Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Science ; 377(6601): 47-56, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35771919

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway.


Assuntos
Dieta , Leucina , Fígado , Alvo Mecanístico do Complexo 1 de Rapamicina , Sestrinas , Tecido Adiposo Branco/enzimologia , Animais , Leucina/metabolismo , Fígado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Sestrinas/metabolismo , Transdução de Sinais
2.
J Cell Biol ; 221(2)2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34889941

RESUMO

Prostate cancer aggressiveness and metastatic potential are influenced by gene expression and genomic aberrations, features that can be influenced by the 3D structure of chromosomes inside the nucleus. Using chromosome conformation capture (Hi-C), we conducted a systematic genome architecture comparison on a cohort of cell lines that model prostate cancer progression, from normal epithelium to bone metastasis. We describe spatial compartment identity (A-open versus B-closed) changes with progression in these cell lines and their relation to gene expression changes in both cell lines and patient samples. In particular, 48 gene clusters switch from the B to the A compartment, including androgen receptor, WNT5A, and CDK14. These switches are accompanied by changes in the structure, size, and boundaries of topologically associating domains (TADs). Further, compartment changes in chromosome 21 are exacerbated with progression and may explain, in part, the genesis of the TMPRSS2-ERG translocation. These results suggest that discrete 3D genome structure changes play a deleterious role in prostate cancer progression. .


Assuntos
Cromossomos Humanos/metabolismo , Progressão da Doença , Modelos Biológicos , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Cromatina/metabolismo , Estudos de Coortes , Genes Neoplásicos , Genoma Humano , Humanos , Masculino , Metástase Neoplásica , Neoplasias da Próstata/genética , Serina Endopeptidases/metabolismo , Ativação Transcricional/genética
3.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33483422

RESUMO

In mammalian cells, nutrients and growth factors signal through an array of upstream proteins to regulate the mTORC1 growth control pathway. Because the full complement of these proteins has not been systematically identified, we developed a FACS-based CRISPR-Cas9 genetic screening strategy to pinpoint genes that regulate mTORC1 activity. Along with almost all known positive components of the mTORC1 pathway, we identified many genes that impact mTORC1 activity, including DCAF7, CSNK2B, SRSF2, IRS4, CCDC43, and HSD17B10 Using the genome-wide screening data, we generated a focused sublibrary containing single guide RNAs (sgRNAs) targeting hundreds of genes and carried out epistasis screens in cells lacking nutrient- and stress-responsive mTORC1 modulators, including GATOR1, AMPK, GCN2, and ATF4. From these data, we pinpointed mitochondrial function as a particularly important input into mTORC1 signaling. While it is well appreciated that mitochondria signal to mTORC1, the mechanisms are not completely clear. We find that the kinases AMPK and HRI signal, with varying kinetics, mitochondrial distress to mTORC1, and that HRI acts through the ATF4-dependent up-regulation of both Sestrin2 and Redd1. Loss of both AMPK and HRI is sufficient to render mTORC1 signaling largely resistant to mitochondrial dysfunction induced by the ATP synthase inhibitor oligomycin as well as the electron transport chain inhibitors piericidin and antimycin. Taken together, our data reveal a catalog of genes that impact the mTORC1 pathway and clarify the multifaceted ways in which mTORC1 senses mitochondrial dysfunction.


Assuntos
Fator 4 Ativador da Transcrição/genética , Edição de Genes/métodos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mitocôndrias/genética , Proteínas Serina-Treonina Quinases/genética , 3-Hidroxiacil-CoA Desidrogenases/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aminoácidos/deficiência , Aminoácidos/farmacologia , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Meios de Cultura/química , Meios de Cultura/farmacologia , Regulação da Expressão Gênica , Genoma Humano , Glucose/deficiência , Glucose/farmacologia , Células HEK293 , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligomicinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Transdução de Sinais , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
4.
Endocr Relat Cancer ; 27(7): R255-R265, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32369771

RESUMO

Many solid tumors metastasize to bone, but only prostate cancer has bone as a single, dominant metastatic site. Recently, the FGF axis has been implicated in cancer progression in some tumors and mounting evidence indicate that it mediates prostate cancer bone metastases. The FGF axis has an important role in bone biology and mediates cell-to-cell communication. Therefore, we discuss here basic concepts of bone biology, FGF signaling axis, and FGF axis function in adult bone, to integrate these concepts in our current understanding of the role of FGF axis in bone metastases.


Assuntos
Neoplasias Ósseas/secundário , Fatores de Crescimento de Fibroblastos/metabolismo , Feminino , Humanos , Masculino , Transdução de Sinais
5.
ACS Chem Biol ; 13(9): 2438-2448, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30102854

RESUMO

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 µM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.


Assuntos
Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Benzodiazepinonas/química , Benzodiazepinonas/farmacologia , Proteínas de Ciclo Celular , Cristalografia por Raios X , Células HeLa , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/química , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Polifarmacologia , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
6.
Nat Chem Biol ; 14(5): 431-441, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29581585

RESUMO

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.


Assuntos
Sistemas CRISPR-Cas , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína 1A de Ligação a Tacrolimo/química , Fatores de Transcrição/genética , Alelos , Animais , Proteínas de Ciclo Celular , Proliferação de Células , Citoplasma/metabolismo , Dimerização , Técnicas de Introdução de Genes , Células HEK293 , Homeostase , Humanos , Ligantes , Camundongos , Mutação , Células NIH 3T3 , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos , Proteólise , Proteômica , Transdução de Sinais , Transgenes
7.
Nat Commun ; 9(1): 53, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29302039

RESUMO

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin's favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.


Assuntos
Benzamidas/farmacologia , Proteínas Correpressoras/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Melanoma/tratamento farmacológico , Proteínas do Tecido Nervoso/antagonistas & inibidores , Piridinas/farmacologia , Tranilcipromina/farmacologia , Idoso , Animais , Antineoplásicos , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Correpressoras/metabolismo , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Histona Desacetilases/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Elife ; 62017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28926338

RESUMO

Thorough preclinical target validation is essential for the success of drug discovery efforts. In this study, we combined chemical and genetic perturbants, including the development of a novel selective maternal embryonic leucine zipper kinase (MELK) inhibitor HTH-01-091, CRISPR/Cas9-mediated MELK knockout, a novel chemical-induced protein degradation strategy, RNA interference and CRISPR interference to validate MELK as a therapeutic target in basal-like breast cancers (BBC). In common culture conditions, we found that small molecule inhibition, genetic deletion, or acute depletion of MELK did not significantly affect cellular growth. This discrepancy to previous findings illuminated selectivity issues of the widely used MELK inhibitor OTSSP167, and potential off-target effects of MELK-targeting short hairpins. The different genetic and chemical tools developed here allow for the identification and validation of any causal roles MELK may play in cancer biology, which will be required to guide future MELK drug discovery efforts. Furthermore, our study provides a general framework for preclinical target validation.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células , Proteínas Serina-Treonina Quinases/análise , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética
9.
Mol Cell ; 67(1): 5-18.e19, 2017 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-28673542

RESUMO

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular , Quinase 9 Dependente de Ciclina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação Leucêmica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Complexos Multiproteicos , Proteínas Nucleares/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Estabilidade Proteica , Proteólise , RNA Polimerase II/metabolismo , Fatores de Tempo , Elongação da Transcrição Genética/efeitos dos fármacos , Fatores de Transcrição/genética , Transfecção , Ubiquitina-Proteína Ligases , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Oncotarget ; 8(27): 44447-44464, 2017 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-28591703

RESUMO

Whether vitamin D is chemopreventive and/or has potential therapeutically in prostate cancer is unresolved. One confounding factor is that many prostate cancers express a TMPRSS2:ERG fusion gene whose expression is increased both by androgens and by vitamin D receptor (VDR) activation. Two challenges that limit VDR agonist use clinically are hypercalcemia and the cooperation of VDR with ERG to hyper-induce the 1α,25-dihydroxyvitamin D3 metabolizing enzyme, CYP24A1, thus reducing VDR activity. Using the VCaP TMPRSS2:ERG positive cell line as a model, we found that a nonsecosteroidal CYP24A1 resistant VDR agonist, VDRM2, substantially reduces growth of xenograft tumors without inducing hypercalcemia. Utilizing next generation RNA sequencing, we found a very high overlap of 1,25D(OH)2D3 and VDRM2 regulated genes and by drawing upon previously published datasets to create an ERG signature, we found activation of VDR does not induce ERG activity above the already high basal levels present in VCaP cells. Moreover, we found VDR activation opposes 8 of the 10 most significant ERG regulated Hallmark gene set collection pathways from Gene Set Enrichment Analysis (GSEA). Thus, a CYP24A1 resistant VDR agonist may be beneficial for treatment of TMPRSS2:ERG positive prostate cancer; one negative consequence of TMPRSS2:ERG expression is inactivation of VDR signaling.


Assuntos
Regulação Neoplásica da Expressão Gênica , Receptores de Calcitriol/agonistas , Serina Endopeptidases/genética , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Masculino , Camundongos , Modelos Biológicos , Proteína Oncogênica p55(v-myc)/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma , Carga Tumoral , Vitamina D/análogos & derivados , Vitamina D/química , Vitamina D/farmacologia , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nature ; 543(7644): 270-274, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28241139

RESUMO

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.


Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia/genética , Leucemia/metabolismo , Domínios Proteicos , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Edição de Genes , Genoma/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteólise , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética
12.
Isr J Chem ; 57(3-4): 319-330, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30760938

RESUMO

The synthesis of biotinylated conjugates of synthetic analogues of the potent and selective histone deacetylase (HDAC) inhibitor largazole is reported. The thiazole moiety of the parent compound's cap group was derivatized to allow the chemical conjugation to biotin. The derivatized largazole analogues were assayed across a panel of HDACs 1-9 and retained potent and selective inhibitory activity towards the class I HDAC isoforms. The biotinylated conjugate was further shown to pull down HDACs 1, 2, and 3.

13.
ChemMedChem ; 11(23): 2575-2581, 2016 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-27862999

RESUMO

Evaluating the engagement of a small molecule ligand with a protein target in cells provides useful information for chemical probe optimization and pharmaceutical development. While several techniques exist that can be performed in a low-throughput manner, systematic evaluation of large compound libraries remains a challenge. In-cell engagement measurements are especially useful when evaluating compound classes suspected to target multiple cellular factors. In this study we used a bioluminescent resonant energy transfer assay to assess bromodomain engagement by a compound series containing bromodomain- and kinase-biasing polypharmacophores based on the known dual BRD4 bromodomain/PLK1 kinase inhibitor BI2536. With this assay, we discovered several novel agents with bromodomain-selective specificity profiles and cellular activity. Thus, this platform aids in distinguishing molecules whose cellular activity is difficult to assess due to polypharmacologic effects.


Assuntos
Proteínas Nucleares/metabolismo , Pteridinas/química , Fatores de Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Humanos , Medições Luminescentes , Proteínas Nucleares/antagonistas & inibidores , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/metabolismo , Pteridinas/toxicidade , Fatores de Transcrição/antagonistas & inibidores , Quinase 1 Polo-Like
14.
Nat Chem Biol ; 12(12): 1089-1096, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27775715

RESUMO

Cellular signaling is often propagated by multivalent interactions. Multivalency creates avidity, allowing stable biophysical recognition. Multivalency is an attractive strategy for achieving potent binding to protein targets, as the affinity of bivalent ligands is often greater than the sum of monovalent affinities. The bromodomain and extraterminal domain (BET) family of transcriptional coactivators features tandem bromodomains through which BET proteins bind acetylated histones and transcription factors. All reported antagonists of the BET protein BRD4 bind in a monovalent fashion. Here we describe, to our knowledge for the first time, a bivalent BET bromodomain inhibitor-MT1-which has unprecedented potency. Biophysical and biochemical studies suggest MT1 is an intramolecular bivalent BRD4 binder that is more than 100-fold more potent, in cellular assays, than the corresponding monovalent antagonist, JQ1. MT1 significantly (P < 0.05) delayed leukemia progression in mice, as compared to JQ1. These data qualify a powerful chemical probe for BET bromodomains and a rationale for further development of multidomain inhibitors of epigenetic reader proteins.


Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Desenho de Fármacos , Leucemia/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Azepinas/administração & dosagem , Azepinas/química , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Leucemia/patologia , Ligantes , Camundongos , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Triazóis/administração & dosagem , Triazóis/química
15.
Nature ; 529(7586): 413-417, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26735014

RESUMO

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.


Assuntos
Azepinas/farmacologia , Azepinas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Nucleares/antagonistas & inibidores , Estrutura Terciária de Proteína/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologia , Triazóis/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Ligação Competitiva/efeitos dos fármacos , Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cromatina/genética , Cromatina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Humanos , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Proteômica , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Protein Sci ; 25(4): 852-64, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26808198

RESUMO

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra- and intermolecular binding assays of proteins containing the domains.


Assuntos
Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bases de Dados de Ácidos Nucleicos , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Desdobramento de Proteína
17.
Curr Protoc Chem Biol ; 7(4): 263-278, 2015 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-26629616

RESUMO

Bromodomain-containing proteins have emerged as desirable targets for anti-neoplastic and anti-inflammatory drug discovery. Toward the development of selective inhibitors of the BET family of bromodomains, we optimized bead-based assays to detect interactions between bromodomains and poly-acetylated histone peptides. Donor and acceptor beads bound to target and ligand are brought into proximity by this protein-protein interaction. After laser illumination, singlet oxygen evolved from donor beads travels to the spatially close acceptor beads, resulting in chemiluminesence. This AlphaScreen assay has proven amendable to high-throughput screening, secondary validation, and specificity profiling during lead discovery and optimization. Here we report our protocol for assay development to measure inhibition of ligand binding to bromodomain-containing protein 4 (BRD4). We discuss the discovery of an appropriate probe, optimization of bead, probe, and protein concentrations, and the derivation of protein-probe inhibition curves. Finally, we explore the implementation of this technology for high-throughput screening of potential BRD4 inhibitors.


Assuntos
Sondas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Azepinas/química , Azepinas/metabolismo , Biotina/química , Proteínas de Ciclo Celular , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Ligantes , Sondas Moleculares/química , Proteínas Nucleares/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Triazóis/química , Triazóis/metabolismo
18.
Oncotarget ; 6(31): 31997-2012, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26378018

RESUMO

Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.


Assuntos
Processamento Alternativo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Metabolômica , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Apoptose , Western Blotting , Movimento Celular , Proliferação de Células , Humanos , Técnicas Imunoenzimáticas , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição Gênica , Células Tumorais Cultivadas
19.
Pharm Pat Anal ; 4(4): 261-84, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26174566

RESUMO

Gene regulatory pathways comprise an emerging and active area of chemical probe discovery and investigational drug development. Emerging insights from cancer genome sequencing and chromatin biology have identified leveraged opportunities for development of chromatin-directed small molecules as cancer therapies. At present, only six agents in two epigenetic target classes have been approved by the US FDA, limited to treatment of hematological malignancies. Recently, new classes of epigenetic inhibitors have appeared in literatures. First-in-class compounds have successfully transitioned to clinical investigation, importantly also in solid tumors and pediatric malignancies. This review considers patent applications for small-molecule inhibitors of selected epigenetic targets from 2010 to 2014. Included are exemplary classes of chromatin-associated epigenomic writers (DOT1L and EZH2), erasers (LSD1) and readers (BRD4).


Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Epigenômica/métodos , Neoplasias/tratamento farmacológico , Patentes como Assunto , Animais , Antineoplásicos/química , Sistemas de Liberação de Medicamentos/tendências , Epigenômica/tendências , Humanos , Neoplasias/genética
20.
Science ; 348(6241): 1376-81, 2015 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-25999370

RESUMO

The development of effective pharmacological inhibitors of multidomain scaffold proteins, notably transcription factors, is a particularly challenging problem. In part, this is because many small-molecule antagonists disrupt the activity of only one domain in the target protein. We devised a chemical strategy that promotes ligand-dependent target protein degradation using as an example the transcriptional coactivator BRD4, a protein critical for cancer cell growth and survival. We appended a competitive antagonist of BET bromodomains to a phthalimide moiety to hijack the cereblon E3 ubiquitin ligase complex. The resultant compound, dBET1, induced highly selective cereblon-dependent BET protein degradation in vitro and in vivo and delayed leukemia progression in mice. A second series of probes resulted in selective degradation of the cytosolic protein FKBP12. This chemical strategy for controlling target protein stability may have implications for therapeutically targeting previously intractable proteins.


Assuntos
Azepinas/farmacologia , Desenho de Fármacos , Proteínas Nucleares/metabolismo , Peptídeo Hidrolases/metabolismo , Ftalimidas/química , Proteólise/efeitos dos fármacos , Talidomida/análogos & derivados , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Azepinas/química , Azepinas/uso terapêutico , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cristalografia por Raios X , Modelos Animais de Doenças , Leucemia Promielocítica Aguda/tratamento farmacológico , Ligantes , Camundongos , Terapia de Alvo Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteína 1A de Ligação a Tacrolimo/metabolismo , Talidomida/química , Talidomida/farmacologia , Talidomida/uso terapêutico , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Ubiquitina-Proteína Ligases/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...