid cells.

CCR5 is a chemokine receptor expressed on leukocytes and a coreceptor used by HIV-1 to enter CD4(+) T lymphocytes and macrophages. Stimulation of CCR5 by chemokines triggers internalization of chemokine-bound CCR5 molecules in a process called down-modulation, which contributes to the anti-HIV activity of chemokines. Recent studies have shown that CCR5 conformational heterogeneity influences chemokine-CCR5 interactions and HIV-1 entry in transfected cells or activated CD4(+) T lymphocytes. However, the effect of CCR5 conformations on other cell types and on the process of down-modulation remains unclear. We used mAbs, some already shown to detect distinct CCR5 conformations, to compare the behavior of CCR5 on in vitro generated human T cell blasts, monocytes and MDMs and CHO-CCR5 transfectants. All human cells express distinct antigenic forms of CCR5 not detected on CHO-CCR5 cells. The recognizable populations of CCR5 receptors exhibit different patterns of down-modulation on T lymphocytes compared with myeloid cells. On T cell blasts, CCR5 is recognized by all antibodies and undergoes rapid chemokine-mediated internalization, whereas on monocytes and MDMs, a pool of CCR5 molecules is recognized by a subset of antibodies and is not removed from the cell surface. We demonstrate that this cell surface-retained form of CCR5 responds to prolonged treatment with more-potent chemokine analogs and acts as an HIV-1 coreceptor. Our findings indicate that the regulation of CCR5 is highly specific to cell type and provide a potential explanation for the observation that native chemokines are less-effective HIV-entry inhibitors on macrophages compared with T lymphocytes.

Potent Anti-HIV Chemokine Analogs Direct Post-Endocytic Sorting of CCR5.

G protein-coupled receptors (GPCRs) are desensitized and internalized following activation. They are then subjected to post-endocytic sorting (degradation, slow recycling or fast recycling). The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors. This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV. Using Chinese Hamster Ovary cells stably expressing CCR5 we show that two different anti-HIV chemokine analogs, PSC-RANTES and 5P14-RANTES, direct receptor trafficking into two distinct subcellular compartments: the trans-Golgi network and the endosome recycling compartment, respectively. Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1), with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.

Targeting Spare CC Chemokine Receptor 5 (CCR5) as a Principle to Inhibit HIV-1 Entry

CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of β-arrestin 2·CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade.

Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C–C chemokine receptor 5 with RANTES/CCL5 analogues

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150 nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues.

Blockade of CXCR2 signalling: A potential therapeutic target for preventing neutrophil-mediated inflammatory diseases

Polymorphonuclear neutrophils (PMN) play a key role in host innate immune responses by migrating to the sites of inflammation. Furthermore, PMN recruitment also plays a significant role in the pathophysiology of a plethora of inflammatory disorders such as chronic obstructive pulmonary disease (COPD), gram negative sepsis, inflammatory bowel disease (IBD), lung injury, and arthritis. Of note, chemokine-dependent signalling is implicated in the amplification of immune responses by virtue of its role in PMN chemotaxis in most of the inflammatory diseases. It has been clinically established that impediment of PMN recruitment ameliorates disease severity and provides relief in majority of other immune-associated disorders. This review focuses on different novel approaches clinically proven to be effective in blocking chemokine signalling associated with PMN recruitment that includes CXCR2 antagonists, chemokine analogs, anti-CXCR2 monoclonal antibodies, and CXCR2 knock-out models. It also highlights the significance of the utility of nanoparticles in drugs used for blocking migration of PMN to the sites of inflammation.

CCR5 Conformations Are Dynamic and Modulated by Localization, Trafficking and G Protein Association

CCR5 acts as the principal coreceptor during HIV-1 transmission and early stages of infection. Efficient HIV-1 entry requires a series of processes, many dependent on the conformational state of both viral envelope protein and cellular receptor. Monoclonal antibodies (MAbs) are able to identify different CCR5 conformations, allowing for their use as probes to distinguish CCR5 populations. Not all CCR5 MAbs are able to reduce HIV-1 infection, suggesting the use of select CCR5 populations for entry. In the U87.CD4.CCR5-GFP cell line, we used such HIV-1-restricting MAbs to probe the relation between localization, trafficking and G protein association for individual CCR5 conformations. We find that CCR5 conformations not only exhibit different localization and abundance patterns throughout the cell, but that they also display distinct sensitivities to endocytosis inhibition. Using chemokine analogs that vary in their HIV-1 inhibitory mechanisms, we also illustrate that responses to ligand engagement are conformation-specific. Additionally, we provide supporting evidence for the select sensitivity of conformations to G protein association. Characterizing the link between the function and dynamics of CCR5 populations has implications for understanding their selective targeting by HIV-1 and for the development of inhibitors that will block CCR5 utilization by the virus.