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1.
Am J Surg ; 205(5): 581-4; discussion 584, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23592166

RESUMO

BACKGROUND: The current practice of completion axillary lymph node dissection (ALND) for patients with a positive sentinel lymph node (SLN) is being questioned. This led us to examine the outcomes of patients with positive SLNs undergoing mastectomy who underwent ALND compared with those who did not. METHODS: A retrospective review of cancer registry data identified 561 women with stages 1 to 3 breast cancer with positive SLNs who underwent mastectomy between 2000 and 2010. Four hundred twenty-six women underwent formal ALND and 135 were managed expectantly. Recurrence-free survival was defined as no locoregional or distant metastases. RESULTS: Mean time to recurrence was 29.9 months. Mean follow-up for patients without recurrence was 40.3 months. Survival curves showed no significant difference in recurrence-free survival between the 2 groups (P = .23). CONCLUSIONS: In our experience, there is no significant difference in recurrence-free survival in patients with positive SLNs undergoing mastectomy when completion ALND was not performed, suggesting that a closer look at the indications for ALND in early breast cancer be further explored.


Assuntos
Neoplasias da Mama/cirurgia , Excisão de Linfonodo , Mastectomia , Recidiva Local de Neoplasia/prevenção & controle , Adulto , Idoso , Axila , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Metástase Neoplásica/prevenção & controle , Modelos de Riscos Proporcionais , Sistema de Registros , Estudos Retrospectivos , Biópsia de Linfonodo Sentinela , Resultado do Tratamento
2.
J Med Chem ; 56(1): 345-56, 2013 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-23214979

RESUMO

The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.


Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Ciclopropanos/síntese química , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Pirazinas/síntese química , Pirróis/síntese química , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Células CACO-2 , Cristalografia por Raios X , Ciclopropanos/farmacocinética , Ciclopropanos/farmacologia , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Interleucina-2/fisiologia , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Camundongos , Modelos Moleculares , Pirazinas/farmacocinética , Pirazinas/farmacologia , Pirróis/farmacocinética , Pirróis/farmacologia , RNA Interferente Pequeno/genética , Ratos , Receptores de Interleucina-6/fisiologia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
3.
Protein Sci ; 20(2): 428-36, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21280133

RESUMO

Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10)- and α-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/química , Tirosina Quinase da Agamaglobulinemia , Animais , Camundongos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxazinas/química , Oxazinas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Piridinas/química , Piridinas/metabolismo , Difração de Raios X
4.
Bioorg Med Chem Lett ; 20(17): 5217-20, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20655210

RESUMO

JNK2 and p38alpha are closely related mitogen-activated protein kinases that regulate various cellular activities and are considered drug targets for inflammatory diseases. We have determined the X-ray crystal structure of the clinical phase II p38alpha inhibitor BIRB796 bound to its off-target JNK2. This shows for the first time a JNK subfamily member in the DFG-out conformation. The fully resolved activation loop reveals that BIRB796 inhibits JNK2 activation by stabilizing the loop in a position that does not allow its phosphorylation by upstream kinases. The structure suggests that substituents at the BIRB796 morpholino group and modifications of the t-butyl moiety should further increase the p38alpha to JNK2 potency ratio. For the design of selective DFG-out binding JNK2 inhibitors, the binding pocket of the BIRB796 tolyl group may have the best potential.


Assuntos
Proteína Quinase 9 Ativada por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/química , Naftalenos/química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Cristalografia por Raios X , Desenho de Fármacos , Modelos Moleculares , Estrutura Molecular
5.
Chem Biol Drug Des ; 76(2): 154-63, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20545945

RESUMO

IL-2-inducible T cell kinase plays an essential role in T cell receptor signaling and is considered a drug target for the treatment of Th2-mediated inflammatory diseases. By applying high-throughput protein engineering and crystallization, we have determined the X-ray crystal structures of IL-2-inducible T cell kinase in complex with its selective inhibitor BMS-509744 and the broad-spectrum kinase inhibitors sunitinib and RO5191614. Sunitinib uniquely stabilizes IL-2-inducible T cell kinase in the helix C-in conformation by inducing side chain conformational changes in the ATP-binding site. This preference of sunitinib to bind to an active kinase conformation is reflective of its broad-spectrum kinase activity. BMS-509744 uniquely stabilizes the activation loop in a substrate-blocking inactive conformation, indicating that structural changes described for Src family kinases are also involved in the regulation of IL-2-inducible T cell kinase activity. The observed BMS-509744 binding mode allows rationalization of structure-activity relationships reported for this inhibitor class and facilitates further structure-based drug design. Sequence-based analysis of this binding mode provides guidance for the rational design of inhibitor selectivity.


Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Indóis/química , Indóis/farmacologia , Engenharia de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , Sunitinibe , Quinases da Família src/metabolismo
6.
Am J Surg ; 197(5): 633-6, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19306975

RESUMO

BACKGROUND: As the number of breast cancer survivors increases, the appearance of second malignancies and unusual metastatic patterns likely also is increasing. In particular, we and others have observed gastric malignancies in breast cancer survivors. METHODS: We reviewed 3 regional hospital system tumor databases, comprising 19,049 analytic breast cancer cases, to determine the number, types, and outcomes of subsequent gastric malignancies. RESULTS: Twenty-eight patients developed subsequent gastric malignancies, representing .15% of breast cancer survivors; 82% of patients had gastric symptoms. Overall survival for the cohort was 39%. Twenty-four patients (86%) had gastric primaries and 13 died of their second cancers. Four patients had gastric metastases; all had lobular histology in both their primary tumors and metastatic lesions. Five patients had gastrointestinal stromal tumors; all patients underwent resection and currently are alive. CONCLUSION: Gastric symptoms in breast cancer survivors may represent malignant lesions, often second primaries. All gastric metastases in our series were of lobular histology.


Assuntos
Neoplasias da Mama/epidemiologia , Segunda Neoplasia Primária/epidemiologia , Neoplasias Gástricas/epidemiologia , Adenocarcinoma/epidemiologia , Adulto , Idoso , Neoplasias da Mama/patologia , Feminino , Tumores do Estroma Gastrointestinal/epidemiologia , Humanos , Linfoma/epidemiologia , Pessoa de Meia-Idade , Sistema de Registros , Neoplasias Gástricas/secundário
7.
Chem Biol Drug Des ; 73(4): 466-70, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19220318

RESUMO

Spleen tyrosine kinase is considered an attractive drug target for the treatment of allergic and antibody mediated autoimmune diseases. We have determined the co-crystal structures of spleen tyrosine kinase complexed with three known inhibitors: YM193306, a 7-azaindole derivative and R406. The cis-cyclohexyldiamino moiety of YM193306 is forming four hydrophobically shielded polar interactions with the spleen tyrosine kinase protein and is therefore crucial for the high potency of this inhibitor. Its primary amino group is inducing a conformational change of the spleen tyrosine kinase DFG Asp side chain. The crystal structure of the 7-azaindole derivative bound to spleen tyrosine kinase is the first demonstration of a 2-substituted 7-azaindole bound to a protein kinase. Its indole-amide substituent is tightly packed between the N- and C-terminal kinase lobes. The co-crystal structure of the spleen tyrosine kinase-R406 complex shows two main differences to the previously reported structure of spleen tyrosine kinase soaked with R406: (i) the side chain of the highly conserved Lys is disordered and not forming a hydrogen bond to R406 and (ii) the DFG Asp side chain is pointing away from and not towards R406. The novel protein-ligand interactions and protein conformational changes revealed in these structures guide the rational design and structure-based optimization of second-generation spleen tyrosine kinase inhibitors.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Baço/enzimologia , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Ligantes , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinase Syk
8.
Mol Immunol ; 46(7): 1458-66, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19181383

RESUMO

IRAK-1 and IRAK-4 are protein kinases that mediate signaling by Toll/IL1/Plant R (TIR) domain-containing receptors including the IL-1, IL-18, and Toll-like receptors (TLRs). Although well studied in mouse systems, the mechanism by which they function in human systems is less clear. To extend our knowledge of how these proteins regulate inflammatory signaling in human cells, we genetically and pharmacologically manipulated IRAK-1 and IRAK-4 kinase activities in vitro. Ablation of IRAK-4 expression in human umbilical vein endothelial cells (HUVEC) with siRNA suppressed IL-1beta induced IL-6 and IL-8 production whereas IRAK-1 siRNA suppressed TNFalpha induced but not IL-1beta induced cytokine production. Complementation of IRAK-4-depleted cells with a kinase-inactive allele restored IL-1beta induced cytokine gene expression suggesting that the IRAK-4 kinase activity is dispensable relative to its scaffolding function. Consistent with this finding, an IRAK-4 selective kinase inhibitor (RO6245) that inhibited IRAK-1 degradation failed to block IL-1beta induced cytokine production. In contrast, an inhibitor of both IRAK-1 and IRAK-4 (RO0884) reduced IL-1beta induced p38 MAP kinase, c-Jun N-terminal kinase activation, and IL-6 production in HUVEC. RO0884 also antagonized IL-1beta, TNFalpha, and TLR-mediated cytokine production in human fibroblast-like synoviocytes and peripheral blood mononuclear cells. Therefore in human cells the non-kinase functions of IRAK-4 are essential, whereas the kinase activity of IRAK-4 appears redundant with that of IRAK-1. Pharmacologic inhibition of both kinases appears necessary to block pro-inflammatory cytokine production.


Assuntos
Citocinas/genética , Mediadores da Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/fisiologia , Interleucina-1beta/metabolismo , Interleucina-1beta/fisiologia , Camundongos , Modelos Biológicos , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção
9.
J Mol Biol ; 383(4): 885-93, 2008 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-18801372

RESUMO

c-Jun N-terminal kinase (JNK) 2 is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. MAP kinases share a common sequence insertion called "MAP kinase insert", which, for ERK2, has been shown to interact with regulatory proteins and, for p38alpha, has been proposed to be involved in the regulation of catalytic activity. We have determined the crystal structure of human JNK2 complexed with an indazole inhibitor by applying a high-throughput protein engineering and surface-site mutagenesis approach. A novel conformation of the activation loop is observed, which is not compatible with its phosphorylation by upstream kinases. This activation inhibitory conformation of JNK2 is stabilized by the MAP kinase insert that interacts with the activation loop in an induced-fit manner. We therefore suggest that the MAP kinase insert of JNK2 plays a role in the regulation of JNK2 activation, possibly by interacting with intracellular binding partners.


Assuntos
Proteína Quinase 9 Ativada por Mitógeno/química , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Estrutura Terciária de Proteína , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 9 Ativada por Mitógeno/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Engenharia de Proteínas
10.
Biochemistry ; 46(51): 15103-14, 2007 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-18052078

RESUMO

Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that plays an important signaling role in several types of immune cells. To improve our understanding of the enzymology and activation mechanism of Syk, we characterized the steady state kinetics of Syk substrate phosphorylation. A new real time fluorescence kinase assay was employed that utilizes a nonnatural amino acid in the peptide substrate which undergoes an enhancement in fluorescence following phosphorylation. Characterizing the steady state kinetics using a Syk kinase domain construct [Syk(360-635)] revealed that Syk employs a ternary complex kinetic mechanism involving little cooperativity between substrate binding sites and a Km(ATP) of 36 +/- 5 microM and a Km(peptide substrate) of 4.4 +/- 0.9 microM. The order of substrate binding was determined to be either random or ordered with ATP binding first, as determined in substrate analogue inhibitor studies. Utilizing the real time capabilities of the fluorescence assay, we established that Syk demonstrates no lag phase in product formation. Furthermore, a Syk mutant lacking tyrosine in the activation loop (Syk Y525F,Y526F) exhibited activity identical to that of wild-type Syk. These two findings indicate that autophosphorylation of the activation loop of Syk does not regulate Syk(360-635) activity. We also compared the activity of Syk(360-635) to that of full-length Syk and revealed that Syk(360-635) is 10-fold more active, suggesting that residues outside the catalytic domain of Syk suppress kinase activity. The findings presented here provide the first kinetic description of the Syk enzyme mechanism.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Espectrometria de Fluorescência , Especificidade por Substrato , Quinase Syk , Fatores de Tempo
11.
J Biol Chem ; 282(12): 8768-76, 2007 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-17264076

RESUMO

Bruton's tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.


Assuntos
Proteínas Tirosina Quinases/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Tirosina Quinase da Agamaglobulinemia , Sítios de Ligação , Ligação Competitiva , Ativação Enzimática , Humanos , Cinética , Modelos Químicos , Mutação , Peptídeos/química , Ligação Proteica , Estaurosporina/química , Estaurosporina/farmacologia , Especificidade por Substrato , Tirosina/química
12.
J Immunol ; 178(5): 2641-5, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17312103

RESUMO

IL-1R-associated kinase (IRAK)4 plays a central role in innate and adaptive immunity, and is a crucial component in IL-1/TLR signaling. We have determined the crystal structures of the apo and ligand-bound forms of human IRAK4 kinase domain. These structures reveal several features that provide opportunities for the design of selective IRAK4 inhibitors. The N-terminal lobe of the IRAK4 kinase domain is structurally distinctive due to a loop insertion after an extended N-terminal helix. The gatekeeper residue is a tyrosine, a unique feature of the IRAK family. The IRAK4 structures also provide insights into the regulation of its activity. In the apo structure, two conformations coexist, differing in the relative orientation of the two kinase lobes and the position of helix C. In the presence of an ATP analog only one conformation is observed, indicating that this is the active conformation.


Assuntos
Quinases Associadas a Receptores de Interleucina-1/química , Animais , Cristalografia por Raios X , Humanos , Imunidade Inata/imunologia , Interleucina-1/imunologia , Quinases Associadas a Receptores de Interleucina-1/imunologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/imunologia , Relação Estrutura-Atividade , Receptores Toll-Like/imunologia
13.
J Biol Chem ; 277(26): 23573-81, 2002 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-11950839

RESUMO

A small molecule inhibitor of NF-kappaB-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) alpha-induced IkappaB(alpha) degradation in MM6 cells but not the degradation of beta-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNFalpha, interleukin-1beta, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC(50) values below 1 microm. Ro106-9920 also blocked TNFalpha production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require betaTRCP but associates with IkappaB(alpha) and will ubiquitinate IkappaB(alpha) S32E,S36E (IkappaB(alpha)(ee)) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IkappaB(alpha)(ee) ubiquitination with an IC(50) value of 2.3 +/- 0.09 microm. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF(betaTRCP), the putative E3 for IkappaB(alpha) ubiquitination. Ro106-9920 was observed to be selective for IkappaB(alpha)(ee) ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPS- and TNFalpha-induced IkappaB(alpha) degradation and NF-kappaB activation.


Assuntos
Citocinas/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , NF-kappa B/antagonistas & inibidores , Sulfóxidos/farmacologia , Tetrazóis/farmacologia , Ubiquitina/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Inibidor de NF-kappaB alfa , Ratos , Ratos Wistar , Especificidade por Substrato
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