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1.
Front Immunol ; 11: 2094, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32973818

RESUMO

The spread of the novel human respiratory coronavirus (SARS-CoV-2) is a global public health emergency. There is no known successful treatment as of this time, and there is a need for medical options to mitigate this current epidemic. SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) receptor and is primarily trophic for the lower and upper respiratory tract. A number of current studies on COVID-19 have demonstrated the substantial increase in pro-inflammatory factors in the lungs during infection. The virus is also documented in the central nervous system and, particularly in the brainstem, which plays a key role in respiratory and cardiovascular function. Currently, there are few antiviral approaches, and several alternative drugs are under investigation. Two of these are Idelalisib and Ebastine, already proposed as preventive strategies in airways and allergic diseases. The interesting and evolving potential of phosphoinositide 3-kinase δ (PI3Kδ) inhibitors, together with Ebastine, lies in their ability to suppress the release of pro-inflammatory cytokines, such as IL-1ß, IL-8, IL-6, and TNF-α, by T cells. This may represent an optional therapeutic choice for COVID-19 to reduce inflammatory reactions and mortality, enabling patients to recover faster. This concise communication aims to provide new potential therapeutic targets capable of mitigating and alleviating SARS-CoV-2 pandemic infection.


Assuntos
Betacoronavirus , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Terapia de Alvo Molecular/métodos , Pneumonia Viral/tratamento farmacológico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Antirreumáticos/uso terapêutico , Antivirais/uso terapêutico , Butirofenonas/farmacologia , Butirofenonas/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Infecções por Coronavirus/virologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/sangue , Pandemias , Peptidil Dipeptidase A/metabolismo , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Pneumonia Viral/virologia , Purinas/farmacologia , Purinas/uso terapêutico , Quinazolinonas/farmacologia , Quinazolinonas/uso terapêutico
2.
Life Sci ; 259: 118148, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32721465

RESUMO

Pancreatic cancer is a malignant cancer with poor prognosis. This study aimed to explore how O6-methylguanine-DNA methyltransferase (MGMT) affects the gemcitabine resistance of pancreatic cancer cells by the regulatory role of SHH/GLI signaling pathway. MGMT inhibition induced by lomeguatrib (LM) suppressed the proliferation, invasion, migration and autophagy, promoted the apoptosis of PanC-1/GEM cells and up-regulated the GEM inhibition rates for PanC-1/GEM cells. Moreover, MGMT inhibition increased the expression of Caspase-3 and Bax and decreased the expression of Bcl-2, Beclin1 and Atg5 in PanC-1/GEM cells. PVT1 silencing could also produce the similar effects of MGMT inhibition induced by LM on PanC-1/GEM cells. And, PVT1 silencing could inhibit the SHH/GLI signaling pathway in PanC-1/GEM cells by regulating the MGMT expression. miR-409 was demonstrated to be a potential target of PVT1 and SHH was demonstrated to be a potential target of miR-409. Furthermore, GLI overexpression could reverse the effects of PVT1 silencing. In the xenograft model of pancreatic cancer, nude mice were treated with GEM. MGMT inhibition suppressed the tumor growth and autophagy and promoted the apoptosis in tumor tissues. And, PVT1 silencing could inhibit the SHH/GLI signaling pathway in tumor tissues. In conclusion, MGMT inhibition could suppress the proliferation, invasion, migration and autophagy and promote the apoptosis of PanC-1/GEM cells in vitro and in vivo. PVT1 silencing may affect the PanC-1/GEM cells through changing the MGMT expression by inhibiting the SHH/GLI signaling pathway.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Metilases de Modificação do DNA/biossíntese , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/biossíntese , Enzimas Reparadoras do DNA/genética , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Animais , Autofagia/efeitos dos fármacos , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/antagonistas & inibidores , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neoplasias Pancreáticas/patologia , Purinas/farmacologia , Purinas/uso terapêutico , Proteínas Supressoras de Tumor/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Nature ; 585(7824): 293-297, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32494016

RESUMO

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.


Assuntos
Ciclinas/deficiência , Ciclinas/metabolismo , Proteólise/efeitos dos fármacos , Purinas/química , Purinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Purinas/toxicidade , Piridinas/toxicidade , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinação/efeitos dos fármacos
4.
Nat Commun ; 11(1): 2709, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483169

RESUMO

Aberrant immune responses including reactive phagocytes are implicated in the etiology of age-related macular degeneration (AMD), a major cause of blindness in the elderly. The translocator protein (18 kDa) (TSPO) is described as a biomarker for reactive gliosis, but its biological functions in retinal diseases remain elusive. Here, we report that tamoxifen-induced conditional deletion of TSPO in resident microglia using Cx3cr1CreERT2:TSPOfl/fl mice or targeting the protein with the synthetic ligand XBD173 prevents reactivity of phagocytes in the laser-induced mouse model of neovascular AMD. Concomitantly, the subsequent neoangiogenesis and vascular leakage are prevented by TSPO knockout or XBD173 treatment. Using different NADPH oxidase-deficient mice, we show that TSPO is a key regulator of NOX1-dependent neurotoxic ROS production in the retina. These data define a distinct role for TSPO in retinal phagocyte reactivity and highlight the protein as a drug target for immunomodulatory and antioxidant therapies for AMD.


Assuntos
NADPH Oxidase 1/genética , Neovascularização Patológica/genética , Fagócitos/metabolismo , Receptores de GABA/genética , Degeneração Macular Exsudativa/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , NADPH Oxidase 1/metabolismo , Neovascularização Patológica/metabolismo , Fagócitos/efeitos dos fármacos , Purinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de GABA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Degeneração Macular Exsudativa/metabolismo
5.
PLoS One ; 15(6): e0234638, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32569325

RESUMO

Hematopoietic stem cell transplantation is successfully applied since the late 1950s; however, its efficacy still needs to be increased. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an improved ex vivo culture system that supports proliferation and maintains HSC pluripotency would override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro, we optimized the HSC medium composition with a panel of cytokines and valproic acid and used an artificial 3D bone marrow-like scaffold made of polydimethylsiloxane (PDMS). This 3D scaffold offered a suitable platform to amplify human HSCs in vitro and, simultaneously, to support their viability, multipotency and ability for self-renewal. Silicon oxide-covering of PDMS structures further improved amplification of CD34+ cells, although the conservation of naïve HSCs was better on non-covered 3D PDMS. Finally, we found that HSC cultivated on non-covered 3D PDMS generated most pluripotent colonies within colony forming unit assays. In conclusion, by combining biological and biotechnological approaches, we optimized in vitro HSCs culture conditions, resulting in improved amplification, multipotency maintenance and vitality of HSCs.


Assuntos
Materiais Biomiméticos/farmacologia , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/farmacologia , Dimetilpolisiloxanos/farmacologia , Feminino , Fibronectinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Masculino , Purinas/farmacologia , Nicho de Células-Tronco/efeitos dos fármacos , Ácido Valproico/farmacologia
6.
Vet Microbiol ; 242: 108586, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32122590

RESUMO

Brucella as a stealthy intracellular pathogen avoids activation of innate immune response. Here we investigated the contribution of an adenosine receptor, Adora2b, during Brucella infection in professional phagocyte RAW 264.7 cells and in a murine model. Adora2b-deficient cells showed attenuated Brucella internalization and intracellular survival with enhanced release of IL-6, TNF-α, IL-12 and MCP-1. In addition, blockade of Adora2b using MRS 1754 treatment in mice resulted in increased total weight of the spleens but suppressed bacterial burden in these organs accompanied by elevated levels of IL-6, IFN-γ, TNF-α, IL-12 and MCP-1, while reduced IL-10. Overall, we proposed that the Adora2b participates in the successful phagocytic pathway and intracellular survival of Brucella in RAW 264.7 cells, and could be a potential therapeutic target for the treatment of acute brucellosis in animals.


Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Brucelose/tratamento farmacológico , Imunidade Inata , Macrófagos/microbiologia , Receptor A2B de Adenosina/imunologia , Acetamidas/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Aminopiridinas/farmacologia , Animais , Brucella abortus/efeitos dos fármacos , Brucella abortus/fisiologia , Brucelose/microbiologia , Citocinas/imunologia , Feminino , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Purinas/farmacologia , Células RAW 264.7 , Receptor A2B de Adenosina/genética , Transdução de Sinais
7.
Transbound Emerg Dis ; 67 Suppl 1: 40-55, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32174040

RESUMO

Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour-like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite-dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T. annulata isolates identified a conserved CDK1 phosphorylation motif T131 PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA-based experiments using a phospho-threonine-proline (pThr-Pro) antibody revealed, that CDK1-cyclin B specifically phosphorylates T131 , identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T. annulata-infected cells. Further studies demonstrated a nearly complete co-localization of the pThr-Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle-dependent event. Immunostainings of isolated, non-permeabilized schizonts confirmed the presence of the pThr-Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr-Pro signal of the schizont, which was reconstituted by the addition of CDK1-cyclin B. Treatment of T. annulata-infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin-rich cell protrusions and an extension of the schizont, and a dose-dependent reduction of BrdU incorporation and Ki67 staining of T. annulata-infected cells, demonstrating a clear impact on the Theileria-dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria-infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.


Assuntos
Proteína Quinase CDC2/metabolismo , Doenças dos Bovinos/prevenção & controle , Proteínas de Protozoários/metabolismo , Theileria annulata/imunologia , Theileriose/prevenção & controle , Motivos de Aminoácidos , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Bovinos , Doenças dos Bovinos/parasitologia , Proliferação de Células , Ensaio de Imunoadsorção Enzimática/veterinária , Fosforilação , Purinas/farmacologia , Esquizontes , Theileria annulata/metabolismo , Theileriose/parasitologia
8.
PLoS One ; 15(2): e0229490, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32107496

RESUMO

Application of plant growth regulators has become one of the most important means of improving yield and quality of medicinal plants. To understand the molecular basis of phytohormone-regulated oleanolic acid metabolism, RNA-seq was used to analyze global gene expression in Achyranthes bidentata treated with 2.0 mg/L 1-naphthaleneacetic acid (NAA) and 1.0 mg/L 6-benzyladenine (6-BA). Compared with untreated controls, the expression levels of 20,896 genes were significantly altered with phytohormone treatment. We found that 13071 (62.5%) unigenes were up-regulated, and a lot of differentially expressed genes involved in hormone or terpenoid biosynthesis, or transcription factors were significantly up-regulated. These results suggest that oleanolic acid biosynthesis induced by NAA and 6-BA occurs due to the expression of key genes involved in jasmonic acid signal transduction. This study is the first to analyze the production and hormonal regulation of medicinal A. bidentata metabolites at the molecular level. The results herein contribute to a better understanding of the regulation of oleanane-type triterpenoid saponins accumulation and define strategies to improve the yield of these useful metabolites.


Assuntos
Achyranthes/efeitos dos fármacos , Achyranthes/metabolismo , Compostos de Benzil/farmacologia , Ciclopentanos/metabolismo , Ácidos Naftalenoacéticos/farmacologia , Ácido Oleanólico/biossíntese , Oxilipinas/metabolismo , Purinas/farmacologia , Achyranthes/crescimento & desenvolvimento , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Medicina Tradicional Chinesa , Filogenia , Reguladores de Crescimento de Planta/metabolismo , Plantas Medicinais/efeitos dos fármacos , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , RNA-Seq , Saponinas/metabolismo
9.
J Med Chem ; 63(6): 2930-2940, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32068404

RESUMO

The molecular chaperone TRAP1 is the mitochondrial paralog of Hsp90 and is overexpressed in many cancer cells. The orthosteric ATP-binding site of TRAP1 has been considered the primary inhibitor binding location, but TRAP1 allosteric modulators have not yet been investigated. Here, we generated and characterized the Hsp90 inhibitor PU-H71, conjugated to the mitochondrial delivery vehicle triphenylphosphonium (TPP) with a C10 carbon spacer, named SMTIN-C10, to enable dual binding to orthosteric and allosteric sites. In addition to tight binding with the ATP-binding site through the PU-H71 moiety, SMTIN-C10 interacts with the E115 residue in the N-terminal domain through the TPP moiety and subsequently induces structural transition of TRAP1 to a tightly packed closed form. The data indicate the existence of a druggable allosteric site neighboring the orthosteric ATP pocket that can be exploited to develop potent TRAP1 modulators.


Assuntos
Sítio Alostérico/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzodioxóis/química , Benzodioxóis/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Purinas/química , Purinas/farmacologia , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Conformação Proteica/efeitos dos fármacos
10.
J Med Chem ; 63(4): 1717-1723, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32026684

RESUMO

Iodide homeostasis and thyroid hormone metabolism in the brain are potentially related to changes in the activity of the sodium iodide symporter (NIS). No radiotracers are currently available for imaging brain NIS activity. Here, we synthesized 6-[124I]iodo-9-pentylpurine that can noninvasively measure iodide efflux from the brain and showed that the efflux rate of [124I]I- in NIS knockout mice was 84% lower than that of wild-type mice. Thus, 6-[124I]iodo-9-pentylpurine would be useful for imaging brain NIS activity.


Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Purinas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Simportadores/metabolismo , Animais , Iodetos/metabolismo , Radioisótopos do Iodo/química , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tomografia por Emissão de Pósitrons , Purinas/síntese química , Purinas/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Simportadores/genética
11.
Biomed Chromatogr ; 34(3): e4783, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31899811

RESUMO

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS2 spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.


Assuntos
Aminopiridinas , Cromatografia Líquida/métodos , Metaboloma/efeitos dos fármacos , Microssomos Hepáticos , Purinas , Espectrometria de Massas por Ionização por Electrospray/métodos , Aminopiridinas/análise , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Animais , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Purinas/análise , Purinas/metabolismo , Purinas/farmacologia , Ratos
12.
Plant Reprod ; 33(1): 21-34, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31907610

RESUMO

KEY MESSAGE: Cytokinin might be an important factor to regulate floral sex at the very early stage of flower development in sacha inchi. Sacha inchi (Plukenetia volubilis, Euphorbiaceae) is characterized by having female and male flowers in a thyrse with particular differences. The mechanisms involved in the development of unisexual flowers are very poorly understood. In this study, the inflorescence and flower development of P. volubilis were investigated using light microscopy and scanning electron microscopy. We also investigated the effects of cytokinin on flower sex determination by exogenous application of 6-benzyladenine (BA) in P. volubilis. The floral development of P. volubilis was divided into eight stages, and the first morphological divergence between the male and female flowers was found to occur at stage 3. Both female and male flowers can be structurally distinguished by differences in the shape and size of the flower apex after sepal primordia initiation. There are no traces of gynoecia in male flowers or of androecia in female flowers. Exogenous application of BA effectively induced gynoecium primordia initiation and female flower development, especially at the early flower developmental stages. We propose that flower sex is determined earlier and probably occurs before flower initiation, either prior to or at inflorescence development due to the difference in the position of the female and male primordia in the inflorescence and in the time of the female and male primordia being initiated. The influence of cytokinin on female primordia during flower development in P. volubilis strongly suggests a feminization role for cytokinin in sex determination. These results indicate that cytokinin could modify the fate of the apical meristem of male flower and promote the formation of carpel primordia in P. volubilis.


Assuntos
Euphorbiaceae , Flores , Compostos de Benzil/farmacologia , Citocininas/metabolismo , Euphorbiaceae/efeitos dos fármacos , Flores/classificação , Flores/fisiologia , Flores/ultraestrutura , Regulação da Expressão Gênica de Plantas , Inflorescência/ultraestrutura , Reguladores de Crescimento de Planta/farmacologia , Purinas/farmacologia , Processos de Determinação Sexual/efeitos dos fármacos
13.
Cancer Immunol Immunother ; 69(4): 501-512, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31950225

RESUMO

Obinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Interleucina-12/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de IgG/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , MicroRNAs/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Purinas/farmacologia , Quinazolinonas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores
14.
BMC Plant Biol ; 20(1): 44, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996151

RESUMO

BACKGROUND: Exogenous 6-benzyladenine (6-BA) could improve leaf defense system activity. In order to better understand the regulation mechanism of exogenous 6-benzyladenine (6-BA) on waterlogged summer maize, three treatments including control (CK), waterlogging at the third leaf stage for 6 days (V3-6), and application of 100 mg dm- 3 6-BA after waterlogging for 6 days (V3-6-B), were employed using summer maize hybrid DengHai 605 (DH605) as the experimental material. We used a labeling liquid chromatography-based quantitative proteomics approach with tandem mass tags to determine the changes in leaf protein abundance level at the tasseling stage. RESULTS: Waterlogging significantly hindered plant growth and decreased the activities of SOD, POD and CAT. In addition, the activity of LOX was significantly increased after waterlogging. As a result, the content of MDA and H2O2 was significantly increased which incurred serious damages on cell membrane and cellular metabolism of summer maize. And, the leaf emergence rate, plant height and grain yield were significantly decreased by waterlogging. However, application of 6-BA effectively mitigated these adverse effects induced by waterlogging. Compared with V3-6, SOD, POD and CAT activity of V3-6-B were increased by 6.9, 12.4, and 18.5%, LOX were decreased by 13.6%. As a consequence, the contents of MDA and H2O2 in V3-6-B were decreased by 22.1 and 17.2%, respectively, compared to that of V3-6. In addition, the leaf emergence rate, plant height and grain yield were significantly increased by application of 6-BA. Based on proteomics profiling, the proteins involved in protein metabolism, ROS scavenging and fatty acid metabolism were significantly regulated by 6-BA, which suggested that application of 6-BA exaggerated the defensive response of summer maize at proteomic level. CONCLUSIONS: These results demonstrated that 6-BA had contrastive effects on waterlogged summer maize. By regulating key proteins related to ROS scavenging and fatty acid metabolism, 6-BA effectively increased the defense system activity of waterlogged summer maize, then balanced the protein metabolism and improved the plant physiological traits and grain yield.


Assuntos
Antioxidantes/metabolismo , Compostos de Benzil/farmacologia , Imunidade Vegetal/efeitos dos fármacos , Purinas/farmacologia , Zea mays/metabolismo , Catalase/efeitos dos fármacos , Catalase/genética , Catalase/metabolismo , Ontologia Genética , Peróxido de Hidrogênio/metabolismo , Lipoxigenase/efeitos dos fármacos , Lipoxigenase/genética , Lipoxigenase/metabolismo , Peroxidase/efeitos dos fármacos , Peroxidase/genética , Peroxidase/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/efeitos dos fármacos , Proteômica , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Água
15.
Biomolecules ; 10(2)2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31991557

RESUMO

The present study demonstrates hormonal control of Salvia viridis growth and development using four different purine-type cytokinins at different concentrations. The addition of cytokinins significantly increased biomass of cultures, proliferation rate, and, interestingly, secondary metabolite production. The best response in terms of multiplication ratio was recorded on Murashige and Skoog medium supplemented with 0.5 mg/L BPA (N-benzylotetrahydropyranyl adenine), while the greatest biomass accumulation was achieved when supplemented with 1 mg/L m-T (meta-topoline). Quantitative UPLC-DAD analysis of the hydromethanolic extract from S. viridis culture revealed the presence of 12 polyphenols: seven phenolic acids and five phenylethanoids. The highest total content of polyphenolic compounds was found in shoots cultivated on medium with 2 mg/L BPA (18.66 mg/g DW): almost twice that of control shoots. The medium was also the most optimal for the biosynthesis of rosmarinic acid, the predominant phenolic acid. However, the greater phenylethanoid accumulation was stimulated by 1 mg/L m-T: the metabolite content was above three times higher than that found in shoots grown on the control medium (8.03 mg/g DW vs. 2.37 mg/g DW). Hence, it was demonstrated that phytohormones are capable of influencing not only vital physiological processes, but therapeutic potential of plants as well. Therefore, the cytokinin-based sage cultures may be also considered as the alternative sources of bioactive compounds.


Assuntos
Proliferação de Células/efeitos dos fármacos , Citocininas/farmacologia , Purinas/farmacologia , Salvia/efeitos dos fármacos , Cinamatos/metabolismo , Depsídeos/metabolismo , Hidroxibenzoatos/metabolismo , Células Vegetais/efeitos dos fármacos , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Polifenóis/biossíntese , Salvia/crescimento & desenvolvimento
16.
Nat Commun ; 11(1): 414, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964872

RESUMO

WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.


Assuntos
Proteínas Desgrenhadas/metabolismo , Descoberta de Drogas/métodos , Receptores Frizzled/agonistas , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Piridinas/química , Tiofenos/química , Via de Sinalização Wnt/efeitos dos fármacos , Membrana Celular/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular/métodos , Morfolinas/farmacologia , Estudo de Prova de Conceito , Purinas/farmacologia , Piridinas/farmacologia , Receptor Smoothened/agonistas , Relação Estrutura-Atividade , Tiofenos/farmacologia
17.
Physiol Plant ; 168(3): 675-693, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31343748

RESUMO

Potato tuberization is a complicated biological process regulated by multiple phytohormones, in particular cytokinins (CKs). The information available on the molecular mechanisms regulating tuber development by CKs remains largely unclear. Physiological results initially indicated that low 6-benzylaminopurine (BAP) concentration (3 mg l-1 ) advanced the tuberization beginning time and promoted tuber formation. A comparative proteomics approach was applied to investigate the proteome change of tuber development by two-dimensional gel electrophoresis in vitro, subjected to exogenous BAP treatments (0, 3, 6 and 13 mg l-1 ). Quantitative image analysis showed a total of 83 protein spots with significantly altered abundance (>2.5-fold, P < 0.05), and 55 differentially abundant proteins were identified by MALDI-TOF/TOF MS. Among these proteins, 22 proteins exhibited up-regulation with the increase of exogenous BAP concentration, and 31 proteins were upregulated at 3 mg l-1 BAP whereas being downregulated at higher BAP concentrations. These proteins were involved in metabolism and bioenergy, storage, redox homeostasis, cell defense and rescue, transcription and translation, chaperones, signaling and transport. The favorable effects of low BAP concentrations on tuber development were found in various cellular processes, mainly including the stimulation of starch and storage protein accumulation, the enhancement of the glycolysis pathway and ATP synthesis, the cellular homeostasis maintenance, the activation of pathogen defense, the higher efficiency of transcription and translation, as well as the enhanced metabolite transport. However, higher BAP concentration, especially 13 mg l-1 , showed disadvantageous effects. The proposed hypothetical model would explain the interaction of these proteins associated with CK-induced tuber development in vitro.


Assuntos
Citocininas/fisiologia , Proteínas de Plantas/fisiologia , Tubérculos/crescimento & desenvolvimento , Proteoma , Solanum tuberosum/fisiologia , Compostos de Benzil/farmacologia , Regulação da Expressão Gênica de Plantas , Purinas/farmacologia
18.
Eur J Med Chem ; 186: 111888, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31787359

RESUMO

Targeting L858R/T790M/C797S mutant EGFR is a major challenge in the new-generation EGFR tyrosine kinase inhibitors development for conquering drug resistant NSCLC. In this study, a series of novel 9-heterocyclyl substituted 9H-purine derivatives were designed as EGFRL858 R/T790 M/C797S tyrosine kinase inhibitors. Among these compounds, D4, D9, D11 and D12 showed significantly potent anti-proliferation and EGFRL858 R/T790 M/C797S inhibition activity. In particular, the most potent compound D9 showed anti-proliferation against HCC827 and H1975 cell lines with the IC50 values of 0.00088 and 0.20 µM, respectively. And D9 inhibited the EGFRL858R/T790M/C797S with an IC50 value of 18 nM. Furtherly, D9 could significantly suppress the EGFR phosphorylation, induce the apoptosis, arrest cell cycle at G0/G1, and inhibit colony formation in HCC827 cell line by a concentration-dependent manner. Molecular docking indicated that the introduction of a cyclopropylsulfonamide group in D9 led to the formation of additional two hydrogen bonds with mutant Ser797 which played key roles in generating efficient EGFRL858 R/T790 M/C797S inhibitory activity. These findings strongly indicated that 9-heterocyclyl substituted 9H-purine derivatives were promising L858R/T790M/C797S mutant EGFR-TKIs. The introduction of extra hydrogen bond interaction with mutant Ser797 is efficient method for the design of the fourth-generation EGFR-TKIs.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Compostos Heterocíclicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Humanos , Estrutura Molecular , Mutação , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Purinas/síntese química , Purinas/química , Relação Estrutura-Atividade
19.
Mol Pharmacol ; 97(1): 23-34, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707356

RESUMO

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.


Assuntos
Sítios de Ligação/genética , Bioensaio/métodos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Alcaloides de Veratrum/farmacologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Compostos de Boro/química , Cinamatos/farmacologia , Descoberta de Drogas/métodos , Técnicas de Inativação de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Luciferases/química , Morfolinas/farmacologia , Nanoestruturas/química , Purinas/farmacologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
20.
J Cardiovasc Pharmacol ; 75(3): 240-249, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31868827

RESUMO

Recent studies have shown that TRPA1, a nonselective cation channel with high permeability to calcium, is expressed in many tissues of the cardiovascular system and is involved in the pathogenesis of many cardiovascular diseases. However, the role of TRPA1 in cardiac repair after myocardial infarction (MI) has not been clearly defined. The aim of this study was to confirm whether inhibition of TRPA1 could attenuate MI-induced cardiac ischemia injury. The C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery and treated with TRPA1-specific inhibitor HC-030031 (HC) for 4 weeks. Echocardiography was performed to assess cardiac function. The results showed that HC significantly attenuated MI-induced cardiac dysfunction 4 weeks after MI. Similarly, HC reduced cardiac fibrosis and cell apoptosis after MI and significantly increased angiogenesis in the border zone of the infarct. In vitro, we found that HC promoted the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Importantly, HC treatment decreased phosphatase and tensin homolog expression and augmented the expression of phosphorylated Akt in the myocardium post MI and HUVECs. However, treatment of HUVECs with a PI3K inhibitor, LY294002, before HC administration almost completely abolished HC-induced migration in HUVECs. In conclusion, we demonstrate that the inhibition of TRPA1 promotes angiogenesis after MI, thereby alleviating myocardial ischemia injury via mechanisms involving inhibition of phosphatase and tensin homolog expression and subsequent activation of the PI3K/Akt signaling.


Assuntos
Acetanilidas/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Purinas/farmacologia , Canal de Cátion TRPA1/antagonistas & inibidores , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Neovascularização Fisiológica , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Canal de Cátion TRPA1/metabolismo
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