RESUMO
Biochemical, pathogenic, and human genetic data confirm that GSAP (γ-secretase activating protein), a selective γ-secretase modulatory protein, plays important roles in Alzheimer's disease (AD) and Down's syndrome. However, the molecular mechanism(s) underlying GSAP-dependent pathogenesis remains largely elusive. Here, through unbiased proteomics and single-nuclei RNAseq, we identified that GSAP regulates multiple biological pathways, including protein phosphorylation, trafficking, lipid metabolism, and mitochondrial function. We demonstrated that GSAP physically interacts with the Fe65-APP complex to regulate APP trafficking/partitioning. GSAP is enriched in the mitochondria-associated membrane (MAM) and regulates lipid homeostasis through the amyloidogenic processing of APP. GSAP deletion generates a lipid environment unfavorable for AD pathogenesis, leading to improved mitochondrial function and the rescue of cognitive deficits in an AD mouse model. Finally, we identified a novel GSAP single-nucleotide polymorphism that regulates its brain transcript level and is associated with an increased AD risk. Together, our findings indicate that GSAP impairs mitochondrial function through its MAM localization and that lowering GSAP expression reduces pathological effects associated with AD.
Assuntos
Doença de Alzheimer/patologia , Homeostase , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Proteínas/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Sequência de Bases , Modelos Animais de Doenças , Hipocampo/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Teste de Campo Aberto , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas/genética , Transcrição GênicaRESUMO
Neurotoxic amyloid-ß peptides (Aß) are major drivers of Alzheimer's disease (AD) and are formed by sequential cleavage of the amyloid precursor protein (APP) by ß-secretase (BACE) and γ-secretase. Our previous study showed that the anticancer drug Gleevec lowers Aß levels through indirect inhibition of γ-secretase activity. Here we report that Gleevec also achieves its Aß-lowering effects through an additional cellular mechanism. It renders APP less susceptible to proteolysis by BACE without inhibiting BACE enzymatic activity or the processing of other BACE substrates. This effect closely mimics the phenotype of APP A673T, a recently discovered mutation that protects carriers against AD and age-related cognitive decline. In addition, Gleevec induces formation of a specific set of APP C-terminal fragments, also observed in cells expressing the APP protective mutation and in cells exposed to a conventional BACE inhibitor. These Gleevec phenotypes require an intracellular acidic pH and are independent of tyrosine kinase inhibition, given that a related compound lacking tyrosine kinase inhibitory activity, DV2-103, exerts similar effects on APP metabolism. In addition, DV2-103 accumulates at high concentrations in the rodent brain, where it rapidly lowers Aß levels. This study suggests that long-term treatment with drugs that indirectly modulate BACE processing of APP but spare other BACE substrates and achieve therapeutic concentrations in the brain might be effective in preventing or delaying the onset of AD and could be safer than nonselective BACE inhibitor drugs.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacosRESUMO
Amyloidosis is a major problem in over one hundred diseases, including Alzheimer's disease (AD). Using the iDISCO visualization method involving targeted molecular labeling, tissue clearing, and light-sheet microscopy, we studied plaque formation in the intact AD mouse brain at up to 27 months of age. We visualized amyloid plaques in 3D together with tau, microglia, and vasculature. Volume imaging coupled to automated detection and mapping enables precise and fast quantification of plaques within the entire intact mouse brain. The present methodology is also applicable to analysis of frozen human brain samples without specialized preservation. Remarkably, amyloid plaques in human brain tissues showed greater 3D complexity and surprisingly large three-dimensional amyloid patterns, or TAPs. The ability to visualize amyloid in 3D, especially in the context of their micro-environment, and the discovery of large TAPs may have important scientific and medical implications.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Placa Amiloide/patologia , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Imageamento Tridimensional/métodos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Placa Amiloide/metabolismoRESUMO
The components involved in cellular trafficking and protein recycling machinery that have been associated with increased Alzheimer's disease (AD) risk belong to the late secretory compartments for the most part. Here, we hypothesize that these late unavoidable events might be the consequence of earlier complications occurring while amyloid precursor protein (APP) is trafficking through the early secretory pathway. We investigated the relevance to AD of coat protein complex I (COPI)-dependent trafficking, an early step in Golgi-to-endoplasmic reticulum (ER) retrograde transport and one of the very first trafficking steps. Using a complex set of imaging technologies, including inverse fluorescence recovery after photobleaching (iFRAP) and photoactivatable probes, coupled to biochemical experiments, we show that COPI subunit δ (δ-COP) affects the biology of APP, including its subcellular localization and cell surface expression, its trafficking, and its metabolism. These findings demonstrate the crucial role of δ-COP in APP metabolism and, consequently, the generation of amyloid-ß (Aß) peptide, providing previously nondescribed mechanistic explanations of the underlying events.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Proteína Coatomer/metabolismo , Neurônios/metabolismo , Frações Subcelulares/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Transporte Proteico/fisiologiaRESUMO
Cellular trafficking and recycling machineries belonging to late secretory compartments have been associated with increased Alzheimer's disease (AD) risk. We have shown that coat protein complex I (COPI)-dependent trafficking, an early step in Golgi-to-endoplasmic reticulum retrograde transport, affects amyloid precursor protein subcellular localization, cell-surface expression, as well as its metabolism. We present here a set of experiments demonstrating that, by targeting subunit δ-COP function, the moderation of the COPI-dependent trafficking in vivo leads to a significant decrease in amyloid plaques in the cortex and hippocampus of neurological 17 mice crossed with the 2xTg AD mouse model. Remarkably, an improvement of the memory impairments was also observed. Importantly, human genetic association studies of different AD cohorts led to the identification of 12 SNPs and 24 mutations located in COPI genes linked to an increased AD risk. These findings further demonstrate in vivo the importance of early trafficking steps in AD pathogenesis and open new clinical perspectives.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , Progressão da Doença , Placa Amiloide/metabolismo , Frações Subcelulares/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/fisiologiaRESUMO
Increased production of amyloid-ß (Aß)42 peptide, derived from the amyloid-ß protein precursor, and its subsequent aggregation into oligomers and plaques constitutes a hallmark of Alzheimer's disease (AD). We here report on a family of low molecular weight molecules, the Aftins (Amyloid-ß Forty-Two Inducers), which, in cultured cells, dramatically affect the production of extracellular/secreted amyloid peptides. Aftins trigger ß-secretase inhibitor and γ-secretase inhibitors (GSIs) sensitive, robust upregulation of Aß42, and parallel down-regulation of Aß38, while Aß40 levels remain stable. In contrast, intracellular levels of these amyloids appear to remain stable. In terms of their effects on Aß38/Aß40/Aß42 relative abundance, Aftins act opposite to γ-secretase modulators (GSMs). Aß42 upregulation induced by Aftin-5 is unlikely to originate from reduced proteolytic degradation or diminished autophagy. Aftin-5 has little effects on mitochondrial functional parameters (swelling, transmembrane potential loss, cytochrome c release, oxygen consumption) but reversibly alters the ultrastructure of mitochondria. Aftins thus alter the Aß levels in a fashion similar to that described in the brain of AD patients. Aftins therefore constitute new pharmacological tools to investigate this essential aspect of AD, in cell cultures, allowing (1) the detection of inhibitors of Aftin induced action (potential 'anti-AD compounds', including GSIs and GSMs) but also (2) the identification, in the human chemical exposome, of compounds that, like Aftins, might trigger sustained Aß42 production and Aß38 down-regulation (potential 'pro-AD compounds').
Assuntos
Adenina/análogos & derivados , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Modelos Químicos , Fragmentos de Peptídeos/metabolismo , Adenina/farmacologia , Adenina/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , HumanosRESUMO
The pathways leading specifically to the toxic Aß42 peptide production, a key event in Alzheimer's disease (AD), are unknown. While searching for pathways that mediate pathological increases of Aß42, we identified Aftin-4, a new compound that selectively and potently increases Aß42 compared to DMSO (N2a cells: 7-fold; primary neurons: 4-fold; brain lysates: 2-fold) with an EC(50) of 30 µM. These results were confirmed by ELISA and IP-WB. Using affinity chromatography and mass spectrometry, we identified 3 proteins (VDAC1, prohibitin, and mitofilin) relevant to AD that interact with Aftin-4, but not with a structurally similar but inactive molecule. Electron microscopy studies demonstrated that Aftin-4 induces a reversible mitochondrial phenotype reminiscent of the one observed in AD brains. Sucrose gradient fractionation showed that Aftin-4 perturbs the subcellular localization of γ-secretase components and could, therefore, modify γ-secretase specificity by locally altering its membrane environment. Remarkably, Aftin-4 shares all these properties with two other "AD accelerator" compounds. In summary, treatment with three Aß42 raising agents induced similar biochemical alterations that lead to comparable cellular phenotypes in vitro, suggesting a common mechanism of action involving three structural cellular targets.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Encéfalo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Fragmentos de Peptídeos/biossíntese , Adenina/análogos & derivados , Adenina/química , Adenina/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Western Blotting , Encéfalo/metabolismo , Celecoxib , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Fenofibrato/metabolismo , Fenofibrato/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Neurônios/metabolismo , Compostos Orgânicos/metabolismo , Fragmentos de Peptídeos/genética , Proibitinas , Ligação Proteica/efeitos dos fármacos , Purinas/metabolismo , Purinas/farmacologia , Pirazóis/metabolismo , Pirazóis/farmacologia , Proteínas Repressoras/metabolismo , Roscovitina , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
A new series of 2,6,9-trisubstituted purines, structurally related to the cyclin-dependent kinase (CDK) inhibitor Roscovitine, has been synthesized. These compounds mainly differ by the substituent on the C-2 position which encompasses a diol group. These compounds were screened for kinase inhibitory activities and antiproliferative effects. They were shown to be potent inhibitors of cyclin-dependent kinases but also, for some of them of casein kinase 1 (CK1) and dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). The inhibition of kinases was accompanied by an antiproliferative effect against several tumor cell-lines. The most potent derivatives inhibited SH-SY5Y (neuroblastoma) tumor cell line with an IC(50) < 0.5 µM which means approximately a 30 fold increase compared to Roscovitine. A valine ester was also prepared from the most potent inhibitor to serve as a prodrug.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Purinas/síntese química , Purinas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Accumulation of neurotoxic amyloid-beta is a major hallmark of Alzheimer's disease. Formation of amyloid-beta is catalysed by gamma-secretase, a protease with numerous substrates. Little is known about the molecular mechanisms that confer substrate specificity on this potentially promiscuous enzyme. Knowledge of the mechanisms underlying its selectivity is critical for the development of clinically effective gamma-secretase inhibitors that can reduce amyloid-beta formation without impairing cleavage of other gamma-secretase substrates, especially Notch, which is essential for normal biological functions. Here we report the discovery of a novel gamma-secretase activating protein (GSAP) that drastically and selectively increases amyloid-beta production through a mechanism involving its interactions with both gamma-secretase and its substrate, the amyloid precursor protein carboxy-terminal fragment (APP-CTF). GSAP does not interact with Notch, nor does it affect its cleavage. Recombinant GSAP stimulates amyloid-beta production in vitro. Reducing GSAP concentrations in cell lines decreases amyloid-beta concentrations. Knockdown of GSAP in a mouse model of Alzheimer's disease reduces levels of amyloid-beta and plaque development. GSAP represents a type of gamma-secretase regulator that directs enzyme specificity by interacting with a specific substrate. We demonstrate that imatinib, an anticancer drug previously found to inhibit amyloid-beta formation without affecting Notch cleavage, achieves its amyloid-beta-lowering effect by preventing GSAP interaction with the gamma-secretase substrate, APP-CTF. Thus, GSAP can serve as an amyloid-beta-lowering therapeutic target without affecting other key functions of gamma-secretase.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Benzamidas , Linhagem Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Mesilato de Imatinib , Camundongos , Fragmentos de Peptídeos/metabolismo , Piperazinas/farmacologia , Proteínas/genética , Pirimidinas/farmacologia , Interferência de RNA , Receptor Notch1/metabolismoRESUMO
Neuroblastoma (NB), the most frequent extracranial solid tumor of children accounting for nearly 15% of all childhood cancer mortality, displays overexpression of antiapoptotic Bcl-2 and Mcl-1 in aggressive forms of the disease. The clinical phase 2 drug roscovitine (CYC202, seliciclib), a relatively selective inhibitor of cyclin-dependent kinases (CDKs), and CR8, a recently developed and more potent analog, induce concentration-dependent apoptotic cell death of NB cells (average IC(50) values: 24.2 µM and 0.4 µM for roscovitine and CR8, respectively). Both roscovitine and CR8 trigger rapid down-regulation of the short-lived survival factor Mcl-1 in the 9 investigated human NB cell lines. This effect was further analyzed in the human SH-SY5Y NB cell line. Down-regulation of Mcl-1 appears to depend on inhibition of CDKs rather than on interaction of roscovitine and CR8 with their secondary targets. CR8 is an adenosine triphosphate-competitive inhibitor of CDK9, and the structure of a CDK9/cyclin T/CR8 complex is described. Mcl-1 down-regulation occurs both at the mRNA and protein levels. This effect can be accounted for by a reduction in Mcl-1 protein synthesis, under stable Mcl-1 degradation conditions. Mcl-1 down-regulation is accompanied by a transient increase in free Noxa, a proapoptotic factor. Mcl-1 down-regulation occurs independently of the presence or up-regulation of p53 and of the MYCN status. Taken together, these results suggest that the clinical drug roscovitine and its novel analog CR8 induce apoptotic tumor cell death by down-regulating Mcl-1, a key survival factor expressed in all NB cell lines. CDK inhibition may thus constitute a new approach to treat refractory high-risk NB.
RESUMO
Pharmacological inhibitors of cyclin-dependent kinases (CDKs) have a wide therapeutic potential. Among the CDK inhibitors currently under clinical trials, the 2,6,9-trisubstituted purine (R)-roscovitine displays rather high selectivity, low toxicity, and promising antitumor activity. In an effort to improve this structure, we synthesized several bioisosteres of roscovitine. Surprisingly, one of them, pyrazolo[1,5-a]-1,3,5-triazine 7a (N-&-N1, GP0210), displayed significantly higher potency, compared to (R)-roscovitine and imidazo[2,1-f]-1,2,4-triazine 13 (N-&-N2, GP0212), at inhibiting various CDKs and at inducing cell death in a wide variety of human tumor cell lines. This approach may thus provide second generation analogues with enhanced biomedical potential.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Purinas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Purinas/síntese química , Pirazóis/síntese química , Pirazóis/farmacologia , Roscovitina , Triazinas/síntese química , Triazinas/farmacologiaRESUMO
Glycogen synthase kinase -3 (GSK-3) is a key enzyme involved in numerous physiological events and in major diseases, such as Alzheimer's disease, diabetes, and cardiac hypertrophy. Indirubins are bis-indoles that can be generated from various natural sources or chemically synthesized. While rather potent and selective as GSK-3 inhibitors, most indirubins exhibit low water solubility. To address the issue of solubility, we have designed novel analogues of 6-bromo-indirubin-3'-oxime with increased hydrophilicity based on the GSK-3/indirubins cocrystal structures. The new derivatives with an extended amino side chain attached at position 3' showed potent GSK-3 inhibitory activity, enhanced selectivity, and dramatically increased water solubility. Furthermore, some of them displayed little or no cytotoxicity. The new indirubins inhibit GSK-3 in a cellular reporter model. They alter the circadian period measured in rhythmically expressing cell cultures, suggesting that they might constitute tools to investigate circadian rhythm regulation.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Animais , Sítios de Ligação , Compostos de Bromo/síntese química , Compostos de Bromo/química , Compostos de Bromo/farmacologia , Linhagem Celular , Cristalografia por Raios X , Quinase 3 da Glicogênio Sintase/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Indóis/síntese química , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Ratos , Solubilidade , Relação Estrutura-Atividade , beta Catenina/metabolismoRESUMO
Cyclin-dependent kinases (CDKs) and their regulators show frequent abnormalities in tumors. Ten low molecular weight pharmacologic inhibitors of CDKs are currently in clinical trials against various cancers, including the 2,6,9-trisubstituted purine (R)-roscovitine (CYC202/Seliciclib). We here report the characterization of N-&-N1, a bioisoster of roscovitine displaying improved antitumoral properties. N-&-N1 shows exquisite selectivity for CDKs, with 2- to 3-fold enhanced potency compared with (R)-roscovitine. Inhibition of retinoblastoma protein phosphorylation and RNA polymerase II Ser2 phosphorylation in neuroblastoma SH-SY5Y cells exposed to N-&-N1 indicates that N-&-N1 is able to inhibit CDKs in a cellular context. N-&-N1 also down-regulates the expression of RNA polymerase. Cocrystal structures of N-&-N1 and (R)-roscovitine in complex with CDK2/cyclin A reveal that both inhibitors adopt similar binding modes. A competitive assay shows that, compared with (R)-roscovitine, N-&-N1 has reduced affinity for Erk2 and pyridoxal kinase. N-&-N1 triggers cell death in a panel of diverse cell lines. Cell death is accompanied by events characteristic of apoptosis: cytochrome c release, activation of effector caspases, and poly(ADP-ribose) polymerase cleavage. Induction of p53 and p21CIP1 and down-regulation of the Mcl-1 antiapoptotic factor were also observed. Studies in mice show that N-&-N1 has pharmacokinetics properties similar to those of (R)-roscovitine. Altogether, these results show that analogues of (R)-roscovitine can be designed with improved antitumor potential.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Purinas/química , Roscovitina , Suínos , Extratos de Tecidos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cyclin-dependent kinases (CDKs) and casein kinases 1 (CK1) are involved in the two key molecular features of Alzheimer's disease, production of amyloid-beta peptides (extracellular plaques) and hyper-phosphorylation of Tau (intracellular neurofibrillary tangles). A series of 2,6,9-trisubstituted purines, structurally related to the CDK inhibitor roscovitine, have been synthesized. They mainly differ by the substituent on the C-6 position. These compounds were screened for kinase inhibitory activities and antiproliferative effects. Several biaryl derivatives displayed potent inhibition of both CDKs and CK1. In particular, derivative 13a was a potent inhibitor of CDK1/cyclin B (IC 50: 220 nM), CDK5/p25 (IC 50: 80 nM), and CK1 (IC 50: 14 nM). Modeling of these molecules into the ATP-binding pocket of CK1delta provided a rationale for the increased selectivity toward this kinase. 13a was able to prevent the CK1-dependent production of amyloid-beta in a cell model. CDK/CK1 dual-specificity inhibitors may have important applications in Alzheimer's disease and cancers.
Assuntos
Caseína Quinase I/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Purinas/química , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Inibidores de Proteínas Quinases/análogos & derivados , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Roscovitina , Relação Estrutura-AtividadeRESUMO
Twenty-nine analogs of indirubin, an isomer of indigo, have been synthesized to optimize its promising kinase inhibitory scaffold. These compounds being also pigmented, have been tested for their photoreactivity. Absorption maxima were between 485 nm and 560 nm. Addition of fetal calf serum induced fluorescence and time dependent absorption modifications. Appropriate illumination induced Reactive Oxygen Species (ROS) production for nineteen compounds out of twenty-nine. The relationship between fluorescence and ROS production is discussed. Six compounds showed an important toxicity on F98 cells, a murine glioma cell line. Three of these were found to be also phototoxic, as four other non-toxic compounds. All but one phototoxic compounds were detected as ROS producers by in vitro tests. Photoreactivity assessment is important to anticipate adverse reactions for compounds that might be clinically developed. The experimental assay was found to be the only way to evaluate the photoreactivity of this family of compounds since no predictive criteria on structures could be found. Combining the vascular tumor growth inhibition induced by kinase inhibitors with the massive local blood flow arrest following photodynamic treatment may be an efficient anti-cancer strategy. These data could orientate further syntheses of either non-photoreactive compounds or compounds displaying both kinase inhibitory activity and strong phototoxicity.
Assuntos
Luz , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/efeitos da radiação , Proteínas Quinases/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fluorescência , Humanos , Indóis/química , Indóis/farmacologia , Indóis/efeitos da radiação , Estrutura Molecular , Fotoquímica , Inibidores de Proteínas Quinases/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/efeitos da radiação , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodos , Espectrofotometria Ultravioleta/métodos , Estereoisomerismo , Células Tumorais CultivadasRESUMO
We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Schächtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.; Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors. Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity. Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative disorders, and polycystic kidney disease.
Assuntos
Compostos Aza/síntese química , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Ciclina A/química , Quinase 2 Dependente de Ciclina/química , Indóis/síntese química , Pirimidinas/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Compostos Aza/química , Compostos Aza/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalização , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/química , Indóis/farmacologia , Modelos Moleculares , Pirimidinas/química , Pirimidinas/farmacologia , Esferoides Celulares/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Compostos Aza/química , Compostos Aza/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Células Cultivadas , Cristalografia por Raios X , Ciclina A/química , Ciclina A/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/química , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , Pirimidinas/química , Pirimidinas/metabolismo , Especificidade por Substrato , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Indirubins are kinase inhibitory bis-indoles that can be generated from various plant, mollusk, mammalian, and bacterial sources or chemically synthesized. We here report on the synthesis and biological evaluation of 3'-substituted 7-halogenoindirubins. Molecular modeling and kinase assays suggest that steric hindrance prevents 3'-substituted 7-halogenoindirubins from interacting with classical kinase targets of other indirubins such as cyclin-dependent kinases and glycogen synthase kinase-3. Surprisingly 3'-substituted 7-halogenoindirubins induce cell death in a diversity of human tumor cell lines. Although some 3'-substituted 7-halogenoindirubins appear to induce effector caspase-independent, nonapoptotic cell death, others trigger the landmarks of classical apoptosis. A structure-activity relationship study was performed to optimize 3'-substituted 7-halogenoindirubins with respect to solubility and cell death induction. Despite their unidentified targets, 3'-substituted 7-halogenoindirubins constitute a new promising family of antitumor agents.