Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.930
Filtrar
1.
Int J Nanomedicine ; 14: 5581-5594, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413564

RESUMO

Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder due to the existence of BCR-ABL fusion protein that allows the cells to keep proliferating uncontrollably. Although tyrosine kinase inhibitors can inhibit the activity of BCR-ABL fusion protein to trigger the cells apoptosis, drug resistance or intolerance exists in part of CML patients. Arsenic sulfide in its raw form (r-As4S4) can be orally administrated and certain therapeutic effects have been found out in the treatment of hematologic malignancies through inducing cell apoptosis. Methods: In this work, a water-dissolvable arsenic sulfide nanoformualtion (ee-As4S4) composed of As4S4 particulates with 470 nm in diameter and encapsulated by a kind of hydrophilic polymer was fabricated and applied to the CML cell line K562, K562/AO2 and primary cells from the bone marrow of CML patients. Results: Results showed that instead of inhibiting the activity of BCR-ABL, ee-As4S4 induced direct degradation of BCR-ABL in K562 cells within 6 hr incubation, followed by the occurrence of erythroid differentiation in K562 after 72 hr incubation, evidenced by the significantly upregulated CD235a and benzidine staining, which was not detectable with r-As4S4. The ee-As4S4-induced erythroid differentiation was also observed in K562/AO2 cells and bone marrow mononuclear cells of CML patients. Mechanistic studies indicated that ee-As4S4 induced autophagy by downregulating the level of intracellular ROS and hypoxia-inducible factor-1α significantly, which led to the subsequent degradation of BCR-ABL. When the concentration was increased, ee-As4S4 induced much more significant apoptosis and cell cycle arrest than r-As4S4, and the cytotoxicity of the former was about 178 times of the latter. Conclusion: ee-As4S4 was capable of inducing significant erythroid differentiation of CML cells by inducing the direct degradation of BCR-ABL; the new effect could improve hematopoietic function of CML patients as well as inhibit the leukemic cell proliferation.


Assuntos
Arsenicais/farmacologia , Diferenciação Celular/efeitos dos fármacos , Composição de Medicamentos , Células Eritroides/citologia , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Nanopartículas/química , Proteólise/efeitos dos fármacos , Sulfetos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Eritroides/efeitos dos fármacos , Células Eritroides/ultraestrutura , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Nat Commun ; 10(1): 2572, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189917

RESUMO

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.


Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Retroalimentação Fisiológica/fisiologia , Chaperonas Moleculares/metabolismo , Ataxias Espinocerebelares/patologia , Animais , Retroalimentação Fisiológica/efeitos dos fármacos , Fibroblastos , Células HEK293 , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Hipocampo/patologia , Holoenzimas/metabolismo , Humanos , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ataxias Espinocerebelares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
3.
Nat Chem Biol ; 15(7): 737-746, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209349

RESUMO

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (for example, SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or proteolysis targeting chimeras, PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16-a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases-as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10-40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neosubstrate degradation without substantially perturbing the function of the participating E3 ligase.


Assuntos
Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
4.
Nat Chem Biol ; 15(7): 747-755, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209351

RESUMO

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Limoninas/farmacologia , Proteólise/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Limoninas/química , Limoninas/isolamento & purificação
5.
Mar Drugs ; 17(5)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117253

RESUMO

Among malignancies, lung cancer is the major cause of cancer death. Despite the advance in lung cancer therapy, the five-year survival rate is extremely restricted due to therapeutic failure and disease relapse. Targeted therapies selectively inhibiting certain molecules in cancer cells have been accepted as promising ways to control cancer. In lung cancer, evidence has suggested that the myeloid cell leukemia 1 (Mcl-1) protein, an anti-apoptotic member of the Bcl-2 family, is a target for drug action. Herein, we report the Mcl-1 targeting activity of renieramycin T (RT), a marine-derived tetrahydroisoquinoline alkaloid that was isolated from the Thai blue sponge Xestospongia sp. RT was shown to be dominantly toxic to lung cancer cells compared to the normal cells in the lung. The cytotoxicity of this compound toward lung cancer cells was mainly exerted through apoptosis induction. For the mechanism of action, we found that RT mediated activation of p53 protein and caspase-9 and -3 activations. While others Bcl-2 family proteins (Bcl-2, Bak, and Bax) were minimally changed in response to RT, Mcl-1 protein was dramatically diminished. We further performed the cycloheximide experiment and found that the half-life of Mcl-1 was significantly shortened by RT treatment. When MG132, a potent selective proteasome inhibitor, was utilized, it could restore the Mcl-1 level. Furthermore, immunoprecipitation analysis revealed that RT significantly increased the formation of Mcl-1-ubiquitin complex compared to the non-treated control. In conclusion, we report the potential apoptosis induction of RT with a mechanism of action involving the targeting of Mcl-1 for ubiquitin-proteasomal degradation. As Mcl-1 is critical for cancer cell survival and chemotherapeutic failure, this novel information regarding the Mcl-1-targeted compound would be beneficial for the development of efficient anti-cancer strategies or targeted therapies.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Sistemas de Liberação de Medicamentos , Neoplasias Pulmonares/fisiopatologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Pulmonares/tratamento farmacológico , Poríferos/química , Proteólise/efeitos dos fármacos , Tetra-Hidroisoquinolinas/uso terapêutico , Tetra-Hidroisoquinolinas/toxicidade , Ubiquitinação/efeitos dos fármacos
6.
Eur J Med Chem ; 176: 476-491, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31128449

RESUMO

Tumor suppressor protein p53 is important to the regulation of many cellular processes and the prevention of cancer development. In some cancer cells, the function of p53 is inhibited by murine double minute 2 protein (MDM2). To restore the function of p53, the inhibition or depletion of MDM2 has become a potential therapeutic treatment. We have successfully developed a series of small molecule MDM2 degraders that can promote the proteolysis of MDM2 oncoprotein, thus reactivating tumor suppressor p53. The superior degrader features a nutlin-based MDM2 ligand and a lenalidomide-based cereblon E3 ubiquitin ligase ligand with a short linker between the two ligands. At low nanomolar concentrations in RS4; 11 leukemia cells, this degrader promotes efficient degradation of MDM2. It also inhibits the proliferation of leukemia cells with an IC50 value of 3.2 nM and induces apoptosis effectively. All of these data indicate that MDM2 degraders are promising therapeutics for the treatment of cancers, such as leukemia.


Assuntos
Imidazóis/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Drogas , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/metabolismo , Ligação Proteica , Estereoisomerismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
7.
Lett Appl Microbiol ; 69(2): 88-95, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31102470

RESUMO

Natural enzyme inhibitors have been widely described in literature because of its pharmacological and cosmetic applications. Fungi found in caves represent a promising source of bioactive substances that are still little explored scientifically. Thus, the present work evaluated the presence of enzymatic modulators in a filtrate obtained from the cultivation of the cave fungus Lecanicillium aphanocladii (Family: Cordycipitaceae). Snake venoms from Bothrops alternatus and Bothrops atrox were used as an enzymatic source for the induction of the phospholipase, proteolytic, thrombolytic, cytotoxic and coagulant activities. Compounds present in the fungal filtrate inhibited 50, 23·8, 26·6, 50·9 and 52·5% of the proteolytic, phospholipase, haemolytic, thrombolytic and coagulant activities respectively. The filtrate was not cytotoxic on erythrocytes, but induced partial dissolution of thrombi. Fungal enzyme inhibitors that have low or no toxicity and can be obtained on a large scale and at low cost are relevant in the medical-scientific context. Therefore, the inhibition of phospholipases A2 and proteases observed in the present work highlights the potential of fungal metabolites for the development of drugs that can be used in the treatment of haemostasis and inflammation-related disorders. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, secondary metabolites synthesized by Lecanicillium aphanocladii, a fungus isolated from caves, demonstrated modulating action on proteases and phospholipases A2 present in snake venoms of the Bothrops genus, widely used as tools for the study of pathophysiology processes related to haemostasis and inflammation. The results suggest the possibility of future applications for these metabolites in the development of pharmaceuticals of medical-scientific interest.


Assuntos
Ascomicetos/química , Bothrops/metabolismo , Venenos de Crotalídeos/enzimologia , Peptídeo Hidrolases/metabolismo , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Inibidores de Proteases/farmacologia , Animais , Ascomicetos/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Hemostasia/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Proteólise/efeitos dos fármacos
8.
Am J Chin Med ; 47(3): 657-674, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30974966

RESUMO

Glioblastoma (GBM) is the most commonly occurring tumor in the cerebral hemispheres. Currently, temozolomide (TMZ), an alkylating agent that induces DNA strand breaks, is considered the frontline chemotherapeutic agent for GBM. Despite its frontline status, GBM patients commonly exhibit resistance to TMZ treatment. We have recently established and characterized TMZ-resistant human glioma cells. The aim of this study is to investigate whether curcumin modulates cell apoptosis through the alternation of the connexin 43 (Cx43) protein level in TMZ-resistant GBM. Overexpression of Cx43, but not ATP-binding cassette transporters (ABC transporters), was observed (approximately 2.2-fold) in TMZ-resistant GBM cells compared to the Cx43 levels in parental GBM cells. Furthermore, at a concentration of 10 µ M, curcumin significantly reduced Cx43 protein expression by about 40%. In addition, curcumin did not affect the expression of other connexins like Cx26 or epithelial-to-mesenchymal transition (EMT) proteins such as ß -catenin or α E-catenin. Curcumin treatment led to an increase in TMZ-induced cell apoptosis from 4% to 8%. Importantly, it did not affect the mRNA expression level of Cx43. Concomitant treatment with the translation inhibitor cycloheximide (CHX) exerted additional effects on Cx43 degradation. Treatment with the autophagy inhibitor 3-MA (methyladenine) did not affect the curcumin-induced Cx43 degradation. Interestingly, treatment with the proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) significantly negated the curcumin-induced Cx43 degradation, which suggests that curcumin-induced Cx43 degradation occurs through the ubiquitin-proteasome pathway.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Apoptose/efeitos dos fármacos , Conexina 43/metabolismo , Curcumina/farmacologia , Glioblastoma/genética , Glioblastoma/patologia , Proteólise/efeitos dos fármacos , Temozolomida/farmacologia , Humanos , Estimulação Química , Células Tumorais Cultivadas
9.
Eur J Med Chem ; 174: 159-180, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31035238

RESUMO

Inhibitors and nucleic acid based techniques were two main approaches to interfere with protein signaling and respective cascade in the past. Until recently, a new class of small molecules named proteolysis-targeting chimeras (PROTACs) have emerged. Each contains a target warhead, a linker and an E3 ligand. These bifunctional molecules recruit E3 ligases and target specific proteins for degradation via the ubiquitin (Ub) proteasome system (UPS). The degradation provides several advantages over inhibition in potency, selectivity and drug resistance. Thus, a variety of small molecule PROTACs have been discovered so far. In this review, we summarize the biological mechanism, advantages and recent progress of PROTACs, trying to offer an outlook in development of drugs targeting degradation in future.


Assuntos
Oligopeptídeos/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Ligantes , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas/química , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
10.
Artif Cells Nanomed Biotechnol ; 47(1): 1043-1049, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942091

RESUMO

Osteoarthritis (OA) is a major age-related disease, which may be caused by the accumulation of advanced glycation end-products (AGEs). Excessive degradation of type II collagen and aggrecan by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS) induced by AGEs is a pivotal event in the pathogenesis of osteoarthritis. In addition, activation of the nuclear factor-κB (NF-κB) pathway induces the expression of a cascade of proinflammatory cytokines, such as interleukin (IL)-1ß and tumor necrosis factor-α (TNF-α). In the present study, we investigated the effects of salicin, one of the main constituents of aspirin and a derivative of Alangium chinense, on AGE-induced degradation of the articular extracellular matrix in SW1353 human chondrocytes. Our findings reveal a novel beneficial role of salicin in rescuing degradation of type II collagen and aggrecan, reducing oxidative stress, attenuating expression of proinflammatory cytokines, and inhibiting activation of the NF-κB proinflammatory signaling pathway in chondrocytes stimulated with AGEs. Salicin may thus have potential as a safe and effective therapy against the development and progression of OA.


Assuntos
Agrecanas/metabolismo , Álcoois Benzílicos/farmacologia , Condrócitos/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Glucosídeos/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Osteoartrite/tratamento farmacológico , Proteólise/efeitos dos fármacos , Álcoois Benzílicos/uso terapêutico , Quimiocinas/metabolismo , Condrócitos/metabolismo , Condrócitos/patologia , Glucosídeos/uso terapêutico , Humanos , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Transdução de Sinais/efeitos dos fármacos
11.
Nat Commun ; 10(1): 1844, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015445

RESUMO

Lentiviruses have evolved to acquire an auxiliary protein Vpx to counteract the intrinsic host restriction factor SAMHD1. Although Vpx is phosphorylated, it remains unclear whether such phosphorylation indeed regulates its activity toward SAMHD1. Here we identify the PIM family of serine/threonine protein kinases as the factors responsible for the phosphorylation of Vpx and the promotion of Vpx-mediated SAMHD1 counteraction. Integrated proteomics and subsequent functional analysis reveal that PIM family kinases, PIM1 and PIM3, phosphorylate HIV-2 Vpx at Ser13 and stabilize the interaction of Vpx with SAMHD1 thereby promoting ubiquitin-mediated proteolysis of SAMHD1. Inhibition of the PIM kinases promotes the antiviral activity of SAMHD1, ultimately reducing viral replication. Our results highlight a new mode of virus-host cell interaction in which host PIM kinases facilitate promotion of viral infectivity by counteracting the host antiviral system, and suggest a novel therapeutic strategy involving restoration of SAMHD1-mediated antiviral response.


Assuntos
Infecções por HIV/imunologia , HIV-2/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Imidazóis/farmacologia , Tolerância Imunológica , Simulação de Dinâmica Molecular , Monócitos , Fosforilação/imunologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/imunologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/imunologia , Proteólise/efeitos dos fármacos , Proteômica , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/imunologia , Piridazinas/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/química , Proteína 1 com Domínio SAM e Domínio HD/imunologia , Serina/metabolismo , Tiazolidinas/farmacologia , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia
12.
BMC Cancer ; 19(1): 349, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975087

RESUMO

BACKGROUND: The exact signalling mechanism of the mTOR complex remains a subject of constant debate, even with some evidence that amino acids participate in the same pathway as used for insulin signalling during protein synthesis. Therefore, this work conducted further study of the actions of amino acids, especially leucine, in vivo, in an experimental model of cachexia. We analysed the effects of a leucine-rich diet on the signalling pathway of protein synthesis in muscle during a tumour growth time-course. METHODS: Wistar rats were distributed into groups based on Walker-256 tumour implant and subjected to a leucine-rich diet and euthanised at three different time points following tumour development (the 7th, 14th and 21st day). We assessed the mTOR pathway key-proteins in gastrocnemius muscle, such as RAG-A-GTPase, ERK/MAP4K3, PKB/Akt, mTOR, p70S6K1, Jnk, IRS-1, STAT3, and STAT6 comparing among the experimental groups. Serum WF (proteolysis-induced factor like from Walker-256 tumour) and muscle protein synthesis and degradation were assessed. RESULTS: The tumour-bearing group had increased serum WF content, and the skeletal-muscle showed a reduction in IRS-1 and RAG activation, increased PKB/Akt and Erk/MAP4K3 on the 21st day, and maintenance of p70S6K1, associated with increases in muscle STAT-3 and STAT-6 levels in these tumour-bearing rats. CONCLUSION: Meanwhile, the leucine-rich diet modulated key steps of the mTOR pathway by triggering the increased activation of RAG and mTOR and maintaining JNK, STAT-3 and STAT-6 levels in muscle, leading to an increased muscle protein synthesis, reducing the degradation during tumour evolution in a host, minimising the cancer-induced damages in the cachectic state.


Assuntos
Caquexia/prevenção & controle , Carcinoma 256 de Walker/dietoterapia , Suplementos Nutricionais , Leucina/administração & dosagem , Serina-Treonina Quinases TOR/metabolismo , Animais , Caquexia/etiologia , Carcinoma 256 de Walker/complicações , Carcinoma 256 de Walker/patologia , Feminino , Proteínas Musculares/biossíntese , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteólise/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
13.
Nat Commun ; 10(1): 1402, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926793

RESUMO

Protein-protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, ß-Catenin, and its cognate E3 ligase, SCFß-TrCP. These enhancers potentiate the ubiquitylation of mutant ß-Catenin by ß-TrCP in vitro and induce the degradation of an engineered mutant ß-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of 'molecular glue' presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.


Assuntos
Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo
14.
Methods Mol Biol ; 1953: 105-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30912018

RESUMO

Small molecule-induced targeted protein degradation is a powerful approach for drug target validation given its selectivity, high kinetic resolution, dose dependency, and reversibility. Out of the several methods that have been reported so far, the 12 kDa degradation tag (dTAG) system has the advantage of hijacking a degradation machinery that is ubiquitously expressed in all human tissues. Therefore it is independent of expressing additional, exogenous factors and additionally permits target validation in vivo. Here, we describe the protocol for generation and validation of clones harboring knock-in of a selectable dTAG cassette at the endogenous locus of proteins of interest using the near-haploid cell line KBM7.


Assuntos
Descoberta de Drogas/métodos , Técnicas de Introdução de Genes/métodos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular , Clonagem Molecular/métodos , Edição de Genes/métodos , Vetores Genéticos/genética , Haploidia , Humanos , Plasmídeos/genética , Proteínas/genética , Proteínas/metabolismo
15.
Kaohsiung J Med Sci ; 35(4): 202-208, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30896891

RESUMO

The proto-oncogene MDM2 is a nuclear-localized E3 ubiquitin ligase, which promotes tumor formation by targeting tumor suppressor proteins, such as p53, for proteasomal degradation. In this study, the anti-infective drug nitroxoline (NXQ) was screened out to effectively inhibit cell survival of small-cell lung cancer (SCLC) cells, and induce SCLC cell apoptosis by suppressing antiapoptotic proteins (such as Bcl-2 and MCL1) and upregulating proapoptotic protein Bim. In the mechanistic study, NXQ was found to downregulate MDM2 expression by inducing its proteasomal degradation, and thus upregulated p53 expression, which was a substrate protein of MDM2. Moreover, overexpression of MDM2 decreased the cytotoxicity of NXQ on SCLC cells. These results demonstrated that NXQ displayed anti-SCLC activity by suppressing MDM2 expression, which suggested that anti-infective NXQ had potential for SCLC treatment by targeting the MDM2/p53 axis.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Nitroquinolinas/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Nitroquinolinas/química , Complexo de Endopeptidases do Proteassoma/metabolismo
16.
J Enzyme Inhib Med Chem ; 34(1): 692-702, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30777474

RESUMO

Matriptase is ectopically expressed in neoplastic B-cells, in which matriptase activity is enhanced by negligible expression of its endogenous inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1. HAI-1, however, is also involved in matriptase synthesis and intracellular trafficking. The lack of HAI-1 indicates that other related inhibitor, such as HAI-2, might be expressed. Here, we show that HAI-2 is commonly co-expressed in matriptase-expressing neoplastic B-cells. The level of active matriptase shed after induction of matriptase zymogen activation in 7 different neoplastic B-cells was next determined and characterised. Our data reveal that active matriptase can only be generated and shed by those cells able to activate matriptase and in a rough correlation with the levels of matriptase protein. While HAI-2 can potently inhibit matriptase, the levels of active matriptase are not proportionally suppressed in those cells with high HAI-2. Our survey suggests that matriptase proteolysis might aberrantly remain high in neoplastic B-cells regardless of the levels of HAI-2.


Assuntos
Linfócitos B/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glicoproteínas de Membrana/biossíntese , Proteólise/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Humanos , Glicoproteínas de Membrana/metabolismo , Serina Endopeptidases/biossíntese
17.
Cell Commun Signal ; 17(1): 15, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30786890

RESUMO

BACKGROUND: ErbB2 overexpression identifies a subset of breast cancer as ErbB2-positive and is frequently associated with poor clinical outcomes. As a membrane-embedded receptor tyrosine kinase, cell surface levels of ErbB2 are regulated dynamically by membrane physical properties. The present study aims to investigate the influence of membrane cholesterol contents on ErbB2 status and cellular responses to its tyrosine kinase inhibitors. METHODS: The cholesterol abundance was examined in ErbB2-positive breast cancer cells using filipin staining. Cellular ErbB2 localizations were investigated by immunofluorescence with altered membrane cholesterol contents. The inhibitory effects of the cholesterol-lowering drug lovastatin were assessed using cell proliferation, apoptosis, immunoblotting and immunofluorescence assays. The synergistic effects of lovastatin with the ErbB2 inhibitor lapatinib were evaluated using an ErbB2-positive breast cancer xenograft mouse model. RESULTS: Membrane cholesterol contents positively correlated with cell surface distribution of ErbB2 through increasing the rigidity and decreasing the fluidity of cell membranes. Reduction in cholesterol abundance assisted the internalization and degradation of ErbB2. The cholesterol-lowering drug lovastatin significantly potentiated the inhibitory effects of ErbB2 kinase inhibitors, accompanied with enhanced ErbB2 endocytosis. Lovastatin also synergized with lapatinib to strongly suppress the in vivo growth of ErbB2-positive breast cancer xenografts. CONCLUSION: The cell surface distribution of ErbB2 was closely regulated by membrane physical properties governed by cholesterol contents. The cholesterol-lowering medications can hence be exploited for potential combinatorial therapies with ErbB2 kinase inhibitors in the clinical treatment of ErbB2-positive breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Receptor ErbB-2/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Endocitose/efeitos dos fármacos , Feminino , Filipina/farmacologia , Humanos , Lapatinib/farmacologia , Lovastatina/farmacologia , Camundongos Nus , Modelos Biológicos , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Cell Mol Gastroenterol Hepatol ; 7(2): 297-312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30704981

RESUMO

BACKGROUND & AIMS: Hepatitis B virus (HBV) infection is a major health concern worldwide. Although currently used nucleos(t)ide analogs efficiently inhibit viral replication, viral proteins transcribed from the episomal viral covalently closed circular DNA (cccDNA) minichromosome continue to be expressed long-term. Because high viral RNA or antigen loads may play a biological role during this chronicity, the elimination of viral products is an ultimate goal of HBV treatment. HBV regulatory protein X (HBx) was recently found to promote transcription of cccDNA with degradation of Smc5/6 through the interaction of HBx with the host protein DDB1. Here, this protein-protein interaction was considered as a new molecular target of HBV treatment. METHODS: To identify candidate compounds that target the HBx-DDB1 interaction, a newly constructed split luciferase assay system was applied to comprehensive compound screening. The effects of the identified compounds on HBV transcription and cccDNA maintenance were determined using HBV minicircle DNA, which mimics HBV cccDNA, and the natural HBV infection model of human primary hepatocytes. RESULTS: We show that nitazoxanide (NTZ), a thiazolide anti-infective agent that has been approved by the FDA for protozoan enteritis, efficiently inhibits the HBx-DDB1 protein interaction. NTZ significantly restores Smc5 protein levels and suppresses viral transcription and viral protein production in the HBV minicircle system and in human primary hepatocytes naturally infected with HBV. CONCLUSIONS: These results indicate that NTZ, which targets an HBV-related viral-host protein interaction, may be a promising new therapeutic agent and a step toward a functional HBV cure.


Assuntos
DNA Circular/genética , Proteínas de Ligação a DNA/metabolismo , Vírus da Hepatite B/genética , Tiazóis/farmacologia , Transativadores/metabolismo , Transcrição Genética/efeitos dos fármacos , DNA Viral/genética , Células HEK293 , Células Hep G2 , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética
19.
Genes Cells ; 24(4): 284-296, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30762924

RESUMO

The multisubunit complex transcription factor IIH (TFIIH) has dual functions in transcriptional initiation and nucleotide excision repair (NER). TFIIH is comprised of two subcomplexes, the core subcomplex (seven subunits) including XPB and XPD helicases and the cyclin-dependent kinase (CDK)-activating kinase (CAK) subcomplex (three subunits) containing CDK7 kinase. Recently, it has been reported that spironolactone, an anti-aldosterone drug, inhibits cellular NER by inducing proteasomal degradation of XPB and potentiates the cytotoxicity of platinum-based drugs in cancer cells, suggesting possible drug repositioning. In this study, we have tried to uncover the mechanism underlying the chemical-induced XPB destabilization. Based on siRNA library screening and subsequent analyses, we identified SCFFBXL18 E3 ligase consisting of Skp1, Cul1, F-box protein FBXL18 and Rbx1 responsible for spironolactone-induced XPB polyubiquitination and degradation. In addition, we showed that CDK7 kinase activity is required for this process. Finally, we found that the Ser90 residue of XPB is essential for the chemical-induced destabilization. These results led us to propose a model that spironolactone may trigger the phosphorylation of XPB at Ser90 by CDK7, which promotes the recognition and polyubiquitination of XPB by SCFFBXL18 for proteasomal degradation.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas F-Box/metabolismo , Espironolactona/farmacologia , Fator de Transcrição TFIIH/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteólise/efeitos dos fármacos
20.
Cell Mol Life Sci ; 76(10): 2003-2013, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30747251

RESUMO

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.


Assuntos
Proteínas de Artrópodes/farmacologia , Cistatinas/farmacologia , Imunossupressores/farmacologia , Cistatinas Salivares/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Cristalografia por Raios X , Cistatinas/classificação , Cistatinas/genética , Citocinas/metabolismo , Compostos de Epóxi/metabolismo , Feminino , Imunossupressores/química , Imunossupressores/metabolismo , Ixodes/química , Ixodes/genética , Ixodes/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Filogenia , Proteólise/efeitos dos fármacos , Cistatinas Salivares/química , Cistatinas Salivares/genética , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA