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1.
J Immunol Methods ; 510: 113358, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36126779

RESUMEN

Ferroptosis, a type of iron-dependent necrotic cell death, is specifically associated with increased lipid peroxidation. The dysfunction of the glutathione (GSH) production via the starvation of cysteine or the inhibition of phospholipid hydroperoxide glutathione peroxidase (GPX4) typically results in the accumulation of lipid peroxidation products and, consequently, the development of ferroptosis. We recently reported on the production of a rat monoclonal antibody, referred to as FerAb, against mouse-derived Hepa 1-6 cells that had been cultivated in cystine-deprived medium. Immunocytological analyses by means of fluorescence microscopy revealed that FerAb binds to fixed ferroptotic cells regardless of the species from which they were obtained, but not to apoptotic cells. We report herein on an in-depth characterization of the reactivity of FerAb with respect to unfixed cells by means of flow cytometry. The binding of FerAb to the cells was stimulated by incubating the cells in cystine deprived culture medium or treatment with RSL3, a GPX4 inhibitor, while treatment with staurosporine, an apoptosis inducer, had no effect on its binding to the cells. Supplementation with ferrostatin-1, a ferroptosis inhibitor, effectively suppressed the binding of FerAb to cells that had been cultivated in cystine-deprived medium or treated with RSL3, further confirming the specific binding of FerAb to ferroptotic cells. Thus, FerAb combined with a flow cytometry can be used to distinguish ferroptotic cells from living cells or apoptotic cells without the need for fixation. Applications of this combined technique will enable the quantitative evaluation of ferroptotic cells under a variety of patho-physiological conditions and will contribute to our understanding of the roles of ferroptosis in the body as well as cultured cells.


Asunto(s)
Ferroptosis , Animales , Anticuerpos Monoclonales/farmacología , Muerte Celular , Cisteína , Cistina , Citometría de Flujo , Glutatión/metabolismo , Hierro , Ratones , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Ratas , Estaurosporina/farmacología
2.
Brief Bioinform ; 23(1)2022 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-34962256

RESUMEN

The pharmacological arsenal against the COVID-19 pandemic is largely based on generic anti-inflammatory strategies or poorly scalable solutions. Moreover, as the ongoing vaccination campaign is rolling slower than wished, affordable and effective therapeutics are needed. To this end, there is increasing attention toward computational methods for drug repositioning and de novo drug design. Here, multiple data-driven computational approaches are systematically integrated to perform a virtual screening and prioritize candidate drugs for the treatment of COVID-19. From the list of prioritized drugs, a subset of representative candidates to test in human cells is selected. Two compounds, 7-hydroxystaurosporine and bafetinib, show synergistic antiviral effects in vitro and strongly inhibit viral-induced syncytia formation. Moreover, since existing drug repositioning methods provide limited usable information for de novo drug design, the relevant chemical substructures of the identified drugs are extracted to provide a chemical vocabulary that may help to design new effective drugs.


Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , COVID-19 , Células Gigantes , Pirimidinas/farmacología , SARS-CoV-2/metabolismo , Estaurosporina/análogos & derivados , Células A549 , COVID-19/metabolismo , Biología Computacional , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Células Gigantes/metabolismo , Células Gigantes/virología , Humanos , Estaurosporina/farmacología
3.
Int J Mol Sci ; 22(21)2021 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-34769389

RESUMEN

BACKGROUND: Staurosporine-dependent single and collective cell migration patterns of breast carcinoma cells MDA-MB-231, MCF-7, and SK-BR-3 were analysed to characterise the presence of drug-dependent migration promoting and inhibiting yin-yang effects. METHODS: Migration patterns of various breast cancer cells after staurosporine treatment were investigated using Western blot, cell toxicity assays, single and collective cell migration assays, and video time-lapse. Statistical analyses were performed with Kruskal-Wallis and Fligner-Killeen tests. RESULTS: Application of staurosporine induced the migration of single MCF-7 cells but inhibited collective cell migration. With the exception of low-density SK-BR-3 cells, staurosporine induced the generation of immobile flattened giant cells. Video time-lapse analysis revealed that within the borderline of cell collectives, staurosporine reduced the velocity of individual MDA-MB-231 and SK-BR-3, but not of MCF-7 cells. In individual MCF-7 cells, mainly the directionality of migration became disturbed, which led to an increased migration rate parallel to the borderline, and hereby to an inhibition of the migration of the cell collective as a total. Moreover, the application of staurosporine led to a transient activation of ERK1/2 in all cell lines. CONCLUSION: Dependent on the context (single versus collective cells), a drug may induce opposite effects in the same cell line.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Movimiento Celular , Inhibidores Enzimáticos/farmacología , Estaurosporina/farmacología , Yin-Yang , Apoptosis , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Humanos , Transducción de Señal , Células Tumorales Cultivadas
4.
Anal Chim Acta ; 1168: 338612, 2021 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-34051997

RESUMEN

The process of protein precipitation can be used to decipher the interaction of ligand and protein. For example, the classic Thermal Proteome Profiling (TPP) method uses heating as the driving force for protein precipitation, to discover the drug target protein. Under heating or other denature forces, the target protein that binds with the drug compound will be more resistant to precipitation than the free protein. Similar to thermal stress, mechanical stress can also induce protein precipitation. Upon mechanical stress, protein will gradually precipitate along with protein conformational changes, which can be exploited for the study of the ligand-protein interaction. Herein, we proposed a Mechanical Stress Induced Protein Precipitation (MSIPP) method for drug target deconvolution. Its streamlined workflow allows in situ sample preparation on the surface of microparticles, from protein precipitation to digestion. The mechanical stress was generated by vortexing the slurry of protein solution and microparticle materials. The mechanical stress induced protein precipitate was captured by the microparticles, which guarantees the MSIPP method to be scalable and user-friendly. The MSIPP method was successfully applied to four drug compounds, Methotrexate, Raltitrexed, SHP099, Geldanamycin and a pan-inhibitor of protein kinases, Staurosporine. Besides, DHFR was demonstrated to be a target of Raltitrexed, which has not been revealed by any other modification-free drug target discovery method yet. Thus, MSIPP is a complementary method to other drug target screening methods.


Asunto(s)
Sistemas de Liberación de Medicamentos , Preparaciones Farmacéuticas , Precipitación Química , Evaluación Preclínica de Medicamentos , Estaurosporina , Estrés Mecánico
5.
Minerva Med ; 111(5): 427-442, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32955823

RESUMEN

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene arise in 25-30% of all acute myeloid leukemia (AML) patients. These mutations lead to constitutive activation of the protein product and are divided in two broad types: internal tandem duplication (ITD) of the juxtamembrane domain (25% of cases) and point mutations in the tyrosine kinase domain (TKD). Patients with FLT3 ITD mutations have a high relapse risk and inferior cure rates, whereas the role of FLT3 TKD mutations still remains to be clarified. Additionally, growing research indicates that FLT3 status evolves through a disease continuum (clonal evolution), where AML cases can acquire FLT3 mutations at relapse - not present in the moment of diagnosis. Several FLT3 inhibitors have been tested in patients with FLT3-mutated AML. These drugs exhibit different kinase inhibitory profiles, pharmacokinetics and adverse events. First-generation multi-kinase inhibitors (sorafenib, midostaurin, lestaurtinib) are characterized by a broad-spectrum of drug targets, whereas second-generation inhibitors (quizartinib, crenolanib, gilteritinib) show more potent and specific FLT3 inhibition, and are thereby accompanied by less toxic effects. Notwithstanding, all FLT3 inhibitors face primary and acquired mechanisms of resistance, and therefore the combinations with other drugs (standard chemotherapy, hypomethylating agents, checkpoint inhibitors) and its application in different clinical settings (upfront therapy, maintenance, relapsed or refractory disease) are under study in a myriad of clinical trials. This review focuses on the role of FLT3 mutations in AML, pharmacological features of FLT3 inhibitors, known mechanisms of drug resistance and accumulated evidence for the use of FLT3 inhibitors in different clinical settings.


Asunto(s)
Antineoplásicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Sorafenib/farmacología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genética , Compuestos de Anilina/farmacología , Bencimidazoles/farmacología , Benzotiazoles/farmacología , Carbazoles/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Predicción , Furanos , Trasplante de Células Madre Hematopoyéticas , Humanos , Imidazoles/farmacología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Quimioterapia de Mantención/métodos , Mutación , Compuestos de Fenilurea/farmacología , Piperidinas/farmacología , Mutación Puntual , Pirazinas/farmacología , Piridazinas/farmacología , Recurrencia , Estaurosporina/análogos & derivados , Estaurosporina/farmacología
6.
J Biosci Bioeng ; 130(1): 106-113, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32253091

RESUMEN

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.


Asunto(s)
Actinobacteria/química , Ciclo Celular/efectos de los fármacos , Hongos/química , Inhibidores de Crecimiento/farmacología , Agua de Mar/microbiología , Actinobacteria/genética , Actinobacteria/aislamiento & purificación , Actinobacteria/metabolismo , Animales , Células CHO , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Hongos/genética , Hongos/aislamiento & purificación , Hongos/metabolismo , Inhibidores de Crecimiento/metabolismo , Prodigiosina/análogos & derivados , Prodigiosina/metabolismo , Prodigiosina/farmacología , Estaurosporina/metabolismo , Estaurosporina/farmacología
7.
Anal Chem ; 92(1): 1363-1371, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31794197

RESUMEN

High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDUFV1 and NDUFAB1 were identified for the first time, and the NDUFV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug-target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.


Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Metotrexato/farmacología , NADH Deshidrogenasa/antagonistas & inhibidores , Oxazoles/farmacología , Proteómica , Estaurosporina/farmacología , Tiazoles/farmacología , Ácido Acético/química , Acetona/química , Células Cultivadas , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Etanol/química , Células HEK293 , Proteínas HSP90 de Choque Térmico/química , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Metotrexato/química , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismo , Oxazoles/química , Solventes/química , Estaurosporina/química , Tiazoles/química
8.
Leuk Res ; 87: 106262, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31756575

RESUMEN

INTRODUCTION: Identification of cytogenetic and molecular abnormalities has become vital for the appropriate treatment of acute myeloid leukemia (AML). One of the most common molecular alterations in AML is the constitutive activation by internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3). METHODS: This observational, retrospective, cohort study at the Huntsman Cancer Institute (HCI) had two time periods: 1) a historical pre-midostaurin time period which consisted of the FLT3 mutated (FLT3m) and FLT3 wild type (FLT3wt) cohorts from January 1, 2007, to December 31, 2016, and 2) a post-midostaurin cohort which consisted of the FLT3 mutated midostaurin-user cohort (early mido) from May 01, 2017 to December 31, 2018. RESULTS: In total, 39 patients were included in the FLT3m cohort, 61 in the FLT3wt cohort, and seven in the early mido cohort. FLT3m patients spent fewer days in the hospital during the first consolidation regimen and received fewer consolidation cycles compared to FLT3wt patients. Overall survival (OS) was similar between FLT3m and FLT3wt patients. For patients without hematopoietic stem cell transplant, OS was significantly shorter for FLT3m patients compared to FLT3wt patients. Mean AML related inpatient charges and physician charges for FLT3m patients were significantly higher than FLT3wt patients. CONCLUSION: The FLT3 mutation is historically associated with a shorter time to transplant and increased total health care charges. More information is needed to evaluate the real-world treatment strategies for FLT3-mutated patients in the presence of FLT3 inhibitors and the impact of these treatment strategies on clinical and economic outcomes.


Asunto(s)
Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/economía , Mutación , Estaurosporina/análogos & derivados , Tirosina Quinasa 3 Similar a fms/genética , Adulto , Anciano , Estudios de Cohortes , Atención Integral de Salud/economía , Femenino , Costos de la Atención en Salud , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Pronóstico , Inhibidores de Proteínas Quinasas/economía , Inhibidores de Proteínas Quinasas/uso terapéutico , Estudios Retrospectivos , Estaurosporina/economía , Estaurosporina/uso terapéutico , Resultado del Tratamiento
9.
Bioorg Med Chem Lett ; 29(21): 126641, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31526603

RESUMEN

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.


Asunto(s)
Colorantes Fluorescentes/química , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Estaurosporina/química , Sitios de Unión , Unión Competitiva , Técnicas Biosensibles , Evaluación Preclínica de Medicamentos/métodos , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/síntesis química , Humanos , Enlace de Hidrógeno , Metilaminas/química , Estructura Molecular , Unión Proteica , Sensibilidad y Especificidad , Estaurosporina/síntesis química , Relación Estructura-Actividad
10.
Cancer ; 125(21): 3755-3766, 2019 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-31310323

RESUMEN

BACKGROUND: The addition of midostaurin to induction chemotherapy improves survival in younger patients with newly diagnosed, FLT3-mutated acute myeloid leukemia (AML). Sorafenib is a potent multikinase inhibitor with efficacy when given as monotherapy. The authors investigated whether the addition of sorafenib to intensive induction chemotherapy improves outcomes in patients with FLT3-internal tandem duplication (ITD)-mutated AML. METHODS: In total, 183 patients who were newly diagnosed with FLT3-ITD-mutated AML between February 2001 and December 2017 were identified. Of these, 79 patients (43%) underwent intensive chemotherapy with the addition of sorafenib, and 104 (57%) received intensive chemotherapy alone. Propensity score matching identified 42 patients in each cohort. RESULTS: The overall response rate was 98% in the sorafenib cohort and 83% in the intensive chemotherapy cohort (P = .057). The median follow-up was 54 months. The median event-free survival was 35 months in the sorafenib cohort and 8 months in the intensive chemotherapy cohort (P = .019), and the median overall survival was 42 and 13 months, respectively (P = .026). With censoring at the time of allogeneic stem cell transplantation, the median event-free survival was 31 and 8 months in the sorafenib and intensive therapy cohorts, respectively (P = .031), and the median overall survival was not reached and 10 months, respectively (P = .001). Multivariate Cox proportional hazards models confirmed that treatment with sorafenib was a favorable prognostic factor (P = .009; hazard ratio, 0.558; 95% CI, 0.360-0.865). CONCLUSIONS: The addition of sorafenib improves survival in patients with FLT3-ITD-mutated AML regardless of whether they undergo allogeneic stem cell transplantation.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia Mieloide/terapia , Mutación , Secuencias Repetidas en Tándem/genética , Tirosina Quinasa 3 Similar a fms/genética , Enfermedad Aguda , Adulto , Estudios de Cohortes , Terapia Combinada , Femenino , Humanos , Quimioterapia de Inducción/métodos , Estimación de Kaplan-Meier , Leucemia Mieloide/genética , Masculino , Persona de Mediana Edad , Inducción de Remisión , Sorafenib/administración & dosificación , Estaurosporina/administración & dosificación , Estaurosporina/análogos & derivados , Trasplante Homólogo , Adulto Joven
11.
Blood ; 134(9): 741-745, 2019 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-31243041

RESUMEN

The natural history of FLT3-mutated AML is changing after the approval of midostaurin for frontline therapy and gilteritinib for relapsed or refractory patients. Recently reported, positive randomized trials of the drugs gilteritinib, quizartinib, and sorafenib predict even wider use of FLT3 inhibitors going forward. FLT3 inhibitors now emerge as an important, if not indispensable, part of therapy for a large subset of high-risk patients.


Asunto(s)
Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Animales , Antineoplásicos/farmacología , Benzotiazoles/farmacología , Benzotiazoles/uso terapéutico , Ensayos Clínicos como Asunto , Humanos , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Pirazinas/uso terapéutico , Sorafenib/farmacología , Sorafenib/uso terapéutico , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Estaurosporina/uso terapéutico
12.
Molecules ; 23(12)2018 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-30501110

RESUMEN

Protein kinases are deeply involved in immune-related diseases and various cancers. They are a potential target for structure-based drug discovery, since the general structure and characteristics of kinase domains are relatively well-known. However, the ATP binding sites in protein kinases, which serve as target sites, are highly conserved, and thus it is difficult to develop selective kinase inhibitors. To resolve this problem, we performed molecular dynamics simulations on 26 kinases in the aqueous solution, and analyzed topological water networks (TWNs) in their ATP binding sites. Repositioning of a known kinase inhibitor in the ATP binding sites of kinases that exhibited a TWN similar to interleukin-1 receptor-associated kinase 4 (IRAK4) allowed us to identify a hit molecule. Another hit molecule was obtained from a commercial chemical library using pharmacophore-based virtual screening and molecular docking approaches. Pharmacophoric features of the hit molecules were hybridized to design a novel compound that inhibited IRAK4 at low nanomolar levels in the in vitro assay.


Asunto(s)
Diseño de Fármacos , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Agua/química , Sitios de Unión , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Estaurosporina/química , Estaurosporina/farmacología
13.
Adv Exp Med Biol ; 1074: 649-653, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29721999

RESUMEN

Talaumidin, a tetrahydrofuran neolignan isolated from the root of Aristolochia arcuata, was an interesting small molecule with neurotrophic activity in the cultured neuron. Talaumidin can promote neurite outgrowth from neurons. However, the mechanism by which talaumidin exerts its neurotrophic actions on retinal neurons has not been elucidated to date. In this study, we describe that talaumidin has neurotrophic properties such as neurite outgrowth in neuroretinal cell line, RGC-5. Talaumidin promotes staurosporine-induced neurite outgrowth in RGC-5 cells. The neurite outgrowth effect of talaumidin was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, but not by Erk inhibitor, PD98059. These data suggest that talaumidin promotes neurite outgrowth through PI3K/Akt pathway and that the potential of talaumidin serves as a promising lead compound for the treatment of retinal degenerative disorders.


Asunto(s)
Furanos/farmacología , Proyección Neuronal/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas c-akt/fisiología , Células Ganglionares de la Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Ratones , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fitoterapia , Inhibidores de Proteínas Quinasas/farmacología , Degeneración Retiniana/tratamiento farmacológico , Células Ganglionares de la Retina/ultraestructura , Estaurosporina/farmacología
15.
Mol Cancer Ther ; 16(1): 88-101, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-28062706

RESUMEN

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR.


Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Terapia Molecular Dirigida , Animales , Antígenos CD , Línea Celular Tumoral , Biología Computacional/métodos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Interacciones Farmacológicas , Humanos , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Proteína Proto-Oncogénica c-fli-1/antagonistas & inhibidores , Proteína EWS de Unión a ARN/antagonistas & inhibidores , Receptor IGF Tipo 1 , Receptor de Insulina/antagonistas & inhibidores , Receptores de Somatomedina/antagonistas & inhibidores , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Transducción de Señal/efectos de los fármacos , Estaurosporina/análogos & derivados , Estaurosporina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
16.
Skeletal Radiol ; 46(1): 23-33, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27815598

RESUMEN

OBJECTIVE: To determine the concentrations exhibiting toxicity of a cartilage-targeted magnetic resonance imaging contrast agent compared with gadopentetate dimeglumine (Gd-DT-PA) in chondrocyte cultures. MATERIALS AND METHODS: A long-term Swarm rat chondrosarcoma chondrocyte-like cell line was exposed for 48 h to 1.0-20 mM concentrations of diaminobutyl-linked nitroxide (DAB4-DLN) citrate, 1.0-20 mM Gd-DTPA, 1.0 µM staurosporine (positive control), or left untreated. Cell appearance, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays of metabolic activity, quantitative PicoGreen assays of DNA content, and calcein-AM viability assays were compared. RESULTS: At 1.0-7.5 mM, minimal decrease in cell proliferation was found for both agents. At all doses of both agents, cell culture appearances were similar after 24 h of treatment. At the higher doses, differences in cell culture appearance were found after 48 h of treatment, with dose-dependent declines in chondrocyte populations for both agents. Concentration-dependent declines in DNA content and calcein fluorescence were found after 48 h of treatment, but beginning at a lower dose of DAB4-DLN citrate than Gd-DTPA. Dose-dependent decreases in MTT staining (cell metabolism) were apparent for both agents, but larger effects were evident at a lower dose for DAB-DLN citrate. Poor MTT staining of cells exposed for 48 h to 20 mM DAB4-DLN citrate probably indicates dead or dying cells. CONCLUSION: The minimal effect of the long-term exposure of model chondrocyte cell cultures to DAB4-DLN citrate and Gd-DTPA concentrations up to 7.5 mM (3x typical arthrographic administration) is supporting evidence that these doses are acceptable for MR arthrography. The findings are reassuring given that the experimental exposure to the contrast agents at sustained concentrations was much longer than when used clinically.


Asunto(s)
Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Medios de Contraste/toxicidad , Gadolinio DTPA/toxicidad , Animales , Cartílago Articular/metabolismo , Cartílago Articular/patología , Proliferación Celular , Condrocitos/metabolismo , Condrocitos/patología , Medios de Contraste/administración & dosificación , Dendrímeros/administración & dosificación , Dendrímeros/toxicidad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Gadolinio DTPA/administración & dosificación , Imagen por Resonancia Magnética , Ratas , Estaurosporina , Células Tumorales Cultivadas/efectos de los fármacos
17.
Virol J ; 13(1): 177, 2016 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-27769267

RESUMEN

BACKGROUND: HIV-1 latency is a major obstacle for HIV-1 eradication. Extensive efforts are being directed toward the reactivation of latent HIV reservoirs with the aim of eliminating latently infected cells via the host immune system and/or virus-mediated cell lysis. RESULTS: We screened over 1,500 small molecules and kinase inhibitors and found that a small molecule, PKC412 (midostaurin, a broad-spectrum kinase inhibitor), can stimulate viral transcription and expression from the HIV-1 latently infected ACH2 cell line and primary resting CD4+ T cells. PKC412 reactivated HIV-1 expression in ACH2 cells in a dose- and time-dependent manner. Our results also suggest that the nuclear factor κB (NF-κB) signaling could be one of cellular pathways activated during PKC412-mediated activation of latent HIV-1 expression. Additionally, combining PKC412 with the HDAC inhibitor vorinostat (VOR) had an additive effect on HIV-1 reactivation in both ACH2 cells and infected resting CD4+ T cells. CONCLUSIONS: These studies provide evidence that PKC412 is a new compound with the potential for optimization as a latency-reactivator to eradicate HIV-1 infection.


Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , VIH-1/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Estaurosporina/análogos & derivados , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Evaluación Preclínica de Medicamentos , VIH-1/fisiología , Humanos , Estaurosporina/metabolismo
18.
ACS Chem Neurosci ; 7(7): 912-23, 2016 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-27213824

RESUMEN

Alzheimer's disease (AD) is the most common brain disorder worldwide. Aberrant tau hyperphosphorylation and accumulation play critical roles in the formation of neurofibrillary tangles highly associated with neuronal dysfunction and cognitive impairment in AD pathogenesis. Glycogen synthase kinase-3ß (GSK3ß) is a key kinase responsible for tau hyperphosphorylation. Selective inhibition of GSK3ß is a promising strategy in AD therapy. Corn silks (CS, Zea mays L.) have been traditionally used as a medicinal herb and recently noted for their potentially cognitive benefits. However, the neuroprotective components of CS and their molecular mechanism have received little attention to date. As part of our effort screening phytochemicals against a broad panel of kinases targeting AD tauopathy, we found inhibition of GSK3ß by CS extracts. Subsequent bioassay-guided fractionation led to the isolation and identification of two 6-C-glycosylflavones, isoorientin (1) and 3'-methoxymaysin (2), with selective inhibition against GSK3ß in vitro. Enzyme kinetics and molecular docking studies demonstrated that 1 specifically inhibited GSK3ß via an ATP noncompetitive mechanism, acting as a substrate competitive inhibitor of GSK3ß. Further in vitro cellular studies demonstrated that 1 effectively attenuated tau phosphorylation mediated by GSK3ß and was neuroprotective against ß-amyloid-induced tau hyperphosphorylation and neurotoxicity in SH-SY5Y cells. The C-glycosylflavones represent new lead candidates with a novel mechanism of action for the development of AD phytopharmaceuticals.


Asunto(s)
Péptidos beta-Amiloides/farmacología , Inhibidores Enzimáticos/farmacología , Flavonoides/química , Flavonoides/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas tau/metabolismo , Adenosina Trifosfato/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Isoflavonas/química , Isoflavonas/farmacología , Luteolina/química , Luteolina/farmacología , Modelos Químicos , Neuroblastoma/patología , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Estaurosporina/farmacología , Proteínas tau/efectos de los fármacos
19.
J Agric Food Chem ; 64(7): 1528-39, 2016 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-26832452

RESUMEN

Increased apoptosis of retinal ganglion cells (RGCs) contributes to the gradual loss of retinal neurons at the early phase of diabetic retinopathy (DR). There is an urgent need to search for drugs with neuroprotective effects against apoptosis of RGCs for the early treatment of DR. This study aimed to investigate the neuroprotective effects of saponins extracted from Panax notoginseng, a traditional Chinese medicine, on apoptosis of RGCs stimulated by palmitate, a metabolic factor for the development of diabetes and its complications, and to explore the potential molecular mechanism. We showed that crude saponins of P. notoginseng (CSPN) inhibited the increased apoptosis and loss of postsynaptic protein PSD-95 by palmitate in staurosporine-differentiated RGC-5 cells. Moreover, CSPN suppressed palmitate-induced reactive oxygen species generation and endoplasmic reticulum stress-associated eIF2α/ATF4/CHOP and caspase 12 pathways. Thus, our findings address the potential therapeutic significance of CSPN for the early stage of DR.


Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Palmitatos/efectos adversos , Panax notoginseng/química , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Saponinas/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Estrés Oxidativo/efectos de los fármacos , Proteínas Asociadas a SAP90-PSD95 , Estaurosporina/farmacología
20.
Planta Med ; 82(3): 205-10, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26544120

RESUMEN

Many herbs, and recently their biomass from in vitro cultures, are essential for the treatment of diseases. The aim of this study was to determine the optimal growth of Bacopa monnieri (water hyssop) in an in vitro culture and to examine if extracts of the B. monnieri biomass from the in vitro culture would affect hydrogen peroxide- and staurosporine-induced injury of the human neuroblastoma SH-SY5Y cell line. It has been found that B. monnieri at concentrations of 25, 50, and 100 µg/mL inhibited both hydrogen peroxide-induced efflux of lactate dehydrogenase from damaged cells to culture medium and increased cell viability determined by an MTT assay. Moreover, B. monnieri at concentrations of 10, 25, and 50 µg/mL decreased staurosporine-induced activity of an executive apoptotic enzyme-caspase-3 and protected mitochondrial membrane potential. The obtained data indicate that the biomass from the in vitro culture of B. monnieri prevented SH-SY5Y cell damage related to oxidative stress and had the ability to inhibit the apoptotic process. Thus, this study supports the traditional use of B. monnieri as a neuroprotective therapy, and further in vivo studies on the effects of this preparation on morphology and function of nerve cells could lead to its wider application.


Asunto(s)
Bacopa/química , Peróxido de Hidrógeno/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Estaurosporina/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Neuroblastoma
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