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1.
Cell Chem Biol ; 26(10): 1380-1392.e6, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31378711

RESUMO

Gene expression signature-based inference of functional connectivity within and between genetic perturbations, chemical perturbations, and disease status can lead to the development of actionable hypotheses for gene function, chemical modes of action, and disease treatment strategies. Here, we report a FuSiOn-based genome-wide integration of hypomorphic cellular phenotypes that enables functional annotation of gene network topology, assignment of mechanistic hypotheses to genes of unknown function, and detection of cooperativity among cell regulatory systems. Dovetailing genetic perturbation data with chemical perturbation phenotypes allowed simultaneous generation of mechanism of action hypotheses for thousands of uncharacterized natural products fractions (NPFs). The predicted mechanism of actions span a broad spectrum of cellular mechanisms, many of which are not currently recognized as "druggable." To enable use of FuSiOn as a hypothesis generation resource, all associations and analyses are available within an open source web-based GUI (http://fusion.yuhs.ac).

2.
J Nat Prod ; 82(5): 1301-1311, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31084028

RESUMO

Natural products continue to provide a platform to study biological systems. A bioguided study of cancer cell models led us to a new member of the jatrophane natural products from Jatropha gossypiifolia, which was independently identified and characterized as jatrogossone A (1). Purification and structure elucidation was performed by column chromatography and high-performance liquid chromatography-mass spectrometry and NMR techniques, and the structure was confirmed via X-ray crystallography. The unique molecular scaffold of jatrogossone A prompted an evaluation of its mode of action. Cytotoxicity assays demonstrated that jatrogossone A displays selective antiproliferative activity against cancer cell models in the low micromolar range with a therapeutic window. Jatrogossone A (1) affects mitochondrial membrane potential (ΔΨm) in a time- and dose-dependent manner. This natural product induces radical oxygen species (ROS) selectively in cancer cellular models, with minimal ROS induction in noncancerous cells. Compound 1 induces ROS in the mitochondria, as determined by colocalization studies, and it induces mitophagy. It promotes also in vitro cell death by causing cell arrest at the G2/M stage, caspase (3/7) activation, and PARP-1 cleavage. The combined findings provide a potential mechanism by which 1 relies on upregulation of mitochondrial ROS to potentiate cytotoxic effects through intracellular signaling.

3.
Eur J Med Chem ; 164: 391-398, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30611980

RESUMO

Although pediatric leukemia is generally treatable, certain leukemic subtypes face poor prognosis in the clinic suggesting new selective therapeutic agents are needed. Thus, to identify selective apoptosis inducers, a small-molecule library screening approach was conducted using an isogenic leukemic murine p185+ B-ALL cell line pair (BCR-ABL-WT and the BAX/BAK deficient BCR-ABL-DKO). Gratifyingly, the investigation revealed several compounds featuring substituted aromatic five-membered-ring heterocycles with significant activity against murine and human leukemic cellular models. The identified compounds represent potentially novel antileukemic molecular scaffolds exemplified by compounds 1, 2 and 7, which demonstrated EC50 values in the nanomolar and low micromolar range against various leukemia subtypes (SUP-B15, KOPN-8, NALM-06, UoC-B1 cellular models) and pro-apoptotic properties in solid tumor cell models (MDA-MB-231, SUM149) with ample therapeutic index in normal cells. Herein, we highlight compounds 1, 2 and 7 which promote cell death mediated by caspase 3/7 induction. Our study establishes a strategic platform for the development of potent and selective anti-leukemic agents.


Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Leucemia/tratamento farmacológico , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Caspases/genética , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Indução Enzimática/efeitos dos fármacos , Compostos Heterocíclicos/química , Humanos , Camundongos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Índice Terapêutico
4.
Mar Drugs ; 15(3)2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-28294973

RESUMO

Chemicals found in nature have evolved over geological time scales to productively interact with biological molecules, and thus represent an effective resource for pharmaceutical development. Marine-derived bacteria are rich sources of chemically diverse, bioactive secondary metabolites, but harnessing this diversity for biomedical benefit is limited by challenges associated with natural product purification and determination of biochemical mechanism. Using Functional Signature Ontology (FUSION), we report the parallel isolation and characterization of a marine-derived natural product, N6,N6-dimethyladenosine, that robustly inhibits AKT signaling in a variety of non-small cell lung cancer cell lines. Upon validation of the elucidated structure by comparison with a commercially available sample, experiments were initiated to understand the small molecule's breadth of effect in a biological setting. One such experiment, a reverse phase protein array (RPPA) analysis of >50 kinases, indicated a specific cellular response to treatment. In all, leveraging the FUSION platform allowed for the rapid generation and validation of a biological mechanism of action hypothesis for an unknown natural product and permitted accelerated purification of the bioactive component from a chemically complex fraction.


Assuntos
Organismos Aquáticos/química , Bactérias/química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ontologias Biológicas , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico
5.
Bioorg Med Chem Lett ; 26(16): 3923-7, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27426302

RESUMO

The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/toxicidade , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/toxicidade , Relação Estrutura-Atividade , Temperatura de Transição , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Nat Chem Biol ; 11(6): 401-8, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25867045

RESUMO

Modern cancer treatment employs many effective chemotherapeutic agents originally discovered from natural sources. The cyclic depsipeptide didemnin B has demonstrated impressive anticancer activity in preclinical models. Clinical use has been approved but is limited by sparse patient responses combined with toxicity risk and an unclear mechanism of action. From a broad-scale effort to match antineoplastic natural products to their cellular activities, we found that didemnin B selectively induces rapid and wholesale apoptosis through dual inhibition of PPT1 and EEF1A1. Furthermore, empirical discovery of a small panel of exceptional responders to didemnin B allowed the generation of a regularized regression model to extract a sparse-feature genetic biomarker capable of predicting sensitivity to didemnin B. This may facilitate patient selection in a fashion that could enhance and expand the therapeutic application of didemnin B against neoplastic disease.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Depsipeptídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Farmacogenética , Apoptose/genética , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Estudo de Associação Genômica Ampla , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Tioléster Hidrolases , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
7.
Cell ; 160(4): 715-728, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25679763

RESUMO

AMP-activated protein kinase (AMPK) is a master sensor and regulator of cellular energy status. Upon metabolic stress, AMPK suppresses anabolic and promotes catabolic processes to regain energy homeostasis. Cancer cells can occasionally suppress the growth-restrictive AMPK pathway by mutation of an upstream regulatory kinase. Here, we describe a widespread mechanism to suppress AMPK through its ubiquitination and degradation by the cancer-specific MAGE-A3/6-TRIM28 ubiquitin ligase. MAGE-A3 and MAGE-A6 are highly similar proteins normally expressed only in the male germline but frequently re-activated in human cancers. MAGE-A3/6 are necessary for cancer cell viability and are sufficient to drive tumorigenic properties of non-cancerous cells. Screening for targets of MAGE-A3/6-TRIM28 revealed that it ubiquitinates and degrades AMPKα1. This leads to inhibition of autophagy, activation of mTOR signaling, and hypersensitization to AMPK agonists, such as metformin. These findings elucidate a germline mechanism commonly hijacked in cancer to suppress AMPK.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antígenos de Neoplasias/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Metabolismo Energético , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Alinhamento de Sequência , Transdução de Sinais , Testículo/metabolismo
8.
Sci Signal ; 6(297): ra90, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24129700

RESUMO

A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA (miRNA) libraries. With this strategy, we matched proteins and miRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected siRNAs, miRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets.


Assuntos
Produtos Biológicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Transcriptoma/efeitos dos fármacos , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Bactérias/química , Bactérias/classificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Linhagem Celular Tumoral , Células Cultivadas , Análise por Conglomerados , Biologia Computacional/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Células HCT116 , Humanos , Invertebrados/química , Células MCF-7 , Biologia Marinha , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Estrutura Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA
9.
J Am Chem Soc ; 135(36): 13387-92, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-23984625

RESUMO

Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase involved in a variety of cellular response pathways, including regulation of cell growth, proliferation, and motility. Using a newly developed platform to identify the signaling pathway/molecular target of natural products, we identified a family of alkaloid natural products, discoipyrroles A-D (1-4), from Bacillus hunanensis that inhibit the DDR2 signaling pathway. The structure of 1-4, determined by detailed two-dimensional (2D) NMR methods and confirmed by X-ray crystallographic analysis has an unusual 3H-benzo[d]pyrrolo][1,3]oxazine-3,5-dione core. Discoipyrroles A-D potently inhibit DDR2 dependent migration of BR5 fibroblasts and show selective cytotoxicity to DDR2 mutant lung cancer cell lines (IC50 120-400 nM). Examination of the biosynthesis has led to the conclusion that the discoipyrroles are formed through a nonenzymatic process, leading to a one-pot total synthesis of 1.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Bacillus/química , Produtos Biológicos/farmacologia , Fibroblastos/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Pirrolidinonas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Mitogênicos/antagonistas & inibidores , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Receptores com Domínio Discoidina , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/citologia , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/isolamento & purificação , Humanos , Modelos Moleculares , Estrutura Molecular , Pirrolidinonas/química , Pirrolidinonas/isolamento & purificação , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Mitogênicos/genética , Receptores Mitogênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade
10.
Nat Rev Cancer ; 11(1): 50-8, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21150934

RESUMO

Cancer is a complex disease in which cells have circumvented normal restraints on tissue growth and have acquired complex abnormalities in their genomes, posing a considerable challenge to identifying the pathways and mechanisms that drive fundamental aspects of the malignant phenotype. Genetic analyses of the normal development of the nematode Caenorhabditis elegans have revealed evolutionarily conserved mechanisms through which individual cells establish their fates, and how they make and execute the decision to survive or undergo programmed cell death. The pathways identified through these studies have mammalian counterparts that are co-opted by malignant cells. Effective cancer drugs now target some of these pathways, and more are likely to be discovered.


Assuntos
Caenorhabditis elegans/fisiologia , Morte Celular , Linhagem da Célula , Animais , Pesquisa Biomédica , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Oncologia
11.
Dev Biol ; 329(2): 374-85, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19254707

RESUMO

Mutations that aberrantly activate trithorax-group proteins, Hox transcription factors and TALE-class Hox cofactors promote leukemogenesis, but their target genes critical for leukemogenesis remain largely unknown. Through genetic analyses in C. elegans, we find that the trithorax-group gene lin-59 and the TALE-class Hox cofactor unc-62 are required for survival of the VC motor neurons. With the goal of providing a model for how aberrantly active Hox complexes might promote leukemia, we elucidate the mechanism through which these new inhibitors of programmed cell death act: lin-59 maintains transcription of the Hox gene lin-39, while unc-62 promotes nuclear localization of the TALE-class Hox cofactor ceh-20. A LIN-39/CEH-20 complex binds the promoter of the pro-apoptotic BH3-only gene egl-1, repressing its transcription and ensuring survival of the VC neurons. In the absence of this regulatory mechanism, egl-1 is transcribed and the VC neurons die. Furthermore, ectopic expression of the Hox gene lin-39, as occurs for human Hox genes in leukemia, is sufficient to block death of some cells. This work identifies BH3-only pro-apoptotic genes as targets of Hox-mediated repression and suggests that aberrant activation of Hox networks may promote leukemia in part by inhibiting apoptosis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Sobrevivência Celular/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas Repressoras/genética , Apoptose/genética , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Homeodomínio/genética , Humanos , Neurônios Motores/citologia , Mutagênese Sítio-Dirigida , Transcrição Genética
12.
Development ; 133(4): 641-50, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16421192

RESUMO

Hox genes are crucial determinants of cell fates and of body morphology of animals; mutations affecting these genes result in abnormal patterns of programmed cell death. How Hox genes regulate programmed cell death is an important and poorly understood aspect of normal development. In the nematode C. elegans, the Hox gene mab-5 is required for the programmed cell deaths of two lineally related cells generated in the P11 and P12 lineages. We show here that in the P11 lineage, a complex between MAB-5 and the Pbx homolog CEH-20 directly regulates transcription of the BH3 domain gene egl-1 to initiate programmed cell death; in the P12 lineage, mab-5 and ceh-20 apparently act indirectly to initiate programmed cell death. Direct regulation of programmed cell death may be an evolutionarily ancient and conserved function of Hox genes.


Assuntos
Apoptose/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Mutação , Proteínas Repressoras/genética , Fatores de Transcrição/genética
13.
J Cell Biol ; 171(6): 925-30, 2005 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-16344307

RESUMO

Overexpression studies have identified X-linked inhibitor of apoptosis protein (XIAP) as a potent inhibitor of caspases. However, the exact function of endogenous XIAP in regulating mammalian apoptosis is less clear. Endogenous XIAP strictly regulates cytochrome c-dependent caspase activation in sympathetic neurons but not in many mitotic cells. We report that postmitotic cardiomyocytes, unlike fibroblasts, are remarkably resistant to cytosolic microinjection of cytochrome c. The cardiomyocyte resistance to cytochrome c is mediated by endogenous XIAP, as XIAP-deficient cardiomyocytes die rapidly with cytosolic cytochrome c alone. Importantly, we found that cardiomyocytes, like neurons, have markedly reduced Apaf-1 levels and that this decrease in Apaf-1 is directly linked to the tight regulation of caspase activation by XIAP. These data identify an important function of XIAP in cardiomyocytes and point to a striking similarity in the regulation of apoptosis in postmitotic cells.


Assuntos
Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Fator Apoptótico 1 Ativador de Proteases , Caspases/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Citocromos c/farmacologia , Citosol/metabolismo , Camundongos , Microinjeções , Microscopia de Fluorescência , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Ratos , Fatores de Tempo , Transfecção , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/farmacologia
14.
Mol Cell Biol ; 24(23): 10289-99, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15542838

RESUMO

Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.


Assuntos
Apoptose , Citocromos c/metabolismo , Proteínas de Fusão bcr-abl/fisiologia , Mitocôndrias/metabolismo , Animais , Fator Apoptótico 1 Ativador de Proteases , Western Blotting , Caspase 9 , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Citosol/metabolismo , Ativação Enzimática , Fibroblastos/metabolismo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HL-60 , Humanos , Imunoprecipitação , Células K562 , Camundongos , Fosfotirosina/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Temperatura , Fatores de Tempo , Xenopus
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