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1.
Eur J Med Chem ; 187: 112004, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31881458

RESUMO

Protein phosphorylation by kinases is of critical importance for the regulation of many cellular functions. When kinases are deregulated numerous biological processes are affected, which may cause a variety of diseases. Therefore, kinase inhibition plays an important role for therapeutic intervention. A number of kinase inhibitors have been approved as drugs, initially in oncology where promiscuous (multi-kinase) inhibitors were most efficacious. Exploring kinase inhibitor selectivity and promiscuity for therapy is among the most challenging aspects of kinase drug discovery. Herein, we thoroughly analyze a kinase profiling experiment in which 637 designated inhibitors of p38α MAP kinase (p38α) were tested against a panel of 60 kinases distributed across the human kinome. In this experiment, only 19% of the inhibitors were found to be promiscuous when the median p38α inhibition level was applied as an activity threshold. Promiscuous inhibitors had a median value of two targets per compound, and many of these inhibitors were only active against the p38α and closely related JNK3 enzymes. Promiscuity cliffs were identified and analyzed in a network representation revealing structural modifications that were implicated in triggering compound promiscuity. Taken together, the findings revealed a high degree of selectivity of designated p38α directed inhibitors although they target the ATP binding site that is largely conserved across the human kinome.


Assuntos
Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Relação Dose-Resposta a Droga , Desenho de Drogas , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
2.
Eur J Med Chem ; 182: 111624, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31445234

RESUMO

This work describes the rational discovery of novel chemotypes of p38α MAPK inhibitors using a funnel approach consisting of several computer-aided drug discovery methods and biological experiments. Among the identified hits, four compounds belonging to different chemical families showed IC50 values lower than 10 µM. In particular, the 1,4-benzodioxane derivative 5 turned out to be a potent and efficient p38α MAPK inhibitor having IC50 = 0.07 µM, and LEexp and LipE values of 0.38 and 4.8, respectively; noteworthy, the compound had also a promising kinase selectivity profile and the capability to suppress p38α MAPK effects in human immune cells. Overall, the collected findings highlight that the applied strategy has been successful in generating chemical novelty in the inhibitor kinase field, providing suitable chemical candidates for further inhibitor optimization.


Assuntos
Descoberta de Drogas , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Voluntários Saudáveis , Humanos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
3.
Nat Commun ; 10(1): 2968, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273206

RESUMO

NMDA receptor-dependent long-term depression (LTD) in the hippocampus is a well-known form of synaptic plasticity that has been linked to different cognitive functions. The core mechanism for this form of plasticity is thought to be entirely neuronal. However, we now demonstrate that astrocytic activity drives LTD at CA3-CA1 synapses. We have found that LTD induction enhances astrocyte-to-neuron communication mediated by glutamate, and that Ca2+ signaling and SNARE-dependent vesicular release from the astrocyte are required for LTD expression. In addition, using optogenetic techniques, we show that low-frequency astrocytic activation, in the absence of presynaptic activity, is sufficient to induce postsynaptic AMPA receptor removal and LTD expression. Using cell-type-specific gene deletion, we show that astrocytic p38α MAPK is required for the increased astrocytic glutamate release and astrocyte-to-neuron communication during low-frequency stimulation. Accordingly, removal of astrocytic (but not neuronal) p38α abolishes LTD expression. Finally, this mechanism modulates long-term memory in vivo.


Assuntos
Astrócitos/enzimologia , Hipocampo/fisiologia , Memória de Longo Prazo/fisiologia , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Comportamento Animal/fisiologia , Medo/fisiologia , Feminino , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Depressão Sináptica de Longo Prazo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Optogenética , Técnicas de Patch-Clamp , Potenciais Sinápticos/fisiologia
4.
Nat Commun ; 10(1): 3071, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296856

RESUMO

The formation of new blood vessels is essential for normal development, tissue repair and tumor growth. Here we show that inhibition of the kinase p38α enhances angiogenesis in human and mouse colon tumors. Mesenchymal cells can contribute to tumor angiogenesis by regulating proliferation and migration of endothelial cells. We show that p38α negatively regulates an angiogenic program in mesenchymal stem/stromal cells (MSCs), multipotent progenitors found in perivascular locations. This program includes the acquisition of an endothelial phenotype by MSCs mediated by both TGF-ß and JNK, and negatively regulated by p38α. Abrogation of p38α in mesenchymal cells increases tumorigenesis, which correlates with enhanced angiogenesis. Using genetic models, we show that p38α regulates the acquisition of an endothelial-like phenotype by mesenchymal cells in colon tumors and damage tissue. Taken together, our results indicate that p38α in mesenchymal cells restrains a TGF-ß-induced angiogenesis program including their ability to transdifferentiate into endothelial cells.


Assuntos
Neoplasias do Colo/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Proliferação de Células , Transdiferenciação Celular , Células Endoteliais/patologia , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Transição Epitelial-Mesenquimal , Técnicas de Silenciamento de Genes , Células HT29 , Humanos , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Quinase 14 Ativada por Mitógeno/genética , Neoplasias Experimentais/induzido quimicamente , RNA Interferente Pequeno/metabolismo
5.
Eur J Med Chem ; 178: 530-543, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31212132

RESUMO

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 µM for ERK5; IC50 > 120 µM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.


Assuntos
Antineoplásicos/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Disponibilidade Biológica , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo
6.
PLoS One ; 14(5): e0216948, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31141540

RESUMO

As an important part of the comprehensive treatment methods, the urate-lowering Chinese herbs could provide favorable clinical effects on hyperuricemia in its ability to invigorate spleen and remove dampness. Owing to the long-term duration, it brought up the potential adverse reactions (ADRs) and concerns about the drug-induced liver injury from these herbs. To address this problem, the bioinformatics approaches which combined the network pharmacology, computer simulation and molecular biology experiments were undertaken to elucidate the underlying drug-induced liver injury molecular mechanisms of urate-lowering Chinese herbs. Several electronic databases were searched to identify the potential liver injury compounds in published research. Then, the putative target profile of liver injury was predicted, and the interaction network was constructed based on the links between the compounds, corresponding targets and core pathways. Accordingly, the molecular docking simulation was performed to recognize the representative compounds with hepatotoxicity. Finally, the cell experiments were conducted to investigate the biochemical indicators and expression of the crucial protein that were closely associated with liver injury. In conclusion, the current research revealed that the compounds with potential liver injury including diosgenin, baicalin, saikosaponin D, tetrandrine, rutaecarpine and evodiamine from urate-lowering Chinese herbs, could lead to decline the survival rate of L-02 cell, increase the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) in cell-culture medium, enhance the expression of p-p38/p38, while the p38 inhibitor could achieve the trend of regulating and controlling liver injury. These research findings bring further support to the growing evidence that the mechanism of the liver injury induced by the compounds from urate-lowering Chinese herbs may be associated with the activation of p38α.


Assuntos
Antimetabólitos/efeitos adversos , Medicamentos de Ervas Chinesas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Supressores da Gota/efeitos adversos , Proteína Quinase 14 Ativada por Mitógeno/química , Alanina Transaminase/genética , Alanina Transaminase/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Antimetabólitos/química , Antimetabólitos/isolamento & purificação , Antimetabólitos/farmacologia , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Benzilisoquinolinas/efeitos adversos , Benzilisoquinolinas/química , Benzilisoquinolinas/isolamento & purificação , Benzilisoquinolinas/farmacologia , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Biologia Computacional/métodos , Flavonoides/efeitos adversos , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Supressores da Gota/química , Supressores da Gota/isolamento & purificação , Supressores da Gota/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Hiperuricemia/tratamento farmacológico , Hiperuricemia/fisiopatologia , Alcaloides Indólicos/efeitos adversos , Alcaloides Indólicos/química , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/farmacologia , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Quinazolinas/efeitos adversos , Quinazolinas/química , Quinazolinas/isolamento & purificação , Quinazolinas/farmacologia , Saponinas/efeitos adversos , Saponinas/química
7.
Life Sci Alliance ; 2(2)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30940732

RESUMO

Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here, we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification. The activation of MAPK14 induced MAPK7 phosphorylation in cultured SSCs, and targeted deletion of Mapk14 or Mapk7 resulted in significant SSC deficiency after spermatogonial transplantation. The activation of this signaling pathway not only induced Nox1 but also increased ROS levels. Chemical screening of MAPK7 targets revealed many ROS-dependent spermatogonial transcription factors, of which BCL6B was found to initiate ROS production by increasing Nox1 expression via ETV5-induced nuclear translocation. Because hydrogen peroxide or Nox1 transfection also induced BCL6B nuclear translocation, our results suggest that BCL6B initiates and amplifies ROS signals to activate ROS-dependent spermatogonial transcription factors by forming a positive feedback loop.


Assuntos
Células-Tronco Germinativas Adultas/fisiologia , Autorrenovação Celular/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Benzodiazepinonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Retroalimentação Fisiológica/fisiologia , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
8.
Cell Physiol Biochem ; 52(4): 758-768, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30933440

RESUMO

BACKGROUND/AIMS: Bromodomain-containing protein 4 (BRD4) and phosphatidylinositol 3-kinase (PI3K) are key oncogenic cascades in colorectal cancer (CRC). SF1126 is a novel and potent PI3K-BRD4 dual inhibitor. METHODS: CRC cells and human colon epithelial cells were treated with SF1126. Cell survival was tested by MTT and soft agar colony formation assays. Cell proliferation was tested by BrdU ELISA method. Cell apoptosis was tested by a TUNEL staining method and Histone DNA ELISA. Western blotting was utilized to test the signaling proteins. A HT-29 xenograft mice model was established to study the anti-tumor activity of SF1126 in vivo. RESULTS: SF1126 potently inhibited the survival, proliferation, and progression of the cell cycle in an established CRC cell line (HT-29) and primary human colon cancer cells. Significant activation of apoptosis was detected in SF1126-treated CRC cells. In CRC cells, SF1126 blocked Akt-mammalian target of rapamycin (mTOR) complex1/2 signaling and downregulated BRD4 target proteins (Myc and cyclin D1). Further studies showed that SF1126 activated p38 signaling in CRC cells. In contrast, the p38 inhibitors or p38 short hairpin RNA inhibited SF1126-induced cytotoxicity and apoptosis in CRC cells. In vivo, subcutaneous administration of SF1126 significantly inhibited HT-29 xenograft tumor growth in nude mice. CONCLUSION: SF1126 inhibits CRC cell growth possibly by targeting PI3K-Akt-mTOR, BRD4, and p38 signaling.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Oligopeptídeos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromonas/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteínas Nucleares/metabolismo , Oligopeptídeos/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transplante Heterólogo
9.
Ann Nucl Med ; 33(5): 333-343, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30953245

RESUMO

OBJECTIVE: p38 mitogen-activated protein (MAP) kinase (p38α) has drawn attention as a new target molecule for the treatment and diagnosis of cancer, and its overexpression and activation have been reported in various types of cancer. In this study, a single photon emission computed tomography (SPECT) imaging probe of p38α was developed to noninvasively image p38α activity for effective qualitative diagnosis of cancer. METHODS: Pyrrolepyridine derivatives, m-YTM and p-YTM, were designed and synthesized based on the structure of the p38α-selective inhibitor. Radioactive iodine-labeled m-YTM, [125I]m-YTM, was synthesized because m-YTM greatly inhibited the phosphorylation of p38α upon examining the inhibitory effects of the compounds. After investigating the binding affinity of [125I]m-YTM to the recombinant p38α, a saturation binding experiment using activated p38α and inactive p38α was performed to determine the binding site. Uptake of [125I]m-YTM into various cancer cell lines was investigated, and the pharmacokinetics was evaluated using tumor-bearing mice. RESULTS: The inhibitory activity of m-YTM was approximately 13 times higher than that of SB203580, a p38α-selective inhibitor. The binding site of [125I]m-YTM was estimated to be the p38α activating site, similar to that of SB203580, because the [125I]m-YTM bound strongly to both activated p38α and inactive p38α. Various different cancer cells incorporated [125I]m-YTM; however, its accumulation was significantly reduced by treatment with SB203580. Pharmacokinetics study of [125I]m-YTM in B-16 tumor-bearing mice was examined which revealed high accumulation of radioactivity in tumor tissues. The ratios of radioactivity in the B-16 tumor to that in blood were 3.1 and 50 after 1 and 24 h, respectively. The ratio of radioactivity in the tumor to that in blood in the tumor-bearing mice generated using other cancer cell lines was also ≥ 1 at 1 h after the administration of the probe. CONCLUSIONS: This study suggests that [123I]m-YTM has potential as a p38α imaging probe effective for various cancer types.


Assuntos
Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Pirróis/química , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Pirróis/metabolismo , Pirróis/farmacocinética , Pirróis/farmacologia , Distribuição Tecidual
10.
Basic Clin Pharmacol Toxicol ; 125(3): 304-314, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30861304

RESUMO

Reactive oxygen species (ROS) is regarded as a critical denominator in nanoparticle toxicology and inflammation. Previously, we have shown that silica nanoparticles sized 50 nm (Si50) induce release of CXCL8 and IL-6 from BEAS-2B cells, via mechanisms involving NFκB, p38 MAP kinase and TGF-α-activated EGF receptor. In the present study, the role of ROS-mediated mechanisms in the concentration-dependent Si50 induction of CXCL8 and IL-6 responses was examined. Si50 (200 µg/mL) induced a time-dependent ROS formation and a postponed increase in expression of haem oxygenase (HO-1) mRNA and protein. Pre-treatment with the ROS inhibitors N-acetyl cysteine (NAC) and diphenyleneiodonium (DPI) partially attenuated CXCL8 and IL-6 responses to 200 µg/mL, but not to 100 µg/mL Si50. The release of TGF-α induced by Si50 (200 µg/mL) was significantly reduced by NAC, but not by DPI nor siRNA against NADPH oxidase DUOX-1 (siDUOX-1). Furthermore, siDUOX-1 reduced Si50-induced CXCL8, but not IL-6. Both p38 and p65 phosphorylations were inhibited by siDUOX-1, but for NAC only p65 phosphorylation reached a significant reduction. Neither NAC nor DPI reduced Si50-induced CXCL8 and IL-6 gene expressions. In conclusion, Si50-induced CXCL8 and IL-6 involved both ROS-dependent and ROS-independent mechanisms. Notably, the role of ROS seemed restricted to effects of higher concentrations of Si50 and not mediated via the gene expression.


Assuntos
Brônquios/efeitos dos fármacos , Nanopartículas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Dióxido de Silício/toxicidade , Brônquios/citologia , Brônquios/imunologia , Linhagem Celular , Oxidases Duais/genética , Oxidases Duais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-6/imunologia , Interleucina-6/metabolismo , Interleucina-8/imunologia , Interleucina-8/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Tamanho da Partícula , Fosforilação/efeitos dos fármacos , Fosforilação/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/imunologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Transcrição RelA/metabolismo
11.
Photochem Photobiol Sci ; 18(6): 1398-1407, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-30924488

RESUMO

In photopharmacology, photoswitchable compounds including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacologically less potent. Herein we report the design, synthesis and photochemical/inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were observed. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible reduction of the azo group to the corresponding hydrazine was observed. Independent experiments revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biological assays were responsible. Two further sources of error are the concentration dependence of the E-Z-switching efficiency and artefacts due to incomplete exclusion of light during testing. Our findings may also apply to a number of previously investigated azobenzene-based photoswitchable inhibitors.


Assuntos
Azocinas/farmacologia , Caseína Quinase Idelta/antagonistas & inibidores , Imidazóis/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Azocinas/química , Caseína Quinase Idelta/metabolismo , Relação Dose-Resposta a Droga , Imidazóis/química , Ligantes , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Processos Fotoquímicos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tiazóis/química
12.
Biochem Biophys Res Commun ; 511(3): 579-586, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30824186

RESUMO

p38α mitogen-activated protein kinase (MAPK) is an attracting pharmacological target in inflammatory diseases and cancer. Searching for new and more efficient p38-MAPK inhibitors, two recently developed pyrazolobenzothiazine-based (COXP4M12 and COXH11) compounds were investigated in this study using a cellular model of p38 activation. This consisted of HT29 human colorectal adenocarcinoma cells exposed to H2O2 or lipopolysaccharide (LPS). Immunoblot data confirmed the inhibitory effect of COXP4M12 and COXH11 on p38 substrate phosphorylation (MAPK-APK2 and ATF2 transcription factor). Compound cytotoxicity was very low and apparent efficacy of these inhibitors was comparable with that of SB203580, a commercially available type I inhibitor of p38. All these compounds also inhibit upstream kinases that promote p38-MAPK phosphorylation and co-activate the stress-activated protein kinase JNK, while ERK1/2 MAPK phosphorylation was unaffected. Compound-target kinase interaction was investigated by means of co-crystallization experiments that provided further structural and molecular insight on the inhibitory mechanism and optimization strategy of this new class of p38-MAPK inhibitors.


Assuntos
Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tiazinas/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Proteína Quinase 14 Ativada por Mitógeno/química , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirazóis/farmacologia , Tiazinas/química
13.
Bioorg Med Chem ; 27(7): 1308-1319, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30792101

RESUMO

Two new series of furochromone and benzofuran derivatives were designed, synthesized and evaluated for their in vitro anticancer activity against MCF-7 and MDA231 breast cancer cell lines. Compounds 5, 6, 7, 9, 15a, 16, 17a and 18 exhibited the best antiproliferative activities with IC50 values ranging from 1.19 to 2.78 µM against MCF-7 superior to lapatinib as reference standard (IC50; 4.69 µM). Compounds 15a and 18 revealed significant cytotoxic activity against MCF-7 and MDA231, therefore their inhibitory potencies against p38α MAP kinase were evaluated. Remarkably they exhibited significant IC50 of 0.04 µM comparable to SB203580 (IC50; 0.50 µM) as a reference standard. These promising results of cytotoxic activity and significant inhibition of p38α MAP kinase, were confirmed by exploring the effect of benzofuran derivative (18) on the apoptotic induction and cell cycle progression of MCF-7 cell line. Compound 18 induced preG1 apoptosis and cell growth arrest at G2/M phase preventing the mitotic cycle. Moreover it activated the caspase-7 which executes apoptosis. Molecular docking study was carried out using GOLD program to predict the mode of binding interaction of the synthesized compounds into the target p38α MAPK. Additionally, the physicochemical properties and ADME parameters of compound 18 were examined in silico to investigate its drug-likeness.


Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Desenho de Drogas , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Produtos Biológicos/síntese química , Produtos Biológicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Células MCF-7 , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Oxigênio/química , Oxigênio/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
14.
Redox Biol ; 22: 101137, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30771750

RESUMO

Injury-induced stenosis is a serious vascular complication. We previously reported that p38α (MAPK14), a redox-regulated p38MAPK family member was a negative regulator of the VSMC contractile phenotype in vitro. Here we evaluated the function of VSMC-MAPK14 in vivo in injury-induced neointima hyperplasia and the underlying mechanism using an inducible SMC-MAPK14 knockout mouse line (iSMC-MAPK14-/-). We show that MAPK14 expression and activity were induced in VSMCs after carotid artery ligation injury in mice and ex vivo cultured human saphenous veins. While the vasculature from iSMC-MAPK14-/- mice was indistinguishable from wildtype littermate controls at baseline, these mice exhibited reduced neointima formation following carotid artery ligation injury. Concomitantly, there was an increased VSMC contractile protein expression in the injured vessels and a decrease in proliferating cells. Blockade of MAPK14 through a selective inhibitor suppressed, while activation of MAPK14 by forced expression of an upstream MAPK14 kinase promoted VSMC proliferation in cultured VSMCs. Genome wide RNA array combined with VSMC lineage tracing studies uncovered that vascular injury evoked robust inflammatory responses including the activation of proinflammatory gene expression and accumulation of CD45 positive inflammatory cells, which were attenuated in iSMC-MAPK14-/- mice. Using multiple pharmacological and molecular approaches to manipulate MAPK14 pathway, we further confirmed the critical role of MAPK14 in activating proinflammatory gene expression in cultured VSMCs, which occurs in a p65/NFkB-dependent pathway. Finally, we found that NOX4 contributes to MAPK14 suppression of the VSMC contractile phenotype. Our results revealed that VSMC-MAPK14 is required for injury-induced neointima formation, likely through suppressing VSMC differentiation and promoting VSMC proliferation and inflammation. Our study will provide mechanistic insights into therapeutic strategies for mitigation of vascular stenosis.


Assuntos
Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Neointima/patologia , Animais , Biomarcadores , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Diferenciação Celular , Proliferação de Células , Imunofluorescência , Expressão Gênica , Humanos , Hiperplasia , Imuno-Histoquímica , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação , Camundongos , Proteína Quinase 14 Ativada por Mitógeno/genética , Miócitos de Músculo Liso/citologia , NADPH Oxidase 4/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição RelA/metabolismo
15.
Mar Drugs ; 17(1)2019 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-30642059

RESUMO

Marine animals and plants provide abundant secondary metabolites with antitumor activity. Itampolin A is a brominated natural tyrosine secondary metabolite that is isolated from the sponge Iotrochota purpurea. Recently, we have achieved the first total synthesis of this brominated tyrosine secondary metabolite, which was found to be a potent p38α inhibitor exhibiting anticancer effects. A fragment-based drug design (FBDD) was carried out to optimize itampolin A. Forty-five brominated tyrosine derivatives were synthesized with interesting biological activities. Then, a QSAR study was carried out to explore the structural determinants responsible for the activity of brominated tyrosine skeleton p38α inhibitors. The lead compound was optimized by a FBDD method, then three series of brominated tyrosine derivatives were synthesized and evaluated for their inhibitory activities against p38α and tumor cells. Compound 6o (IC50 = 0.66 µM) exhibited significant antitumor activity against non-small cell lung A549 cells (A549). This also demonstrated the feasibility of the FBDD method of structural optimization.


Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Desenho de Drogas , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Poríferos , Células A549 , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios Enzimáticos , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/patologia , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade
16.
Mol Cell Endocrinol ; 484: 25-33, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30682387

RESUMO

Unexplained hyperandrogenic oligoanovulation is a main feature of polycystic ovary syndrome (PCOS). P450c17 phosphorylation selectively increases 17,20-lyase activity and androgen biosynthesis but minimally affects 17α-hydroxylase. Studies have recently identified mitogen-activated protein kinase 14 (MAPK14, p38α) as the kinase responsible for enhancing 17,20-lyase activity through P450c17 phosphorylation. We investigated whether oxidant-induced oxidative stress increases 17,20-lyase activity through oxidant-sensitive p38α signaling pathways. NCI-H295R adrenal cells were treated with three oxidants, palmitate, H2O2 and 4-hydroxy-2-nonenal (HNE), to simulate the excessive oxidative stress of PCOS. Oxidant exposure significantly induced dehydroepiandrosterone production and increased p38α phosphorylation and activation, but the effect on 17α-hydroxyprogesterone production was far less clear. None of the treatments altered the expression of P450c17 or its necessary factors POR and b5. LC-MS/MS revealed increased DHEA production in NCI-H295R cells. Both p38α inhibition and siRNA-mediated silencing attenuated H2O2- or 0.45-0.75 mM PA-mediated augmentation of DHEA production with relatively stable 17OHP levels, indicating that activated p38α mediates oxidative stress-induced 17,20-lyase activation and androgen synthesis stimulation, which may underlie hyperandrogenism in PCOS.


Assuntos
Hiperandrogenismo/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Estresse Oxidativo , Esteroide 17-alfa-Hidroxilase/metabolismo , Aldeídos/efeitos adversos , Linhagem Celular , Desidroepiandrosterona/metabolismo , Ativação Enzimática , Humanos , Peróxido de Hidrogênio/efeitos adversos , Ácido Palmítico/efeitos adversos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
17.
FEBS J ; 286(5): 1030-1052, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30663248

RESUMO

Bistability (coexistence of two stable steady states in a dynamical system) is a key mechanism of cellular decision-making and has been observed in many biochemical reaction networks such as mitogen-activated protein kinase (MAPK) signaling pathways. Theoretical studies have shown that bistability can arise in a single two-site MAPK phosphorylation and dephosphorylation cycle. However, the bistable behavior mostly relies on the kinetic mechanisms and parameters of this two-site modification. In exploring the system-level properties of MAPK regulation, most models to date focus on two limiting reaction regimes, distributive and processive, and are characterized by high levels of parametric uncertainty. Here, we developed a combined kinetic method which applies a continuous spectrophotometric enzyme-coupled assay incorporated with the viscosity approach, to perform detailed kinetic analyses of p38α MAPK dual phosphorylation by MKK6. Almost all kinetic rate constants for the first and second phosphorylation steps in p38α activation have been quantitatively determined, supporting that the phosphorylation occurs randomly in the first step, albeit preferring the tyrosine residue. The release rates of monophosphorylated p38α from MKK6, either as the product in the first modification or as the substrate in the second step, were comparable to the respective adjacent phosphoryl transfer steps. These results indicated that dual phosphorylation of p38α by MKK6 involves a random, partially processive mechanism. Based on the experimentally determined models and parameters, dynamics of the p38α-MKK6-MKP5 system were explored, demonstrating for the first time that bistability can arise with this model at biologically feasible parameter values. ENZYMES: p38α (EC 2.7.11.24); MKK6 (EC 2.7.12.2).


Assuntos
MAP Quinase Quinase 6/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Humanos , Cinética , Fosforilação , Espectrofotometria/métodos , Especificidade por Substrato
18.
Gene ; 689: 18-23, 2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30521886

RESUMO

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Aberrant expression of HOXA5 results in various diseases, including cancers. However, the specific function and molecular mechanism of HOXA5 in osteosarcoma is not fully understood. In the present study, we focused on HOXA5 in U2OS and MG63 cells in vitro. We observed lower expression of HOXA5 in U2OS, MG63, and SaOS2 human osteosarcoma cells, compared with hFOB1.19 human osteoblastic cells. HOXA5 overexpression in U2OS and MG63 cells markedly reduced cell survival and proliferation and elevated cell apoptosis and caspase-3 activity. HOXA5 also activated the p38α MAPK pathway by increasing p53. Treating U2OS and MG63 cells with the p53 inhibitor α-pifithrin or the p38α MAPK inhibitor SB203580 led to higher cell survival and proliferation and lower cell apoptosis, compared with the pcDNA3.1-HOXA5 group. In conclusion, our study showed that the p53 and p38α MAPK signal axis facilitated HOXA5's role in inhibiting growth and stimulating apoptosis of osteosarcoma cells.


Assuntos
Apoptose/genética , Neoplasias Ósseas/patologia , Proteínas de Homeodomínio/genética , Osteossarcoma/patologia , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Osteossarcoma/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/genética
19.
Drug Res (Stuttg) ; 69(2): 100-110, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30041258

RESUMO

In this study, the optimized 4-(4-hydroxybenzyl)-2-amino-6-hydroxypyrimidine-5-carboxamide derivative was formulated as nanoparticles to evaluate for their anticancer activity. The response surface methodology (RSM) was performed with utilization of Box-Behnken statistical design (BBSD) to optimize the experimental conditions for identification of significant synthetic methodology. To explore the stability of the derivative was done by density functional theory (DFT). Graph theoretical analysis was introduced to identify the drug target p38α MAP Kinases and then insilico modeling was performed to provide straightforward information for further structural optimization. The experimental results under optimal experimental conditions obtained 74.55-76% yield of 4-(4-hydroxybenzyl)-2-amino-6-hydroxypyrimidine-5-carboxamide, 127oC melting point and Rf value 0.59 were well matched with the predicted results and this was gaining 95% of confidence level and suitability of RSM. The spectral data were reliable with the assigned structures of synthetic yields. The formulated nanoparticles were exhibited a good anticancer activity against used cancer cell line MCF7. Amusingly the observed docking scores and in-vitro anticancer activity was proving the compound significance and potential as a potent p38α inhibitor. Further, we have elucidated the mechanism of action at its functional level using label-free quantitative proteomics. Interestingly the observed results were indicating that the derived proteomics data involving in the alteration process in cancer-related regulatory pathways.


Assuntos
Antineoplásicos/química , Portadores de Fármacos/química , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Modelos Moleculares , Pirimidinas/química , Antineoplásicos/farmacologia , Química Farmacêutica , Composição de Medicamentos/métodos , Desenho de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Proteômica/métodos , Pirimidinas/farmacologia , Software
20.
Med Sci Monit ; 24: 8804-8811, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30517088

RESUMO

BACKGROUND Fractalkine is widely expressed throughout the brain and spinal cord, where it can exert effects on pain enhancement and hyperalgesia by activating microglia through CX3C chemokine receptor 1 (CX3CR1), which triggers the release of several pro-inflammatory cytokines in the spinal cord. Fractalkine has also been shown to increase cytosolic calcium ([Ca2+]i) in microglia. MATERIAL AND METHODS Based on the characteristics of CX3CR1, a G protein-coupled receptor, we explored the role of inositol 1,4,5-trisphosphate (IP3) signaling in fractalkine-induced inflammatory response in BV-2 cells in vitro. The effect and the underlying mechanism induced by fractalkine in the brain were observed using a mouse model with intracerebroventricular (i.c.v.) injection of exogenous fractalkine. RESULTS [Ca2+]i was significantly increased and IL-1ß and TNF-α levels were higher in the fractalkine-treated cell groups than in the farctalkine+ 2-APB groups. We found that i.c.v. injection of fractalkine significantly increased p-p38MAPK, IL-1ß, and TNF-α expression in the brain, while i.c.v. injection of a fractalkine-neutralizing antibody (anti-CX3CR1), trisphosphate receptor (IP3R) antagonist (2-APB), or p38MAPK inhibitor (SB203580) prior to fractalkine addition yielded an effective and reliable anti-allodynia effect, following the reduction of p-p38MAPK, IL-1ß, and TNF-α expression. CONCLUSIONS Our results suggest that fractalkine leads to hyperalgesia, and the underlying mechanism may be associated with IP3/p38MAPK-mediated calcium signaling and its phlogogenic properties.


Assuntos
Receptor 1 de Quimiocina CX3C/efeitos dos fármacos , Quimiocina CX3CL1/fisiologia , Hiperalgesia/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Quimiocina CX3CL1/metabolismo , China , Injeções Espinhais , Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-1beta/metabolismo , Ativação de Macrófagos , Camundongos , Microglia/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Dor/tratamento farmacológico , Receptores de Quimiocinas , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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