RESUMEN
The bromodomain and extraterminal (BET) family of bromodomain-containing proteins are important regulators of the epigenome through their ability to recognize N-acetyl lysine (KAc) post-translational modifications on histone tails. These interactions have been implicated in various disease states and, consequently, disruption of BET-KAc binding has emerged as an attractive therapeutic strategy with a number of small molecule inhibitors now under investigation in the clinic. However, until the utility of these advanced candidates is fully assessed by these trials, there remains scope for the discovery of inhibitors from new chemotypes with alternative physicochemical, pharmacokinetic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library technology, with an N-methyl pyridone series identified through fragment screening. Optimization via structure- and property-based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is projected to have a low human efficacious dose.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/farmacología , Bencimidazoles/química , Bencimidazoles/farmacocinética , Bencimidazoles/farmacología , Quimiocina CCL2/biosíntesis , Cristalografía por Rayos X , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Humanos , Interleucina-6/antagonistas & inhibidores , Leucocitos/efectos de los fármacos , Masculino , Ratones , Modelos Moleculares , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Bibliotecas de Moléculas PequeñasRESUMEN
The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.
Asunto(s)
Benzodiazepinas/uso terapéutico , Química Clic , Sistemas de Liberación de Medicamentos , Epigenómica , Leucemia/tratamiento farmacológico , Animales , Benzodiazepinas/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Leucemia/patología , Ratones , Medicina de Precisión , Distribución Tisular , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay.
Asunto(s)
Naftiridinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Disponibilidad Biológica , Cristalografía por Rayos X , Humanos , Pruebas de Mutagenicidad , Naftiridinas/administración & dosificación , Naftiridinas/farmacocinética , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Histonas/genética , Leucemia Bifenotípica Aguda/genética , Metiltransferasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Acetilación , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Proteínas de Ciclo Celular , Proliferación Celular , Cromatina/química , Cromatina/metabolismo , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Femenino , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Leucemia Bifenotípica Aguda/metabolismo , Leucemia Bifenotípica Aguda/patología , Masculino , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , Unión Proteica , Proteómica/métodos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores Enzimáticos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Oxazepinas/farmacología , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Sinergismo Farmacológico , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Humanos , Leucemia Mieloide Aguda/enzimología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Oxazepinas/administración & dosificación , Oxazepinas/química , Estructura Terciaria de Proteína , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Transcripción p300-CBP/químicaRESUMEN
Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/ß-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.
Asunto(s)
Benzodiazepinas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Animales , Azepinas/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Células Clonales/patología , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes myc/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Terapia Molecular Dirigida , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Triazoles/farmacología , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Acetiltransferasa E N-Terminal/metabolismo , Acetiltransferasas N-Terminal , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Unión Proteica , Isoformas de Proteínas , Transporte de Proteínas , Factores de Transcripción/genética , Carga Tumoral/efectos de los fármacosRESUMEN
OBJECTIVE: Intestinal inflammation resulting from manipulation-induced mast cell activation is a crucial mechanism in the pathophysiology of postoperative ileus (POI). Recently it has been shown that spleen tyrosine kinase (Syk) is involved in mast cell degranulation. Therefore, we have evaluated the effect of the Syk-inhibitor GSK compound 143 (GSK143) as potential treatment to shorten POI. DESIGN: In vivo: in a mouse model of POI, the effect of the Syk inhibitor (GSK143) was evaluated on gastrointestinal transit, muscular inflammation and cytokine production. In vitro: the effect of GSK143 and doxantrazole were evaluated on cultured peritoneal mast cells (PMCs) and bone marrow derived macrophages. RESULTS: In vivo: intestinal manipulation resulted in a delay in gastrointestinal transit at t=24 h (Geometric Center (GC): 4.4 ± 0.3). Doxantrazole and GSK143 significantly increased gastrointestinal transit (GC doxantrazole (10 mg/kg): 7.2 ± 0.7; GSK143 (1 mg/kg): 7.6 ± 0.6), reduced inflammation and prevented recruitment of immune cells in the intestinal muscularis. In vitro: in PMCs, substance P (0-90 µM) and trinitrophenyl (0-4 µg/ml) induced a concentration-dependent release of ß-hexosaminidase. Pretreatment with doxantrazole and GSK143 (0.03-10 µM) concentration dependently blocked substance P and trinitrophenyl induced ß-hexosaminidase release. In addition, GSK143 was able to reduce cytokine expression in endotoxin-treated bone marrow derived macrophages in a concentration-dependent manner. CONCLUSIONS: The Syk inhibitor GSK143 reduces macrophage activation and mast cell degranulation in vitro. In addition, it inhibits manipulation-induced intestinal muscular inflammation and restores intestinal transit in mice. These findings suggest that Syk inhibition may be a new tool to shorten POI.
Asunto(s)
Compuestos de Anilina/uso terapéutico , Ileus/prevención & control , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Complicaciones Posoperatorias/prevención & control , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Compuestos de Anilina/administración & dosificación , Compuestos de Anilina/farmacología , Animales , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Tránsito Gastrointestinal/efectos de los fármacos , Ileus/fisiopatología , Activación de Macrófagos/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/antagonistas & inhibidores , Ovalbúmina/farmacología , Inhibidores de Fosfodiesterasa/uso terapéutico , Complicaciones Posoperatorias/fisiopatología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Sustancia P/antagonistas & inhibidores , Sustancia P/farmacología , Quinasa Syk , Tioxantenos/uso terapéutico , Xantonas/uso terapéuticoRESUMEN
Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC and the polymerase-associated factor complex (PAFc), are associated with the BET family of acetyl-lysine recognizing, chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias.