The non-ELR CXC chemokine encoded by human cytomegalovirus UL146 genotype 5 contains a C-terminal β-hairpin and induces neutrophil migration as a selective CXCR2 agonist

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1_GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1_GT5 and vCXCL1_GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1_GT5, the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1_GT5 recruited β-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1_GT1 stimulated G protein signaling, recruited β-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1_GT1 and vCXCL1_GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1_GT4 and non-ELR vCXCL1_GT6 activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1_GT1, vCXCL1_GT4, vCXCL1_GT5, and vCXCL1_GT6) forms a novel β-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1_GT1 supported the existence of two stable β-strands. C-terminal deletion of vCXCL1_GT1 resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes.     

The highly O-glycosylated glycoprotein gp2 of equine herpesvirus 1 is encoded by gene 71.

There have been conflicting reports regarding the gene assignment of the high-molecular-mass envelope glycoprotein gp2 (gp300) of equine herpesvirus 1. Here, we provide an unequivocal demonstration that gp2 is encoded by gene 71. gp2 that was purified with a defining monoclonal antibody was cleaved internally to yield a 42-kDa protein encoded by gene 71. Amino acid composition data and N-terminal sequence analysis of a tryptic peptide identified gp2 as the product of equine herpesvirus 1 gene 71 with the SWISS-PROT database. Analysis of gp2's monosaccharide composition and the 42-kDa subunit showed that the high level of O glycosylation occurs on the serine/threonine-rich region upstream of the cleavage site.     

The synthesis and biological activity of a highly selective adenosine A2a receptor agonist

Three novel nucleosides 1, 2, and 3 were prepared that contained side chains at the 2-position of adenosine. Compound 1 was shown to be the most selective A2a receptor agonist reported to date having an A1/A2 ratio of 2400. In addition, compound 1 was shown to reduce blood pressure in rats and dogs with only minimal effects on heart rate.     

Salvinorin A: a novel and highly selective kappa-opioid receptor agonist

kappa-opioid receptors (KORs) represent the principal site of action of dynorphin and related neuropeptides. Recently, Salvinorin A--a naturally occurring neoclerodane diterpene hallucinogen was identified to be a highly selective KOR agonist. In this brief review we summarize the known chemistry, pharmacology and biology of salvinorin A. Because salvinorin A profoundly alters human consciousness and perception, a study of how salvinorin A exerts its actions on KORs may yield novel insights into the molecular and cellular basis of uniquely human higher cortical functions.     

Design and synthesis of a highly selective EP2-receptor agonist.

EP2-receptor selective agonist 3 was identified by the structural hybridization of butaprost 1a and PGE(2) 2a. Based on this information, a chemically more stabilized 4 was discovered as another highly selective EP2-receptor agonist, iv administration of which to anesthetized rats suppressed uterine motility, while PGE(2) 2a stimulated uterine motility.     

Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.     

Minor structural modifications convert a selective PPARalpha agonist into a potent, highly selective PPARdelta agonist

We report the solid-phase synthesis and pharmacological evaluation of a new series of small-molecule agonists of the human peroxisome proliferator-activated receptor delta (PPARdelta) based on a lead structure from our PPARalpha program. Compound 33 showed good pharmacokinetics.     

The CC chemokine CCL20 and its receptor CCR6

CCL20, alternatively named liver and activation-regulated chemokine (LARC), macrophage inflammatory protein-3alpha (MIP-3alpha) or Exodus-1, is the only chemokine known to interact with CC chemokine receptor 6 (CCR6), a property shared with the antimicrobial beta-defensins. The ligand-receptor pair CCL20-CCR6 is responsible for the chemoattraction of immature dendritic cells (DC), effector/memory T-cells and B-cells and plays a role at skin and mucosal surfaces under homeostatic and inflammatory conditions, as well as in pathology, including cancer and rheumatoid arthritis. In this review, the discovery, the gene and protein structure, the in vitro biological activities, the cell and inducer specific expression and the tissue distribution of CCL20 and CCR6 are discussed.     

Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.     

Identification and analysis of a novel heparin-binding glycoprotein encoded by human herpesvirus 7

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3') end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)(+) RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such as N-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.     

Suppression of CCR5- but not CXCR4-tropic HIV-1 in lymphoid tissue by human herpesvirus 6.

HIV-1 infects target cells via a receptor complex formed by CD4 and a chemokine receptor, primarily CCR5 or CXCR4 (ref. 1). Commonly, HIV-1 transmission is mediated by CCR5-tropic variants, also designated slow/low, non-syncytia-inducer or macrophage-tropic, which dominate the early stages of HIV-1 infection and frequently persist during the entire course of the disease. In contrast, HIV-1 variants that use CXCR4 are typically detected at the later stages, and are associated with a rapid decline in CD4+ T cells and progression to AIDS (refs. 2,7-11). Disease progression is also associated with the emergence of concurrent infections that may affect the course of HIV disease by unknown mechanisms. A lymphotropic agent frequently reactivated in HIV-infected patients is human herpesvirus 6 (HHV-6), which has been proposed as a cofactor in AIDS progression. Here we show that in human lymphoid tissue ex vivo, HHV-6 affects HIV-1 infection in a coreceptor-dependent manner, suppressing CCR5-tropic but not CXCR4-tropic HIV-1 replication, as shown with both uncloned viral isolates and isogenic molecular chimeras. Furthermore, we demonstrate that HHV-6 increases the production of the CCR5 ligand RANTES ('regulated upon activation, normal T-cell expressed and secreted'), the most potent HIV-inhibitory CC chemokine, and that exogenous RANTES mimics the effects of HHV-6 on HIV-1, providing a mechanism for the selective blockade of CCR5-tropic HIV-1. Our data suggest that HHV-6 may profoundly influence the course of HIV-1 infection.     

A TNF receptor 2 selective agonist rescues human neurons from oxidative stress-induced cell death

Tumor necrosis factor (TNF) plays a dual role in neurodegenerative diseases. Whereas TNF receptor (TNFR) 1 is predominantly associated with neurodegeneration, TNFR2 is involved in tissue regeneration and neuroprotection. Accordingly, the availability of TNFR2-selective agonists could allow the development of new therapeutic treatments of neurodegenerative diseases. We constructed a soluble, human TNFR2 agonist (TNC-scTNF(R2)) by genetic fusion of the trimerization domain of tenascin C to a TNFR2-selective single-chain TNF molecule, which is comprised of three TNF domains connected by short peptide linkers. TNC-scTNF(R2) specifically activated TNFR2 and possessed membrane-TNF mimetic activity, resulting in TNFR2 signaling complex formation and activation of downstream signaling pathways. Protection from neurodegeneration was assessed using the human dopaminergic neuronal cell line LUHMES. First we show that TNC-scTNF(R2) interfered with cell death pathways subsequent to H(2)O(2) exposure. Protection from cell death was dependent on TNFR2 activation of the PI3K-PKB/Akt pathway, evident from restoration of H(2)O(2) sensitivity in the presence of PI3K inhibitor LY294002. Second, in an in vitro model of Parkinson disease, TNC-scTNF(R2) rescues neurons after induction of cell death by 6-OHDA. Since TNFR2 is not only promoting anti-apoptotic responses but also plays an important role in tissue regeneration, activation of TNFR2 signaling by TNC-scTNF(R2) appears a promising strategy to ameliorate neurodegenerative processes.     

A highly selective kappa-opioid receptor agonist with low addictive potential and dependence liability

Buprenorphine analogs have been synthesized. In the studies of analgesic and addictive effects in mice and [(35)S]GTPgammaS binding assay in human brain tissue, an analog of buprenorphine where the tert-butyl is replaced by a cyclobutyl moiety (16) has been identified as a selective kappa-partial agonist which gives antinociceptive effects, but has low abuse potential. The results may lead to lower degrees of dysphoria than full kappa-agonists.     

Infection of primary human fetal astrocytes by human herpesvirus 6.

Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus which productively infects human CD4+ T cells and monocytes. HHV-6 is the etiologic agent for exanthem subitum (roseola), and it is well-known that central nervous system complications occur frequently during the course of HHV-6-associated disease. In addition, HHV-6 has been associated with encephalitis or encephalopathy. However, very little is known about its tropism for neural cells. There are reports that HHV-6 may infect some glial cell lines, but whether it can infect any primary neural cells is not known. Our studies show that both HHV-6A (GS) and HHV-6B (Z-29) can infect highly purified primary fetal astrocytes in vitro. Infected cells showed cytopathic effects, forming giant syncytia. In dual immunofluorescence assays, the infected cells were detected by antibodies against the HHV-6 p41 nuclear antigen and glial fibrillary acidic protein, indicating that the infected cells are indeed astrocytes. PCR and Northern (RNA) blot analyses also confirmed that the astrocytes are infected by HHV-6. The progeny virus did not alter its host range and could reinfect T cells as well as primary astrocytes. These findings suggest that infection of primary human astrocytes may play a role in the neuropathogenesis of HHV-6.     

Induction of T-cell apoptosis by human herpesvirus 6.

The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection.     

Structural basis for the interaction of CCR5 with a small molecule, functionally selective CCR5 agonist

The chemokine receptor CCR5 is an attractive target for HIV-1 drug development, as individuals whose cells lack surface CCR5 expression are highly resistant to HIV-1 infection. CCR5 ligands, such as CCL5/RANTES, effectively inhibit HIV-1 infection by competing for binding opportunities to the CCR5 and inducing its internalization. However, the inherent proinflammatory activity of the chemotactic response of CCR5 ligands has limited their clinical use. In this study, we found that a novel small molecule, functionally selective CCR5 agonist, 2,2-dichloro-1-(triphenylphosphonio)vinyl formamide perchlorate (YM-370749), down-modulates CCR5 from the cell surface without inducing a chemotactic response and inhibits HIV-1 replication. In molecular docking studies of YM-370749 and a three-dimensional model of CCR5 based on the rhodopsin crystal structure as well as binding and functional studies using various CCR5 mutants, the amino acid residues necessary for interaction with YM-370749 were marked. These results provide a structural basis for understanding the activation mechanism of CCR5 and for designing functionally selective agonists as a novel class of anti-HIV-1 agents.     

Transactivation of human immunodeficiency virus promoter by human herpesvirus 6.

Patients with acquired immunodeficiency syndrome (AIDS) are often infected with a number of other heterologous viruses in addition to the initial human immunodeficiency virus (HIV) infection, and these agents could act as potential reactivating agents of latent HIV. A new antigenically distinct herpesvirus, designated human herpesvirus 6 (HHV-6), has recently been isolated from patients with AIDS and has been shown to infect a number of different human cells, specifically human T cells, B cells, and glial cells. Since these are some of the same cells that harbor the AIDS virus, it is quite important to determine any interaction between this new herpesvirus and HIV. In this report, we demonstrate that HHV-6 can trans-activate the HIV promoter in human T-cell lines as measured by the expression of the bacterial gene chloramphenicol acetyltransferase. This indicates that stimulation of HIV gene expression by HHV-6 could play a role in HIV pathogenesis.     

A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues

Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.     

Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice

The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2--15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3--10% of CD4 and 10--40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5(+) and CCR2(+) T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1alpha and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC(50) of 0.09 and 0.6--1.0 microg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.