RESUMEN
The development of a new therapeutic drug is a complex, lengthy and expensive process. On average, only one out of 10,000 - 30,000 originally synthesized compounds will clear all the hurdles on the way to becoming a commercially available drug. The process of early and full preclinical discovery and clinical development for a new drug can take twelve to fifteen years to complete, and cost approximately 800 million dollars. The field of bioinformatics has become a major part of the drug discovery pipeline playing a key role in improvement and acceleration of this time and money consuming process. Here we reviewed the application of the EIIP/ISM bioinformatics concept for the development of new drugs. This approach, connecting the electron-ion interaction potential of organic molecules and their biological properties, can significantly reduce development time through (i) identification of promising lead compounds that have some activity against a disease by fast virtual screening of the large molecular libraries, (ii) refinement of selected lead compounds in order to increase their biological activity, and (iii) identification of domains of proteins and nucleotide sequences representing potential targets for therapy. Special attention is paid in this review to the application of the EIIP/ISM bioinformatics platform along with other experimental techniques (screening of a phage displayed peptide libraries, testing selected peptides and small molecules for antiviral activity in vitro) in development of HIV entry inhibitors, representing a new generation of the AIDS drugs.
Asunto(s)
Biología Computacional/métodos , Diseño de Fármacos , Fármacos Anti-VIH , Evaluación Preclínica de Medicamentos/métodos , Humanos , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
The HIV-1 co-receptor CCR5 has been thought a relevant target for small interfering RNA (siRNA)-based therapeutics. However, recent findings suggest that siRNA can stimulate innate cytokine responses in mammals. All siRNA agents tested were able to down-regulate the expression of CCR5, albeit with different efficiency (51-74% down-regulation), block HIV-induced syncytia formation between HIV-1 BaL-infected and uninfected CD4(+) cells or block single-round HIV-1 infection as measured by a luciferase reporter assay (46-83% inhibition). Conversely, siRNA directed against CCR5 did not affect replication of a vesicular stomatitis virus (VSV) pseudotyped virus, suggesting that inhibition of HIV replication was specific to CCR5 down-regulation. However, two of four siRNA tested were able to induce the production of interleukin (IL) IL-6 (sixfold induction) and IL-8 (ninefold induction) but no interferon (IFN)-alpha, IFN-beta, IFN-gamma, tumour necrosis factor (TNF)-alpha, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, IL-1beta, IL-10 or IL-12p70 cytokine induction was noted. In the absence of detectable IFN-alpha, IL-6 or IL-8 may represent markers of non-specific effects triggered by siRNA.
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Linfocitos T CD4-Positivos/inmunología , Interleucina-6/inmunología , Interleucina-8/inmunología , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores CCR5/genética , Secuencia de Bases , Linfocitos T CD4-Positivos/virología , Línea Celular , Citocinas/inmunología , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Infecciones por VIH/inmunología , Infecciones por VIH/terapia , VIH-1/genética , VIH-1/fisiología , Humanos , Inmunoterapia , Datos de Secuencia Molecular , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección/métodos , Virus de la Estomatitis Vesicular Indiana/fisiología , Replicación ViralRESUMEN
During inflammation, interleukin (IL)-12 and IL-18 are produced by macrophages and other cell types such as neutrophils (IL-12), keratinocytes and damaged endothelial cells (IL-18). To explore the role of IL-12 and IL-18 in inflammatory innate immune responses we investigated their impact on human peripheral blood monocytes and mature bronchoalveolar lavage (BAL) macrophages. IL-12 and IL-18 together, but not alone, prevented spontaneous apoptosis of cultured monocytes, promoted monocyte clustering and subsequent differentiation into macrophages. These morphological changes were accompanied by increased secretion of CXC chemokine ligands (CXCL)9, CXCL10 (up to 100-fold, P < 0.001) and CXCL8 (up to 10-fold, P < 0.001) but not CCL3, CCL4 or CCL5. Mature macrophages (from BALs) expressed high basal levels of CXCL8, that were no modified upon stimulation with IL-12 and IL-18. In contrast, the basal production of CXCL9 and CXCL10 by BALs was increased by 10-fold (P < 0.001) in the presence of either IL-12 or IL-18 alone and by 50-fold in the presence of both cytokines. In conclusion, our results indicate a relevant role for IL-12 and IL-18 in the activation and resolution of inflammatory immune responses, by increasing the survival of monocytes and by inducing the production of chemokines. In particular, those that may regulate angiogenesis and promote the recruitment of monocytes, activated T cells (CXCL9 and CXCL10) and granulocytes (CXCL8).
Asunto(s)
Quimiocinas CXC/biosíntesis , Interleucina-12/farmacología , Interleucina-18/farmacología , Macrófagos Alveolares/inmunología , Monocitos/inmunología , Análisis de Varianza , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL10 , Quimiocina CXCL9 , Humanos , Inmunidad Innata , Interleucina-8 , Fagocitosis , Factor de Transcripción STAT4/análisis , Estimulación QuímicaRESUMEN
A compilation of combinatorial chemistry techniques applied to anti-HIV drug development is presented in this review. This synthetic strategy together with high throughput screening assays has allowed the discovery and optimization of novel lead anti-HIV compounds.
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Fármacos Anti-VIH/síntesis química , Técnicas Químicas Combinatorias/métodos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , VIH/efectos de los fármacos , VIH/fisiología , Inhibidores de Fusión de VIH/síntesis química , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de la Proteasa del VIH/síntesis química , Humanos , Estructura Molecular , Inhibidores de la Transcriptasa Inversa/síntesis químicaRESUMEN
The pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) has been linked to an overexpression of the chemokine receptor CXCR4 and increased in vitro functional response to its natural ligand CXCL12 (SDF-1). The CXCR4/SDF-1 system appears to be important for tissue localization and increased survival of B-CLL cells. The aim of our study was to examine if CXCR4 expression and SDF-1 blood levels were correlated to clinical and pathological stage of B-CLL. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) techniques were used to determine CXCR4 expression and SDF-1 plasma levels, respectively, in a cohort of 51 patients diagnosed with B-CLL to correlate these measurements with several parameters that define the clinical stage of the disease. We confirmed that CXCR4 was consistently expressed on circulating B-CLL cells with a fluorescence intensity that was five-fold greater than in cells from healthy volunteers. There was a correlation between CXCR4 expression and leukocyte count ( r: 0.55, p<0.01), and CD19(+)/CD5(+ )cells ( r: 0.63, p<0.01). Interestingly, the group of B-CLL patients showed lower SDF-1 plasma levels compared to the control group. However, there was no correlation between CXCR4 or SDF-1 expression and the clinical stage of disease or the pattern of bone marrow infiltration. The results obtained suggest that other factors, and not only alteration in the SDF-1/CXCR4 chemokine system, must account for marrow infiltration of neoplastic cells observed in B-CLL and that CXCR4 could be involved in other features that exhibit malignant B cells, such as increased survival, rather than in their homing or migration to the bone marrow.
Asunto(s)
Quimiocinas CXC/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Receptores CXCR4/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Quimiocina CCL5/sangre , Quimiocina CXCL12 , Quimiocinas CXC/sangre , Femenino , Humanos , Interleucina-7/sangre , Masculino , Persona de Mediana Edad , Estadificación de NeoplasiasRESUMEN
Human immunodeficiency virus type 1 (HIV-1) primary infection is characterized by the use of CCR5 as a coreceptor for viral entry, which is associated with the non-syncytium-inducing (NSI) phenotype in lymphoid cells. Syncytium-inducing (SI) variants of HIV-1 appear in advanced stages of HIV-1 infection and are characterized by the use of CXCR4 as a coreceptor. The emergence of SI variants is accompanied by a rapid decrease in the number of T cells. However, it is unclear why SI variants emerge and what factors trigger the evolution of HIV from R5 to X4 variants. Interleukin-7 (IL-7), a cytokine produced by stromal cells of the thymus and bone marrow and by keratin, is known to play a key role in T-cell development. We evaluated IL-7 levels in plasma of healthy donors and HIV-positive patients and found significantly higher levels in HIV-positive patients. There was a negative correlation between circulating IL-7 levels and CD4(+) T-cell count in HIV-positive patients (r = -0.621; P < 0.001), suggesting that IL-7 may be involved in HIV-induced T-cell depletion and disease progression. IL-7 levels were higher in individuals who harbored SI variants and who had progressed to having CD4 cell counts of lower than 200 cells/microl than in individuals with NSI variants at a similar stage of disease. IL-7 induced T-cell proliferation and up-regulated CXCR4 expression in peripheral blood mononuclear cells in vitro. Taken together, our results suggest a role for IL-7 in the maintenance of T-cell regeneration and depletion by HIV in infected individuals and a possible relationship between IL-7 levels and the emergence of SI variants.
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Síndrome de Inmunodeficiencia Adquirida/inmunología , Recuento de Linfocito CD4 , VIH-1/clasificación , Interleucina-7/sangre , Síndrome de Inmunodeficiencia Adquirida/virología , Biomarcadores , Humanos , Interleucina-7/fisiología , Estudios Longitudinales , Activación de Linfocitos , Receptores CXCR4/biosíntesis , Linfocitos T/inmunologíaRESUMEN
In a correlative study, the mean plasma level of the chemokine stromal-cell-derived factor 1 (SDF-1) was lower in subjects with syncytium-inducing (SI) than in subjects with non-syncytium-inducing (NSI) HIV isolates, regardless of the CD4 cell count or when compared with HIV-negative individuals. Individuals with high SDF-1 had an 81% probability of having an NSI virus phenotype compared with individuals with lower SDF-1. Increased expression of SDF-1 may help explain why the more pathogenic SI HIV-1 variants do not appear in some individuals.
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Quimiocinas CXC/sangre , Células Gigantes , Infecciones por VIH/virología , VIH-1/fisiología , Quimiocina CXCL12 , Quimiocinas CXC/fisiología , Infecciones por VIH/sangre , Infecciones por VIH/patología , VIH-1/clasificación , Humanos , FenotipoRESUMEN
Syncytia arising from the fusion of cells expressing a lymphotropic human immunodeficiency virus (HIV)-1-encoded envelope glycoprotein complex (Env) gene with cells expressing the CD4/CXCR4 complex undergo apoptosis through a mitochondrion-controlled pathway initiated by the upregulation of Bax. In syncytial apoptosis, phosphorylation of p53 on serine 15 (p53S15) precedes Bax upregulation, the apoptosis-linked conformational change of Bax, the insertion of Bax in mitochondrial membranes, subsequent release of cytochrome c, caspase activation, and apoptosis. p53S15 phosphorylation also occurs in vivo, in HIV-1(+) donors, where it can be detected in preapoptotic and apoptotic syncytia in lymph nodes, as well as in peripheral blood mononuclear cells, correlating with viral load. Syncytium-induced p53S15 phosphorylation is mediated by the upregulation/activation of mammalian target of rapamycin (mTOR), also called FKBP12-rapamycin-associated protein (FRAP), which coimmunoprecipitates with p53. Inhibition of mTOR/FRAP by rapamycin reduces apoptosis in several paradigms of syncytium-dependent death, including in primary CD4(+) lymphoblasts infected by HIV-1. Concomitantly, rapamycin inhibits p53S15 phosphorylation, mitochondrial translocation of Bax, loss of the mitochondrial transmembrane potential, mitochondrial release of cytochrome c, and nuclear chromatin condensation. Transfection with dominant negative p53 has a similar antiapoptotic action as rapamycin, upstream of the Bax upregulation/translocation. In summary, we demonstrate that phosphorylation of p53S15 by mTOR/FRAP plays a critical role in syncytial apoptosis driven by HIV-1 Env.
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Apoptosis/inmunología , Proteínas Portadoras , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Inmunofilinas/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol) , Proteína p53 Supresora de Tumor/inmunología , Animales , Células Gigantes , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , Células HeLa , Humanos , Mamíferos , Fosforilación , Serina/metabolismo , Serina-Treonina Quinasas TOR , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
TAK-779 is a CCR5 antagonist under investigation by Takeda and Kagoshima University for the potential treatment of HIV [324663], [342114]. TAK-779 inhibits chemokine binding to the CCR5 receptor at nanomolar concentrations. However, it has no effect on the binding of chemokines to the CXCR4 receptor [346835]. A US IND for injectable TAK-779 was filed in june 1999, with Takeda initially planning to commence phase I trials in August 1999. However, the FDA did not clear the IND and recommended that Takeda altered the study protocol to include non-invasive measurement of local toxicities and to evaluate other routes of administration. By September 1999, Takeda had conducted some studies in response to the FDA's recommendations and had made efforts to develop an oral formulation. At this time, the company planned to file a new IND application upon completion of the oral formulation [342114]. In August 1995, Lehman Brothers predicted potential worldwide peak sales of US $300 million in
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Amidas/farmacología , Fármacos Anti-VIH/farmacología , Antagonistas de los Receptores CCR5 , Drogas en Investigación , Compuestos de Amonio Cuaternario/farmacología , Amidas/química , Amidas/toxicidad , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/toxicidad , Quimiocinas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Predicción , Estructura Molecular , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/toxicidadRESUMEN
We have evaluated the death of CD4(+) and CD8(+) T cells during in vitro human immunodeficiency virus (HIV) infection of peripheral blood mononuclear cells (PBMC) and tonsilar tissue. Acute infections with several X4 and R5 HIV isolates induced a decrease in cell viability that was higher in infections with X4 viruses and correlated with an increased rate of CD4(+) T-cell death. In CD4(+) T cells, the primary X4 isolate AOM induced higher levels of death than the laboratory X4 isolates IIIB and NL4-3 or the R5 isolates BaL and MDM. An effect on CD8(+) T-cell viability was exclusively observed in infections by X4 viruses, including the NL4-3 strain, in both PBMC and tonsilar tissue. This effect was dependent on the env gene of the infecting isolate and required productive HIV replication in CD4(+) but not in CD8(+) T cells. Our results suggest that X4 and R5 HIV isolates depleted CD4(+) T cells to a different extent and that CD8(+) T-cell viability may also be affected by mechanisms other than those acting in CD4(+) T cells.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , VIH-1/fisiología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Muerte Celular , Supervivencia Celular , Células Cultivadas , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/aislamiento & purificación , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/virología , Tejido Linfoide/citología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Replicación ViralRESUMEN
The retrovirus sections of the meeting focused on fundamental, preclinical aspects of the development of antiretroviral agents, although some clinical developments were also discussed. Novel nucleoside analogs, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, as well as preclinical data on new molecules that target early stages of HIV-1 infection (HIV integrase), were discussed. The meeting comprised plenary presentations, short oral communications and over 180 oral or poster presentations, of which more than 70 dealt with inhibition of retrovirus replication with an emphasis on the human immunodeficiency virus (HIV).
RESUMEN
Syncytia arising from the fusion of cells expressing a lymphotropic HIV type 1-encoded envelope glycoprotein complex (Env) with cells expressing the CD4/CXC chemokine receptor 4 complex spontaneously undergo cell death. Here we show that this process is accompanied by caspase activation and signs of mitochondrial membrane permeabilization (MMP), including the release of intermembrane proteins such as cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF) from mitochondria. In Env-induced syncytia, caspase inhibition did not suppress AIF- and Cyt-c translocation, yet it prevented all signs of nuclear apoptosis. Translocation of Bax to mitochondria led to MMP, which was inhibited by microinjected Bcl-2 protein or bcl-2 transfection. Bcl-2 also prevented the subsequent nuclear chromatin condensation and DNA fragmentation. The release of AIF occurred before that of Cyt-c and before caspase activation. Microinjection of AIF into syncytia sufficed to trigger rapid, caspase-independent Cyt-c release. Neutralization of endogenous AIF by injection of an antibody prevented all signs of spontaneous apoptosis occurring in syncytia, including the Cyt-c release and nuclear apoptosis. In contrast, Cyt-c neutralization only prevented nuclear apoptosis, and did not affect AIF release. Our results establish that the following molecular sequence governs apoptosis of Env-induced syncytia: Bax-mediated/Bcl-2-inhibited MMP --> AIF release --> Cyt-c release --> caspase activation --> nuclear apoptosis.
Asunto(s)
Apoptosis/fisiología , Antígenos CD4/fisiología , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Productos del Gen env/metabolismo , Células Gigantes/virología , VIH-1/fisiología , Mitocondrias/fisiología , Linfocitos T CD4-Positivos/fisiología , Fusión Celular , Técnicas de Cocultivo , Genes env , Células Gigantes/citología , Células Gigantes/fisiología , Células HeLa , Humanos , Membranas Intracelulares/fisiología , Cinética , Permeabilidad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , TransfecciónRESUMEN
Conjugates of L-arginine with aminoglycosides have already been described as potent in vitro inhibitors of the HIV-1 Tat-trans-activation responsive element interaction. The polycationic nature of these agents leads us to suggest that they may be active against HIV-1 replication by inhibiting earlier stages of the virus life cycle. We have found that R4K and R3G, kanamycin A, and gentamicin C, conjugated with arginine, inhibited HIV-1 NL4-3 replication at EC50 values of 15 and 30 microM for R3G and R4K, respectively, without a detectable tonic effect on MT-4 cells at concentrations higher than 4000 and about 1000 microM, respectively. Both compounds inhibited the binding of a monoclonal antibody (12G5) directed to CXCR4 as well as the intracellular Ca2+ signal induced by the chemokine SDF-1alpha on CXCR4+ cells, suggesting that aminoglycoside-arginine conjugates interact with CXCR4, the coreceptor used by T-tropic, X4 strains of HIV-1. On the other hand, CB4K, a conjugate of kanamycin A with gamma-guanidinobutyric acid, structurally similar to R4K, failed to display any anti-HIV activity of CXCR4 antagonist activity. An HIV-1 strain that was made resistant to the known CXCR4 antagonist AMD3100 was cross-resistant to both R4K and R3G. However, unlike SDF-1alpha and R4K, R3G inhibited the binding of HIV-1 to MT-4 cells. Aminoglycoside-arginine conjugates inhibit HIV replication by interrupting the early phase of the virus life cycle, namely virus binding to CD4 cells and interaction with CXCR4. R3G and R4K may serve as prototypes of novel anti-HIV agents and should be further studied.
Asunto(s)
Fármacos Anti-VIH/farmacología , Arginina/farmacología , VIH-1/efectos de los fármacos , Kanamicina/farmacología , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Anticuerpos Monoclonales , Arginina/química , Arginina/metabolismo , Bencilaminas , Linfocitos T CD4-Positivos/virología , Señalización del Calcio/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclamas , Citometría de Flujo , VIH-1/fisiología , Compuestos Heterocíclicos/farmacología , Humanos , Kanamicina/química , Kanamicina/metabolismo , Receptores CXCR4/inmunología , Receptores CXCR4/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Overproduction of beta-chemokines and genetic variations in chemokine receptors have been correlated with protection against infection by HIV-1 or slow progression to AIDS in infected individuals. STUDY DESIGN AND METHODS: The protective role of chemokines and their receptors was evaluated in a group of seven uninfected (seronegative) hemophiliacs transfused with hemoderivatives presumably contaminated with HIV-1. This group was compared to a group of seven infected (seropositive) hemophiliacs and a group of healthy donors (controls). The CD4+ cell count, intracellular cytokine levels, beta-chemokine levels in plasma, beta-chemokine production by PBMNCs, and expression of chemokine receptors CCR5 and CXCR4 in CD4+ cells were evaluated. The occurrence of protective genotypes in CCR5, CCR2b, and SDF-1 (stromal cell-derived factor 1) genes and susceptibility to infection by HIV-1 were also studied. RESULTS: Significant differences in the production and plasma levels of beta-chemokines among the three groups were not detected. Lower IL-2 and IFN-gamma production was observed in the uninfected exposed hemophiliacs than in the controls. Genetic analysis of CCR5, CCR2b, and SDF-1 showed several polymorphisms associated with resistance in some HIV-exposed uninfected hemophiliacs. However, these genetic features cannot explain the protection of all exposed hemophiliacs. In fact, only one patient, carrying two copies of CCR5 from which 32 bp was deleted, showed low CCR5 expression and low susceptibility to infection by a CCR5-using HIV-1 strain. In contrast, PBMNCs from all other individuals supported infection in vitro by both CCR5- and CXCR4-using HIV-1 strains. CONCLUSION: It is not possible to assign to beta-chemo-kines and polymorphisms in chemokine receptors a central role in preventing HIV-1 infection. Natural protection against HIV-1 infection is likely to be due to a multiplicity of factors.
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Quimiocinas CC/fisiología , Infecciones por VIH/prevención & control , VIH-1 , Hemofilia A/sangre , Estudios de Evaluación como Asunto , Infecciones por VIH/sangre , Hemofilia A/virología , HumanosRESUMEN
Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.
Asunto(s)
Bacteriófago lambda/efectos de los fármacos , Proteasa del VIH/metabolismo , Inhibidores de Proteasas/farmacología , Secuencia de Aminoácidos , Bacteriófago lambda/enzimología , Bacteriófago lambda/genética , Relación Dosis-Respuesta a Droga , Farmacorresistencia Microbiana , Vectores Genéticos , Proteasa del VIH/efectos de los fármacos , Proteasa del VIH/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis , Homología de Secuencia de Aminoácido , Replicación ViralRESUMEN
In most examples of physiological or pathological cell death, mitochondrial membrane permeabilization (MMP) constitutes an early critical event of the lethal process. Signs of MMP that precede nuclear apoptosis include the translocation of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to an extra-mitochondrial localization, as well as the dissipation of the mitochondrial transmembrane potential. MMP also occurs in HIV-1-induced apoptosis. Different HIV-1 encoded proteins (Env, Vpr, Tat, PR) can directly or indirectly trigger MMP, thereby causing cell death. The gp120/gp41 Env complex constitutes an example for an indirect MMP inducer. Env expressed on the plasma membrane of HIV-1 infected (or Env-transfected) cells mediates cell fusion with CD4/CXCR4-expressing uninfected cells. After a cell type-dependent latency period, syncytia then undergo MMP and apoptosis. Vpr exemplifies a direct MMP inducer. Vpr binds to the adenine nucleotide translocator (ANT), a mitochondrial inner membrane protein which also interacts with apoptosis-regulatory proteins from the Bcl-2/Bax family. Binding of Vpr to ANT favors formation of a non-specific pore leading to MMP. The structural motifs of the Vpr protein involved in MMP are conserved among most pathogenic HIV-1 isolates and determine the cytotoxic effect of Vpr. These data suggest the possibility that viruses employ multiple strategies to regulate host cell apoptosis by targeting mitochondria.
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Apoptosis/fisiología , Grupo Citocromo c/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Mitocondrias/metabolismo , Proteínas de los Retroviridae/metabolismo , Animales , Factor Inductor de la Apoptosis , Línea Celular , Flavoproteínas/metabolismo , Infecciones por VIH/patología , VIH-1/genética , Humanos , Linfocitos/metabolismo , Proteínas de la Membrana/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death by apoptosis in infected and uninfected cells. The envelope glycoprotein complex ([gp120/gp41](n)) of X4 HIV-1 isolates is involved in both infected and uninfected cell death via its interaction with cellular receptors CD4 and CXCR4. We studied the effect of the blockade of CXCR4 receptors by the agonist stromal derived factor (SDF-1alpha) and the antagonist bicyclam AMD3100 on apoptotic cell death of CD4(+) cells in different models of HIV infection. In HIV-infected CEM or SUP-T1 cultures, AMD3100 showed antiapoptotic activity even when added 24 h after infection. In contrast, other antiviral agents, such as zidovudine, failed to block apoptosis under these conditions. The antiapoptotic activity of AMD3100 was also studied in coculture of peripheral blood mononuclear cells or CD4(+) cell lines with chronically infected H9/IIIB cells. AMD3100 was found to inhibit both syncytium formation and apoptosis induction with 50% inhibitory concentrations ranging from 0.009 to 0.24 microg/ml, depending on the cell type. When compared to SDF-1alpha, AMD3100 showed higher inhibitory potency in all cell lines tested. Our data indicate that the bicyclam AMD3100 not only inhibits HIV replication but also efficiently blocks cell-surface-expressed HIV-1 envelope-induced apoptosis in uninfected cells.
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Fármacos Anti-VIH/farmacología , Apoptosis/efectos de los fármacos , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Proteínas del Envoltorio Viral/fisiología , Bencilaminas , Línea Celular , Ciclamas , Dipeptidil Peptidasa 4/análisis , VIH-1/fisiología , Humanos , Receptores CXCR4/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Negatively charged albumins (NCAs) have been identified as potent inhibitors of HIV-1 replication in vitro. Time of addition studies suggest that succinylated and aconitylated human serum albumin (Suc-HSA and Aco-HSA) act at an early stage of the virus life cycle, and surface plasmon resonance (BIAcore) experiments have confirmed a direct interaction of NCAs with HIV-1 gp120. Resistance to Suc-HSA and Aco-HSA was analyzed by characterizing HIV-1 variants that were selected in cell culture after serial passage of the NL4-3 strain in the presence of the compounds. After 24 passages (126 days) we isolated variants that were resistant to Suc-HSA (>27-fold) and Aco-HSA (37-fold), as compared with the wild-type NL4-3 virus. The binding of the NCA-resistant HIV strains to CD4+ MT-4 cells could no longer be inhibited by either Suc- or Aco-HSA. The emergence of mutations in the envelope gp120 of the resistant virus paralleled the emergence of the resistant phenotype. The Suc-HSA-resistant strain was 100-fold cross-resistant to the G quartet-containing oligonucleotide AR177 (Zintevir, an HIV-binding inhibitor), and partially cross-resistant to dextran sulfate, but remained sensitive to the bicyclam AMD3100 and the chemokine SDF-1alpha, which block HIV replication by interaction with the chemokine receptor CXCR4. Furthermore, neither Suc-HSA nor Aco-HSA inhibited the binding of monoclonal antibodies 12G5 and 2D7 (directed to CXCR4 and CCR5, respectively) in SUPT-1 cells or THP-1 cells. These results confirm that NCAs bind primarily to gp120 and do not interact directly with the HIV chemokine receptor but block the binding of the virus particles (through gp120) with CD4+ cells.
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Ácido Aconítico/análogos & derivados , Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/virología , VIH-1/efectos de los fármacos , Albúmina Sérica/farmacología , Replicación Viral/efectos de los fármacos , Ácido Aconítico/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Farmacorresistencia Microbiana , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/inmunología , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Albúmina Sérica HumanaRESUMEN
The emergence of X4 human immunodeficiency virus type 1 (HIV-1) strains in HIV-1-infected individuals has been associated with CD4(+) T-cell depletion, HIV-mediated CD8(+) cell apoptosis, and an impaired humoral response. The bicyclam AMD3100, a selective antagonist of CXCR4, selected for the outgrowth of R5 virus after cultivation of mixtures of the laboratory-adapted R5 (BaL) and X4 (NL4-3) HIV strains in the presence of the compound. The addition of AMD3100 to peripheral blood mononuclear cells infected with X4 or R5X4 clinical HIV isolates displaying the syncytium-inducing phenotype resulted in a complete suppression of X4 variants and a concomitant genotypic change in the V2 and V3 loops of the envelope gp120 glycoprotein. The recovered viruses corresponded genotypically and phenotypically to R5 variants in that they could no longer use CXCR4 as coreceptor or induce syncytium formation in MT-2 cells. Furthermore, the phenotype and genotype of a cloned R5 HIV-1 virus converted to those of the R5X4 virus after prolonged culture in lymphoid cells. However, these changes did not occur when the infected cells were cultured in the presence of AMD3100, despite low levels of virus replication. Our findings indicate that selective blockade of the CXCR4 receptor prevents the switch from the less pathogenic R5 HIV to the more pathogenic X4 HIV strains, a process that heralds the onset of AIDS. In this article, we show that it could be possible to redirect the evolution of HIV so as to impede the emergence of X4 strains or to change the phenotype of already-existing X4 isolates to R5.
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Fármacos Anti-VIH/farmacología , Infecciones por VIH/virología , VIH-1/fisiología , Compuestos Heterocíclicos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Adaptación Biológica , Secuencia de Aminoácidos , Bencilaminas , Ciclamas , Genotipo , Células Gigantes/virología , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fenotipo , Receptores CCR5/metabolismo , Replicación ViralRESUMEN
OBJECTIVE: The envelope glycoprotein complex (gp120/gp41)n of HIV-1 is one of the viral products responsible for increased apoptosis in HIV infection. Here the role of the chemokine receptor CXCR4 in HIV-1 envelope protein-induced apoptosis was investigated. METHODS: Apoptosis occurring in cocultures of chronically HIV-1 IIIB-infected cells with CD4 target cells expressing the CXCR4 receptor was quantified by terminal deoxinucleotidyl transferase dUTP nick end labeling (TUNEL) or propidium iodide staining followed by fluorescent antibody cell sorting, which allows the evaluation of single-cell killing. Moreover global (single cell- and syncytium-associated) apoptosis was quantified by a new radioactive TUNEL-derived assay. RESULTS: By using these different techniques it was shown that single and syncytium-forming CD4 T cells die by apoptosis upon contact with envelope protein expressing cells independently of viral replication. Moreover, both the CXCR4 agonist SDF-1alpha, and the antagonist AMD3100, showed inhibitory effects on HIV-1 envelope protein-induced apoptosis in the CD4 T-cell subset of peripheral blood mononuclear cells and CD4 cell lines. CXCR4 signalling-induced by HIV-1 envelope proteins in CD4 T cells was not detected. Furthermore, it was shown that envelope protein-induced apoptosis can occur after treating target cells with the Gi-protein inhibitor pertussis toxin. CONCLUSIONS: Evidence is provided for a role of CXCR4 in the mechanisms of HIV envelope protein-induced pathogenesis, contributing to selective CD4 cell killing. The results suggest that CXCR4 is involved in HIV-1-induced apoptosis; however, this role does not appear to involve G-protein-mediated CXCR4 signalling.