gp120 induces cell death in human neuroblastoma cells through the CXCR4 and CCR5 chemokine receptors

To infect target cells, the human immunodeficiency virus (HIV) type I (HIV-1) must engage not only the well-known CD4 molecule, but it also requires one of several recently described coreceptors. In particular, the CXCR4 (LESTR/fusin) receptor allows fusion and entry of T-tropic strains of HIV, whereas CCR5 is the major coreceptor used by primary HIV-1 strains that infect macrophages and CD4(+) T-helper cells (M-tropic viruses). In addition, the alpha chemokine SDF1alpha and the beta chemokines MIP1alpha, MIP1beta, and RANTES, natural ligands of CXCR4 and CCR5, respectively, are potent soluble inhibitors of HIV infection by blocking the binding between the viral envelope glycoprotein gp120 and the coreceptors. Approximately two-thirds of individuals with acquired immunodeficiency syndrome (AIDS) show neurologic complications, which are referred to a syndrome called AIDS dementia complex or HIV-1-associated cognitive/motor complex. The HIV-1 coat glycoprotein gp120 has been proposed as the major etiologic agent for neuronal damage, mediating both direct and indirect effects on the CNS. Furthermore, recent findings showing the presence of chemokine receptors on the surface of different cell types resident in the CNS raise the possibility that the association of gp120 with these receptors may contribute to the pathogenesis of neurological dysfunction. Here, we address the possible role of alpha and beta chemokines in inhibiting gp120-mediated neurotoxicity using the human neuroblastoma CHP100 cell line as an experimental model. We have previously shown that, in CHP100 cells, picomolar concentrations of gp120 produce a significant increase in cell death, which seems to proceed through a Ca(2+) - and NMDA receptor-dependent cascade. In this study, we gained insight into the mechanism(s) of neurotoxicity elicited by the viral glycoprotein. We found that CHP100 cells constitutively express both CXCR4 and CCR5 receptors and that stimulation with phorbol 12-myristate 13-acetate down-regulates their expression, thus preventing gp120-induced cell death. Furthermore, all the natural ligands of these receptors exerted protective effects against gp120-mediated neuronal damage, although with different efficiencies. These findings, together with our previous reports, suggest that the neuronal injury observed in HIV-1 infection could be due to direct (or indirect) interactions between the viral protein gp120 and chemokine and/or NMDA receptors.     

Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. Here, we provide the first evidence that hypoxia inhibits CCR5 chemokine receptor expression in mouse macrophages. CCR5 was constitutively expressed in macrophages and upregulated by IFNgamma. Hypoxia downregulated both constitutive and IFNgamma-induced CCR5 mRNA and protein. Reoxygenation of hypoxic cells reverted CCR5 inhibition. CCR5 upregulation by IL-10, LPS, and IL-4 was also antagonized by hypoxia. CCR5 inhibition may be a way to retain/concentrate recruited macrophages at hypoxic sites or a feedback mechanism to control the autocrine activation of macrophages which produce CCR5 ligands.     

Design and synthesis of pyridin-2-yloxymethylpiperidin-1-ylbutyl amide CCR5 antagonists that are potent inhibitors of M-tropic (R5) HIV-1 replication

A novel series of CCR5 antagonists were identified based on the redesign of Schering C. An SAR was established based on inhibition of CCR5 (RANTES) binding and these compounds exhibited potent inhibition of R5 HIV-1 replication in peripheral blood mononuclear cells.     

Design and synthesis of pyridin-2-ylmethylaminopiperidin-1-ylbutyl amide CCR5 antagonists that are potent inhibitors of M-tropic (R5) HIV-1 replication

A series of CCR5 antagonists were optimized for potent inhibition of R5 HIV-1 replication in peripheral blood mononuclear cells. Compounds that met acceptable ADME criteria, selectivity, human plasma protein binding, potency shift in the presence of α-glycoprotein were evaluated in rat and dog pharmacokinetics.     

Discovery of a novel series of cyclic urea as potent CCR5 antagonists

A novel series of cyclic urea-based CCR5 antagonists was designed aiming to resolve instability issue in the fasted simulated intestinal fluid (FSIF) associated with the acyclic urea moiety in 1. This class of CCR5 compounds demonstrated high antiviral activities against HIV-1 infection in both HOS and PBL assays. Further evaluation of these compounds indicated that 16-R not only substantially enhanced its stability, but also exhibited excellent pharmacokinetics properties.     

pH-independent endocytic cycling of the chemokine receptor CCR5

Following agonist activation, the chemokine receptor CCR5 is internalised through clathrin-coated pits and delivered to recycling endosomes. Subsequently, ligand- free and resensitised receptors are recycled to the cell surface. Currently little is known of the mechanisms regulating resensitisation and recycling of this G-protein coupled receptor. Here we show that raising the pH of endocytic compartments, using bafilomycin A, monensin or NH(4)Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well-characterised G protein coupled receptor, the beta(2)-adrenergic receptor, following agonist-induced internalisation. CCR5-bound RANTES (CCL5) and MIP-1beta (CCL4) only exhibit pH-dependent dissociation at pH < 4.0, below the values normally found in endocytic organelles. Although receptor-agonist dissociation is not dependent on low pH, the subsequent degradation of released chemokine is inhibited in the presence of reagents that raise endosomal pH. Our data show that exposure to low pH is not required for RANTES or MIP-1beta dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.     

Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1

To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.     

Spiropiperidine CCR5 antagonists

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.     

CCR5 N-terminus peptides enhance X4 HIV-1 infection by CXCR4 up-regulation

The HIV-1 envelope glycoprotein gp120 interacts consecutively with CD4 and CCR5 to mediate the entry of R5-HIV-1 strains into target cells. The N-terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in gp120-CCR5 binding and, consequently, in viral entry. Here, we demonstrate that a tyrosine sulfated peptide, reproducing the entire N-terminal extracellular region of CCR5, its unsulfated analogue, and a point-mutated peptide are unable to inhibit R5-HIV-1 mediated infection, competing with the entire CCR5 in the formation of gp120-CD4-CCR5 complex. Surprisingly, these peptides show the capability of enhancing HIV-1 infection caused by X4 strains through the up-regulation of both CD4 and CXCR4 receptors.     

Discovery of novel pyridyl carboxamides as potent CCR5 antagonists and optimization of their pharmacokinetic profile in rats

A novel series of pyridyl carboxamide-based CCR5 inhibitors was designed, synthesized, and demonstrated to be highly potent against HIV-1 infection in both HOS and PBL assays. Attempts to evaluate this series of compounds in a rat PK model revealed its instability in rat plasma. A hypothesis for this liability was proposed, and strategies to overcome this issue were pursued, leading to discovery of highly potent 40 and 41, which featured dramatically improved rat PK profiles.     

趋化因子CCR7受体在子宫内膜组织上的表达水平及意义

目的通过观察趋化因子CCR7受体在子宫内膜异位症组织上的表达,探讨趋化因子CCR7受体在子宫内膜异位症发病中的作用。方法采用SABC免疫组化染色法观察CCR7在正常对照的子宫内膜和AM的异位子宫内膜及OEM的异位子宫内膜、在位子宫内膜腺上皮组织中表达水平。结果 CCR7主要在腺上皮细胞的胞浆和胞膜表达,CCR7受体的表达水平在AM组及OEM组的异位、在位子宫内膜显著高于正常子宫内膜(P0.05)。CCR7受体的表达水平在AM组及OEM组的异位内膜间差异无统计学意义(P0.05)。结论     

HIV chemokine receptor inhibitors as novel anti-HIV drugs

The chemokine receptors CXCR4 and CCR5 are the main coreceptors used by the T-cell-tropic (CXCR4-using, X4) and macrophage-tropic (CCR5-using, R5) HIV-1 strains, respectively, for entering their CD4⁺ target cells. In this review, we focus on the function of these chemokine receptors in HIV infection and their role as novel targets for viral inhibition. Besides some modified chemokines with antiviral activity, several low-molecular weight CCR5 and CXCR4 antagonistic compounds have been described with potent antiviral activity. The best CXCR4 antagonists described are the bicyclam derivatives, which consistently block X4 but also R5/X4 viral replication in PBMCs. We believe that chemokine receptor antagonists will become important new antiviral drugs to combat AIDS. Both CXCR4 and CCR5 chemokine receptor inhibitors will be needed in combination and even in combinations of antiviral drugs that also target other aspects of the HIV replication cycle to obtain optimum antiviral therapeutic effects.     

Spirodiketopiperazine-based CCR5 antagonist: discovery of an antiretroviral drug candidate

Following the discovery that hydroxylated derivative 3 (Fig. 1) was one of the oxidative metabolites of the original lead 1, it was found that hydroxylated compound 4 possesses higher in vitro anti-HIV potency than the corresponding non-hydroxylated compound 2. Structural hybridation of 4 with the orally available analog 5 resulted in another orally-available spirodiketopiperazine CCR5 antagonist 6a that possesses more favorable pharmaceutical profile for use as a drug candidate.     

Cell membrane hybrid bilayers containing the G-protein-coupled receptor CCR5

A hybrid bilayer membrane is a planar model membrane that is formed at an alkanethiol monolayer-coated gold surface by the spontaneous reorganization of phospholipid vesicles. Membrane vesicles from monkey kidney COS-1 cells also reorganize at an alkanethiol/lipid monolayer-coated surface resulting in the formation of a cell membrane hybrid bilayer. Atomic force microscopy and spectroscopic ellipsometry indicate that the cell membrane layer is equivalent to the thickness of one leaflet of the membrane and is continuous over large areas. Cell membrane hybrid bilayers were formed from membrane vesicles from COS-1 cells that were transiently transfected with a synthetic human CCR5 chemokine receptor gene. Preparations that contained "inside out" and "right side out" membrane vesicles were used. Binding of monoclonal antibodies to either the amino- or carboxyl-terminus of CCR5 was observed by surface plasmon resonance and confirmed the presence and the random orientation of these integral membrane receptors. Specific and concentration-dependent binding of the beta-chemokine RANTES to the cell membrane hybrid confirmed that CCR5 retained ligand-binding activity. The ability to form cell membrane hybrid bilayers that contain functional G-protein-coupled or other multispanning receptors without requiring protein isolation, purification, and reconstitution offers a promising method for the rapid screening of potential ligands.     

Alkylsulfone-containing trisubstituted cyclohexanes as potent and bioavailable chemokine receptor 2 (CCR2) antagonists

We describe novel alkylsulfones as potent CCR2 antagonists with reduced hERG channel activity and improved pharmacokinetics over our previously described antagonists. Several of these new alkylsulfones have a profile that includes functional antagonism of CCR2, in vitro microsomal stability, and oral bioavailability. With this improved profile, we demonstrate that two of these antagonists, 2 and 12, are orally efficacious in an animal model of inflammatory recruitment.     

The HIV-1 co-receptor CCR5 binds to alpha-catenin, a component of the cellular cytoskeleton

The chemokine receptors CCR5 and CXCR4 belong to the family of seven transmembrane-spanning G protein-coupled receptors, which have diverse functions in host cell defense and are associated with numerous diseases. CCR5 and CXCR4 are known as co-receptors for entry of HIV-1. In this study the intracellular carboxy-terminus of CCR5, which is deleted in HIV-infected long-term non-progressors, was shown to interact with the carboxy-terminus of alpha-catenin, a component of the cytoskeleton, in a yeast two-hybrid screen. This interaction was verified in mammalian cells. Furthermore, the interaction of alpha-catenin with CCR5 and CXCR4 at endogenous protein levels was demonstrated in PM1 T-lymphocytes, a host cell line of HIV-1. Our results suggest that alpha-catenin links CCR5 and CXCR4 to the cytoskeleton and is involved in the organization of these receptors at the membrane, thereby possibly affecting HIV-1 infection.     

Discovery of INCB10820/PF-4178903, a potent, selective, and orally bioavailable dual CCR2 and CCR5 antagonist

We report the discovery of a potent, selective, and orally bioavailable dual CCR2 and CCR5 antagonist (3S,4S)-N-[(1R,3S)-3-isopropyl-3-({4-[4-(trifluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-3-methoxytetrahydro-2H-pyran-4-amine (19). After evaluation in 28-day toxicology studies, compound 19 (INCB10820/PF-4178903) was selected as a clinical candidate.     

CCR5 interactions with the variable 3 loop of gp120

The G-protein coupled receptor CCR5 functions pathologically as the primary co-receptor for macrophage tropic (R5) strains of HIV-1. The interactions responsible for co-receptor activity are unknown. Molecular-dynamics simulations of the extracellular and adjacent transmembrane domains of CCR5 were performed with explicit solvation utilizing a rhodopsin-based homology model. The functional unit of co-receptor binding was constructed via docking and molecular-dynamics simulation of CCR5 and the variable 3 loop of gp120, which is a dominant determinant of co-receptor utilization. The variable 3 loop was demonstrated to interact primarily with the amino terminus and the second extracellular loop of CCR5, providing novel structural information regarding the co-receptor-binding site. Alanine mutants that alter chemokine binding and co-receptor activity were examined. Molecular-dynamics simulations with and without the variable 3 loop of gp120 were able to rationalize the activities of these mutants successfully, providing support for the proposed model. Based on these results, the global complex of CCR5, gp120 including the V3 loop and CD4, was investigated. The utilization of computational analysis, in combination with molecular biological data, provides a powerful approach for understanding the use of CCR5 as a co-receptor by HIV-1.     

Genetic diversity and prevalence of CCR2-CCR5 gene polymorphisms in the Omani population

Polymorphisms in the regulatory region of the CCR5 gene affect protein expression and modulate the progress of HIV-1 disease. Because of this prominent role, variations in this gene have been under differential pressure and their frequencies vary among human populations. The CCR2V64I mutation is tightly linked to certain polymorphisms in the CCR5 gene. The current Omani population is genetically diverse, a reflection of their history as traders who ruled extensive regions around the Indian Ocean. In this study, we examined the CCR2-CCR5 haplotypes in Omanis and compared the patterns of genetic diversity with those of other populations. Blood samples were collected from 115 Omani adults and genomic DNA was screened to identify the polymorphic sites in the CCR5 gene and the CCR2V64I mutation. Four minor alleles were common: CCR5-2554T and CCR5-2086G showed frequencies of 49% and 46%, respectively, whereas CCR5-2459A and CCR5-2135C both had a frequency of 36%. These alleles showed moderate levels of heterozygosity, indicating that they were under balancing selection. However, the well-known allele CCR5Δ32 was relatively rare. Eleven haplotypes were identified, four of which were common: HHC (46%), HHE (20%), HHA (14%) and HHF*2 (12%).