RESUMO
Cancer cells can invade three-dimensional matrices by distinct mechanisms, recently defined by their dependence on extracellular proteases, including matrix metalloproteinases. Upon treatment with protease inhibitors, some tumour cells undergo a 'mesenchymal to amoeboid' transition that allows invasion in the absence of pericellular proteolysis and matrix degradation. We show here that in HT1080 cells, this transition is associated with weakened integrin-dependent adhesion, consistently reduced cell surface expression of the alpha2beta1 integrin collagen receptor and impaired signalling downstream, as judged by reduced autophosphorylation of focal adhesion kinase (FAK). On examining cancer cells that use defined invasion strategies, we show that distinct from mesenchymal invasion, amoeboid invasion is independent of intracellular calpain 2 proteolytic activity that is usually needed for turnover of integrin-linked adhesions during two-dimensional planar migration. Moreover, an inhibitor of Rho/ROCK signalling, which specifically impairs amoeboid-like invasion, restores cell surface expression of alpha2beta1 integrin, downstream FAK autophosphorylation and calpain 2 sensitivity--features of mesenchymal invasion. These findings link weakened integrin function to a lack of requirement for calpain 2-mediated integrin adhesion turnover during amoeboid invasion. In keeping with the need for integrin adhesion turnover, mesenchymal invasion is uniquely sensitive to Src inhibitors. Thus, the need for a major pathway that controls integrin adhesion turnover defines and distinguishes cancer cell invasion strategies.
Assuntos
Calpaína/metabolismo , Integrinas/fisiologia , Quinases da Família src/metabolismo , Sequência de Bases , Calpaína/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular , Linhagem Celular Tumoral , Fibrossarcoma/patologia , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/patologia , Mesoderma/enzimologia , Mesoderma/fisiologia , Mutação de Sentido Incorreto , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Quinases da Família src/genéticaRESUMO
Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process.
Assuntos
Adenocarcinoma/fisiopatologia , Movimento Celular/fisiologia , Neoplasias do Colo/fisiopatologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Quinases da Família src/genética , Quinases da Família src/metabolismo , Adenocarcinoma/enzimologia , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Plasmídeos , RNA Interferente Pequeno/genéticaRESUMO
Protein phosphorylation has been exploited by Nature in profound ways to control various aspects of cell proliferation, differentiation, metabolism, survival, motility and gene transcription. Cellular signal transduction pathways involve protein kinases, protein phosphatases, and phosphoprotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3-3) containing cellular proteins to provide multidimensional, dynamic and reversible regulation of many biological activities. Knowledge of cellular signal transduction pathways has led to the identification of promising therapeutic targets amongst these superfamilies of enzymes and adapter proteins which have been linked to various cancers as well as inflammatory, immune, metabolic and bone diseases. This review focuses on protein kinase, protein phosphatase and phosphoprotein-interacting cellular protein therapeutic targets with an emphasis on small-molecule drug discovery from a chemistry perspective. Noteworthy studies related to molecular genetics, signal transduction pathways, structural biology, and drug design for several of these therapeutic targets are highlighted. Some exemplary proof-of-concept lead compounds, clinical candidates and/or breakthrough medicines are further detailed to illustrate achievements as well as challenges in the generation, optimization and development of small-molecule inhibitors of protein kinases, protein phosphatases or phosphoprotein-interacting domain containing cellular proteins.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Fosfoproteínas Fosfatases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Quinases/efeitos dos fármacos , Proteínas/metabolismo , Inibidores Enzimáticos/farmacologia , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Transdução de Sinais , Relação Estrutura-AtividadeAssuntos
Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Peptídeos/química , Peptídeos/uso terapêutico , Proteômica/métodos , Cristalografia/métodos , Bases de Dados de Proteínas , Genômica/métodos , Genômica/tendências , Humanos , Peptídeos/genética , Conformação Proteica , Engenharia de Proteínas/métodos , Proteômica/tendênciasRESUMO
A series of compounds was designed and prepared as inhibitors of interleukin-1beta converting enzyme (ICE), also known as caspase-1. These inhibitors, which employ a diphenyl ether sulfonamide, were designed to improve potency by forming favorable interactions between the diphenyl ether rings and the prime side hydrophobic region. An X-ray crystal structure of a representative member of the diphenyl ether sulfonamide series bound to the active site of caspase-1 was obtained.
Assuntos
Inibidores de Caspase , Inibidores Enzimáticos/síntese química , Éteres/síntese química , Sulfonamidas/síntese química , Caspase 1/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Éteres/química , Éteres/farmacologia , Modelos Moleculares , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
The novel phosphotyrosine (pTyr) mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp) has been designed and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60(c-Src) (Src) tyrosine kinase. A 2.2 A X-ray crystal structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure-activity relationships and supports the design concept of this new pTyr mimetic.
Assuntos
Benzamidas/química , Benzamidas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Organofosfonatos/química , Organofosfonatos/farmacologia , Fosfotirosina/química , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Domínios de Homologia de src/efeitos dos fármacos , Animais , Sítios de Ligação , Reabsorção Óssea , Cristalografia por Raios X , Dentina/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Espectroscopia de Ressonância Magnética , Mimetismo Molecular , Mutação , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Coelhos , Relação Estrutura-AtividadeAssuntos
Benzamidas/síntese química , Osso e Ossos/enzimologia , Ácido Cítrico/química , Cicloexanos/síntese química , Inibidores Enzimáticos/síntese química , Compostos Organofosforados/síntese química , Proteínas Proto-Oncogênicas pp60(c-src)/química , Domínios de Homologia de src , Animais , Benzamidas/química , Benzamidas/farmacologia , Reabsorção Óssea/tratamento farmacológico , Cristalografia por Raios X , Cicloexanos/química , Cicloexanos/farmacologia , Durapatita/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligantes , Modelos Moleculares , Mimetismo Molecular , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Osteoclastos/patologia , Fosfotirosina/química , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , CoelhosAssuntos
Genes/genética , Preparações Farmacêuticas , Farmacologia/métodos , Animais , Biotecnologia/métodos , Genômica , HumanosRESUMO
Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.
Assuntos
Reabsorção Óssea/metabolismo , Difosfonatos/metabolismo , Osteoclastos/metabolismo , Domínios de Homologia de src/fisiologia , Quinases da Família src/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Dentina/metabolismo , Difosfonatos/química , Difosfonatos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Humanos , Ligantes , Mamíferos , Camundongos , Dados de Sequência Molecular , Osteoclastos/citologia , Osteoporose/metabolismo , Coelhos , Ensaio Radioligante , Ratos , Trítio , Técnicas do Sistema de Duplo-Híbrido , Quinases da Família src/antagonistas & inibidoresRESUMO
Two new species of Korotnevella Goodkov, 1988, Korotnevella hemistylolepis n. sp. and Korotnevella monacantholepis n. sp., are described from mesohaline ecosystems. The amoebae are characterized on the basis of light and electron microscopy with special emphasis on the structure of the basket scales, which have species-specific architecture. The two new species are the second and third ones recovered from environments other than freshwater. In terms of scale morphology they most closely resemble a freshwater species, Korotnevella bulla (Schaeffer, 1926) Goodkov, 1988. Two genus names, Dactylamoeba Korotnev, 1880 and Korotnevella Goodkov, 1988, are in current use. The latter name is preferred, pending rediscovery and characterization of Dactylamoeba elongata Korotnev, 1880, the type species of the genus. Korotnevella species can be divided into three groups on the basis of scale morphology, suggesting that the genus may not be monophyletic. A key to species is provided.
Assuntos
Amébidos/classificação , Água do Mar/parasitologia , Amébidos/ultraestrutura , Animais , Criopreservação , Dimetil Sulfóxido , Ecossistema , Microscopia Eletrônica , Cloreto de SódioRESUMO
Using a novel, solid-phase parallel synthetic route and a computational docking program, a series of phosphorylated nonpeptides were generated to determine their structure-activity relationships (SAR) for binding at the SH2 domain of pp60src (Src). A functionalized benzoic acid intermediate was attached to solid support via Rink amide linkage, which upon acid cleavage generated the desired benzamide template-based nonpeptides in a facile manner. Compounds were synthesized using a combination of solid- and solution-phase techniques. Purification using reversed-phase, semipreparative HPLC allowed for quantitative SAR studies. Specifically, this work focused on functional group modifications, in a parallel fashion, designed to explore hydrophobic binding at the pY+3 pocket of the Src SH2 domain.
Assuntos
Benzamidas/síntese química , Sítios de Ligação , Proteínas Proto-Oncogênicas pp60(c-src)/química , Domínios de Homologia de src , Benzamidas/química , Cristalografia por Raios X , Desenho de Fármacos , Fosforilação , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The observations that Src(-/-) mice develop osteopetrosis and Src family tyrosine kinase inhibitors decrease osteoclast-mediated resorption of bone have implicated Src in the regulation of osteoclast-resorptive activity. We have designed and synthesized a compound, AP22161, that binds selectively to the Src SH2 domain and demonstrated that it inhibits Src-dependent cellular activity and inhibits osteoclast-mediated resorption. RESULTS: AP22161 was designed to bind selectively to the Src SH2 domain by targeting a cysteine residue within the highly conserved phosphotyrosine-binding pocket. AP22161 was tested in vitro for binding to SH2 domains and was found to bind selectively and with high affinity to the Src SH2 domain. AP22161 was further tested in mechanism-based cellular assays and found to block Src SH2 binding to peptide ligands, inhibit Src-dependent cellular activity and diminish osteoclast resorptive activity. CONCLUSIONS: These results indicate that a compound that selectively inhibits Src SH2 binding can be used to inhibit osteoclast resorption. Furthermore, AP22161 has the potential to be further developed for treating osteoporosis.
Assuntos
Benzoatos/farmacologia , Reabsorção Óssea/prevenção & controle , Inibidores Enzimáticos/farmacologia , Osteoclastos/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Benzoatos/síntese química , Benzoatos/química , Sítios de Ligação/genética , Reabsorção Óssea/etiologia , Reabsorção Óssea/fisiopatologia , Células Cultivadas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Técnicas In Vitro , Ligantes , Camundongos , Dados de Sequência Molecular , Osteoclastos/fisiologia , Coelhos , Ratos , Homologia de Sequência de Aminoácidos , Transformação Genética , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de src/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Signal transduction targets include catalytic and/or non-catalytic domains, which are critical to various aspects of cell growth, differentiation, metabolism and function, mitogenesis, motility and gene transcription. Specific examples of molecular targets include the catalytic domains of protein tyrosine kinases (PTKs) and of protein tyrosine phosphatases (PTPases), as well as related protein-protein interaction motifs (eg, SH2, PTB and SH3 domains). From the relationship of tyrosine phosphorylation to intracellular pathway regulation by PTKs and PTPases, the dynamic and reversible binding interactions of SH2 and PTB domain-containing proteins with their cognate phosphotyrosine (pTyr)-containing proteins, provide an additional dimension to the modulation of signal transduction pathways which exist as a result of pTyr formation, degradation and molecular recognition events. This review focuses on our current understanding of key relative to recent reports which have provided further insight into their three-dimensional structure and mechanism. This review also highlights recent progress in the design and optimization of molecular mechanism-based signal transduction inhibitors.
RESUMO
The synthesis of a series of 1,2,4-oxadiazole analogs is discussed along with their ZAP-70 SH2 inhibitory activity. The tyrosine moiety in the original series has been replaced with nonpeptidic functional groups without a substantial loss of binding affinity.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Domínios de Homologia de src , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Polarização de Fluorescência , Ligação Proteica , Proteína-Tirosina Quinase ZAP-70RESUMO
A series of 1,2,4-oxadiazole analogues has been shown to be potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70, a potential therapeutic target for immune suppression. These compounds typically are 200-400-fold more potent than the native, monophosphorylated tetrapeptide sequences. When compared with the high-affinity zeta-1-ITAM peptide (Ac-NQL-pYNELNLGRREE-pYDVLD-NH(2), wherein pY refers to phosphotyrosine) some of the best 1,2, 4-oxadiazole analogues are approximately 1 order of magnitude less active. This series of compounds displays an unprecedented level of selectivity over the closely related tyrosine kinase Syk, as well as other SH2-containing proteins such as Src and Grb2. Gel shift studies using a protein construct consisting only of C-terminal ZAP-70 SH2 demonstrate that these compounds can effectively engage this particular SH2 domain.