RESUMEN
Abnormal calcium signaling is a central pathological component of Alzheimer's disease (AD). Here, we describe the identification of a class of compounds called ReS19-T, which are able to restore calcium homeostasis in cell-based models of tau pathology. Aberrant tau accumulation leads to uncontrolled activation of store-operated calcium channels (SOCCs) by remodeling septin filaments at the cell cortex. Binding of ReS19-T to septins restores filament assembly in the disease state and restrains calcium entry through SOCCs. In amyloid-ß and tau-driven mouse models of disease, ReS19-T agents restored synaptic plasticity, normalized brain network activity, and attenuated the development of both amyloid-ß and tau pathology. Our findings identify the septin cytoskeleton as a potential therapeutic target for the development of disease-modifying AD treatments.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Calcio , Homeostasis , Fármacos Neuroprotectores , Septinas , Proteínas tau , Animales , Humanos , Ratones , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/efectos de los fármacos , Modelos Animales de Enfermedad , Plasticidad Neuronal/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Septinas/metabolismo , Proteínas tau/metabolismoRESUMEN
BACKGROUND: Neuronal Ca2+ dyshomeostasis and hyperactivity play a central role in Alzheimer's disease pathology and progression. Amyloid-beta together with non-genetic risk-factors of Alzheimer's disease contributes to increased Ca2+ influx and aberrant neuronal activity, which accelerates neurodegeneration in a feed-forward fashion. As such, identifying new targets and drugs to modulate excessive Ca2+ signalling and neuronal hyperactivity, without overly suppressing them, has promising therapeutic potential. METHODS: Here we show, using biochemical, electrophysiological, imaging, and behavioural tools, that pharmacological modulation of Rap1 signalling by inhibiting its interaction with Pde6δ normalises disease associated Ca2+ aberrations and neuronal activity, conferring neuroprotection in models of Alzheimer's disease. RESULTS: The newly identified inhibitors of the Rap1-Pde6δ interaction counteract AD phenotypes, by reconfiguring Rap1 signalling underlying synaptic efficacy, Ca2+ influx, and neuronal repolarisation, without adverse effects in-cellulo or in-vivo. Thus, modulation of Rap1 by Pde6δ accommodates key mechanisms underlying neuronal activity, and therefore represents a promising new drug target for early or late intervention in neurodegenerative disorders. CONCLUSION: Targeting the Pde6δ-Rap1 interaction has promising therapeutic potential for disorders characterised by neuronal hyperactivity, such as Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Neuroprotección/fisiología , Fármacos Neuroprotectores/uso terapéutico , Complejo Shelterina , Transducción de Señal/fisiologíaRESUMEN
Although a wide variety of genetic and nongenetic Alzheimer's disease (AD) risk factors have been identified, their role in onset and/or progression of neuronal degeneration remains elusive. Systematic analysis of AD risk factors revealed that perturbations of intraneuronal signalling pathways comprise a common mechanistic denominator in both familial and sporadic AD and that such alterations lead to increases in Aß oligomers (Aßo) formation and phosphorylation of TAU. Conversely, Aßo and TAU impact intracellular signalling directly. This feature entails binding of Aßo to membrane receptors, whereas TAU functionally interacts with downstream transducers. Accordingly, we postulate a positive feedback mechanism in which AD risk factors or genes trigger perturbations of intraneuronal signalling leading to enhanced Aßo formation and TAU phosphorylation which in turn further derange signalling. Ultimately intraneuronal signalling becomes deregulated to the extent that neuronal function and survival cannot be sustained, whereas the resulting elevated levels of amyloidogenic Aßo and phosphorylated TAU species self-polymerizes into the AD plaques and tangles, respectively.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Neuronas/metabolismo , Transducción de Señal , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/genética , Animales , Endocitosis , Humanos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/patología , Fosforilación , Factores de Riesgo , Proteínas tau/metabolismoRESUMEN
The redesign of azamacrocyclic CXCR4 chemokine receptor antagonists resulted in the discovery of novel, small molecule, orally bioavailable compounds that retained T-tropic (CXCR4 using, X4) anti-HIV-1 activity. A structure-activity relationship (SAR) was determined on the basis of the inhibition of replication of X4 HIV-1 NL4.3 in MT-4 cells. As a result of lead optimization, we identified (S)-N'-((1H-benzo[d]imidazol-2-yl)methyl)-N'-(5,6,7,8-tetrahydroquinolin-8-yl)butane-1,4-diamine (AMD070) 2 as a potent and selective antagonist of CXCR4 with an IC(50) value of 13 nM in a CXCR4 125I-SDF inhibition binding assay. Compound 2 inhibited the replication of T-tropic HIV-1 (NL4.3 strain) in MT-4 cells and PBMCs with an IC(50) of 2 and 26 nM, respectively, while remaining noncytotoxic to cells at concentrations exceeding 23 microM. The pharmacokinetics of 2 was evaluated in rat and dog, and good oral bioavailability was observed in both species. This compound represents the first small molecule orally bioavailable CXCR4 antagonist that was developed for the treatment of HIV-1 infection.
Asunto(s)
Fármacos Anti-VIH/síntesis química , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos con 1 Anillo/síntesis química , Receptores CXCR4/antagonistas & inhibidores , Administración Oral , Aminoquinolinas , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Bencimidazoles , Disponibilidad Biológica , Butilaminas , Calcio/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular , Perros , VIH-1/fisiología , Compuestos Heterocíclicos con 1 Anillo/química , Compuestos Heterocíclicos con 1 Anillo/farmacología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Ensayo de Unión Radioligante , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Bis-tetraazamacrocycles such as the bicyclam AMD3100 (1) are a class of potent and selective anti-HIV-1 agents that inhibit virus replication by binding to the chemokine receptor CXCR4, the coreceptor for entry of X4 viruses. By sequential replacement and/or deletion of the amino groups within the azamacrocyclic ring systems, we have determined the minimum structural features required for potent antiviral activity in this class of compounds. All eight amino groups are not required for activity, the critical amino groups on a per ring basis are nonidentical, and the overall charge at physiological pH can be reduced without compromising potency. This approach led to the identification of several single ring azamacrocyclic analogues such as AMD3465 (3d), 36, and 40, which exhibit EC(50)'s against the cytopathic effects of HIV-1 of 9.0, 1.0, and 4.0 nM, respectively, antiviral potencies that are comparable to 1 (EC(50) against HIV-1 of 4.0 nM). More importantly, however, the key structural elements of 1 required for antiviral activity may facilitate the design of nonmacrocyclic CXCR4 antagonists suitable for HIV treatment via oral administration.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/síntesis química , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/síntesis química , Receptores CXCR4/antagonistas & inhibidores , Linfocitos T , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , Compuestos Heterocíclicos/farmacología , Humanos , Modelos Químicos , Estructura Molecular , Piridinas/química , Piridinas/farmacología , Relación Estructura-ActividadRESUMEN
The chemokine receptor CXCR4 functions as one of the HIV-1 coreceptors and can be considered as an attractive target for the development of novel anti-HIV drugs. Here, we investigated the effect of its two known N-glycosylation sites g1 (NYT) and g2 (NVS) on the antiviral potential of several classes of entry inhibitors. The lack of g1 clearly affected the binding of the amino-terminal directed 2B11 mAb, but not the 12G5 mAb. No dramatic effects on CXCL-12 binding and CXCL-12-induced intracellular calcium responses were observed. Importantly, the anti-HIV-1 activity and antagonistic activity of the prototype compound of CXCR4 inhibitors, AMD3100, were not affected by the presence or absence of the CXCR4 N-glycans. Since CXCR4 N-glycans play a less important role in viral entry compared to the N-glycans on the HIV envelope, cells expressing CXCR4 N-glycosylation mutants might be no relevant alternative to allow HIV-1 escape from antivirals.
Asunto(s)
Quimiocinas CXC/metabolismo , VIH-1/fisiología , VIH-1/patogenicidad , Receptores CXCR4/metabolismo , Receptores del VIH/metabolismo , Secuencia de Aminoácidos , Aminoácidos/fisiología , Fármacos Anti-VIH/farmacología , Anticuerpos/metabolismo , Anticuerpos/farmacología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Bencilaminas , Sitios de Unión/fisiología , Línea Celular Tumoral , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Ciclamas , Farmacorresistencia Viral , Glicosilación , Inhibidores de Fusión de VIH/farmacología , Compuestos Heterocíclicos/metabolismo , Compuestos Heterocíclicos/farmacología , Humanos , Lectinas/metabolismo , Lectinas/farmacología , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Receptores del VIH/genética , Transducción de Señal , Virulencia , Replicación ViralRESUMEN
The chemokine receptor CXCR3 can exhibit weak coreceptor function for several human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains and clinical isolates. These viruses produced microscopically visible cytopathicity in U87.CD4.CXCR3 cell cultures, whereas untransfected (CXCR3-negative) U87.CD4 cells remained uninfected. Depending on the particular virus, the coreceptor efficiency of CXCR3 was 100- to >10,000-fold lower compared to that of CXCR4. A CXCR3 variant carrying the CXCR4 binding pocket was constructed by simultaneous lysine-to-alanine and serine-to-glutamate substitutions at positions 300 and 304 of the CXCR3 receptor. This mutant receptor (CXCR3[K300A, S304E]) showed markedly enhanced HIV coreceptor function compared to the wild-type receptor (CXCR3[WT]). Moreover, the CXCR4 antagonist AMD3100 exhibited antagonistic and anti-HIV activities in U87.CD4.CXCR3[K300A, S304E] cells but not in U87.CD4.CXCR3[WT] cells.
Asunto(s)
VIH/inmunología , Imitación Molecular/inmunología , Receptores CXCR4/inmunología , Receptores CXCR4/metabolismo , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Receptores del VIH/inmunología , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , VIH/metabolismo , Humanos , Ligandos , Datos de Secuencia Molecular , Receptores CXCR3 , Receptores CXCR4/química , Receptores de Quimiocina/química , Receptores de Quimiocina/genética , Receptores del VIH/química , Receptores del VIH/genética , Receptores del VIH/metabolismoRESUMEN
The chemokine receptors CXCR4 and CCR5 are the main coreceptors used by the T-cell-tropic (CXCR4-using, X4) and macrophage-tropic (CCR5-using, R5) HIV-1 strains, respectively, for entering their CD4+ target cells. In this review, we focus on the function of these chemokine receptors in HIV infection and their role as novel targets for viral inhibition. Besides some modified chemokines with antiviral activity, several low-molecular weight CCR5 and CXCR4 antagonistic compounds have been described with potent antiviral activity. The best CXCR4 antagonists described are the bicyclam derivatives, which consistently block X4 but also R5/X4 viral replication in PBMCs. We believe that chemokine receptor antagonists will become important new antiviral drugs to combat AIDS. Both CXCR4 and CCR5 chemokine receptor inhibitors will be needed in combination and even in combinations of antiviral drugs that also target other aspects of the HIV replication cycle to obtain optimum antiviral therapeutic effects.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Receptores de Quimiocina/antagonistas & inhibidores , Animales , Fármacos Anti-VIH/uso terapéutico , Antagonistas de los Receptores CCR5 , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Humanos , Receptores CCR5/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Receptores de Quimiocina/metabolismoRESUMEN
The chemokine receptors CCR5 and CXCR4 function as coreceptors for human immunodeficiency virus (HIV) and are attractive targets for the development of anti-HIV drugs. The most potent CXCR4 antagonists described until today are the bicyclams. The prototype compound, AMD3100, exhibits potent and selective anti-HIV activity against CXCR4-using (X4) viruses and showed antiviral efficacy in X4 HIV-1-infected persons in a phase II clinical trial. However, AMD3100 lacks oral bioavailability due to its high overall positive charge. Initial structure-activity relationship studies with bicyclam analogues suggested that the bis-macrocyclic structure was a prerequisite for anti-HIV activity. Now, we report that the N-pyridinylmethylene cyclam AMD3465, which lacks the structural constraints mentioned above, fully conserves all the biological properties of AMD3100. Like AMD3100, AMD3465 blocked the cell surface binding of both CXCL12 (the natural CXCR4 ligand), and the specific anti-CXCR4 monoclonal antibody 12G5. AMD3465 dose-dependently inhibited intracellular calcium signaling, chemotaxis, CXCR4 endocytosis and mitogen-activated protein kinase phosphorylation induced by CXCL12. Compared to the bicyclam AMD3100, AMD3465 was even 10-fold more effective as a CXCR4 antagonist, while showing no interaction whatsoever with CCR5. As expected, AMD3465 proved highly potent against X4 HIV strains (IC50: 1-10 nM), but completely failed to inhibit the replication of CCR5-using (R5) viruses. In conclusion, AMD3465 is a novel, monomacrocyclic anti-HIV agent that specifically blocks the interaction of HIV gp120 with CXCR4. Although oral bioavailability is not yet achieved, the monocyclams, with their decreased molecular charge as compared to the bicyclams, embody an important step forward in the design of oral CXCR4 antagonists that can be clinically used as anti-HIV drugs.
Asunto(s)
Fármacos Anti-VIH/farmacología , Piridinas/farmacología , Receptores CXCR4/antagonistas & inhibidores , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: To evaluate the anti-HIV-1 activity of the cyclotriazadisulfonamide CADA against primary isolates in vitro and the combination of CADA with approved anti-HIV drugs for potential synergy. METHODS: Peripheral blood mononuclear cells (PBMC) were treated with CADA and infected with 16 different clinical isolates. After 8 days of infection, the median inhibitory concentration (IC50) was calculated from the p24 viral antigen content in the supernatant. MT-4 cells were infected with HIV-1NL4.3 and then cultured with CADA or other antiretroviral drugs (i.e., several reverse transcriptase, protease and entry inhibitors), alone and in combination. After 4 days, IC50 was determined for the various drugs in replicate assays. Analysis of combined effects was performed using the median effect principle (CalcuSyn; Biosoft). RESULTS: The entry inhibitor CADA exerted a potent and consistent anti-HIV-1 activity against a wide range of R5, R5/X4 and X4 primary isolates in PBMC. From the two-drug studies, combination indices showed synergy between CADA and reverse transcriptase inhibitors (zidovudine, stavudine, lamivudine, zalcitabine, didanosine, abacavir, tenofovir, nevirapine, delavirdine and efavirenz), and protease inhibitors (lopinavir, saquinavir, indinavir, nelfinavir, amprenavir and ritonavir). In addition, the combination of CADA with the gp41 fusion inhibitor T-20 (enfuvirtide), the CXCR4 antagonist AMD3100 and the gp120-specific interacting plant lectins from Galanthus nivalis (GNA) and Hippeastrum hybrid (HHA) also resulted in a synergistic inhibition. CONCLUSIONS: Compounds that can specifically downmodulate the CD4 receptor in PBMC have broad-spectrum anti-HIV activity against primary isolates and act synergistically when used in conjunction with currently available antiretroviral drugs. They deserve further study as potential candidate anti-HIV drugs.
Asunto(s)
Fármacos Anti-VIH/farmacología , Antígenos CD4/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , VIH-1 , Compuestos Heterocíclicos/farmacología , Leucocitos Mononucleares/inmunología , Inhibidores de la Transcriptasa Inversa/farmacología , Terapia Antirretroviral Altamente Activa , Línea Celular , Sinergismo Farmacológico , Citometría de Flujo , Infecciones por VIH/inmunología , Compuestos Heterocíclicos/química , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Pruebas de Sensibilidad MicrobianaRESUMEN
Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC(50), 1.8 to 7.3 microM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.
Asunto(s)
Fármacos Anti-VIH/farmacología , Antagonistas de los Receptores CCR5 , VIH/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Piridinas/farmacología , Receptores CXCR4/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Calcio/metabolismo , Quimiocina CXCL12 , Quimiocinas CC/antagonistas & inhibidores , Quimiocinas CXC/antagonistas & inhibidores , Quimiotaxis/efectos de los fármacos , VIH/fisiología , HumanosRESUMEN
BACKGROUND: Chemokines drive the migration of leukocytes via interaction with specific G protein-coupled 7-transmembrane receptors. The chemokine ligand/receptor pair stromal cell-derived factor-1 (SDF-1, CXCL12)/CXCR4 is gaining increasing interest because of its involvement in the metastasis of several types of cancer and in certain inflammatory autoimmune disorders such as rheumatoid arthritis. In addition, CXCR4 serves as an important coreceptor for cellular entry of T-tropic strains of human immunodeficiency virus (HIV). Therefore, potent and specific CXCR4 antagonists may have therapeutic potential as anti-HIV, anti-cancer, and anti-inflammatory drugs. METHODS AND RESULTS: Chemokine receptor antagonists can be identified by their ability to inhibit ligand binding to the receptor protein. Until now, chemokine binding assays were mostly performed with radiolabeled chemokine ligands such as [(125)I]CXCL12. To overcome the practical problems associated with such radioactive chemokine binding assays, we have developed a flow cytometric technique using a new, commercially available Alexa Fluor 647 conjugate of CXCL12 (CXCL12(AF647)). Calcium flux, chemotaxis, and p44/42 mitogen-activated protein kinase phosphorylation assays showed that the agonistic activity of the fluorescent CXCL12 was unchanged as compared with that of unlabeled CXCL12. Human T-lymphoid (CXCR4(+)) SupT1 cells and CXCR4-transfected, but not CCR5- or CXCR3-transfected, human astroglioma U87.CD4 cells specifically bound CXCL12(AF647) in a concentration-dependent manner. Unlabeled CXCL12 and the well-known CXCR4 inhibitors, AMD3100 and T22, blocked the binding of CXCL12(AF647) to SupT1 cells with 50% inhibitory concentrations of 92, 13, and 8 ng/ml, respectively. We have also used this method to evaluate CXCL12 binding and CXCR4 expression level in different subsets of human peripheral blood mononuclear cells. CONCLUSION: CXCL12(AF647) is a valuable, more convenient alternative for [(125)I]CXCL12 in ligand/receptor interaction studies.
Asunto(s)
Quimiocinas CXC/química , Quimiocinas CXC/farmacología , Citometría de Flujo/métodos , Colorantes Fluorescentes/farmacología , Receptores CXCR4/química , Calcio/metabolismo , Línea Celular Tumoral , Quimiocina CXCL12 , Quimiocinas/metabolismo , Quimiotaxis , Relación Dosis-Respuesta a Droga , Citometría de Flujo/instrumentación , Humanos , Ligandos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Unión Proteica , Receptores CCR5/metabolismo , Receptores CXCR3 , Receptores de Quimiocina/metabolismo , Linfocitos T/inmunología , TransfecciónRESUMEN
The plant lectins derived from Galanthus nivalis (Snowdrop) (GNA) and Hippeastrum hybrid (Amaryllis) (HHA) selectively inhibited a wide variety of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains and clinical (CXCR4- and CCR5-using) isolates in different cell types. They also efficiently inhibited infection of T lymphocytes by a variety of mutant virus strains. GNA and HHA markedly prevented syncytium formation between persistently infected HUT-78/HIV cells and uninfected T lymphocytes. The plant lectins did not measurably affect the antiviral activity of other clinically approved anti-HIV drugs used in the clinic when combined with these drugs. Short exposure of the lectins to cell-free virus particles or persistently HIV-infected HUT-78 cells markedly decreased HIV infectivity and increased the protective (microbicidal) activity of the plant lectins. Flow cytometric analysis and monoclonal antibody binding studies and a PCR-based assay revealed that GNA and HHA do not interfere with CD4, CXCR4, CCR5, and DC-SIGN and do not specifically bind with the membrane of uninfected cells. Instead, GNA and HHA likely interrupt the virus entry process by interfering with the virus envelope glycoprotein. HHA and GNA are odorless, colorless, and tasteless, and they are not cytotoxic, antimetabolically active, or mitogenic to human primary T lymphocytes at concentrations that exceed their antivirally active concentrations by 2 to 3 orders of magnitude. GNA and HHA proved stable at high temperature (50 degrees C) and low pH (5.0) for prolonged time periods and can be easily formulated in gel preparations for microbicidal use; they did not agglutinate human erythrocytes and were not toxic to mice when administered intravenously.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Galanthus/química , VIH-1/efectos de los fármacos , Liliaceae/química , Manosa/uso terapéutico , Aglutininas/farmacología , Animales , Fármacos Anti-VIH/química , Anticuerpos Monoclonales/química , Señalización del Calcio , División Celular/efectos de los fármacos , Sistema Libre de Células , Células Cultivadas , Técnicas de Cocultivo , Eritrocitos/efectos de los fármacos , Citometría de Flujo , Geles , Células Gigantes/citología , Células Gigantes/virología , VIH-1/genética , VIH-1/crecimiento & desarrollo , Humanos , Lectinas/uso terapéutico , Manosa/química , Ratones , Mitógenos/farmacología , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TemperaturaRESUMEN
BACKGROUND: CCR5 and CXCR4 are the two main coreceptors essential for HIV entry. Therefore, these chemokine receptors have become important targets in the search for anti-HIV agents. Here, we describe the establishment of a novel CD4+ cell line, U87.CD4.CCR5.CXCR4, stably expressing both CCR5 and CXCR4 at the cell surface. RESULTS: In these cells, intracellular calcium signalling through both receptors can be measured in a single experiment upon the sequential addition of CXCR4- and CCR5-directed chemokines. The U87.CD4.CCR5.CXCR4 cell line reliably supported HIV-1 infection of diverse laboratory-adapted strains and primary isolates with varying coreceptor usage (R5, X4 and R5/X4) and allows to investigate the antiviral efficacy of combined CCR5 and CXCR4 blockade. The antiviral effects recorded in these cells with the CCR5 antagonist SCH-C and the CXCR4 antagonist AMD3100 were similar to those noted in the single CCR5- or CXCR4-transfected U87.CD4 cells. Furthermore, the combination of both inhibitors blocked the infection of all evaluated HIV-1 strains and isolates. CONCLUSIONS: Thus, the U87.CD4.CCR5.CXCR4 cell line should be useful in the evaluation of CCR5 and CXCR4 antagonists with therapeutic potential and combinations thereof.
Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Bencilaminas , Antagonistas de los Receptores CCR5 , Linfocitos T CD4-Positivos/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Línea Celular , Línea Celular Tumoral , Quimiocinas/farmacología , Ciclamas , Óxidos N-Cíclicos/farmacología , Compuestos Heterocíclicos/farmacología , Humanos , Células Jurkat , Oximas , Piperidinas/farmacología , Piridinas/farmacología , Receptores CXCR4/antagonistas & inhibidoresRESUMEN
The interaction of the CXCR4 antagonist AMD3100 with its target is greatly influenced by specific aspartate residues in the receptor protein, including Asp(171) and Asp(262). We have now found that aspartate-to-asparagine substitutions at these positions differentially affect the binding of four different anti-CXCR4 monoclonal antibodies as well as the infectivity of diverse human immunodeficiency virus type 1 (HIV-1) strains and clinical isolates. Mutation of Asp(262) strongly decreased the coreceptor efficiency of CXCR4 for wild-type but not for AMD3100-resistant HIV-1 NL4.3. Thus, resistance of HIV-1 NL4.3 to AMD3100 is associated with a decreased dependence of the viral gp120 on Asp(262) of CXCR4, pointing to a different mode of interaction of wild-type versus AMD3100-resistant virus with CXCR4.
Asunto(s)
VIH-1/fisiología , Compuestos Heterocíclicos/metabolismo , Mutación , Receptores CXCR4/metabolismo , Secuencia de Aminoácidos , Bencilaminas , Sitios de Unión , Ciclamas , Citometría de Flujo , Humanos , Fusión de Membrana , Datos de Secuencia Molecular , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/química , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Células Tumorales CultivadasRESUMEN
BACKGROUND: The chemokine receptors CXCR4 and CCR5 are the main coreceptors for human immunodeficiency virus (HIV) 1 to enter its target cells. The antiviral activity of their natural ligands (stromal cell-derived factor 1 [SDF-1], regulated on activation normal T-cell expressed and secreted, (RANTES) and macrophage inflammatory proteins 1alpha and 1beta, MIP-1alpha and MIP-1beta) and the finding that individuals deficient in CCR5 are relatively resistant to HIV infection led to the concept that chemokine receptor antagonists can play an important role in anti-HIV therapy. AMD3100, the prototype compound of the bicyclams, is one of the most potent and selective CXCR4 antagonists described to date. The search for new chemokine receptor antagonists involves the evaluation of compounds for their ability to block the specific chemokine-induced transient intracellular Ca(2+) flux. We evaluated two cell-based-fluorescent methods with the use of the Fluorometric Imaging Plate Reader (FLIPR) system and a flow cytometric assay to measure the SDF-1-induced intracellular Ca(2+) mobilization via CXCR4. Both assay systems were compared for their sensitivity, advantages, and system-dependent limitations. METHODS: CXCR4(+) lymphocytic and monocytic cell lines commonly used in laboratories studying acquired immunodeficiency syndrome and freshly isolated peripheral blood mononuclear cells (PBMCs) were loaded with the Ca(2+) indicator Fluo-3. After washing, the cells were preincubated with the CXCR4 antagonist AMD3100. Then the increase in intracellular Ca(2+) concentration after stimulation with SDF-1 was examined by the FLIPR and the flow cytometer, which monitored the change in green fluorescence intensity of Ca(2+)-bound Fluo-3. Surface CXCR4 expression was also determined flow cytometrically. RESULTS: In all five CXCR4(+) cell lines, SDF-1 elicited a transient increase in intracellular Ca(2+) concentration. For each cell line, the magnitude of response was related to the level of CXCR4 expression: the cells with the highest CXCR4 level showed the strongest Ca(2+) response. AMD3100 effected a dose-dependent inhibition of the SDF-1-induced Ca(2+) flux in all cell lines examined. The FLIPR was more sensitive than the flow cytometer in detecting minor Ca(2+) responses. In freshly isolated PBMCs, the Ca(2+) response was due solely to the stimulation of monocytes and granulocytes, whereas the lymphocyte population, although expressing CXCR4, did not respond at all. This phenomenon could be observed with the flow cytometer and not with the FLIPR. CONCLUSIONS: The FLIPR system is a most adequate to monitor intracellular Ca(2+) mobilization and to evaluate chemokine receptor antagonists. However, flow cytometry and its multiparameter analysis allow additional characterization of the cells involved in the chemokine receptor-mediated signal transduction.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/análisis , Citometría de Flujo/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Compuestos de Anilina , Bencilaminas , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacología , Ciclamas , Citometría de Flujo/instrumentación , Fluorometría/instrumentación , Fluorometría/métodos , Compuestos Heterocíclicos/farmacología , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Reproducibilidad de los Resultados , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , XantenosRESUMEN
The chemokine receptor CXCR4 is the main coreceptor used by T-tropic X4 HIV-1 strains to infect its target T cells. It has been proven that the CXCR4 expression level in T cells is strongly up-regulated by interleukin (IL)-4, a Th2-type cytokine that is secreted preferentially in HIV-infected patients in a later stage of disease. This results in an enhancement of HIV-1 replication in CD4+ T-lymphocytes. We have now evaluated the potency of the CXCR4 antagonist AMD3100 in phytohemagglutinin (PHA)/IL-2- versus PHA/IL-4-activated T cells in order to determine whether the compound has comparable CXCR4-antagonistic and anti-HIV-1 effects under these different cytokine treatments. We analyzed the CXCR4 expression level and the dose-dependent inhibition of CXCR4 expression by AMD3100, by monitoring the binding of an anti-CXCR4 monoclonal antibody (clone 12G5). We also determined stromal cell-derived factor (SDF)-1-induced intracellular calcium signaling and HIV-1 replication in these cells in the absence and presence of AMD3100. The CXCR4 expression level in PHA/IL-4-stimulated cells was much higher than in PHA/IL-2-stimulated cells. However, the potency of the bicyclam AMD3100 to block anti-CXCR4 mAb binding, SDF-1-induced intracellular calcium signaling, and HIV-1 replication of the X4 NL4.3 strain and three primary isolates remained unchanged. Our data indicate that CXCR4 antagonists such as AMD3100 act independently of the HIV-1 coreceptor expression level. These compounds should therefore be useful in suppressing HIV-1 infection in all stages of the disease.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Receptores CXCR4/metabolismo , Receptores Virales/metabolismo , Bencilaminas , Ciclamas , Citocinas/farmacología , Expresión Génica/efectos de los fármacos , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Receptores Virales/genéticaRESUMEN
The novel antiviral agent cyclotriazadisulfonamide (CADA) inhibited human immunodeficiency virus (HIV) (IC50, 0.3-3.2 microM) and human herpesvirus 7 (HHV-7) infection (IC50, 0.3-1.5 microM) in T-cell lines and PBMCs. When T-cells were pretreated with CADA for 24 h, they became markedly protected from viral infection. Flow cytometric analysis revealed a significant decrease in the expression of the CD4 glycoprotein, the primary receptor needed for entry of both viruses. Moreover, the antiviral activity of CADA correlated with its ability to down-modulate the CD4 receptor. CADA did not alter the expression of any other cellular receptor (or HIV coreceptor) examined. Time course experiments showed that CD4 down-modulation by CADA differs in mechanism from the effects of aurintricarboxylic acid, which binds directly to CD4, and phorbol myristate acetate, which activates protein kinase C. Further analysis of CD4 mRNA levels suggested that CADA was not involved in the regulation of CD4 expression at a transcriptional level, but very likely at (post) translational levels. This unique mechanism of action makes CADA an important lead in developing new drugs for treatment of AIDS, autoimmune diseases, and inflammatory disorders.
Asunto(s)
Antiinfecciosos/farmacología , Antígenos CD4/metabolismo , Regulación hacia Abajo , VIH-1/efectos de los fármacos , Herpesvirus Humano 7/efectos de los fármacos , Sulfonamidas/farmacología , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Línea Celular , VIH-1/patogenicidad , VIH-1/fisiología , Herpesvirus Humano 7/fisiología , Humanos , Leucocitos Mononucleares/virología , Sulfonamidas/síntesis química , Sulfonamidas/química , Linfocitos T/virología , Replicación Viral/efectos de los fármacosRESUMEN
This study was undertaken to demonstrate the unique specificity of the chemokine receptor CXCR4 antagonist AMD3100. Calcium flux assays with selected chemokine/cell combinations, affording distinct chemokine receptor specificities, revealed no interaction of AMD3100 with any of the chemokine receptors CXCR1 through CXCR3, or CCR1 through CCR9. In contrast, AMD3100 potently inhibited CXCR4-mediated calcium signaling and chemotaxis in a concentration-dependent manner in different cell types. Also, AMD3100 inhibited stromal cell-derived factor (SDF)-1-induced endocytosis of CXCR4, but did not affect phorbol ester-induced receptor internalization. Importantly, AMD3100 by itself was unable to elicit intracellular calcium fluxes, to induce chemotaxis, or to trigger CXCR4 internalization, indicating that the compound does not act as a CXCR4 agonist. Specific small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of human immunodeficiency virus infections and many other pathological processes that are dependent on SDF-1/CXCR4 interactions (e.g. rheumatoid arthritis, atherosclerosis, asthma and breast cancer metastasis).