RESUMO
Encephalitis rarely occurs during acute Trypanosoma cruzi infection. However, the central nervous system (CNS) is the major site of infection reactivation in immunocompromised patients. We show that the acute T. cruzi-triggered CD8-enriched meningoencephalitis paralleled the in situ expression of CCL3/MIP-1alpha and CCL5/RANTES mRNA. The frequency of CCR5-bearing cells was increased among peripheral blood mononuclear cells (PBMC) of infected mice. Further, CCL5/RANTES-driven in vitro PBMC migration was partially abrogated by the CCR1/CCR5 antagonist Met-RANTES. However, Met-RANTES treatment of infected mice altered neither parasitism nor intensity and nature of the CNS inflammation, indicating that T. cruzi-elicited meningoencephalitis is a CCR1/CCR5 independent process.
Assuntos
Inflamação/etiologia , Meningoencefalite/etiologia , Receptores CCR5/fisiologia , Receptores de Quimiocinas/fisiologia , Trypanosoma cruzi/patogenicidade , Animais , Antígenos CD8/metabolismo , Movimento Celular/efeitos dos fármacos , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica , Imuno-Histoquímica , Inflamação/tratamento farmacológico , Meningoencefalite/complicações , Meningoencefalite/tratamento farmacológico , Meningoencefalite/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Receptores CCR1RESUMO
Development of topical microbicides that prevent sexual transmission of HIV is an active area of investigation. The purpose of this study was to test the ability of the potent membrane fusion inhibitor C34, an HIV gp41 antagonist, to block HIV infection of human Langerhans cells (LCs) in an epithelial environment that mimics a major route of HIV infection. We incubated freshly isolated epidermal explants containing LCs with various doses of C34 before, during, and after exposing explants to HIV. Although C34 only partially blocked HIV infection of LCs when pre-incubated with skin, it displayed full, dose-dependent inhibition when present during and after viral exposure. The poor protection from HIV infectivity in pre-incubated samples is consistent with mechanism of C34 inhibition and starkly contrasts to the full protection provided by PSC-RANTES, an entry inhibitor that prevents HIV gp120 interaction with its co-receptor CCR5. Real-time PCR confirmed that C34 blocked HIV infection of LCs before reverse transcription and inhibited LC-mediated transfer of virus to T cells. We conclude that C34, if used topically at susceptible mucosal sites, and if continually present, has the potential to block sexual transmission of HIV.
Assuntos
Antígenos CD34/administração & dosagem , Infecções por HIV/tratamento farmacológico , Infecções por HIV/transmissão , HIV/efeitos dos fármacos , Células de Langerhans/virologia , Linfócitos T/virologia , Fármacos Anti-HIV/farmacologia , Células Cultivadas , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/farmacologia , Técnicas de Cocultura , Células Epidérmicas , Epiderme/imunologia , Produtos do Gene gag/genética , HIV/genética , Proteína gp41 do Envelope de HIV/genética , Infecções por HIV/imunologia , Humanos , Células de Langerhans/citologia , Células de Langerhans/imunologia , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Integração Viral/efeitos dos fármacos , Zidovudina/farmacologiaRESUMO
N-terminal proteolytic processing modulates the biological activity and receptor specificity of RANTES/CCL5. Previously, we showed that an unidentified protease associated with monocytes and neutrophils digests RANTES into a variant lacking three N-terminal residues (4-68 RANTES). This variant binds CCR5 but exhibits lower chemotactic and antiviral activities than unprocessed RANTES. In this study, we characterize cathepsin G as the enzyme responsible for this processing. Cell-mediated production of the 4-68 variant was abrogated by Eglin C, a leukocyte elastase and cathepsin G inhibitor, but not by the elastase inhibitor elastatinal. Further, anti-cathepsin G antibodies abrogated RANTES digestion in neutrophil cultures. In accordance, reagent cathepsin G specifically digested recombinant RANTES into the 4-68 variant. AOP-RANTES and Met-RANTES were also converted into the 4-68 variant upon exposure to cathepsin G or neutrophils, while PSC-RANTES was resistant to such cleavage. Similarly, macaque cervicovaginal lavage samples digested Met-RANTES and AOP-RANTES, but not PSC-RANTES, into the 4-68 variant and this processing was also inhibited by anti-cathepsin G antibodies. These findings suggest that cathepsin G mediates a novel pathway for regulating RANTES activity and may be relevant to the role of RANTES and its analogs in preventing HIV infection.
Assuntos
Catepsinas/metabolismo , Quimiocina CCL5/análogos & derivados , Infecções por HIV/metabolismo , Neutrófilos/enzimologia , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/metabolismo , Anticorpos/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Catepsina G , Catepsinas/antagonistas & inibidores , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacologia , Quimiocina CCL5/uso terapêutico , Quimiotaxia/efeitos dos fármacos , Infecções por HIV/prevenção & controle , Humanos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas/farmacologia , Receptores CCR5/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Inibidores de Serina Proteinase/farmacologiaRESUMO
CCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+ and CD8+ cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.
Assuntos
Quimiocina CCL5/análogos & derivados , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/metabolismo , Pneumonia Viral/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Quimiocina CCL5/farmacologia , Quimiocinas CC/genética , Eosinofilia/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Vírus Sinciciais Respiratórios , Células Th2/imunologiaRESUMO
BACKGROUND: During colitis, epithelial function is impaired, leading to increased bacterial translocation. Recent studies have shown the important role of proinflammatory cytokines and chemokines, including RANTES (regulated on activation, normal T-cell expressed and secreted), in inflammatory bowel diseases (IBDs). In this study, we evaluated the role of Met-RANTES, an antagonist of the RANTES receptor, on the impairment of bacterial translocation in a rat model of colitis. METHODS: Rats were randomly assigned to 3 groups. Group 1 = control, group 2 = experimental colitis, and group 3 = colitis plus Met-RANTES treatment. On day 7 after colitis was induced, plasma tumor necrosis factor-alpha colon tissue myeloperoxidase and portal blood endotoxin levels were measured. Lymph node, liver, and spleen culture quantified bacterial translocation. RESULTS: Met-RANTES treatment resulted in significant decreases in colonic damage as well as bacterial translocation in experimental colitis. CONCLUSIONS: These results suggest that chemokine receptor antagonists may potentially be useful in the treatment of IBDs.
Assuntos
Anti-Inflamatórios/farmacologia , Translocação Bacteriana/efeitos dos fármacos , Quimiocina CCL5/análogos & derivados , Colite/fisiopatologia , Colo/efeitos dos fármacos , Animais , Quimiocina CCL5/farmacologia , Colite/induzido quimicamente , Colite/microbiologia , Modelos Animais de Doenças , Masculino , Noxas/efeitos adversos , Ratos , Ratos Wistar , Ácido Trinitrobenzenossulfônico/efeitos adversosRESUMO
Tuberculosis (TB) enhances human immunodeficiency virus-1 (HIV-1) activity in patients with dual HIV-1/TB infection. Therapies that control augmentations of HIV-1 activity at sites of Mycobacterium tuberculosis (MTB) infection may be useful in inhibition of viral expansion. Regulated upon activation, normal T-cell expressed and secreted (RANTES) analogues (AOP and NNY) are potent in inhibiting the entry of primary HIV-1 isolates into host mononuclear cells. These analogues were used to inhibit MTB-induced HIV-1 entry in blood monunuclear cells (PBMC) from patients with pulmonary TB, and pleural fluid mononuclear cells (PFMC) from patients with pleural TB. PBMC or PFMC were cultured with and without MTB in presence and absence of RANTES analogues. HIV-1 strong stop DNA was assessed by real-time polymerase chain reaction (PCR) as a measure of infection. CCR5 mRNA was assessed by real-time reverse transcription (RT)-PCR and by immunostaining and FACS analysis. HIV-1 infection was induced by MTB in vitro in PBMC from the majority (14 of 20) of HIV-1/TB subjects, and new infection was inhibited by AOP- or NNY-RANTES. HIV-1 infection was also inhibited by these reagents in MTB-induced PFMC from three of three patients with pleural TB. Expression of CCR5 mRNA was significantly induced by MTB in PBMC from patients with pulmonary TB. Further, expression of CCR5 was higher in PFMC compared to PBMC from patients with pleural TB. Also, CCR5 was fourfold higher on CD14(+) pleural mononuclear cells than on CD4(+) lymphocytes. Blocking new HIV-1 infection of mononuclear cells may be useful in control of HIV-1 during dual HIV-1/TB infection.
Assuntos
Quimiocina CCL5/farmacologia , Quimiocinas CC/imunologia , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , Tuberculose/imunologia , Adulto , Células Cultivadas , Quimiocina CCL5/análogos & derivados , DNA Viral/análise , Feminino , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/isolamento & purificação , HIV-1/patogenicidade , Humanos , Masculino , RNA Mensageiro/genética , Receptores CCR5/biossíntese , Receptores CCR5/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tuberculose/complicações , Tuberculose Pleural/complicações , Tuberculose Pleural/imunologia , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/imunologiaRESUMO
Entry of HIV and SIV into susceptible cells is mediated by CD4 and chemokine receptors, which act as coreceptors. To study cell entry of SIV, we constructed a cell line, xKLuSIV, derived from non-susceptible human K562 cells, that express the firefly luciferase reporter gene under control of a minimal SIV long terminal repeat (LTR). Using these susceptible cells, we studied the entry of a well-characterized molecularly cloned macrophage-tropic SIV. xKLuSIV cells that express rhesus macaque CD4 and/or the rhesus chemokine receptor CCR5 are susceptible to infection with the macrophage-tropic, neurovirulent strain SIV/17E-Fr, but only xKLuSIV cells expressing both CCR5 and CD4 were susceptible to infection by the macrophage-tropic, non-neurovirulent strain SIV/17E-Cl. CCR5-dependent, CD4-independent infection by SIV/17E-Fr was abrogated by pre-incubation of the cells with AOP-RANTES, a ligand for CCR5. In addition to viral entry occurring by a CD4-independent mechanism, neutralization of SIV/17E-Fr with rhesus mAbs from 3 different neutralization groups blocked entry into x KLuSIV cells by both CD4-dependent and -independent mechanisms. Triggering the env glycoprotein of SIV-17 EFr with soluble CD4 had no significant effect in infectivity, but triggering of the same glycoprotein of SIV/17E-Cl allowed it to enter cells in a CD4-independent fashion. Using mutant molecular clones, we studied the determinants for CD4 independence, all of which are confined to the env gene. We report here that truncation of the TM at amino acid 764 and changing a single amino acid (R751G) in the SIV envelope transmembrane protein (TM) conferred the observed CD4-independent phenotype. Our data suggest that the envelope from the neurovirulent SIV/17E-Fr interacts with CCR5 in a CD4-independent manner, and changes in the TM protein of this virus are important components that contribute to neurovirulence in SIV.
Assuntos
Receptores CCR5/fisiologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Substituição de Aminoácidos , Animais , Anticorpos Antivirais , Antígenos Virais/genética , Antígenos CD4/fisiologia , Linhagem Celular , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/farmacologia , Mapeamento de Epitopos , Genes Virais , Humanos , Células K562 , Macaca mulatta , Glicoproteínas de Membrana/fisiologia , Testes de Neutralização , Proteínas dos Retroviridae/genética , Proteínas dos Retroviridae/fisiologia , Deleção de Sequência , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/imunologia , Solubilidade , Proteínas do Envelope Viral/fisiologiaRESUMO
Lymphocyte trafficking is controlled in part by the actions of chemokines. In rat experimental autoimmune uveitis (EAU) we observed differential therapeutic effects of Met-RANTES, a CCR1/CCR5 receptor antagonist, depending on the retinal antigen peptides inducing the disease and the time of application during the afferent or efferent immune response. CCR1 and/or CCR5 blockade may have inhibitory effects on different phases of the autoimmune response, depending on the antigen specificity of T cells in EAU. In contrast, Met-RANTES enhanced therapeutic oral tolerance independently of orally applied antigen.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/uso terapêutico , Quimiocinas CC/antagonistas & inibidores , Uveíte/tratamento farmacológico , Animais , Arrestina/química , Arrestina/toxicidade , Doenças Autoimunes/induzido quimicamente , Citocinas/metabolismo , Interações Medicamentosas , Ectodisplasinas , Olho/efeitos dos fármacos , Olho/metabolismo , Olho/patologia , Proteínas do Olho/química , Proteínas do Olho/toxicidade , Imuno-Histoquímica/métodos , Proteínas de Membrana/metabolismo , Peptídeos/toxicidade , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/toxicidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Índice de Gravidade de Doença , Linfócitos T/efeitos dos fármacos , Fatores de Tempo , Uveíte/induzido quimicamente , Vacinação/métodosRESUMO
The comprehension of the pathogenesis of Trypanosoma cruzi-elicited myocarditis is crucial to delineate new therapeutic strategies aiming to ameliorate the inflammation that leads to heart dysfunction, without hampering parasite control. The augmented expression of CCL5/RANTES and CCL3/MIP-1alpha, and their receptor CCR5, in the heart of T. cruzi-infected mice suggests a role for CC-chemokines and their receptors in the pathogenesis of T. cruzi-elicited myocarditis. Herein, we discuss our recent results using a CC-chemokine receptor inhibitor (Met-RANTES), showing the participation of CC-chemokines in T. cruzi infection and unraveling CC-chemokine receptors as an attractive therapeutic target for further evaluation in Chagas disease.
Assuntos
Cardiomiopatia Chagásica/tratamento farmacológico , Quimiocina CCL5/análogos & derivados , Quimiocinas CC/metabolismo , Miocardite/tratamento farmacológico , Receptores de Quimiocinas/antagonistas & inibidores , Trypanosoma cruzi , Animais , Relação CD4-CD8 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/metabolismo , Quimiocina CCL5/uso terapêutico , Quimiotaxia de Leucócito/imunologia , Camundongos , Camundongos Endogâmicos C3H , Miocardite/imunologia , Miocardite/metabolismo , Miocardite/parasitologia , Trypanosoma cruzi/imunologiaRESUMO
Since the discovery that to enter target cells HIV uses receptors for the class of proteins known as chemokines, attempts have been made to generate anti-HIV molecules based on the chemokine ligands. A significant level of knowledge of the structure-activity relationships of chemokines has been amassed since the beginning of the 1990s. This, together with work that has elucidated the mechanisms underlying the inhibitory activity of chemokines, has guided not only the rational design of anti-HIV chemokine analogues, but also strategies by which chemokine variants with potent anti-HIV activity can be isolated from large libraries by phage display. This review summarizes the current knowledge about the structure-activity relationships and receptor biology of chemokines that is relevant to the development of analogues with anti-HIV activity. We present specific examples of engineered chemokine analogues with potent anti-HIV activity and describe the challenges that will need to be faced if these molecules are to be further developed for clinical applications. Finally, we discuss how these challenges might be met through further engineering of the molecules.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Quimiocina CCL5/análogos & derivados , Quimiocinas/química , Quimiocinas/farmacologia , Engenharia de Proteínas/métodos , Quimiocina CCL5/química , Quimiocina CCL5/farmacologia , Quimiocinas/classificação , Quimiocinas/fisiologia , Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Receptores de Quimiocinas/fisiologia , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal-modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1alpha (MIP-1alpha; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat. METHODS: Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA. RESULTS: Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1alpha and MIP-1beta at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor alpha, interleukin-1beta, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint. CONCLUSION: The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Quimiocina CCL5/análogos & derivados , Animais , Movimento Celular/imunologia , Quimiocina CCL5/uso terapêutico , Quimiocinas CC/imunologia , Macrófagos/imunologia , Ratos , Receptores de Quimiocinas/imunologia , Células Th1/imunologiaRESUMO
We describe the generation of two genetically related human immunodeficiency virus type 1 (HIV-1) isolates highly (>20,000-fold) resistant to the small molecule CCR5 inhibitor, SCH-417690 (formerly SCH-D). Both viruses were cross-resistant to other small molecules targeting entry via CCR5, but they were inhibited by some MAbs against the same coreceptor on primary CD4+ T-cells. The resistant isolates remained sensitive to inhibitors of other stages of virus entry, and to replication inhibitors acting post-entry. Neither escape mutant could replicate detectably in peripheral blood mononuclear cells (PBMC) from two donors homozygous for the CCR5-Delta32 allele and both were insensitive to the CXCR4-specific inhibitor, AMD3100. Hence, the SCH-D escape mutants retained the R5 phenotype. One of the resistant isolates was, however, capable of replication in U87.CD4.CXCR4 cells and, after expansion in those cells, was sensitive to AMD3100 in primary CD4+ T-cells. Hence, some X4 variants may be present in this escape mutant swarm. A notable observation was that the SCH-D escape mutants were also cross-resistant to PSC-RANTES and AOP-RANTES, chemokine derivatives that are reported to down-regulate cell surface CCR5 almost completely. However, the extent to which CCR5 is down-regulated was dependent upon the detection MAb. Hence, the escape mutants may be using a CCR5 configuration that is only detected by some anti-CCR5 MAbs. Finally, two SCH-D-resistant clonal viruses revealed no amino acid changes in the gp120 V3 region relative to the parental viruses, in marked contrast to clones resistant to the AD101 small molecule CCR5 inhibitor that possess 4 such sequence changes. Several sequence changes elsewhere in gp120 (V2, C3 and V4) were present in the SCH-D-resistant clones. Their influence on the resistant phenotype remains to be determined.
Assuntos
Antagonistas dos Receptores CCR5 , HIV-1/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Alelos , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Anticorpos Monoclonais , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/farmacologia , DNA Viral/genética , Farmacorresistência Viral/genética , Expressão Gênica , Produtos do Gene env/genética , Genes env , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/genética , Fenótipo , Receptores CCR5/genética , Receptores CCR5/imunologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
A synthetic strategy that allows for the site-specific attachment of polymers such as poly(ethylene glycol) (PEG) to protein pharmaceuticals is described. PEG was attached to a 67-amino acid fully synthetic CCL-5 (RANTES) analogue at its GAG binding site both to reduce aggregation and to increase the circulating lifetime. Effective protection of an Aoaa chemoselective linker during peptide assembly, total chemical protein synthesis, and protein folding was achieved with an isopropylidene group. Mild deprotection of the resulting folded synthetic protein and subsequent polymer attachment occur without interference with the native folded structure and activity.
Assuntos
Quimiocina CCL5/análogos & derivados , Quimiocinas CC/química , Oximas/química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Quimiocina CCL5/química , Quimiocina CCL5/farmacologia , Quimiocinas CC/farmacologia , Glicina/química , HIV-1/efeitos dos fármacos , Humanos , Modelos Moleculares , Polietilenoglicóis/química , Dobramento de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizAssuntos
Fármacos Anti-HIV/uso terapêutico , Anti-Infecciosos Locais/uso terapêutico , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/uso terapêutico , Infecções por HIV/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Administração Intravaginal , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Macaca mulatta , Vírus da Imunodeficiência Símia/efeitos dos fármacosRESUMO
We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl1]RANTES(2-68), known as AOP-RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure-activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES [N-nonanoyl, des-Ser1[L-thioproline2, L-cyclohexylglycine3]-RANTES(2-68)], which is 50 times more potent than AOP-RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.
Assuntos
Fármacos Anti-HIV/síntese química , Quimiocina CCL5/análogos & derivados , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células CHO , Quimiocina CCL5/síntese química , Quimiocina CCL5/química , Quimiocina CCL5/farmacologia , Cricetinae , Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , Humanos , Leucócitos Mononucleares/transplante , Camundongos , Camundongos SCID , RNA Viral/sangue , Receptores CCR5/metabolismo , Relação Estrutura-AtividadeAssuntos
Fármacos Anti-HIV/uso terapêutico , Anti-Infecciosos Locais/uso terapêutico , Antagonistas dos Receptores CCR5 , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/uso terapêutico , Infecções por HIV/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Administração Intravaginal , Animais , Fármacos Anti-HIV/administração & dosagem , Anti-Infecciosos Locais/administração & dosagem , Quimiocina CCL5/administração & dosagem , Feminino , HIV/efeitos dos fármacos , Infecções por HIV/transmissão , Haplorrinos , Humanos , Masculino , Receptores CCR5/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vagina/virologiaRESUMO
Topical agents, such as microbicides, that can protect against human immunodeficiency virus (HIV) transmission are urgently needed. Using a chimeric simian/human immunodeficiency virus (SHIV SF162), which is tropic for the chemokine receptor CCR5, we report that topical application of high doses of PSC-RANTES, an amino terminus-modified analog of the chemokine RANTES, provided potent protection against vaginal challenge in rhesus macaques. These experimental findings have potentially important implications for understanding vaginal transmission of HIV and the design of strategies for its prevention.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Anti-Infecciosos Locais/uso terapêutico , Antagonistas dos Receptores CCR5 , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/uso terapêutico , Infecções por HIV/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vagina/virologia , Administração Intravaginal , Animais , Fármacos Anti-HIV/administração & dosagem , Anti-Infecciosos Locais/administração & dosagem , Anticorpos Antivirais/sangue , Quimiocina CCL5/administração & dosagem , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Infecções por HIV/transmissão , HIV-1/efeitos dos fármacos , Macaca mulatta , Receptores CCR5/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/imunologiaAssuntos
Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/uso terapêutico , Quimiocinas/antagonistas & inibidores , Miocardite/tratamento farmacológico , Animais , Cardiomiopatia Chagásica/tratamento farmacológico , Quimiocina CCL4 , Quimiocinas/fisiologia , Quimiocinas CC/antagonistas & inibidores , Quimiocinas CC/fisiologia , Humanos , Leucócitos/fisiologia , Proteínas Inflamatórias de Macrófagos/antagonistas & inibidores , Proteínas Inflamatórias de Macrófagos/fisiologia , Camundongos , Miocardite/metabolismoRESUMO
BACKGROUND: Comprehension of the pathogenesis of Trypanosoma cruzi-elicited myocarditis is crucial to delineate strategies aimed at ameliorating the inflammation associated with heart dysfunction. The augmented expression of CC chemokines, especially CCL5/RANTES and CCL3/MIP-1alpha, in the hearts of infected mice suggests a role for CC chemokines and their receptors in the pathogenesis of T cruzi-elicited myocarditis. METHODS AND RESULTS: We report that during the early phase of infection in C3H/HeJ mice infected with 100 blood trypomastigotes of T cruzi, most of the inflammatory cells invading the heart tissue were CD8+ cells and expressed CCR5, a CCL5/RANTES, and CCL3/MIP1-alpha receptor. Furthermore, peripheral blood CD8+ T lymphocytes displayed increased expression of CCR5. These findings led us to use Met-RANTES, a selective CCR1 and CCR5 antagonist, to modulate the acute T cruzi-elicited myocarditis. Met-RANTES treatment did not interfere with parasitism but significantly decreased the numbers of CD4+ and CD8+ T cells, CCR5+, and interleukin-4+ cells invading the heart, paralleling the diminished deposition of fibronectin. Moreover, Met-RANTES treatment resulted in increased survival of infected animals, compared with saline treatment. CONCLUSIONS: These results indicate that the massive influx of CCR5+ cells into cardiac tissue is not crucial for cell-mediated anti-T cruzi immunity but appears to be critical for pathogenesis of T cruzi-elicited myocarditis. Thus, CC chemokine receptors might become an attractive therapeutic target for further evaluation during T cruzi infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Cardiomiopatia Chagásica/tratamento farmacológico , Quimiocina CCL5/análogos & derivados , Quimiocina CCL5/metabolismo , Quimiocina CCL5/uso terapêutico , Ativação Linfocitária , Proteínas Inflamatórias de Macrófagos/metabolismo , Miocardite/tratamento farmacológico , Animais , Antagonistas dos Receptores CCR5 , Linfócitos T CD4-Positivos/imunologia , Cardiomiopatia Chagásica/sangue , Cardiomiopatia Chagásica/fisiopatologia , Quimiocina CCL2/biossíntese , Quimiocina CCL2/genética , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/antagonistas & inibidores , Quimiocinas CC/biossíntese , Quimiocinas CC/genética , Quimiotaxia de Leucócito , Feminino , Fibronectinas/análise , Interleucina-4/análise , Proteínas Inflamatórias de Macrófagos/biossíntese , Proteínas Inflamatórias de Macrófagos/genética , Camundongos , Camundongos Endogâmicos C3H , Miocardite/sangue , Miocardite/parasitologia , Miocardite/fisiopatologia , RNA Mensageiro/biossíntese , Receptores CCR1 , Receptores CCR5/biossíntese , Receptores CCR5/genética , Receptores CCR5/fisiologia , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/fisiologiaRESUMO
BACKGROUND: Proliferation and apoptosis of mesangial cells (MC) are important mechanisms during nephrogenesis, for the maintenance of glomerular homeostasis as well as in renal disease and glomerular regeneration. Expression of chemokines and chemokine receptors by intrinsic renal cells, e.g. SLC/CCL21 on podocytes and CCR7 on MC is suggested to play a pivotal role during these processes. Therefore the effect of selected chemokines on MC proliferation and apoptosis was studied. METHODS: Proliferation assays, cell death assays including cell cycle analysis, hoechst stain and measurement of caspase-3 activity were performed. RESULTS: A dose-dependent, mesangioproliferative effect of the chemokine SLC/CCL21, which is constitutively expressed on human podocytes was seen via activation of the chemokine receptor CCR7, which is constitutively expressed on MC. In addition, in cultured MC SLC/CCL21 had a protective effect on cell survival in Fas-mediated apoptosis. The CXCR3 ligands IP-10/CXCL10 and Mig/CXCL9 revealed a proproliferative effect but did not influence apoptosis of MC. Both the CCR1 ligand RANTES/CCL5 and the amino-terminally modified RANTES analogue Met-RANTES which blocks CCR1 signalling had no effect on proliferation and apoptosis. CONCLUSIONS: The different effects of chemokines and their respective receptors on proliferation and apoptosis of MC suggest highly regulated, novel biological functions of chemokine/chemokine receptor pairs in processes involved in renal inflammation, regeneration and glomerular homeostasis.