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1.
J Physiol ; 602(17): 4291-4307, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39106251

RESUMEN

ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.


Asunto(s)
Canales de Cloruro , Endocitosis , Xenopus laevis , Animales , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Endocitosis/fisiología , Ratones , Túbulos Renales Distales/metabolismo , Hiperplasia , Humanos , Femenino , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo , Ratones Endogámicos C57BL , Células HEK293 , Oocitos/metabolismo , Proteínas de Transporte de Anión
2.
Hum Mol Genet ; 30(17): 1649-1665, 2021 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-34100078

RESUMEN

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.


Asunto(s)
Quistes/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Receptores Acoplados a Proteínas G/genética , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neurona-Glia/genética , Moléculas de Adhesión Celular Neurona-Glia/metabolismo , Proteínas de Ciclo Celular/genética , Canales de Cloruro/genética , Quistes/metabolismo , Células HEK293 , Células HeLa , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Leucoencefalopatías/genética , Leucoencefalopatías/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Malformaciones del Sistema Nervioso/metabolismo , Transporte de Proteínas , Receptores Acoplados a Proteínas G/metabolismo
3.
Neurobiol Dis ; 119: 88-99, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30076890

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes. Previous work indicated that chloride currents mediated by the volume-regulated anion channel (VRAC) and ClC-2 channels were affected in astrocytes deficient in either Mlc1 or Glialcam. ClC-2 forms a ternary complex with GlialCAM and MLC1. LRRC8 proteins have been identified recently as the molecular components of VRAC, but the relationship between MLC and LRRC8 proteins is unknown. Here, we first demonstrate that LRRC8 and MLC1 are functionally linked, as MLC1 cannot potentiate VRAC currents when LRRC8A, the main subunit of VRAC, is knocked down. We determine that LRRC8A and MLC1 do not co-localize or interact and, in Xenopus oocytes, MLC1 does not potentiate LRRC8-mediated VRAC currents, indicating that VRAC modulation in astrocytes by MLC1 may be indirect. Investigating the mechanism of modulation, we find that a lack of MLC1 does not influence either mRNA or total and plasma membrane protein levels of LRRC8A; and neither does it affect LRRC8A subcellular localization. In agreement with recent results that indicated that overexpression of MLC1 decreases the phosphorylation of extracellular signal-regulated kinases (ERK), we find that astrocytes lacking MLC1 show an increase in ERK phosphorylation. In astrocytes with reduced or increased levels of MLC1 we observe changes in the phosphorylation state of the VRAC subunit LRRC8C. Our results thus reinforce previous suggestions that indicated that GlialCAM/MLC1 might modify signal transduction pathways that influence the activity of different proteins, such as VRAC.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Astrocitos/metabolismo , Quistes/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Astrocitos/química , Astrocitos/patología , Proteínas de Ciclo Celular , Células Cultivadas , Quistes/patología , Células HeLa , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas/análisis , Proteínas/genética , Ratas , Xenopus
4.
Muscle Nerve ; 2018 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-29424939

RESUMEN

INTRODUCTION: Mutations in CLCN1 cause recessive or dominant forms of myotonia congenita (MC). Some mutations have been found to exhibit both patterns of inheritance but the mechanism explaining this behavior is unknown. METHODS: A known recessive missense mutation, A493E, was identified in a family with dominant MC. The mutant p.A493E alone or in co-expression with wild-type (WT) ClC-1 was expressed in Xenopus oocytes. Currents were measured and biochemical assays were performed. RESULTS: The mutant showed no significant activity and reduced total and plasma membrane (PM) protein levels. Co-expression with the mutant reduced the activity and PM levels of an engineered lower expression variant of ClC-1, whereas no effect was observed on a higher expression variant. DISCUSSION: Our results suggest that the dominant effect of some CLCN1 mutations showing recessive or dominant inheritance patterns may be due to a dose-dependent defect in PM delivery of the WT channel. Muscle Nerve, 2018.

5.
Eur J Med Genet ; 61(1): 50-60, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29079544

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.


Asunto(s)
Quistes/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Proteínas/genética , Animales , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Quistes/patología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica , Proteínas/química , Proteínas/metabolismo
6.
Curr Alzheimer Res ; 14(12): 1327-1334, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28474567

RESUMEN

BACKGROUND: It is well established that mitochondrial damage plays a role in the pathophysiology of Alzheimer's disease (AD). However, studies carried out in humans barely contemplate regional differences with disease progression. OBJECTIVE: To study the expression of selected nuclear genes encoding subunits of the mitochondrial complexes and the activity of mitochondrial complexes in AD, in two regions: the entorhinal cortex (EC) and frontal cortex area 8 (FC). METHODS: Frozen samples from 148 cases processed for gene expression by qRT-PCR and determination of individual activities of mitochondrial complexes I, II, IV and V using commercial kits and home-made assays. RESULTS: Decreased expression of NDUFA2, NDUFB3, UQCR11, COX7C, ATPD, ATP5L and ATP50, covering subunits of complex I, II, IV and V, occurs in total homogenates of the EC in AD stages V-VI when compared with stages I-II. However reduced activity of complexes I, II and V of isolated mitochondria occurs as early as stages I-II when compared with middle-aged individuals in the EC. In contrast, no alterations in the expression of the same genes and no alterations in the activity of mitochondrial complexes are found in the FC in the same series. CONCLUSION: Different mechanisms of impaired energy metabolism may occur in AD, one of them, represented by the EC, is the result of primary and early alteration of mitochondria; the other one is probably the result, at least in part, of decreased functional input and is represented by hypometabolism in the FC in AD patients aged 86 or younger.


Asunto(s)
Enfermedad de Alzheimer/patología , Corteza Entorrinal/metabolismo , Corteza Entorrinal/ultraestructura , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Femenino , Lóbulo Frontal/metabolismo , Humanos , Masculino , Proteínas Mitocondriales/genética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , ARN Mensajero/metabolismo
7.
Hum Mol Genet ; 26(13): 2436-2450, 2017 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-28398517

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.


Asunto(s)
Moléculas de Adhesión Celular Neurona-Glia/metabolismo , Quistes/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Encefalopatías/patología , Canales de Cloruro CLC-2 , Canales de Calcio Tipo L/genética , Canales de Cloruro , Quistes/genética , Células HEK293 , Células HeLa , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Transporte de Proteínas/genética
8.
J Neuropathol Exp Neurol ; 74(10): 975-99, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26360374

RESUMEN

Tau P301S transgenic mice (PS19 line) are used as a model of frontotemporal lobar degeneration (FTLD)-tau. Behavioral alterations in these mice begin at approximately 4 months of age. We analyzed molecular changes related to disease progression in these mice. Hyperphosphorylated 4Rtau increased in neurons from 1 month of age in entorhinal and piriform cortices to the neocortex and other regions. A small percentage of neurons developed an abnormal tau conformation, tau truncation, and ubiquitination only at 9/10 months of age. Astrocytosis, microgliosis, and increased inflammatory cytokine and immune mediator expression also occurred at this late stage; hippocampi were the most markedly affected. Altered mitochondrial function, increased reactive oxygen species production, and limited protein oxidative damage were observed in advanced disease. Tau oligomers were only present in P301S mice, they were found in somatosensory cortex and hippocampi at the age of 3 months, and they increased across time in the somatosensory cortex and were higher and sustained in hippocampi. Age-related modifications in lipid composition occurred in both P301S and wild-type mice with regional and phenotypic differences; however, changes of total lipids did not seem to have pathogenic implications. Apoptosis only occurred in restricted regions in late disease. The complex tau pathology, mitochondrial alterations, oxidative stress damage, glial reactions, neuroinflammation, and cell death in P301S mice likely parallel those in FTLD-tau. Thus, therapies should focus first on abnormal tau rather than secondary events that appear late in the course of FTLD-tau.


Asunto(s)
Degeneración Lobar Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/patología , Mitocondrias/patología , Estrés Oxidativo/fisiología , Proteínas tau/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Proteínas tau/genética
9.
J Alzheimers Dis ; 45(2): 407-21, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25790933

RESUMEN

Neuroprotection of erythropoietin (EPO) following long-term administration is hampered by the associated undesirable effects on hematopoiesis and body weight. For this reason, we tested carbamylated-EPO (CEPO), which has no effect on erythropoiesis, and compared it with EPO in the AßPP/PS1 mouse model of familial Alzheimer's disease. Groups of 5-month old wild type (WT) and transgenic mice received chronic treatment consisting of CEPO (2,500 or 5,000 UI/kg) or EPO (2,500 U I/kg) 3 days/week for 4 weeks. Memory at the end of treatment was assessed with the object recognition test. Microarray analysis and quantitative-PCR were used for gene expression studies. No alterations in erythropoiesis were observed in CEPO-treated WT and AßPP/PS1 transgenic mice. EPO and CEPO improved memory in AßPP/PS1 animals. However, only EPO decreased amyloid-ß (Aß)plaque burden and soluble Aß(40). Microarray analysis of gene expression revealed a limited number of common genes modulated by EPO and CEPO. CEPO but not EPO significantly increased gene expression of dopamine receptors 1 and 2, and adenosine receptor 2a, and significantly down-regulated adrenergic receptor 1D and gastrin releasing peptide. CEPO treatment resulted in higher protein levels of dopamine receptors 1 and 2 in WT and AßPP/PS1 animals, whereas the adenosine receptor 2a was reduced in WT animals. The present results suggest that the improved behavior observed in AßPP/PS1 transgenic mice after CEPO treatment may be mediated, at least in part, by the observed modulation of the expression of molecules involved in neurotransmission.


Asunto(s)
Enfermedad de Alzheimer/complicaciones , Eritropoyetina/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/etiología , Sinapsis/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Modelos Animales de Enfermedad , Eritropoyetina/uso terapéutico , Péptido Liberador de Gastrina/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Fragmentos de Péptidos/metabolismo , Presenilina-1/genética , Receptores de Catecolaminas/metabolismo , Sinapsis/genética , Factores de Tiempo
10.
J Antimicrob Chemother ; 65(3): 417-24, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20067983

RESUMEN

OBJECTIVES: Identification of CCR5 as an antiretroviral target led to the development of several CCR5 antagonists in clinical trials and the approval of maraviroc. Evaluating the mechanism of drug resistance to CCR5 agents may have implications in the clinical development of this class of agents. We have analysed the resistance profile of two R5 HIV-1 strains [BaL and a clinical isolate (CI)] after long-term passage in cell culture in the presence of TAK-779, the first developed non-peptidic small molecule targeting CCR5. METHODS: Genotypic and phenotypic tests were used to evaluate the resistance of virus isolated from cell culture in the presence of the CCR5 inhibitor TAK-779. RESULTS: Mutations conferring resistance appeared in the gp120 sequence but were not confined to the V3 loop region, and both strains had a different mutation pattern. Recombination of the env gene of the BaL-derived resistant virus into the HIV-1 HXB2 wild-type backbone conferred resistance to TAK-779 and cross-resistance to maraviroc, with 63- and 11-fold changes in their EC(50) (50% effective concentration), respectively, together with an apparent reduction of the maximal plateau inhibition (MPI) of TAK-779 but not of maraviroc. Conversely, the resistant CI viruses showed an approximately 50% reduction in MPI for both TAK-779 and maraviroc. CONCLUSIONS: We confirm that different pathways to the generation of CCR5 drug resistance/cross-resistance may occur that strongly depend on cell culture conditions, CCR5 availability and the genetic background of the HIV strain. Our study provides complementary information to understand the complexity of resistance to CCR5 antagonists.


Asunto(s)
Amidas/farmacología , Fármacos Anti-VIH/farmacología , Antagonistas de los Receptores CCR5 , Farmacorresistencia Viral , VIH-1/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Línea Celular , Células Cultivadas , Ciclohexanos/farmacología , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Humanos , Concentración 50 Inhibidora , Leucocitos Mononucleares/virología , Maraviroc , Pruebas de Sensibilidad Microbiana , Triazoles/farmacología
11.
J Med Chem ; 52(3): 840-51, 2009 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-19140683

RESUMEN

The role played by stereochemistry in the C2-substituent (left part) on the S-DABO scaffold for anti-HIV-1 activity has been investigated for the first time. A series of S-DABO analogues, where the double bond in the C2-substituent is replaced by an enantiopure isosteric cyclopropyl moiety, has been synthesized, leading to the identification of a potent lead compound endowed with picomolar activity against RT (wt) and nanomolar activity against selected drug-resistant mutants. Molecular modeling calculation, enzymatic studies, and surface plasmon resonance experiments allowed us to rationalize the biological behavior of the synthesized compounds, which act as mixed-type inhibitors of HIV-1 RT K103N, with a preferential association to the enzyme-substrate complex. Taken together, our data show that the right combination of stereochemistry on the left and right parts (C6-substituent) of the S-DABO scaffold plays a key role in the inhibition of both wild-type and drug-resistant enzymes, especially the K103N mutant.


Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , Pirimidinonas/síntesis química , Pirimidinonas/farmacología , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacología , Sulfuros/síntesis química , Sulfuros/farmacología , Línea Celular Tumoral , Simulación por Computador , Diseño de Fármacos , Farmacorresistencia Viral , Humanos , Cinética , Modelos Moleculares , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras , Estereoisomerismo , Resonancia por Plasmón de Superficie
12.
Bioorg Med Chem Lett ; 18(21): 5777-80, 2008 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-18842407

RESUMEN

A small family of S-DABO cytosine analogs (S-DABOCs) has been synthesized and biologically evaluated as HIV-1 inhibitor both on wild type (wt) and drug-resistant mutants leading to the identification of an interesting compound (5d). Molecular modeling studies have been finally performed in order to rationalize the results.


Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Citosina/análogos & derivados , Fármacos Anti-VIH/química , Citosina/síntesis química , Citosina/química , Citosina/farmacología , VIH-1/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares
13.
ChemMedChem ; 3(10): 1549-57, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18671217

RESUMEN

HIV cell fusion and entry have been validated as targets for therapeutic intervention against infection. Bicyclams were the first low-molecular-weight compounds to show specific interaction with CXCR4. The most potent bicyclam was AMD3100, in which the two cyclam moieties are tethered by a 1,4-phenylenebis(methylene) bridge. It was withdrawn from clinical trials owing to its lack of oral bioavailability and cardiotoxicity. We have designed a combinatorial library of non-cyclam polynitrogenated compounds by preserving the main features of AMD3100. At least two nitrogen atoms on each side of the p-phenylene moiety, one in the benzylic position and the other(s) in the heterocyclic system were maintained, and the distances between them were similar to the nitrogen atom distances in cyclam. A selection of diverse compounds from this library were prepared, and their in vitro activity was tested in cell cultures against HIV strains. This led to the identification of novel potent CXCR4 coreceptor inhibitors without cytotoxicity at the tested concentrations.


Asunto(s)
Antivirales/química , Receptores CXCR4/antagonistas & inhibidores , Animales , Antivirales/farmacología , Bencilaminas , Células Cultivadas , Ciclamas , Diseño de Fármacos , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Concentración 50 Inhibidora , Ratones , Modelos Químicos , Estructura Terciaria de Proteína , Receptores CXCR4/química
14.
J Med Chem ; 51(15): 4641-52, 2008 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-18630898

RESUMEN

A series of dihydro-alkylthio-benzyl-oxopyrimidines (S-DABOs) bearing a 2-aryl-2-oxoethylsulfanyl chain at pyrimidine C2, an alkyl group at C5, and a 2,6-dichloro-, 2-chloro-6-fluoro-, and 2,6-difluoro-benzyl substitution at C6 (oxophenethyl- S-DABOs, 6-8) is here described. The new compounds showed low micromolar to low nanomolar (in one case subnanomolar) inhibitory activity against wt HIV-1. Against clinically relevant HIV-1 mutants (K103N, Y181C, and Y188L) as well as in enzyme (wt and K103N, Y181I, and L100I mutated RTs) assays, compounds carrying an ethyl/ iso-propyl group at C5 and a 2,6-dichloro-/2-chloro-6-fluoro-benzyl moiety at C6 were the most potent derivatives, also characterized by low fold resistance ratio. Interestingly, the structure-activity relationship (SAR) data drawn from this DABO series are more related to HEPT than to DABO derivatives. These findings were at least in part rationalized by the description of a fair superimposition between the 6-8 and TNK-651 (a HEPT analogue) binding modes in both WT and Y181C RTs.


Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Benceno/química , Pirimidinonas/síntesis química , Pirimidinonas/farmacología , Compuestos de Azufre/síntesis química , Compuestos de Azufre/farmacología , Alquilación , Fármacos Anti-VIH/química , Fenómenos Químicos , Química Física , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , VIH-1/genética , Hidrógeno/química , Modelos Moleculares , Estructura Molecular , Mutación/genética , Oxígeno/química , Unión Proteica , Pirimidinonas/química , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Relación Estructura-Actividad , Compuestos de Azufre/química
15.
ChemMedChem ; 3(9): 1412-8, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18465760

RESUMEN

We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT.


Asunto(s)
Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , Pirimidinonas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Unión Competitiva/efectos de los fármacos , Catálisis , Relación Dosis-Respuesta a Droga , Farmacorresistencia Viral , Fluorobencenos , Perfilación de la Expresión Génica , Transcriptasa Inversa del VIH/química , Cinética , Estructura Molecular , Mutación , Reacción en Cadena de la Polimerasa , Pirimidinonas/síntesis química , Pirimidinonas/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por Sustrato , Factores de Tiempo
17.
Mol Pharmacol ; 73(4): 1264-73, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18182480

RESUMEN

We have studied the mechanism of action of Arg(*)-Arg-Nal(2)-Cys(1x)-Tyr-Gln-Lys-(d-Pro)-Pro-Tyr-Arg-Cit-Cys(1x)-Arg-Gly-(d-Pro)(*) (POL3026), a novel specific beta-hairpin mimetic CXC chemokine receptor (CXCR)4 antagonist. POL3026 specifically blocked the binding of anti-CXCR4 monoclonal antibody 12G5 and the intracellular Ca(2+) signal induced by CXC chemokine ligand 12. POL3026 consistently blocked the replication of human immunodeficiency virus (HIV), including a wide panel of X4 and dualtropic strains and subtypes in several culture models, with 50% effective concentrations (EC(50)) at the subnanomolar range, making POL3026 the most potent CXCR4 antagonist described to date. However, 1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)-resistant and stromal cell-derived factor-1alpha-resistant HIV-1 strains were cross-resistant to POL3026. Time of addition experiments and a multiparametric evaluation of HIV envelope function in the presence of test compounds confirmed the activity of POL3026 at an early step of virus replication: interaction with the coreceptor. Generation of HIV-1 resistance to POL3026 led to the selection of viruses 12- and 25-fold less sensitive and with mutations in gp120, including the V3 loop region. However, POL3026 prevented the emergence of CXCR4-using variants from an R5 HIV-1 strain that may occur in the presence of anti-HIV agents targeting CC chemokine receptor 5.


Asunto(s)
Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Péptidos Cíclicos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Fármacos Anti-VIH/química , Anticuerpos Monoclonales , Bencilaminas , Señalización del Calcio/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Quimiocina CXCL12/farmacología , Quimiotaxis/efectos de los fármacos , Ciclamas , VIH/efectos de los fármacos , VIH/fisiología , Compuestos Heterocíclicos/farmacología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/virología , Macrófagos/efectos de los fármacos , Macrófagos/virología , Péptidos Cíclicos/química , Factores de Tiempo , Proteínas del Envoltorio Viral/metabolismo , Replicación Viral/efectos de los fármacos
18.
ChemMedChem ; 3(4): 573-93, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18081133

RESUMEN

Among the FDA approved drugs for the treatment of AIDS, non-nucleoside reverse transcriptase inhibitors (NNRTIs) are essential components of first-line anti-HIV-1 therapy because of the less-severe adverse effects associated with NNRTIs administration in comparison to therapies based on other anti-HIV-1 agents. In this contest, 3,4-dihydro-2-alkoxy-6-benzyl-4-oxypyrimidines (DABOs) have been the object of many studies aimed at identifying novel analogues endowed with potent inhibitory activity towards HIV-1 wild type and especially drug-resistant mutants. Accordingly, based on the encouraging results obtained from the biological screening of our internal collection of S-DABO derivatives, we started with the systematic functionalization of the pyrimidine scaffold to identify the minimal required structural features for RT inhibition. Herein, we describe how the combination of synthetic, biological, and molecular modeling studies led to the identification of two novel subclasses of S-DABO analogues: S-DABO cytosine analogues (S-DABOCs) and 4-dimethyamino-6-vinylpyrimidines (DAVPs).


Asunto(s)
Diseño de Fármacos , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/síntesis química , Transcriptasa Inversa del VIH/genética , VIH-1/genética , Humanos , Mutación , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad
19.
AIDS ; 22(1): 23-31, 2008 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-18090388

RESUMEN

BACKGROUND: HIV-1 coreceptor switch from CCR5 to CXCR4 is associated with disease progression and AIDS. Selection of resistant HIV-1 to CCR5 agents in cell culture has often occurred in the absence of coreceptor switch. With CCR5 antagonists currently in clinical trials, their impact on coreceptor use is still in doubt. METHODS: Six R5 HIV-1 strains were passaged in lymphoid cells expressing high CXCR4 and low CCR5, in the absence or presence of CCR5 inhibitors (TAK-779, mAb 2D7 and CCL5). AMD3100, zidovudine and lamivudine were used as controls. Phenotype and genotype changes as well as virus coreceptor use were evaluated. RESULTS: In the absence of drug pressure, three out of six strains expanded their coreceptor use to CXCR4 at different times, suggesting that not all virus strains had the capacity to do so. Lowering the replication rate with a suboptimal concentration of different anti-HIV agents (reverse transcriptase inhibitors or CCR5 agents) delayed coreceptor switch. However, virus breakthrough was observed earlier in the presence of CCR5-targeting agents than in presence of reverse transcriptase inhibitors and was associated with a change in sensitivity to TAK-779 or AMD3100, virus coreceptor expansion to CXCR4 and changes in the V3 loop region of gp120. CONCLUSION: Our results suggest that HIV-1 may escape CCR5 drug pressure through coreceptor switch. Experimental conditions strongly determine the outcome of CCR5 drug pressure in cell culture. A cell culture model of the evolution of HIV-1 coreceptor use may be relevant to assess the propensity of clinical isolates to develop resistance through coreceptor change.


Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Amidas/farmacología , Secuencia de Aminoácidos , Bencilaminas , Antagonistas de los Receptores CCR5 , Línea Celular Transformada , Línea Celular Tumoral , Células Cultivadas , Ciclamas , Evolución Molecular , Variación Genética , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Leucocitos Mononucleares , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Compuestos de Amonio Cuaternario/farmacología , Receptores CXCR4/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Alineación de Secuencia , Factores de Tiempo , Acoplamiento Viral/efectos de los fármacos
20.
Virology ; 372(2): 421-9, 2008 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-18068205

RESUMEN

We generated a lymphoid cell line (Sup-T1-Rev/Env) that stably expresses a 19-bp short hairpin RNA (shRNA) targeting a conserved region of HIV-1 encoding for the Envelope and Rev proteins, which potently inhibited viral replication. However, continuous passage of HIV-1 in Sup-T1-Rev/Env generated virus mutants able to overcome the RNAi restriction. Sequence analysis of the emerging viruses showed that mutations were located at positions 5 and 17 of the target sequence. Both mutations are silent in the Env frame, but the mutation 5 generated an amino acid change (V47M) in the Rev reading frame. We have analyzed the impact of these two mutations on the RNAi mechanism, showing a more crucial role of the mutation 17 in the resistance to RNAi. We show that even targeting a conserved region of the HIV-1 genome involved in the biosynthesis of two essential genes, env and rev, the virus could evolve to escape by single point mutations in the target sequence, without a significant fitness cost.


Asunto(s)
Farmacorresistencia Viral , VIH-1/genética , VIH-1/fisiología , Interferencia de ARN , Línea Celular , Genes env/genética , VIH-1/efectos de los fármacos , Humanos , Mutación , Replicación Viral/efectos de los fármacos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética
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