RESUMEN

Depending on its occurrence in the germline or somatic context, a single point mutation, S707Y, of phospholipase C-γ2 (PLCγ2) gives rise to two distinct human disease states: acquired resistance of chronic lymphocytic leukemia cells (CLL) to inhibitors of Brutons´s tyrosine kinase (Btk) and dominantly inherited autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation, APLAID, respectively. The functional relationships of the PLCγ2S707Y mutation to other PLCG2 mutations causing (i) Btk inhibitor resistance of CLL cells and (ii) the APLAID-related human disease PLCγ2-associated antibody deficiency and immune dysregulation, PLAID, revealing different clinical characteristics including cold-induced urticaria, respectively, are currently incompletely understood. Here, we show that PLCγ2S707 point mutants displayed much higher activities at 37° C than the CLL Btk inhibitor resistance mutants R665W and L845F and the two PLAID mutants, PLCγ2Δ19 and PLCγ2Δ20-22. Combinations of CLL Btk inhibitor resistance mutations synergized to enhance PLCγ2 activity, with distinct functional consequences for different temporal orders of the individual mutations. Enhanced activity of PLCγ2S707Y was not observed in a cell-free system, suggesting that PLCγ2 activation in intact cells is dependent on regulatory rather than mutant-enzyme-inherent influences. Unlike the two PLAID mutants, PLCγ2S707Y was insensitive to activation by cooling and retained marked hyperresponsiveness to activated Rac upon cooling. In contrast to the PLAID mutants, which are insensitive to activation by endogenously expressed EGF receptors, the S707Y mutation markedly enhanced the stimulatory effect of EGF, explaining some of the pathophysiological discrepancies between immune cells of PLAID and APLAID patients in response to receptor-tyrosine-kinase activation.

RESUMEN

Rac GTPases have oncogenic roles in cell growth, survival, and migration. We tested response to the Rac inhibitor EHT1864 in a panel of breast cancer cell lines. EHT1864-induced growth inhibition was associated with dual inhibition of the PI3K/AKT/mTORC1 and MEK/ERK pathways. Breast cancer cells harboring PIK3CA mutations or HER2 overexpression were most sensitive to Rac inhibition, suggesting that such oncogenic alterations link Rac activation with PI3K/AKT/mTORC1 and MEK/ERK signaling. Interestingly, EHT1864 decreased activation of the mTORC1 substrate p70S6K earlier than AKT inhibition, suggesting that Rac may activate mTORC1/p70S6K independently of AKT. Comparison of the growth-inhibitory profile of EHT1864 to 137 other anti-cancer drugs across 656 cancer cell lines revealed significant correlation with the p70S6K inhibitor PF-4708671. We confirmed that Rac complexes contain MEK1/2 and ERK1/2, but also contain p70S6K; these interactions were disrupted by EHT1864. Pharmacokinetic profiles revealed that EHT1864 was present in mouse plasma at concentrations effective in vitro for approximately 1 h after intraperitoneal administration. EHT1864 suppressed growth of HER2+ tumors, and enhanced response to anti-estrogen treatment in ER+ tumors. Further therapeutic development of Rac inhibitors for HER2+ and PIK3CA-mutant cancers is warranted.

Asunto(s)

Neoplasias de la Mama/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Resistencia a Antineoplásicos/fisiología , Femenino , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Methyl 9-anilinothiazolo[5,4-f]quinazoline-2-carbimidates 1 (EHT 5372) and 2 (EHT 1610) are strong inhibitors of DYRK's family kinases. The crystal structures of the complex revealed a noncanonical binding mode of compounds 1 and 2 in DYRK2, explaining the remarkable selectivity and potency of these inhibitors. The structural data and comparison presented here provide therefore a template for further improvement of this inhibitor class and for the development of novel inhibitors selectively targeting DYRK kinases.

Asunto(s)

Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinazolinas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Fosforilación , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Tirosina Quinasas/metabolismo , Quinazolinas/síntesis química , Quinazolinas/química , Relación Estructura-Actividad

RESUMEN

Mutations in the gene encoding phospholipase C-γ2 (PLCγ2) have been shown to be associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Bruton's tyrosine kinase inhibitor ibrutinib. The fact that two of these mutations, R665W and L845F, imparted upon PLCγ2 an ∼2-3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca2+ following ligation of the B cell antigen receptor (BCR) led to the assumption that the two mutants exhibit constitutively enhanced intrinsic activity. Here, we show that the two PLCγ2 mutants are strikingly hypersensitive to activation by Rac2 such that even wild-type Rac2 suffices to activate the mutant enzymes upon its introduction into intact cells. Enhanced "basal" activity of PLCγ2 in intact cells is shown using the pharmacologic Rac inhibitor EHT 1864 and the PLCγ2F897Q mutation mediating Rac resistance to be caused by Rac-stimulated rather than by constitutively enhanced PLCγ2 activity. We suggest that R665W and L845F be referred to as allomorphic rather than hypermorphic mutations of PLCG2 Rerouting of the transmembrane signals emanating from BCR and converging on PLCγ2 through Rac in ibrutinib-resistant CLL cells may provide novel drug treatment strategies to overcome ibrutinib resistance mediated by PLCG2 mutations or to prevent its development in ibrutinib-treated CLL patients.

Asunto(s)

Resistencia a Antineoplásicos , Leucemia Linfocítica Crónica de Células B , Mutación Missense , Proteínas de Neoplasias , Fosfolipasa C gamma , Pirazoles/farmacología , Pirimidinas/farmacología , Transducción de Señal , Proteínas de Unión al GTP rac , Adenina/análogos & derivados , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfolipasa C gamma/antagonistas & inhibidores , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/metabolismo , Piperidinas , Pironas/farmacología , Quinolinas/farmacología , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína RCA2 de Unión a GTP

RESUMEN

We developed and evaluated a multiplex antibody detection-based immunoassay for the diagnosis of prosthetic joint infections (PJIs). Sixteen protein antigens from three Staphylococcusspecies (Staphylococcus aureus,Staphylococcus epidermidis, and Staphylococcus lugdunensis) (8 antigens),Streptococcus agalactiae(4 antigens), and Propionibacterium acnes(4 antigens) were selected by comparative immune proteomics using serum samples from PJI cases versus controls. A bead-based multiplex immunoassay that measured serum IgG against purified, recombinant forms of each of the 16 antigens was developed. We conducted a prospective study to evaluate the performance of the assay. A PJI was defined by the presence of a sinus tract and/or positive intraoperative sample cultures (at least one sample yielding a virulent organism or at least two samples yielding the same organism). A total of 455 consecutive patients undergoing revision or resection arthroplasty (hip, 66.3%; knee, 29.7%; shoulder, 4%) at two French reference centers for the management of PJI were included: 176 patients (38.7%) were infected and 279 (61.3%) were not. About 60% of the infections involved at least one of the species targeted by the assay. The sensitivity/specificity values were 72.3%/80.7% for targeted staphylococci, 75%/92.6% forS. agalactiae, and 38.5%/84.8% forP. acnes The assay was more sensitive for infections occurring >3 months after arthroplasty and for patients with an elevated C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR). However, it detected 64.3% and 58.3% of targeted staphylococcal infections associated with normal CRP and ESR values, respectively. This new multiplex immunoassay approach is a novel noninvasive tool to evaluate patients suspected of having PJIs and provides information complementary to that from inflammatory marker values.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Artritis Infecciosa/diagnóstico , Infecciones Bacterianas/diagnóstico , Infecciones Relacionadas con Prótesis/diagnóstico , Pruebas Serológicas/métodos , Anciano , Anciano de 80 o más Años , Antígenos Bacterianos/inmunología , Femenino , Francia , Humanos , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad

RESUMEN

Pre-B and pre-T lymphocytes must orchestrate a transition from a highly proliferative state to a quiescent one during development. Cyclin D3 is essential for these cells' proliferation, but little is known about its posttranslational regulation at this stage. Here, we show that the dual specificity tyrosine-regulated kinase 1A (DYRK1A) restrains Cyclin D3 protein levels by phosphorylating T283 to induce its degradation. Loss of DYRK1A activity, via genetic inactivation or pharmacologic inhibition in mice, caused accumulation of Cyclin D3 protein, incomplete repression of E2F-mediated gene transcription, and failure to properly couple cell cycle exit with differentiation. Expression of a nonphosphorylatable Cyclin D3 T283A mutant recapitulated these defects, whereas inhibition of Cyclin D:CDK4/6 mitigated the effects of DYRK1A inhibition or loss. These data uncover a previously unknown role for DYRK1A in lymphopoiesis, and demonstrate how Cyclin D3 protein stability is negatively regulated during exit from the proliferative phases of B and T cell development.

Asunto(s)

Ciclina D3/metabolismo , Linfopoyesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Alelos , Animales , Linfocitos B/citología , Células de la Médula Ósea/citología , Proliferación Celular , Factores de Transcripción E2F/metabolismo , Exones , Femenino , Citometría de Flujo , Biblioteca de Genes , Genotipo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Procesamiento Proteico-Postraduccional , Transducción de Señal , Linfocitos T/citología , Transcripción Genética , Quinasas DyrK

RESUMEN

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene is located within the Down Syndrome (DS) critical region on chromosome 21 and is implicated in the generation of Tau and amyloid pathologies that are associated with the early onset Alzheimer's Disease (AD) observed in DS. DYRK1A is also found associated with neurofibrillary tangles in sporadic AD and phosphorylates key AD players (Tau, amyloid precursor, protein, etc). Thus, DYRK1A may be an important therapeutic target to modify the course of Tau and amyloid beta (Aß) pathologies. Here, we describe EHT 5372 (methyl 9-(2,4-dichlorophenylamino) thiazolo[5,4-f]quinazoline-2-carbimidate), a novel, highly potent (IC50 = 0.22 nM) DYRK1A inhibitor with a high degree of selectivity over 339 kinases. Models in which inhibition of DYRK1A by siRNA reduced and DYRK1A over-expression induced Tau phosphorylation or Aß production were used. EHT 5372 inhibits DYRK1A-induced Tau phosphorylation at multiple AD-relevant sites in biochemical and cellular assays. EHT 5372 also normalizes both Aß-induced Tau phosphorylation and DYRK1A-stimulated Aß production. DYRK1A is thus as a key element of Aß-mediated Tau hyperphosphorylation, which links Tau and amyloid pathologies. EHT 5372 and other compounds in its class warrant in vivo investigation as a novel, high-potential therapy for AD and other Tau opathies. Inhibition of the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is a new high-potential therapeutic approach for Alzheimer disease. Here we describe EHT 5372, a novel potent and selective DYRK1A inhibitor. EHT 5372 inhibits DYRK1A-induced Tau phosphorylation, Aß production and Aß effects on phospho-Tau, including Tau aggregation.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/biosíntesis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas tau/biosíntesis , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Células Cultivadas , Células HEK293 , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ratas , Ratas Wistar , Resultado del Tratamiento , Quinasas DyrK

RESUMEN

The convenient synthesis of a library of novel 6,6,5-tricyclic thiazolo[5,4-f] quinazolines (forty molecules) was achieved mainly under microwave irradiation. Dimroth rearrangement and 4,5-dichloro-1,2,3,-dithiazolium chloride (Appel salt) chemistry were associated for the synthesis of a novel 6-aminobenzo[d]thiazole-2,7-dicarbonitrile (16) a versatile molecular platform for the synthesis of various bioactive derivatives. Kinase inhibition of the final compounds was evaluated on a panel of four Ser/Thr kinases (DYRK1A, CDK5, CK1 and GSK3) chosen for their strong implications in various regulation processes, especially Alzheimer's disease (AD). In view of the results of this preliminary screening, thiazolo[5,4-f]quinazoline scaffolds constitutes a promising source of inspiration for the synthesis of novel bioactive molecules. Among the compounds of this novel chemolibrary, 7i, 8i and 9i inhibited DYRK1A with IC50 values ranging in the double-digit nanomolar range (40, 47 and 50 nM, respectively).

Asunto(s)

Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química , Quinazolinas/química , Técnicas de Química Sintética , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinazolinas/síntesis química , Quinazolinas/farmacología , Relación Estructura-Actividad , Tiazoles , Quinasas DyrK

RESUMEN

The convenient synthesis of a focused library (forty molecules) of novel 6,6,5-tricyclic thiazolo[5,4-f]quinazolines was realized mainly under microwave irradiation. A novel 6-aminobenzo[d]thiazole-2,7-dicarbonitrile (1) was used as a versatile molecular platform for the synthesis of various derivatives. Kinase inhibition, of the obtained final compounds, was evaluated on a panel of two kinases (DYRK1A/1B) together with some known reference DYRK1A and DYRK1B inhibitors (harmine, TG003, NCGC-00189310 and leucettine L41). Compound IC50 values were obtained and compared. Five of the novel thiazolo[5,4-f]quinazoline derivatives prepared, EHT 5372 (8c), EHT 6840 (8h), EHT 1610 (8i), EHT 9851 (8k) and EHT 3356 (9b) displayed single-digit nanomolar or subnanomolar IC50 values and are among the most potent DYRK1A/1B inhibitors disclosed to date. DYRK1A/1B kinases are known to be involved in the regulation of various molecular pathways associated with oncology, neurodegenerative diseases (such as Alzheimer disease, AD, or other tauopathies), genetic diseases (such as Down Syndrome, DS), as well as diseases involved in abnormal pre-mRNA splicing. The compounds described in this communication constitute a highly potent set of novel molecular probes to evaluate the biology/pharmacology of DYR1A/1B in such diseases.

Asunto(s)

Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/química , Quinazolinas/química , Quinazolinas/farmacología , Técnicas de Química Sintética , Activación Enzimática/efectos de los fármacos , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinazolinas/síntesis química , Quinasas DyrK

RESUMEN

Tumor blood vessels are an important emerging target for anticancer therapy. Here, we characterize the in vitro antiproliferative and antiangiogenic properties of the synthetic small molecule, 7-ethoxy-4-(3,4,5-trimethoxybenzyl)isoquinolin-8-amine dihydrochloride, EHT 6706, a novel microtubule-disrupting agent that targets the colchicine-binding site to inhibit tubulin polymerization. At low nM concentrations, EHT 6706 exhibits highly potent antiproliferative activity on more than 60 human tumor cell lines, even those described as being drug resistant. EHT 6706 also shows strong efficacy as a vascular-disrupting agent, since it prevents endothelial cell tube formation and disrupts pre-established vessels, changes the permeability of endothelial cell monolayers and inhibits endothelial cell migration. Genome-wide transcriptomic analysis of EHT 6706 effects on human endothelial cells shows that the antiangiogenic activity elicits gene deregulations of antiangiogenic pathways. These findings indicate that EHT 6706 is a promising tubulin-binding compound with potentially broad clinical antitumor efficacy.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Isoquinolinas/farmacología , Microtúbulos/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Moduladores de Tubulina/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colchicina/metabolismo , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/patología , Resistencia a Múltiples Medicamentos , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Tubulina (Proteína)/metabolismo

RESUMEN

Monitoring the genomic expression of patients in clinical trials for Alzheimer's disease (AD) can assist trial design and treatment response analysis. Here, we report on the identification in AD patients of blood-based transcriptomic signatures associated with treatment response of EHT 0202, a new compound with potential disease-modifying and symptomatic properties, in a 3-month, placebo-controlled, Phase IIA study aimed at determining the clinical safety, tolerability, and exploratory efficacy of EHT 0202 (40 and 80 mg bid) as adjunctive therapy to one cholinesterase inhibitor in mild to moderate AD patients. Genome-wide transcriptomic profiling was performed on blood samples taken prior to treatment and at study completion in a subpopulation of 60 AD patients selected as either the 10 worst disease decliners or the 10 best improvers of each treatment group, using ADAS-Cog scores as measure of disease severity. In the patients responding to EHT 0202, a pre-treatment (baseline) transcriptomic signature showed activation of pathways related to AD, CNS disorders, diabetes, inflammation, and autoimmunity, while a post-treatment signature indicated reduced activation of these pathways with induced metabolic and transcription stimulation. This pilot study demonstrates the utility of blood transcriptomic signatures used as biomarkers for predicting patient response or monitoring efficacy, for an administered therapeutic drug in a complex disease such as AD. For EHT 0202 or other AD drugs, such biomarkers may help to improve strategies to better identify appropriate patient populations for treatment, understand the drug mechanism of efficacy, and/or clarify the inherent subjectivity in most clinical endpoints used in this disease.

Asunto(s)

Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/genética , Inhibidores de la Colinesterasa/administración & dosificación , Transcriptoma/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Biomarcadores/sangre , Estudios de Cohortes , Método Doble Ciego , Quimioterapia Combinada , Humanos , Proyectos Piloto , Transcriptoma/efectos de los fármacos , Resultado del Tratamiento

RESUMEN

Biomarkers have gained an increased importance in the past years in helping physicians to diagnose Alzheimer's disease (AD). This study was designed to identify a blood-based, transcriptomic signature that can differentiate AD patients from control subjects. The performance of the signature was then evaluated for robustness in an independent blinded sample population. RNA was extracted from 177 blood samples (90 AD patients and 87 controls) and gene expression profiles were generated using the human Genome-Wide Splice Array™. These profiles were used to establish a signature to differentiate AD patients from controls. Subsequently, prediction results were optimized by establishing grey zone boundaries that discount prediction scores near the disease status threshold. Signature validation was then performed on a blinded independent cohort of 209 individuals (111 AD and 98 controls). The AclarusDx™ signature consists of 170 probesets which map to 136 annotated genes, a significant number of which are associated with inflammatory, gene expression, and cell death pathways. Additional signature genes are known to interact with pathways involved in amyloid and tau metabolism. The validation sample set, after removal of 45 individuals with prediction profile scores within the grey zone, consisted of 164 subjects. The AclarusDx™ performance on this validation cohort had a sensitivity of 81.3% (95% CI: [73.3%; 89.3%]); and a specificity of 67.1% (95% CI: [56.3%; 77.9%]). AclarusDx™ is a non-invasive blood-based transcriptomic test that, in combination with standard assessments, can provide physicians with objective information to support the diagnosis of AD.

Asunto(s)

Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/diagnóstico , Transcriptoma/fisiología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Biomarcadores , Estudios Transversales , Interpretación Estadística de Datos , Demografía , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Progresión de la Enfermedad , Femenino , Humanos , Inflamación/patología , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Estudios Prospectivos , ARN/química , ARN/genética , ARN/aislamiento & purificación , Reproducibilidad de los Resultados

RESUMEN

The potential of EHT 6706, a novel tubulin-binding agent, was investigated in combination with ionizing radiation (IR) and with conventional cytotoxic chemotherapy agents. Cell proliferation, cell cycle, apoptosis and clonogenic assays were performed in five human cancer cell lines: H460 (non small cell lung carcinoma, NSCLC), HCT116 and HCT116 p53-/- (colorectal cancer), MDA-MB-231 (breast cancer), and MiaPaca2 cells (pancreatic cancer). The drug inhibited cell proliferation in all cell lines. This effect was associated with G2/M arrest and activation of apoptosis in a dose-dependent manner. The drug was then tested in combination with chemotherapy and IR in vitro. Effects on proliferation and clonogenic survival were analyzed. EHT 6706 treatment inhibited clonogenic survival synergistically with IR in H460 and MiaPaca2 cell lines. In the remaining cell lines, the effects of EHT 6706 and IR were additive. For H460 and MiaPaca2 cell lines, the highest effect was seen when cells were exposed for 20 h to EHT 6706 before being irradiated. EHT 6706 also exerted additive inhibition of proliferation when given in combination with conventional chemotherapy agents, such as oxaliplatin, cisplatin and gemcitabine in H460 and MiaPaca2 tumor cell lines. These data show that EHT 6706 could act synergistically with IR and additively with chemotherapy in tumor cell lines in vitro. This provides a good rationale to further assess EHT 6706 in combination protocols and confirm these effects in vivo.

Asunto(s)

Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Desoxicitidina/análogos & derivados , Isoquinolinas/farmacología , Compuestos Organoplatinos/administración & dosificación , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quimioradioterapia , Desoxicitidina/administración & dosificación , Humanos , Oxaliplatino , Radiación Ionizante , Tubulina (Proteína)/metabolismo , Gemcitabina

RESUMEN

Cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons display neurofibrillary tangles (NFTs) during Alzheimer's disease (AD) progression, yet the mechanisms underlying this selective vulnerability are currently unclear. Rac1, a member of the Rho family of GTPases, may interact with the proapoptotic pan-neurotrophin receptor p75(NTR) to induce neuronal cytoskeletal abnormalities in AD NB neurons. Herein, we examined the expression of Rac1b, a constitutively active splice variant of Rac1, in NB cholinergic neurons during AD progression. CBF tissues harvested from people who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment, or AD were immunolabeled for both p75(NTR) and Rac1b. Rac1b appeared as cytoplasmic diffuse granules, loosely aggregated filaments, or compact spheres in p75(NTR)-positive NB neurons. Although Rac1b colocalized with tau cytoskeletal markers, the percentage of p75(NTR)-immunoreactive neurons expressing Rac1b was significantly increased only in AD compared with both mild cognitive impairment and NCI. Furthermore, single-cell gene expression profiling with custom-designed microarrays showed down-regulation of caveolin 2, GNB4, and lipase A in AD Rac1b-positive/p75(NTR)-labeled NB neurons compared with Rac1b-negative/p75(NTR)-positive perikarya in NCI. These proteins are involved in Rac1 pathway/cell cycle progression and lipid metabolism. These data suggest that Rac1b expression acts as a modulator or transducer of various signaling pathways that lead to NFT formation and membrane dysfunction in a subgroup of CBF NB neurons in AD.

Asunto(s)

Núcleo Basal de Meynert/metabolismo , Neuronas Colinérgicas/metabolismo , Tauopatías/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Cadáver , Caveolina 2/genética , Células Cultivadas , Corteza Cerebelosa/metabolismo , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Subunidades beta de la Proteína de Unión al GTP/genética , Humanos , Masculino , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal/fisiología , Esterol Esterasa/genética , Tauopatías/genética , Proteínas tau

RESUMEN

OBJECTIVE: The goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI. METHODS AND RESULTS: We compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI- and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1. CONCLUSIONS: We demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI- and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.

Asunto(s)

Activación Plaquetaria/fisiología , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Retroalimentación Fisiológica/fisiología , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratones , Ratones Noqueados , Modelos Animales , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Purinérgicos P2Y12/deficiencia , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo

RESUMEN

Rac1, a Rho GTPase, modulates diverse cellular processes and is hyperactive in some cancers. Estrogen receptor-alpha (ERα) in concert with intracellular signaling pathways regulates genes associated with cell proliferation, tumor development, and breast cancer cell survival. Therefore, we examined the possibility of Rac1 and ERα crosstalk in breast cancer cells. We found that Rac1 enhanced ERα transcriptional activity in breast cancer cells. Vav3, a Rho guanine nucleotide exchange factor that activates Rac1, was an upstream mediator, and P21/Cdc42/Rac1 activating kinase-1 (Pak-1) was a downstream effector of Rac1 enhancement of ERα activity. These results suggest that Rac1 may prove to be a therapeutic target. To test this hypothesis, we used a small molecule Rac inhibitor, EHT 1864, and found that EHT 1864 inhibited ERα transcriptional activity. Furthermore, EHT 1864 inhibited estrogen-induced cell proliferation in breast cancer cells and decreased tamoxifen-resistant breast cancer cell growth. EHT 1864 decreased activity of the promoter of the ERα gene resulting in down-regulation of ERα mRNA and protein levels. Therefore, ERα down-regulation by EHT 1864 is the likely mechanism of EHT 1864-mediated inhibition of ERα activity and estrogen-stimulated breast cancer cell proliferation. Since ERα plays a critical role in the pathogenesis of breast cancer and the Rac inhibitor EHT 1864 down-regulates ERα expression and breast cancer cell proliferation, further investigation of the therapeutic potential of Rac1 targeting in the treatment of breast cancer is warranted.

Asunto(s)

Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Western Blotting , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Proteínas Proto-Oncogénicas c-vav/metabolismo , Pironas/farmacología , Quinolinas/farmacología , ARN/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.

Asunto(s)

Actinas/metabolismo , Plaquetas/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Microdominios de Membrana/metabolismo , Agregación Plaquetaria/fisiología , Adenosina Difosfato/metabolismo , Secuencias de Aminoácidos , Activación Enzimática/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosforilación/fisiología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Transporte de Proteínas/fisiología , Proteínas Tirosina Quinasas/metabolismo , Quinasa Syk , Tromboxano A2/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Familia-src Quinasas/metabolismo

RESUMEN

The synthesis of a series of berberine, phenantridine and isoquinoline derivatives was realized to explore their Rho GTPase nucleotide inhibitory activity. The compounds were evaluated in a nucleotide binding competition assay against Rac1, Rac1b, Cdc42 and in a cellular Rac GTPase activation assay. The insertion of 19 AA in the splice variant Rac1b is shown to be sufficient to introduce a conformational difference that allows compounds 4, 21, 22, and 26 to exhibit selective inhibition of Rac 1b over Rac1.

Asunto(s)

Inhibidores Enzimáticos/química , Nucleótidos/química , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Secuencia de Aminoácidos , Berberina/síntesis química , Berberina/química , Berberina/farmacología , Sitios de Unión , Simulación por Computador , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Isoquinolinas/síntesis química , Isoquinolinas/química , Isoquinolinas/farmacología , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Relación Estructura-Actividad , Proteína de Unión al GTP rac1/metabolismo

RESUMEN

Pharmacological modulation of the GABA(A) receptor has gained increasing attention as a potential treatment for central processes affected in Alzheimer disease (AD), including neuronal survival and cognition. The proteolytic cleavage of the amyloid precursor protein (APP) through the alpha-secretase pathway decreases in AD, concurrent with cognitive impairment. This APP cleavage occurs within the beta-amyloid peptide (Abeta) sequence, precluding formation of amyloidogenic peptides and leading to the release of the soluble N-terminal APP fragment (sAPPalpha) which is neurotrophic and procognitive. In this study, we show that at nanomolar-low micromolar concentrations, etazolate, a selective GABA(A) receptor modulator, stimulates sAPPalpha production in rat cortical neurons and in guinea pig brains. Etazolate (20 nM-2 microM) dose-dependently protected rat cortical neurons against Abeta-induced toxicity. The neuroprotective effects of etazolate were fully blocked by GABA(A) receptor antagonists indicating that this neuroprotection was due to GABA(A) receptor signalling. Baclofen, a GABA(B) receptor agonist failed to inhibit the Abeta-induced neuronal death. Furthermore, both pharmacological alpha-secretase pathway inhibition and sAPPalpha immunoneutralization approaches prevented etazolate neuroprotection against Abeta, indicating that etazolate exerts its neuroprotective effect via sAPPalpha induction. Our findings therefore indicate a relationship between GABA(A) receptor signalling, the alpha-secretase pathway and neuroprotection, documenting a new therapeutic approach for AD treatment.

Asunto(s)

Precursor de Proteína beta-Amiloide/metabolismo , Corteza Cerebral/metabolismo , Etazolato/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Receptores de GABA-A/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Secretasas de la Proteína Precursora del Amiloide/efectos de los fármacos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Péptidos beta-Amiloides/toxicidad , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , GABAérgicos/farmacología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Cobayas , Masculino , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/toxicidad , Inhibidores de Fosfodiesterasa/farmacología , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Wistar , Receptores de GABA-A/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

The incorporation of growth factors into new methylidene malonate 2.1.2-based biocompatible polymeric blends of oligomers and polymers to improve their stability and controlled release was investigated. Five growth factors were used in this study: FGF2, PDGF, TGF-beta, NGF and GM-CSF. Formulation in poly(methylidene malonate 2.1.2) blends was achieved by a four-step optimized process, using different oligomers/polymers ratios. Once dried, formulations could be subsequently stored at 4 or 20 degrees C or immediately subjected to degradation in conditioned cell culture medium. Toxicity of blends and their degradation products were evaluated in several cell lines with MTT. Bioactivity and biospecificity of the formulated growth factors were investigated using MTT and immunohistochemical staining. Combined ELISA and crystal violet colorimetric assays were performed to analyze growth factors release. Limited toxicities were observed for unloaded poly(methylidene malonate 2.1.2) blends. Once optimized, growth factors formulations did not reveal lower bioactivities or loss of biospecificity. Moreover, a sustained release over a 21-day period with more than 90% of preserved bioactivity was reached. To conclude, dual growth factor delivery was made possible by the mean of poly(methylidene malonate 2.1.2) blends. These studies demonstrate the ability of methylidene malonate 2.1.2-based polymeric blends for the delivery of growth factors.

Asunto(s)

Portadores de Fármacos , Sustancias de Crecimiento/administración & dosificación , Malonatos/química , Polietilenos/química , Animales , Materiales Biocompatibles/química , Línea Celular , Células Cultivadas , Medios de Cultivo/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular , Ensayo de Materiales , Ratones , Prótesis e Implantes , Transducción de Señal