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1.
Cell ; 149(4): 780-94, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22579283

RESUMO

Crosstalk and complexity within signaling pathways and their perturbation by oncogenes limit component-by-component approaches to understanding human disease. Network analysis of how normal and oncogenic signaling can be rewired by drugs may provide opportunities to target tumors with high specificity and efficacy. Using targeted inhibition of oncogenic signaling pathways, combined with DNA-damaging chemotherapy, we report that time-staggered EGFR inhibition, but not simultaneous coadministration, dramatically sensitizes a subset of triple-negative breast cancer cells to genotoxic drugs. Systems-level analysis-using high-density time-dependent measurements of signaling networks, gene expression profiles, and cell phenotypic responses in combination with mathematical modeling-revealed an approach for altering the intrinsic state of the cell through dynamic rewiring of oncogenic signaling pathways. This process converts these cells to a less tumorigenic state that is more susceptible to DNA damage-induced cell death by reactivation of an extrinsic apoptotic pathway whose function is suppressed in the oncogene-addicted state.


Assuntos
Antineoplásicos/administração & dosagem , Apoptose , Neoplasias da Mama/tratamento farmacológico , Quimioterapia Combinada/métodos , Receptores ErbB/antagonistas & inibidores , Transdução de Sinais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspase 8 , Linhagem Celular Tumoral , Dano ao DNA , Receptores ErbB/metabolismo , Feminino , Humanos , Redes e Vias Metabólicas , Modelos Biológicos
2.
Cell ; 139(6): 1109-18, 2009 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-20005804

RESUMO

Phosphorylation is a common mechanism for activating proteins within signaling pathways. Yet, the molecular transitions between the inactive and active conformational states are poorly understood. Here we quantitatively characterize the free-energy landscape of activation of a signaling protein, nitrogen regulatory protein C (NtrC), by connecting functional protein dynamics of phosphorylation-dependent activation to protein folding and show that only a rarely populated, pre-existing active conformation is energetically stabilized by phosphorylation. Using nuclear magnetic resonance (NMR) dynamics, we test an atomic scale pathway for the complex conformational transition, inferred from molecular dynamics simulations (Lei et al., 2009). The data show that the loss of native stabilizing contacts during activation is compensated by non-native transient atomic interactions during the transition. The results unravel atomistic details of native-state protein energy landscapes by expanding the knowledge about ground states to transition landscapes.


Assuntos
Proteínas de Bactérias/química , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Conformação Proteica , Bactérias/química , Bactérias/metabolismo , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Termodinâmica
3.
Semin Cell Dev Biol ; 22(7): 688-95, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21945648

RESUMO

14-3-3 proteins play critical roles in the regulation of cell fate through phospho-dependent binding to a large number of intracellular proteins that are targeted by various classes of protein kinases. 14-3-3 proteins play particularly important roles in coordinating progression of cells through the cell cycle, regulating their response to DNA damage, and influencing life-death decisions following internal injury or external cytokine-mediated cues. This review focuses on 14-3-3-dependent pathways that control cell cycle arrest and recovery, and the influence of 14-3-3 on the apoptotic machinery at multiple levels of regulation. Recognition of 14-3-3 proteins as signaling integrators that connect protein kinase signaling pathways to resulting cellular phenotypes, and their exquisite control through feedforward and feedback loops, identifies new drug targets for human disease, and highlights the emerging importance of using systems-based approaches to understand signal transduction events at the network biology level.


Assuntos
Proteínas 14-3-3/metabolismo , Apoptose/fisiologia , Pontos de Checagem do Ciclo Celular/fisiologia , Ciclo Celular/fisiologia , Animais , Dano ao DNA , Reparo do DNA , Humanos , Mitose , Ligação Proteica , Proteínas Quinases/metabolismo , Transdução de Sinais
4.
PLoS Biol ; 8(1): e1000287, 2010 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-20126263

RESUMO

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Dano ao DNA , Fase G2/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular , Quinase do Ponto de Checagem 2 , Retroalimentação Fisiológica , Humanos , Fosforilação , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Quinase 1 Polo-Like
5.
Clin Cancer Res ; 25(2): 609-618, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30274985

RESUMO

PURPOSE: Gastrointestinal stromal tumors (GIST) are commonly treated with tyrosine kinase inhibitors (TKI). The majority of patients with advanced GIST ultimately become resistant to TKI due to acquisition of secondary KIT mutations, whereas primary resistance is mainly caused by PDGFRA p.D842V mutation. We tested the activity of avapritinib, a potent and highly selective inhibitor of mutated KIT and PDGFRA, in three patient-derived xenograft (PDX) GIST models carrying different KIT mutations, with differential sensitivity to standard TKI.Experimental Design: NMRI nu/nu mice (n = 93) were transplanted with human GIST xenografts with KIT exon 11+17 (UZLX-GIST9 KIT 11+17 ), exon 11 (UZLX-GIST3 KIT 11 ), or exon 9 (UZLX-GIST2B KIT9 ) mutations, respectively. We compared avapritinib (10 and 30 mg/kg/once daily) versus vehicle, imatinib (50 mg/kg/bid) or regorafenib (30 mg/kg/once daily; UZLX-GIST9 KIT11+17 ); avapritinib (10, 30, 100 mg/kg/once daily) versus vehicle or imatinib [UZLX-GIST3 KIT11 ]; and avapritinib (10, 30, 60 mg/kg/once daily) versus vehicle, imatinib (50, 100 mg/kg/twice daily), or sunitinib (40 mg/kg/once daily; UZLX-GIST2B KIT9 ). RESULTS: In all models, avapritinib resulted in reduction of tumor volume, significant inhibition of proliferation, and reduced KIT signaling. In two models, avapritinib led to remarkable histologic responses, increase in apoptosis, and inhibition of MAPK-phosphorylation. Avapritinib showed superior (UZLX-GIST9 KIT 11+17 and -GIST2B KIT 9 ) or equal (UZLX-GIST3 KIT 11 ) antitumor activity to the standard dose of imatinib. In UZLX-GIST9 KIT 11+17 , the antitumor effects of avapritinib were significantly better than with imatinib or regorafenib. CONCLUSIONS: Avapritinib has significant antitumor activity in GIST PDX models characterized by different KIT mutations and sensitivity to established TKI. These data provide strong support for the ongoing clinical trials with avapritinib in patients with GIST (NCT02508532, NCT03465722).


Assuntos
Antineoplásicos/farmacologia , Tumores do Estroma Gastrointestinal/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Alelos , Substituição de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/patologia , Humanos , Imuno-Histoquímica , Camundongos , Terapia de Alvo Molecular , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Leukemia ; 33(5): 1195-1205, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30911112

RESUMO

Advanced systemic mastocytosis (advSM) is characterized by the presence of an acquired KIT D816V mutation in >90% of patients. In the majority of patients, KIT D816V is not only detected in mast cells but also in other hematopoietic lineages. We sought to investigate the effects of the KIT-inhibitors midostaurin and avapritinib on single-cell-derived myeloid progenitor cells using granulocyte-macrophage colony-forming-units of patients with KIT D816V positive advSM. Colonies obtained prior to treatment were incubated in vitro with midostaurin (n = 10) or avapritinib (n = 11) and showed a marked reduction (≥50%) of KIT D816V positive colonies in 3/10 (30%) and 7/11 (64%) patient samples, respectively. Three of those 7 (43%) avapritinib responders were resistant to midostaurin in both, in vitro and in vivo. Colonies from four patients with high-risk molecular profile and aggressive clinical course were resistant to both drugs. The in vitro activity of midostaurin strongly correlated with clinical and molecular responses, e.g., relative reduction of KIT D816V allele burden and the proportion of KIT D816V positive colonies obtained after six months midostaurin-treatment in vivo. We conclude that the colony inhibition assay provides useful information for prediction of responses on midostaurin and that avapritinib has a superior in vitro activity compared to midostaurin.


Assuntos
Alelos , Mastocitose Sistêmica/genética , Mutação , Células Progenitoras Mieloides/efeitos dos fármacos , Células Progenitoras Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Idoso , Antineoplásicos/farmacologia , Biomarcadores , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mastocitose Sistêmica/diagnóstico , Mastocitose Sistêmica/tratamento farmacológico , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologia , Índice de Gravidade de Doença
7.
Mol Cell Oncol ; 5(3): e1435183, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30250891

RESUMO

Cancer genomics and mechanistic studies have revealed that heterogeneous mutations within a single kinase can result in a variety of activation mechanisms. The challenge has been to match these insights with tailored drug discovery strategies to yield potent, highly selective drugs. With optimized drugs in hand, physicians could apply the principles of personalized medicine with an increasing number of options to treat patients with improved precision according to their tumor's molecular genotype.

8.
Methods Enzymol ; 423: 149-65, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17609130

RESUMO

A fundamental concept of phosphorylation-mediated signaling is the precise switching between discrete functional conformations. According to the traditional view, phosphorylation induces a new, active conformation. In this chapter, a series of NMR experiments performed on a response regulator are described that challenge this traditional notion. The combination of NMR relaxation experiments with chemical shift data and the linkage to structure/function reveals a fundamentally different activation mechanism. The NMR data for the response regulator NtrC provide kinetic (rates of interconversion), thermodynamic (relative populations), and structural (chemical shift) information for the conformational exchange process. The results demonstrate that both the inactive and active states are present before phosphorylation, and activation occurs via a shift of this preexisting equilibrium. This concept is in accordance with the energy landscape view of proteins that embraces the existence of conformational substates. We conjecture that this population-shift mechanism is a general paradigm for response regulator activation and possibly more universal for phosphorylation-mediated signaling.


Assuntos
Proteínas de Escherichia coli/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas PII Reguladoras de Nitrogênio/química , Fatores de Transcrição/química , Sítio Alostérico , Cinética , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Salmonella typhimurium/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Termodinâmica , Fatores de Tempo
9.
Sci Transl Med ; 9(414)2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29093181

RESUMO

Targeting oncogenic kinase drivers with small-molecule inhibitors can have marked therapeutic benefit, especially when administered to an appropriate genomically defined patient population. Cancer genomics and mechanistic studies have revealed that heterogeneous mutations within a single kinase can result in various mechanisms of kinase activation. Therapeutic benefit to patients can best be optimized through an in-depth understanding of the disease-driving mutations combined with the ability to match these insights to tailored highly selective drugs. This rationale is presented for BLU-285, a clinical stage inhibitor of oncogenic KIT and PDGFRA alterations, including activation loop mutants that are ineffectively treated by current therapies. BLU-285, designed to preferentially interact with the active conformation of KIT and PDGFRA, potently inhibits activation loop mutants KIT D816V and PDGFRA D842V with subnanomolar potency and also inhibits other well-characterized disease-driving KIT mutants both in vitro and in vivo in preclinical models. Early clinical evaluation of BLU-285 in a phase 1 study has demonstrated marked activity in patients with diseases associated with KIT (aggressive systemic mastocytosis and gastrointestinal stromal tumor) and PDGFRA (gastrointestinal stromal tumor) activation loop mutations.


Assuntos
Mutação/genética , Medicina de Precisão , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/química
10.
J Mol Biol ; 331(1): 245-54, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12875849

RESUMO

Two-component systems, which are comprised of a single histidine-aspartate phosphotransfer module, are the dominant signaling pathways in bacteria and have recently been identified in several eukaryotic organisms as well. A tandem connection of two or more histidine-aspartate motifs forms complex phosphorelays. While response regulators from simple two-component systems have been characterized structurally in their inactive and active forms, we address here the question of whether a response regulator from a phosphorelay has a distinct structural basis of activation. We report the NMR solution structure of BeF(3)(-)-activated Spo0F, the first structure of a response regulator from a phosphorelay in its activated state. Conformational changes were found in regions previously identified to change in simple two-component systems. In addition, a downward shift by half a helical turn in helix 1, located on the opposite side of the common activation surface, was observed as a consequence of BeF(3)(-) activation. Conformational changes in helix 1 can be rationalized by the distinct function of phosphoryl transfer to the second histidine kinase, Spo0B, because helix 1 is known to interact directly with Spo0B and the phosphatase RapB. The identification of structural rearrangements in Spo0F supports the hypothesis of a pre-existing equilibrium between the inactive and active state prior to phosphorylation that was suggested on the basis of previous NMR dynamics studies on Spo0F. A shift of a pre-existing equilibrium is likely a general feature of response regulators.


Assuntos
Proteínas de Bactérias/química , Berílio , Fluoretos , Ressonância Magnética Nuclear Biomolecular , Ácido Aspártico/metabolismo , Bacillus subtilis/química , Proteínas de Bactérias/metabolismo , Histidina/metabolismo , Modelos Moleculares , Fosforilação , Fosfotransferases/química , Fosfotransferases/metabolismo , Conformação Proteica , Transdução de Sinais
11.
Semin Cancer Biol ; 16(3): 173-82, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16678437

RESUMO

14-3-3 proteins are a ubiquitous class of regulatory proteins found in all eukaryotic cells and were the first class of molecules to be recognized as discrete phosphoserine/threonine binding modules. 14-3-3 proteins bind a large number of different substrates to regulate a wide array of cellular signaling events including cell cycle progression and DNA damage responses, programmed cell death, cytoskeletal dynamics, transcriptional control of gene expression, as well as processes directly related to cancer progression. In this review, the structural basis of phosphorylation-dependent binding of 14-3-3 to peptide and protein ligands is discussed along with mechanisms that govern how 14-3-3 regulates the function of its bound ligands. The X-ray crystal structures of all human 14-3-3 proteins bound to peptides have now been solved. Here, we use structural comparisons between isoforms as a framework for discussion of ligand binding by 14-3-3 as well as the mechanisms through which post-translational modification of the different isoforms alters their function.


Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas 14-3-3/genética , Motivos de Aminoácidos , Sequência de Aminoácidos/genética , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cristalografia por Raios X , Citoplasma/metabolismo , Humanos , Ligantes , Modelos Moleculares , Fosforilação , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Relação Estrutura-Atividade
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