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1.
Nucleic Acids Res ; 52(5): 2546-2564, 2024 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-38214235

RESUMEN

Thiolutin is a natural product transcription inhibitor with an unresolved mode of action. Thiolutin and the related dithiolopyrrolone holomycin chelate Zn2+ and previous studies have concluded that RNA Polymerase II (Pol II) inhibition in vivo is indirect. Here, we present chemicogenetic and biochemical approaches to investigate thiolutin's mode of action in Saccharomyces cerevisiae. We identify mutants that alter sensitivity to thiolutin. We provide genetic evidence that thiolutin causes oxidation of thioredoxins in vivo and that thiolutin both induces oxidative stress and interacts functionally with multiple metals including Mn2+ and Cu2+, and not just Zn2+. Finally, we show direct inhibition of RNA polymerase II (Pol II) transcription initiation by thiolutin in vitro in support of classical studies that thiolutin can directly inhibit transcription in vitro. Inhibition requires both Mn2+ and appropriate reduction of thiolutin as excess DTT abrogates its effects. Pause prone, defective elongation can be observed in vitro if inhibition is bypassed. Thiolutin effects on Pol II occupancy in vivo are widespread but major effects are consistent with prior observations for Tor pathway inhibition and stress induction, suggesting that thiolutin use in vivo should be restricted to studies on its modes of action and not as an experimental tool.


Asunto(s)
Pirrolidinonas , ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Pirrolidinonas/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética , Zinc
2.
Chem Commun (Camb) ; 57(75): 9558-9561, 2021 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-34477193

RESUMEN

Amanitin is used extensively as a research tool to inhibit RNA Pol II thereby implicating its role in mRNA transcription. Recently, amanitin has gained traction as a toxic payload for targeted therapy. Here we report the first-ever photocaged amanitin analog, that is non-toxic and can be pre-loaded into cells. Light provides a means to inhibit RNA Pol II and provoke cell death on-demand.


Asunto(s)
Amanitinas/farmacología , Profármacos/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Amanitinas/síntesis química , Amanitinas/química , Animales , Células CHO , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cricetulus , Relación Dosis-Respuesta a Droga , Estructura Molecular , Procesos Fotoquímicos , Profármacos/síntesis química , Profármacos/química , ARN Polimerasa II/metabolismo
3.
Bioorg Chem ; 104: 104318, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33142427

RESUMEN

RNA polymerase II (RNA Pol II) plays a major role in gene transcription for eukaryote. One of the major modes of regulation in eukaryotes is the phosphorylation of the carboxyl-terminal domain (CTD) of RNA Pol II. The current study found that the phosphorylation of Ser2, Ser5, Ser7, Thr4 and Tyr1 among the heptapeptide repeats of CTD plays a key role in the transcription process. We therefore review the biological functions and inhibitors of kinases that phosphorylate these amino acid residues including transcriptional cyclin-dependent protein kinases (CDKs), bromodomain-containing protein 4 (BRD4), Polo-like kinases 3 (Plk3) and Abelson murine leukemia viral oncogene 1 and 2 (c-Abl1/2).


Asunto(s)
Inhibidores Enzimáticos/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Fosforilación/efectos de los fármacos , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo
4.
Mol Carcinog ; 59(9): 1076-1087, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32691884

RESUMEN

The bromodomain and extra-terminal (BET) domain inhibitor JQ1 exerts potent anticancer activity in various cancer cells. However, the resistance to BET inhibitors in leukemia stem cells limits its implication in acute myeloid leukemia (AML). High concentration of triptolide (TPL) presents anticancer activities but with adverse effects. Here, we investigated whether the combination of low-dose TPL with JQ1 could help to circumvent the dilemma of drug resistance and side effect in treating AML. AML cell lines, primary cells from 10 AML patients with different status, as well as AML mice model were subjected to different treatments and apoptotic related protein expression were evaluated. Data showed that low-dose TPL combined with JQ1 effectively killed AML cell lines and primary cells from AML patients without exerting significantly greater lethal activity against normal cells. Mechanism study revealed that low-dose TPL combined with JQ1 triggered reactive oxygen species production and induced mitochondrial-mediated apoptosis in AML cells, in which the inhibition of RNA polymerase II to downregulate c-Myc was mainly responsible for the enhanced activity of TPL in combination with JQ1. In vivo study presented that cotreatment with low-dose TPL and JQ1 significantly reduced tumor burden of the NOD/SCID mice engrafted with MOLM-13 cells. In conclusion, low-dose TPL enhanced the antitumor effect of JQ1 on AML without increasing the side effects, supporting a potential option for AML treatment.


Asunto(s)
Antineoplásicos Alquilantes/farmacología , Azepinas/farmacología , Diterpenos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Fenantrenos/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Triazoles/farmacología , Adulto , Animales , Apoptosis , Biomarcadores de Tumor , Proliferación Celular , Compuestos Epoxi/farmacología , Femenino , Humanos , Técnicas In Vitro , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Persona de Mediana Edad , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Pronóstico , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Nat Chem Biol ; 16(7): 716-724, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32572259

RESUMEN

Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription. Here we review the molecular mechanisms by which specific CDKs are thought to act at discrete steps in the transcription cycle and describe the recent emergence of transcriptional CDKs as promising drug targets in cancer. We emphasize recent advances in understanding the transcriptional CDK network that were facilitated by development and deployment of small-molecule inhibitors with increased selectivity for individual CDKs. Unexpectedly, several of these compounds have also shown selectivity in killing cancer cells, despite the seemingly universal involvement of their target CDKs during transcription in all cells. Finally, we describe remaining and emerging challenges in defining functions of individual CDKs in transcription and co-transcriptional processes and in leveraging CDK inhibition for therapeutic purposes.


Asunto(s)
Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , ARN Polimerasa II/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antineoplásicos/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Modelos Animales de Enfermedad , Humanos , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteolisis , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Transcripción Genética
6.
Cancer Sci ; 111(7): 2361-2373, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32314454

RESUMEN

To elucidate dynamic changes in native BCR-ABL and alternatively spliced tyrosine kinase inhibitor (TKI)-resistant but function-dead BCR-ABLIns35bp variant, following commencement or discontinuation of TKI therapy, each transcript was serially quantified in patients with chronic myeloid leukemia (CML) by deep sequencing. Because both transcripts were amplified together using conventional PCR system for measuring International Scale (IS), deep sequencing method was used for quantifying such BCR-ABL variants. At the initial diagnosis, 7 of 9 patients presented a small fraction of cells possessing BCR-ABLIns35bp , accounting for 0.8% of the total IS BCR-ABL, corresponding to actual BCR-ABLIns35bp value of 1.1539% IS. TKI rapidly decreased native BCR-ABL but not BCR-ABLIns35bp , leading to the initial increase in the proportion of BCR-ABLIns35bp . Thereafter, both native BCR-ABL and BCR-ABLIns35bp gradually decreased in the course of TKI treatment, whereas small populations positive for TKI-resistant BCR-ABLIns35bp continued fluctuating at low levels, possibly underestimating the molecular response (MR). Following TKI discontinuation, sequencing analysis of 54 patients revealed a rapid relapse, apparently derived from native BCR-ABL+ clones. However, IS fluctuating at low levels around MR4.0 marked a predominant persistence of cells expressing function-dead BCR-ABLIns35bp , suggesting that TKI resumption was unnecessary. We clarified the possible mechanism underlying mis-splicing BCR-ABLIns35bp , occurring at the particular pseudo-splice site within intron8, which can be augmented by TKI treatment through inhibition of RNA polymerase II phosphorylation. No mutations were found in spliceosomal genes. Therefore, monitoring IS functional BCR-ABL extracting BCR-ABLIns35bp would lead us to a correct evaluation of MR status, thus determining the adequate therapeutic intervention.


Asunto(s)
Empalme Alternativo , Proteínas de Fusión bcr-abl/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , ARN Polimerasa II/metabolismo , Adulto , Anciano , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Intrones , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/uso terapéutico , ARN Polimerasa II/antagonistas & inhibidores , Análisis de la Célula Individual
7.
Cancer Lett ; 469: 78-88, 2020 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-31629931

RESUMEN

Selective estrogen receptor modulators (SERMs) are a class of structurally diverse compounds, which have been extensively used to treat hormone-responsive cancers due to their unique partially agonistic and antagonistic properties toward estrogen receptors. Our previous studies have identified a three-dimensional SERM, oxabicycloheptene sulfonate (OBHS), as an estrogen receptor α (ERα) ligand, which is effective for the prevention and treatment of estrogen-dependent endometriosis in vivo. Here, using genome-wide ChIP-seq and RNA-seq analysis, we report that OBHS rapidly induces genome-wide ERα occupancy and acts as a partial agonist and antagonist for ERα. Interestingly, OBHS downregulates the homologous recombination and repair (HRR) modules, resulting in increased DNA damage, apoptosis and cell cycle arrest, inducing synthetic lethality with poly (ADP-ribose) polymerase (PARP) inhibitor olaparib through ERα antagonism. Mechanistically, OBHS impairs the RNA polymerase II (Pol II) loading at the promoters of estrogen-responsive HRR genes. Furthermore, combination therapy of OBHS with olaparib significantly reduces the tumour burden and delays the progression of breast cancer in vivo. Together, our studies not only characterise a novel SERM which uniquely targets the homologous recombination and repair programmes through ERα antagonism but also propose a synthetic lethal strategy by combining OBHS with PARP inhibitor olaparib for ERα-responsive cancers.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Antagonistas del Receptor de Estrógeno/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Ácidos Sulfónicos/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Chlorocebus aethiops , Secuenciación de Inmunoprecipitación de Cromatina , Antagonistas del Receptor de Estrógeno/uso terapéutico , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Ratones , Estructura Molecular , Ftalazinas/farmacología , Ftalazinas/uso terapéutico , Piperazinas/farmacología , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , RNA-Seq , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Relación Estructura-Actividad , Ácidos Sulfónicos/uso terapéutico , Mutaciones Letales Sintéticas/efectos de los fármacos , Células Vero , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Methods Mol Biol ; 1933: 33-48, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30945177

RESUMEN

Noncoding RNAs perform diverse regulatory functions in living cells. In plants, two RNA polymerase II-related enzymes, RNA polymerases IV and V (Pol IV and V), specialize in the synthesis of noncoding RNAs that silence a subset of transposable elements and genes via RNA-directed DNA methylation (RdDM). In this process, Pol IV partners with RNA-dependent RNA polymerase 2 (RDR2) to produce double-stranded RNAs that are then cut by an RNase III enzyme, Dicer-like 3 (DCL3), into 24 nt small interfering RNAs (siRNAs). The siRNAs are loaded into an Argonaute family protein, primarily AGO4, and guide the complex to complementary DNA target sequences where RdDM and repressive chromatin modifications ensue. The dependence of 24 nt siRNA biogenesis on Pol IV and RDR2 has been known for more than a decade, but the elusive pre-siRNA transcripts synthesized by Pol IV and RDR2 have only recently been identified. This chapter describes the approaches that enabled our identification of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) in Arabidopsis thaliana. These included the use of a triple Dicer mutant (dcl2 dcl3 dcl4) to cause P4R2 RNAs to accumulate, genome-wide identification and mapping of P4R2 RNAs using a modified Illumina small RNA-Seq protocol, and multiple bioinformatic pipelines for data analysis and displaying results.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Polimerasa II/genética , Precursores del ARN/genética , ARN de Planta/genética , ARN Interferente Pequeño/genética , Proteínas de Arabidopsis/antagonistas & inhibidores , Biología Computacional/métodos , Regulación de la Expresión Génica de las Plantas , ARN Polimerasa II/antagonistas & inhibidores
9.
Methods ; 159-160: 146-156, 2019 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-30769100

RESUMEN

Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.


Asunto(s)
ARN Polimerasa II/análisis , ARN Polimerasa II/metabolismo , Ubiquitinación , Animales , Daño del ADN , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/genética , Transcripción Genética , Rayos Ultravioleta , Levaduras/enzimología , Levaduras/genética , Levaduras/metabolismo
10.
Nucleic Acids Res ; 47(8): 3921-3936, 2019 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-30805632

RESUMEN

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics. Inhibition of CDK7 using THZ1 caused wide-spread loss of promoter-proximal paused RNA polymerase. This loss of 5' pausing was associated with accumulation of polymerases in the body of a large number of genes. However, there were modest effects on genes regulated by 'super-enhancers'. At the 3' ends of genes, treatment with THZ1 suppressed RNA polymerase 'read through' at the end of the last exon, which resembled a phenotype associated with a mutant RNA polymerase with slower elongation rates. Consistent with this hypothesis, polyA site-sequencing (PolyA-seq) did not detect differences in poly A sites after THZ1 treatment. PROseq analysis after short treatments with THZ1 suggested that these 3' effects were due to altered CDK7 activity at the 5' end of long genes, and were likely to be due to slower rates of elongation.


Asunto(s)
Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/genética , Regulación Leucémica de la Expresión Génica , Fenilendiaminas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , ARN Polimerasa II/genética , Región de Flanqueo 3' , Región de Flanqueo 5'/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Proliferación Celular , Óxidos N-Cíclicos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Flavonoides/farmacología , Humanos , Indolizinas , Células Mieloides/metabolismo , Células Mieloides/patología , Piperazinas/farmacología , Piperidinas/farmacología , Piperidonas/farmacología , Piridinas/farmacología , Compuestos de Piridinio/farmacología , Pirroles/farmacología , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , Translocación Genética , Quinasa Activadora de Quinasas Ciclina-Dependientes
11.
Clin Pharmacokinet ; 58(3): 363-374, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30090974

RESUMEN

BACKGROUND AND OBJECTIVES: Lurbinectedin is an inhibitor of RNA polymerase II currently under clinical development for intravenous administration as a single agent and in combination with other anti-tumor agents for the treatment of several tumor types. The objective of this work was to develop a population-pharmacokinetic model in this patient setting and to elucidate the main predictors to guide the late stages of development. METHODS: Data from 443 patients with solid and hematologic malignancies treated in six phase I and three phase II trials with lurbinectedin as a single agent or combined with other agents were included in the analysis. The potential influence of demographic, co-treatment, and laboratory characteristics on lurbinectedin pharmacokinetics was evaluated. RESULTS: The final population-pharmacokinetic model was an open three-compartment model with linear distribution and linear elimination from the central compartment. Population estimates for total plasma clearance, and apparent volume at steady state were 11.2 L/h and 438 L, respectively. Inter-individual variability was moderate for all parameters, ranging from 20.9 to 51.2%. High α-1-acid glycoprotein and C-reactive protein, and low albumin reduced clearance by 28, 20, and 20%, respectively. Co-administration of cytochrome P450 3A inhibitors reduced clearance by 30%. Combinations with other anti-tumor agents did not modify the pharmacokinetics of lurbinectedin significantly. CONCLUSION: The population-pharmacokinetic model indicated neither a dose nor time dependency, and no clinically meaningful pharmacokinetic differences were found when co-administered with other anticancer agents. A chronic inflammation pattern characterized by decreased albumin and increased C-reactive protein and α-1-acid glycoprotein levels led to high lurbinectedin exposure. Co-administration of cytochrome P450 3A inhibitors increased lurbinectedin exposure.


Asunto(s)
Carbolinas/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Neoplasias/tratamiento farmacológico , ARN Polimerasa II/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Antineoplásicos/administración & dosificación , Proteína C-Reactiva/efectos de los fármacos , Carbolinas/administración & dosificación , Estudios de Casos y Controles , Inhibidores del Citocromo P-450 CYP3A/administración & dosificación , Combinación de Medicamentos , Inhibidores Enzimáticos/administración & dosificación , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/administración & dosificación , Humanos , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Modelos Biológicos , Neoplasias/sangre , Neoplasias/etnología , Neoplasias/fisiopatología , Orosomucoide/efectos de los fármacos , Albúmina Sérica/efectos de los fármacos
12.
World J Gastroenterol ; 24(34): 3834-3848, 2018 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-30228778

RESUMEN

Colorectal cancer (CRC) is often diagnosed at an advanced stage when tumor cell dissemination has taken place. Chemo- and targeted therapies provide only a limited increase of overall survival for these patients. The major reason for clinical outcome finds its origin in therapy resistance. Escape mechanisms to both chemo- and targeted therapy remain the main culprits. Here, we evaluate major resistant mechanisms and elaborate on potential new therapies. Amongst promising therapies is α-amanitin antibody-drug conjugate targeting hemizygous p53 loss. It becomes clear that a dynamic interaction with the tumor microenvironment exists and that this dictates therapeutic outcome. In addition, CRC displays a limited response to checkpoint inhibitors, as only a minority of patients with microsatellite instable high tumors is susceptible. In this review, we highlight new developments with clinical potentials to augment responses to checkpoint inhibitors.


Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Inmunoconjugados/farmacología , Escape del Tumor/efectos de los fármacos , Alfa-Amanitina/farmacología , Alfa-Amanitina/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/mortalidad , Receptores Coestimuladores e Inhibidores de Linfocitos T/antagonistas & inhibidores , Receptores Coestimuladores e Inhibidores de Linfocitos T/inmunología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/inmunología , Humanos , Inmunoconjugados/uso terapéutico , Inmunoterapia/métodos , Inestabilidad de Microsatélites/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/uso terapéutico , ARN Polimerasa II/antagonistas & inhibidores , Resultado del Tratamiento , Escape del Tumor/genética , Escape del Tumor/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Proteína p53 Supresora de Tumor/genética
13.
J Pharm Biomed Anal ; 158: 160-165, 2018 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-29883879

RESUMEN

Lurbinectedin is a novel highly selective inhibitor of RNA polymerase II triggering caspase-dependent apoptosis of cancerous cells. This article describes the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify lurbinectedin in human plasma and urine. Plasma samples were pre-treated with 1 M aqueous ammonia after which they were brought onto supported liquid extraction (SLE) columns. Lurbinectedin was eluted from the columns using tert-butyl methyl ether (TBME). Urine was first diluted in plasma and lurbinectedin was extracted from this matrix by liquid-liquid extraction using TBME. Samples were measured by LC-MS/MS in the positive electron ion spray mode. The method was linear over 0.1-100 ng/mL and 1-1000 ng/mL in plasma and urine, respectively, with accuracies and precisions within ±15% (20% for LLOQ) and below 15% (20% for LLOQ), respectively. The method was developed to support a mass balance study in which patients received a dose of 5 mg lurbinectedin.


Asunto(s)
Antineoplásicos/análisis , Carbolinas/análisis , Compuestos Heterocíclicos de 4 o más Anillos/análisis , Neoplasias Pulmonares/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Carbolinas/farmacocinética , Carbolinas/uso terapéutico , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Ensayos Clínicos Fase III como Asunto , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Límite de Detección , Extracción Líquido-Líquido , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/orina , ARN Polimerasa II/antagonistas & inhibidores , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Carcinoma Pulmonar de Células Pequeñas/sangre , Carcinoma Pulmonar de Células Pequeñas/orina , Espectrometría de Masas en Tándem/instrumentación , Espectrometría de Masas en Tándem/métodos , Factores de Tiempo
14.
J Biol Chem ; 293(19): 7189-7194, 2018 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-29550768

RESUMEN

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.


Asunto(s)
Alfa-Amanitina/química , Inhibidores Enzimáticos/química , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/química , Elongación de la Transcripción Genética/efectos de los fármacos , Alfa-Amanitina/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Microscopía por Crioelectrón , Inhibidores Enzimáticos/farmacología , Enlace de Hidrógeno , Conformación Proteica , Homología de Secuencia de Aminoácido , Porcinos
15.
Nat Commun ; 8(1): 1914, 2017 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-29203770

RESUMEN

TFIIS-like transcript cleavage factors enhance the processivity and fidelity of archaeal and eukaryotic RNA polymerases. Sulfolobus solfataricus TFS1 functions as a bona fide cleavage factor, while the paralogous TFS4 evolved into a potent RNA polymerase inhibitor. TFS4 destabilises the TBP-TFB-RNAP pre-initiation complex and inhibits transcription initiation and elongation. All inhibitory activities are dependent on three lysine residues at the tip of the C-terminal zinc ribbon of TFS4; the inhibition likely involves an allosteric component and is mitigated by the basal transcription factor TFEα/ß. A chimeric variant of yeast TFIIS and TFS4 inhibits RNAPII transcription, suggesting that the molecular basis of inhibition is conserved between archaea and eukaryotes. TFS4 expression in S. solfataricus is induced in response to infection with the S ulfolobus turreted icosahedral virus. Our results reveal a compelling functional diversification of cleavage factors in archaea, and provide novel insights into transcription inhibition in the context of the host-virus relationship.


Asunto(s)
ARN Polimerasa II/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sulfolobus solfataricus/metabolismo , Factores de Transcripción/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción TFII/metabolismo , Transcripción Genética
16.
Proc Natl Acad Sci U S A ; 114(46): 12172-12177, 2017 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-29087308

RESUMEN

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.


Asunto(s)
Antineoplásicos Alquilantes/farmacología , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/química , ARN Polimerasa II/química , Sesquiterpenos/farmacología , Compuestos de Espiro/farmacología , Antineoplásicos Alquilantes/química , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Aductos de ADN/química , Aductos de ADN/metabolismo , Daño del ADN , ADN de Neoplasias/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/patología , Humanos , Cinética , Modelos Moleculares , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Sesquiterpenos/química , Compuestos de Espiro/química
17.
Genome Biol ; 18(1): 134, 2017 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-28687080

RESUMEN

BACKGROUND: Retrotransposons play a central role in plant evolution and could be a powerful endogenous source of genetic and epigenetic variability for crop breeding. To ensure genome integrity several silencing mechanisms have evolved to repress retrotransposon mobility. Even though retrotransposons fully depend on transcriptional activity of the host RNA polymerase II (Pol II) for their mobility, it was so far unclear whether Pol II is directly involved in repressing their activity. RESULTS: Here we show that plants defective in Pol II activity lose DNA methylation at repeat sequences and produce more extrachromosomal retrotransposon DNA upon stress in Arabidopsis and rice. We demonstrate that combined inhibition of both DNA methylation and Pol II activity leads to a strong stress-dependent mobilization of the heat responsive ONSEN retrotransposon in Arabidopsis seedlings. The progenies of these treated plants contain up to 75 new ONSEN insertions in their genome which are stably inherited over three generations of selfing. Repeated application of heat stress in progeny plants containing increased numbers of ONSEN copies does not result in increased activation of this transposon compared to control lines. Progenies with additional ONSEN copies show a broad panel of environment-dependent phenotypic diversity. CONCLUSIONS: We demonstrate that Pol II acts at the root of transposon silencing. This is important because it suggests that Pol II can regulate the speed of plant evolution by fine-tuning the amplitude of transposon mobility. Our findings show that it is now possible to study induced transposon bursts in plants and unlock their use to induce epigenetic and genetic diversity for crop breeding.


Asunto(s)
Fitomejoramiento , ARN Polimerasa II/antagonistas & inhibidores , Retroelementos , Arabidopsis/genética , Metilación de ADN , ADN de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Oryza/genética , ARN Polimerasa II/metabolismo
18.
J Virol ; 91(19)2017 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-28747493

RESUMEN

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.


Asunto(s)
Septicemia Hemorrágica Viral/patología , Novirhabdovirus/crecimiento & desarrollo , Novirhabdovirus/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Replicación Viral/genética , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Cyprinidae , Enfermedades de los Peces/virología , Células HEK293 , Septicemia Hemorrágica Viral/virología , Humanos , Inmunidad Innata/inmunología , Interferón Tipo I/inmunología , Fosforilación/genética , Regiones Promotoras Genéticas/genética , ARN/genética , ARN Polimerasa II/antagonistas & inhibidores , Virus 40 de los Simios/genética , Transcripción Genética/fisiología
19.
Methods ; 124: 89-99, 2017 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-28651964

RESUMEN

In this paper, we introduce a novel computational method for constructing protein networks based on reverse phase protein array (RPPA) data to identify complex patterns in protein signaling. The method is applied to phosphoproteomic profiles of basal expression and activation/phosphorylation of 76 key signaling proteins in three breast cancer cell lines (MCF7, LCC1, and LCC9). Temporal RPPA data are acquired at 48h, 96h, and 144h after knocking down four genes in separate experiments. These genes are selected from a previous study as important determinants for breast cancer survival. Interaction networks are constructed by analyzing the expression levels of protein pairs using a multivariate analysis of variance model. A new scoring criterion is introduced to determine relevant protein pairs. Through a network topology based analysis, we search for wiring patterns to identify key proteins that are associated with significant changes in expression levels across various experimental conditions.


Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Neoplasias/genética , Análisis por Matrices de Proteínas/estadística & datos numéricos , Procesamiento Proteico-Postraduccional , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteína 61 Rica en Cisteína/antagonistas & inhibidores , Proteína 61 Rica en Cisteína/genética , Proteína 61 Rica en Cisteína/metabolismo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7 , Análisis Multivariante , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Polimerasa II/antagonistas & inhibidores , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
20.
Biochemistry ; 56(24): 3008-3018, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28514164

RESUMEN

The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting 1.6 ± 0.5% nominal lesion bypass. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR because each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.


Asunto(s)
Guanidinas/farmacología , Guanosina/análogos & derivados , Hidantoínas/farmacología , ARN Polimerasa II/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Elongación de la Transcripción Genética/efectos de los fármacos , Daño del ADN , Reparación del ADN , Guanidinas/síntesis química , Guanidinas/química , Guanosina/síntesis química , Guanosina/química , Guanosina/farmacología , Células HeLa , Humanos , Hidantoínas/síntesis química , Hidantoínas/química , Conformación Molecular , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-Actividad
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