RESUMO
Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-theta, PKC-iota and PKC-betaII kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC-substrate complex has been determined. In the previously determined PKC-iota complex, residues 533-551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-iota kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0 A resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-iota are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-iota, which is different from the case of the prototypical AGC-branch kinase PKA.
Assuntos
Trifosfato de Adenosina/metabolismo , Isoenzimas/química , Proteína Quinase C/química , Trifosfato de Adenosina/química , Animais , Linhagem Celular , Cristalografia por Raios X , Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteína Quinase C/genética , Proteína Quinase C/isolamento & purificação , Proteína Quinase C/metabolismoRESUMO
MK-1775 is a potent and selective small molecule Wee1 inhibitor. Previously we have shown that it abrogated DNA damaged checkpoints induced by gemcitabine, carboplatin, and cisplatin and enhanced the anti-tumor efficacy of these agents selectively in p53-deficient tumor cells. MK-1775 is currently in Phase I clinical trial in combination with these anti-cancer drugs. In this study, the effects of MK-1775 on 5-fluorouracil (5-FU) and other DNA-damaging agents with different modes of action were determined. MK-1775 enhanced the cytotoxic effects of 5-FU in p53-deficient human colon cancer cells. MK-1775 inhibited CDC2 Y15 phosphorylation in cells, abrogated DNA damaged checkpoints induced by 5-FU treatment, and caused premature entry of mitosis determined by induction of Histone H3 phosphorylation. Enhancement by MK-1775 was specific for p53-deficient cells since this compound did not sensitize p53-wild type human colon cancer cells to 5-FU in vitro. In vivo, MK-1775 potentiated the anti-tumor efficacy of 5-FU or its prodrug, capecitabine, at tolerable doses. These enhancements were well correlated with inhibition of CDC2 phosphorylation and induction of Histone H3 phosphorylation in tumors. In addition, MK-1775 also potentiated the cytotoxic effects of pemetrexed, doxorubicin, camptothecin, and mitomycin C in vitro. These studies support the rationale for testing the combination of MK-1775 with various DNA-damaging agents in cancer patients.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Dano ao DNA/efeitos dos fármacos , Fluoruracila/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Camptotecina/farmacologia , Capecitabina , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Doxorrubicina/farmacologia , Citometria de Fluxo , Fluoruracila/análogos & derivados , Glutamatos/farmacologia , Guanina/análogos & derivados , Guanina/farmacologia , Histonas/metabolismo , Humanos , Técnicas Imunoenzimáticas , Mitomicina/farmacologia , Proteínas Nucleares/metabolismo , Pemetrexede , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Pirimidinonas , Ratos , Ratos Endogâmicos F344 , Ratos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Wee1 is a tyrosine kinase that phosphorylates and inactivates CDC2 and is involved in G(2) checkpoint signaling. Because p53 is a key regulator in the G(1) checkpoint, p53-deficient tumors rely only on the G(2) checkpoint after DNA damage. Hence, such tumors are selectively sensitized to DNA-damaging agents by Wee1 inhibition. Here, we report the discovery of a potent and selective small-molecule inhibitor of Wee1 kinase, MK-1775. This compound inhibits phosphorylation of CDC2 at Tyr15 (CDC2Y15), a direct substrate of Wee1 kinase in cells. MK-1775 abrogates G(2) DNA damage checkpoint, leading to apoptosis in combination with DNA-damaging chemotherapeutic agents such as gemcitabine, carboplatin, and cisplatin selectively in p53-deficient cells. In vivo, MK-1775 potentiates tumor growth inhibition by these agents, and cotreatment does not significantly increase toxicity. The enhancement of antitumor effect by MK-1775 was well correlated with inhibition of CDC2Y15 phosphorylation in tumor tissue and skin hair follicles. Our data indicate that Wee1 inhibition provides a new approach for treatment of multiple human malignancies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Dano ao DNA , Neoplasias/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/deficiência , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2 , Linhagem Celular Tumoral , Ciclina B/metabolismo , Quinases Ciclina-Dependentes , Sinergismo Farmacológico , Citometria de Fluxo , Células HeLa , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Pirimidinonas , Ratos , Ratos Endogâmicos F344 , Ratos Nus , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The synthesis and biological evaluation of new potent opioid receptor-like 1 antagonists are presented. A structure-activity relationship (SAR) study of arylpyrazole lead compound 1 obtained from library screening identified compound 31, (1S,3R)-N-{[1-(3-chloropyridin-2-yl)-5-(5-fluoro-6-methylpyridin-3-yl)-4-methyl-1H-pyrazol-3-yl]methyl}-3-fluorocyclopentanamine, which exhibits high intrinsic potency and selectivity against other opioid receptors and hERG potassium channel.
Assuntos
Ciclopentanos/química , Antagonistas de Entorpecentes , Pirazóis/química , Ciclopentanos/síntese química , Ciclopentanos/farmacologia , Descoberta de Drogas , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Pirazóis/síntese química , Pirazóis/farmacologia , Receptores Opioides/metabolismo , Relação Estrutura-Atividade , Receptor de NociceptinaRESUMO
A structure-activity relationship (SAR) study on the benzimidazole series of opioid receptor-like 1 (ORL1) antagonists related to 1 is described. Optimization of 1 by introduction of a hydrophilic substituent into the thioether part resulted in identification of potent ORL1 antagonists with high selectivity over binding affinity for hERG and other opioid receptors.
Assuntos
Benzimidazóis/química , Cicloexanos/síntese química , Canais de Potássio Éter-A-Go-Go/metabolismo , Antagonistas de Entorpecentes , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Cicloexanos/farmacologia , Canal de Potássio ERG1 , Receptores Opioides/metabolismo , Relação Estrutura-Atividade , Receptor de NociceptinaRESUMO
Based on reported structures, a focused library of biarylmethyl bound to the nitrogen atom of spiropiperidine was designed. Systematic modifications allowed the discovery of a synthetically feasible and highly potent ORL1 antagonist 37, 1'-{[1-(3-chloropyridin-2-yl)-1H-pyrazol-4-yl]methyl}-3H-spiro[2-benzofuran-1,4'-piperidine], which exhibits excellent selectivity to mu, kappa, and human ether-a-go-go related gene potassium channel.
Assuntos
Química Farmacêutica/métodos , Antagonistas de Entorpecentes , Piperidinas/química , Compostos de Espiro/química , Ligação Competitiva , Desenho de Fármacos , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Concentração Inibidora 50 , Modelos Químicos , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Receptores Opioides , Relação Estrutura-Atividade , Receptor de NociceptinaRESUMO
A series of compounds based on 7-{[4-(2-methylphenyl)piperidin-1-yl]methyl}-6,7,8,9-tetrahydro-5 H-cyclohepta[ b]pyridine-9-ol ( (-)-8b), a potent and selective opioid receptor-like 1 (ORL1) antagonist, was prepared and evaluated using structure-activity relationship studies with the aim of removing its affinity to human ether-a-go-go related gene (hERG) K (+) channel. From these studies, 10l was identified as an optimized structure with respect to ORL1 antagonist activity, and affinity to the hERG K (+)channel. Furthermore, 10l showed good in vivo antagonism with a wide therapeutic index in regards to adverse cardiovascular effects.
Assuntos
Cicloparafinas/química , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Antagonistas de Entorpecentes , Piridinas/administração & dosagem , Piridinas/química , Administração Oral , Animais , Fenômenos Químicos , Físico-Química , Cães , Canais de Potássio Éter-A-Go-Go/metabolismo , Hepatócitos/metabolismo , Humanos , Estrutura Molecular , Ligação Proteica , Piridinas/síntese química , Piridinas/classificação , Ratos , Receptores Opioides/metabolismo , Relação Estrutura-Atividade , Receptor de NociceptinaRESUMO
A pale ring of medium-to-large cells surrounding the follicles, namely a marginal zone distribution pattern, is the key criterion for diagnosing nodal marginal zone B-cell lymphoma (NMZBL). The tumor cells of NMZBL occasionally exhibit the morphology of monocytoid B-cells (MBC). However, this condition can be difficult to distinguish from MBC hyperplasia in reactive conditions. We describe the histopathological and immunohistochemical findings of four cases exhibiting florid MBC hyperplasia and resembling NMZBLs. The patients consisted of three males and one female (age range 48-64 years) who had asymptomatic lymphadenopathy in the head and neck area. Histologically, a pale ring surrounding more than 75% of the lymphoid follicles characterizes the lesion. The lymphoid follicles usually had hyperplastic germinal centers, and progressive transformation of germinal center was noted in two cases. The pale ring was composed of medium-to-large cells with indented or round nuclei and relatively abundant pale clear cytoplasm. Numerous plasma cells were observed in one case. The overall histomorphological findings in these four cases were similar to those of NMZBLs. However, immunohistochemical investigations demonstrated that MBCs were CD43- and bcl-2-. Moreover, immunohistochemistry, polymerase chain reaction, and flow cytometry studies demonstrated the polytypic nature of B-lymphocytes. Recognition of this unusual MBC hyperplasia in reactive lymph node lesions is important to avoid confusion with NMZBLs.
Assuntos
Linfócitos B/patologia , Centro Germinativo/patologia , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma de Células B/patologia , Pseudolinfoma/patologia , Linfócitos B/metabolismo , Biomarcadores Tumorais/metabolismo , Diagnóstico Diferencial , Feminino , Centro Germinativo/metabolismo , Humanos , Doenças Linfáticas/patologia , Linfoma de Células B/metabolismo , Linfoma de Zona Marginal Tipo Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Pseudolinfoma/metabolismoRESUMO
We report here a case of mucosa-associated lymphoid tissue (MALT)-type lymphoma arising from the minor salivary gland of the oral cavity exhibiting tumor-forming amyloidosis. The patient was a 64-year-old Japanese woman who presented with 4-year history of a left soft palate mass. Despite multiple and multifocal recurrences including the lip, soft palate, tongue, oral base and vocal code and soft palate, the tumor remained localized in the upper aerodigestive tract, and the patient did not develop multiple myeloma during the course of disease. Histologically, the majority of the lesion was occupied by amyloid deposition. Only the periphery of the lesion contained numerous plasmacytoid cells, along with occasional centrocyte-like cells. In addition, lymphoepithelial lesion and follicular colonization were noted. The present case indicates that primary minor salivary gland MALT-type lymphoma appears to be the cause of tumor-forming amyloidosis of the upper aerodigestive tract including the larynx.
Assuntos
Amiloidose/etiologia , Doenças da Laringe/etiologia , Linfoma de Zona Marginal Tipo Células B/complicações , Palato Mole/patologia , Neoplasias das Glândulas Salivares/complicações , Glândulas Salivares Menores/patologia , Feminino , Humanos , Linfoma de Zona Marginal Tipo Células B/patologia , Pessoa de Meia-Idade , Neoplasias das Glândulas Salivares/patologiaRESUMO
Follicular lymphoid hyperplasia (FLH) of the oral cavity is a rare and poorly understood lymphoproliferative disorder. We present a case of FLH of the oral cavity presenting with progressive transformation of germinal center (PTGC). The patient was a 49-year-old Japanese woman presenting with a hard mass in the right cheek. The resected specimen contained numerous lymphoid follicles with active germinal centers and a portion of the lymphoid follicles exhibited PTGC. The PTGCs contained a few large lymphoid cells resembling lymphocytic and histiocytic Reed-Sternberg cells of nodular lymphocyte-predominant Hodgkin lymphoma. The PTGC was surrounded by groups of epithelioid cells. In situ hybridization studies demonstrated strong expression of Epstein-Barr virus (EBV)-encoded small RNA in scattered large lymphoid cells in the PTGC. Although the etiology of FLH of the oral cavity remains unclear, the present case suggests that a subset of FLH of the oral cavity appears to be an EBV-associated lymphoproliferative disorder.