RESUMEN
Target-protein degradation is an emerging field in drug discovery and development. In particular, the substrate-receptor proteins of the cullin-ubiquitin ligase system play a key role in selective protein degradation, which is an essential component of the anti-myeloma activity of immunomodulatory drugs (IMiDs), such as lenalidomide. Here, we demonstrate that a series of anticancer sulfonamides NSC 719239 (E7820), indisulam, and NSC 339004 (chloroquinoxaline sulfonamide, CQS) induce proteasomal degradation of the U2AF-related splicing factor coactivator of activating protein-1 and estrogen receptors (CAPERα) via CRL4DCAF15 mediated ubiquitination in human cancer cell lines. Both CRISPR-Cas9-based knockout of DCAF15 and a single amino acid substitution of CAPERα conferred resistance against sulfonamide-induced CAPERα degradation and cell-growth inhibition. Thus, these sulfonamides represent selective chemical probes for disrupting CAPERα function and designate DCAFs as promising drug targets for promoting selective protein degradation in cancer therapy.
Asunto(s)
Indoles/farmacología , Proteínas Nucleares/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Sulfonamidas/metabolismo , Antineoplásicos/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteolisis/efectos de los fármacos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: Indisulam possesses anticancer properties through down-regulation of various cell-cycle checkpoint molecules, thereby blocking the phosphorylation of retinoblastoma protein and inducing p53 and p21. Indisulam exhibits synergy with nucleoside analogs and topoisomerase inhibitors. METHODS: The authors designed a phase 2 study of indisulam in combination with idarubicin and cytarabine in patients who had relapsed/refractory acute myeloid leukemia AML and high-risk myelodysplastic syndrome. In stage 1, patients received intravenous indisulam at 400 mg/m2 on days 1 and 8 of a 28-day cycle. If they had no response, then patients received same dose schedule of indisulam followed by intravenous idarubicin 8 mg/m2 daily for 3 days and cytarabine 1.0 g/m2 over 24 hours daily on days 9 through 12 (for those aged < 60 years) or days 9 through 11 (for those aged > 60 years) of a 28-day cycle. Primary endpoints included the overall response rate, and secondary objectives included overall survival. RESULTS: Forty patients were enrolled. Of the 37 evaluable patients, 31 received indisulam with chemotherapy. Of these, 11 (35%) responded for a median duration of 5.3 months. The estimated 1-year overall survival rate was 51% for responders compared with 8 % for nonresponders (P < .001). The most common grade ≥3 nonhematologic toxicities were electrolyte abnormalities (50%) and febrile neutropenia (28%). CONCLUSIONS: The combination of indisulam with idarubicin and cytarabine yielded a 35% response rate in heavily pretreated patients with AML. With emerging data identifying the expression of DCAF15 (DDB1 and CUL4-associated factor 15) as a potential biomarker for activity, the combination of indisulam with idarubicin and cytarabine should be studied in a biomarker-driven trial or in patients who have splicing factor mutations. Cancer 2018;124:2758-65. © 2018 American Cancer Society. Cancer 2018;124:2758-2765. © 2018 American Cancer Society.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inducción de Remisión/métodos , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Citarabina/farmacología , Citarabina/uso terapéutico , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Neutropenia Febril/inducido químicamente , Neutropenia Febril/epidemiología , Femenino , Humanos , Idarrubicina/farmacología , Idarrubicina/uso terapéutico , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Mutación , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Síndromes Mielodisplásicos/patología , Recurrencia Local de Neoplasia/mortalidad , Factores de Empalme de ARN/genética , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Resultado del Tratamiento , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. Some tubulin-binding drugs are known to have antivascular (antiangiogenesis or vascular-disrupting) activities that can target abnormal tumor vessels. Using dynamic contrast-enhanced MRI analyses, here we show that eribulin induces remodeling of tumor vasculature through a novel antivascular activity in MX-1 and MDA-MB-231 human breast cancer xenograft models. Vascular remodeling associated with improved perfusion was shown by Hoechst 33342 staining and by increased microvessel density together with decreased mean vascular areas and fewer branched vessels in tumor tissues, as determined by immunohistochemical staining for endothelial marker CD31. Quantitative RT-PCR analysis of normal host cells in the stroma of xenograft tumors showed that eribulin altered the expression of mouse (host) genes in angiogenesis signaling pathways controlling endothelial cell-pericyte interactions, and in the epithelial-mesenchymal transition pathway in the context of the tumor microenvironment. Eribulin also decreased hypoxia-associated protein expression of mouse (host) vascular endothelial growth factor by ELISA and human CA9 by immunohistochemical analysis. Prior treatment with eribulin enhanced the anti-tumor activity of capecitabine in the MDA-MB-231 xenograft model. These findings suggest that eribulin-induced remodeling of abnormal tumor vasculature leads to a more functional microenvironment that may reduce the aggressiveness of tumors due to elimination of inner tumor hypoxia. Because abnormal tumor microenvironments enhance both drug resistance and metastasis, the apparent ability of eribulin to reverse these aggressive characteristics may contribute to its clinical benefits.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Furanos/farmacología , Cetonas/farmacología , Moduladores de Tubulina/farmacología , Microambiente Tumoral/efectos de los fármacos , Remodelación Vascular/efectos de los fármacos , Animales , Neoplasias de la Mama/patología , Capecitabina , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Femenino , Fluorouracilo/análogos & derivados , Fluorouracilo/farmacología , Humanos , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mass spectrometry is commonly applied to qualitatively and quantitatively profile small molecules, such as peptides, metabolites, or lipids. Modern mass spectrometers provide accurate measurements of mass-to-charge ratios of ions, with errors as low as 1 ppm. Even such high mass accuracy, however, is not sufficient to determine the unique chemical formula of each ion, and additional algorithms are necessary. Here we present a universal software tool for predicting chemical formulas from high-resolution mass spectrometry data, developed within the MZmine 2 framework. The tool is based on the use of a combination of heuristic techniques, including MS/MS fragmentation analysis and isotope pattern matching. The performance of the tool was evaluated using a real metabolomic data set obtained with the Orbitrap MS detector. The true formula was correctly determined as the highest-ranking candidate for 79% of the tested compounds. The novel isotope pattern-scoring algorithm outperformed a previously published method in 64% of the tested Orbitrap spectra. The software described in this manuscript is freely available and its source code can be accessed within the MZmine 2 source code repository.
Asunto(s)
Espectrometría de Masas , Algoritmos , Isótopos de Carbono/química , Cromatografía Líquida de Alta Presión , Schizosaccharomyces/metabolismo , Programas InformáticosRESUMEN
We have developed a practical method for the comprehensive analysis of polar anionic metabolites in biological samples with the use of a nano-LC/MS system. A polyamine-bonded polymer-based apHera NH2 column, which is compatible with ammonium carbonate buffer, effectively retained anionic polar metabolites, such as organic acids, sulfates, and phosphates, but multiply phosphorylated or carboxylated compounds showed highly distorted peak shapes on chromatograms. We found that addition of a trace amount of the metal chelating reagent ethylenediaminetetraacetic acid (EDTA) to the sample solution dramatically improved peak shapes of multiply charged anionic compounds, even though the mass spectra showed no trace of adduct ions in the absence of EDTA. The detection limits of typical polar anionic metabolites in the full-scan mode were from 0.19 to 2.81 pmol. After optimization of all the procedures from sample preparation to nano-LC/MS analysis, we applied our method to real biological samples: Hela cells, mouse brain, human cerebrospinal fluid (CSF), and human plasma. Our results indicated that phosphorylated metabolites were abundant in Hela cells and brain, while plasma and cerebrospinal fluid (CSF) mostly contained organic acids. Phosphorylated compounds might not be secreted into CSF/plasma or might be unstable in CSF/plasma. Finally, the method was used to examine the mode of action of the anticancer drug methotrexate (MTX), which inhibits purine de novo biosynthesis and thymidine biosynthesis. In addition of the expected changes of metabolite levels, we found that a previously unreported metabolite, probably a methylated uridine 5'-triphosphate (UTP), was produced by MTX-treated Hela cells.
Asunto(s)
Ácidos/análisis , Quelantes/química , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Metaboloma , Metabolómica/métodos , Metales/química , Fosfatos/análisis , Ácidos/sangre , Ácidos/líquido cefalorraquídeo , Animales , Metilación de ADN/efectos de los fármacos , Células HeLa , Humanos , Metotrexato/farmacología , Ratones , Nanotecnología , Fosfatos/sangre , Fosfatos/líquido cefalorraquídeoRESUMEN
Highly sensitive and quantitative analytical methods are essential for metabolomics. In this report, we introduce an analytical method focused on endogenous phosphorus metabolites, using nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) and culture-derived isotope-tagged metabolites as global internal standards for quantitative metabolomics. The nanoLC-ESI-MS/MS method employing a stone-arch microcolumn with amino propyl silica gel achieved good separation of phosphorus metabolites with forty- to hundred-fold increase of sensitivity compared with semimicro flow LC-ESI-MS. The quantitative reproducibility of the nanoLC-ESI-MS has been improved to the point where it is useful for studies of cellular metabolism. Focused metabolomics using culture-derived internal standards was employed to monitor 184 phosphorus-related metabolic changes in cancer cells treated with metabolic enzyme inhibitors, methotrexate, fluorouracil, and gemcitabine. We found marked perturbations of cellular metabolism, of which many, though not all, were in line with the known biological activities of these drugs.
Asunto(s)
Cromatografía Liquida/métodos , Metabolómica , Fósforo/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Fluorouracilo/farmacología , Humanos , Metotrexato/farmacología , Estándares de Referencia , Sensibilidad y Especificidad , GemcitabinaRESUMEN
RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.
Asunto(s)
Redes Reguladoras de Genes , Marcación de Gen/métodos , Leucemia Mieloide Aguda/patología , Proteómica/métodos , Proteínas de Unión al ARN/genética , Regulación hacia Arriba , Empalme Alternativo , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Células HL-60 , Proteínas de Homeodominio/genética , Humanos , Células Jurkat , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Ratones , Trasplante de Neoplasias , Pronóstico , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de SupervivenciaRESUMEN
Pladienolide B is a 12-membered macrolide isolated from Streptomyces platensis Mer-11107. It showed potent in vitro and in vivo antitumor activities and is a potential lead for novel antitumor agents. The absolute configurations at ten chiral centers were determined on the basis of spectral data of pladienolide B and its chemical transformation products.
Asunto(s)
Compuestos Epoxi/química , Macrólidos/química , Antineoplásicos/química , Modelos Moleculares , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa por Ionización de Electrospray , Estereoisomerismo , Streptomyces/químicaRESUMEN
Vitamin A deficiency (VAD) caused by malnutrition and certain intestinal diseases induces visual impairments, including night blindness and photoreceptor cell dysfunction as indicated by reduced a and bwaves in an electroretinogram (ERG). The effects of VAD on the inner retinal layer cells, including amacrine and ganglion cells, remain to be elucidated. The functions of these cells are reflected in oscillatory potentials (OPs), another component of the ERG. The present study investigated inner retinal layer cell function in VAD rats by analyzing OPs. In the present study, VAD was induced by feeding Brown Norway rats a vitamin A deficient diet for 10 weeks. A reduced body weight and peripapillary opacification indicative of papilledema without histopathological alterations were observed, which are considered early symptoms of VAD. At this stage, the ERG revealed reduced OPs as well as a and bwaves at various intensities of light stimulation. Further analysis indicated that the ratio of the alterations in OPs was more significant than those of a and bwaves. After 5 weeks of recovery, these changes returned to control levels. These results suggest that OPs are the most sensitive and early marker of VADassociated visual impairment in the ERG.
Asunto(s)
Electrorretinografía/métodos , Trastornos de la Visión/sangre , Deficiencia de Vitamina A/sangre , Animales , Masculino , Oscilometría , Ratas , Ratas Endogámicas BN , Retina/metabolismo , Trastornos de la Visión/diagnóstico , Deficiencia de Vitamina A/complicacionesRESUMEN
IMPORTANCE OF THE FIELD: Metabolomics is increasingly becoming an important field in the pharmaceutical industry to support the discovery and development of therapeutic agents. It allows the comprehensive and simultaneous profiling of hundreds of discrete biologically important molecules, including amino acids, sugars, lipids and exogenous substances from biological fluids and tissues. Metabolomics is the 'omics' field that most represents the interplay of internal biological regulation and external environmental influences on disease, thereby being of particular importance to disease mitigation and management. AREAS COVERED IN THIS REVIEW: Technological advances in the experimental work flow, analytical detection strategies and bioinformatics tools have enabled metabolomics studies to become increasingly comprehensive, robust and informative for the understanding of disease, drug action and the development of biomarkers. This review will focus on the practical aspects of metabolomics studies as they have been applied to the study of mammalian biological systems, specifically targeted to the steps of experimental design with regard to sample preparation, sample analysis and data analysis of both polar and non-polar metabolites. WHAT THE READER WILL GAIN: The reader will gain an overview of the field of metabolomics as it applies to drug development and the practical issues involved with experimental design. We will discuss the various methods of sample preparation and analysis as they apply to different classes of metabolites and highlight recent advances in the field that illustrate these methods. TAKE HOME MESSAGE: The field of metabolomics is a rapidly expanding discipline that is being applied to various aspects of drug development. The large diversity of metabolites found in nature dictates that different methods be developed for the investigation of different classes of metabolites. As the field of metabolomics continues to mature, it is likely that it will play an increasingly important role in the characterization of disease and the future development of biomarkers to assess drug efficacy and safety.
RESUMEN
A new polyacetylenic acid, callyspongynic acid (1), was isolated as an alpha-glucosidase inhibitor from the marine sponge Callyspongia truncata. Its structure was determined by spectroscopic and chemical methods.
Asunto(s)
Acetileno/aislamiento & purificación , Inhibidores Enzimáticos/aislamiento & purificación , Glucosidasas/antagonistas & inhibidores , Polímeros/aislamiento & purificación , Poríferos/química , Acetileno/análogos & derivados , Acetileno/química , Acetileno/farmacología , Alquinos , Animales , Cromatografía en Capa Delgada , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ésteres/química , Ésteres/aislamiento & purificación , Ésteres/farmacología , Estructura Molecular , Países Bajos , Resonancia Magnética Nuclear Biomolecular , Polímeros/química , Polímeros/farmacología , EstereoisomerismoRESUMEN
Three new alpha-glucosidase inhibitors, schulzeines A-C (1-3), were isolated from the marine sponge Penares schulzei. Their structures were elucidated by spectral analysis and chemical degradations to be the isoquinoline alkaloids, encompassing two amino acids, and C(28) fatty acid, the last of which was sulfated. Absolute stereochemistry of schulzeines was determined by application of the modified Mosher analysis to fragments obtained by chemical degradation. Schulzeines A-C inhibit alpha-glucosidase with IC(50) values of 48-170 nM.