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1.
ACS Chem Biol ; 16(7): 1288-1297, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34232635

RESUMO

Inducing the formation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs) represents a potential approach to repairing the loss of myelin observed in multiple sclerosis and other diseases. Recently, we demonstrated that accumulation of specific cholesterol precursors, 8,9-unsaturated sterols, is a dominant mechanism by which dozens of small molecules enhance oligodendrocyte formation. Here, we evaluated a library of 56 sterols and steroids to evaluate whether other classes of bioactive sterol derivatives may also influence mouse oligodendrocyte precursor cell (OPC) differentiation or survival. From this library, we identified U-73343 as a potent enhancer of oligodendrocyte formation that induces 8,9-unsaturated sterol accumulation by inhibition of the cholesterol biosynthesis enzyme sterol 14-reductase. In contrast, we found that mouse OPCs are remarkably vulnerable to treatment with the glycosterol OSW-1, an oxysterol-binding protein (OSBP) modulator that induces Golgi stress and OPC death in the low picomolar range. A subsequent small-molecule suppressor screen identified mTOR signaling as a key effector pathway mediating OSW-1's cytotoxic effects in mouse OPCs. Finally, evaluation of a panel of ER and Golgi stress-inducing small molecules revealed that mouse OPCs are highly sensitive to these perturbations, more so than closely related neural progenitor cells. Together, these studies highlight the wide-ranging influence of sterols and steroids on OPC cell fate, with 8,9-unsaturated sterols positively enhancing differentiation to oligodendrocytes and OSW-1 able to induce lethal Golgi stress with remarkable potency.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Precursoras de Oligodendrócitos/efeitos dos fármacos , Esteróis/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Colestenonas/farmacologia , Colestenonas/toxicidade , Avaliação Pré-Clínica de Medicamentos , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estrenos/farmacologia , Complexo de Golgi/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Células Precursoras de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Pirrolidinonas/farmacologia , Saponinas/farmacologia , Saponinas/toxicidade , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/toxicidade , Esteróis/toxicidade
2.
Biomolecules ; 11(6)2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067463

RESUMO

Submerged mycelial cultures of the ascomycete Colpoma quercinum CCTU A372 were found to produce five previously undescribed tetramic acids, for which we propose the trivial names colposetins A-C (1-3) and colpomenoic acids A and B (4 and 5), along with the known compounds penicillide (6) and monodictyphenone (7). The planar structures of 1-5 were determined by high-resolution electrospray ionization mass spectrometry (HR-ESIMS) and extensive 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Their absolute configurations were determined by the combination of electronic circular dischroism (ECD) analysis, J-based configurational analysis, and a rotating-frame Overhauser effect spectroscopy (ROESY) experiment. Colposetin B displayed weak antimicrobial activity against Bacillus subtilis and Mucor hiemalis (MIC 67 µg/mL).


Assuntos
Ascomicetos/química , Bacillus subtilis/crescimento & desenvolvimento , Mucor/crescimento & desenvolvimento , Micélio/química , Pirrolidinonas , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Irã (Geográfico) , Pirrolidinonas/química , Pirrolidinonas/isolamento & purificação , Pirrolidinonas/farmacologia
3.
Molecules ; 26(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802860

RESUMO

The COVID-19 outbreak continues to spread worldwide at a rapid rate. Currently, the absence of any effective antiviral treatment is the major concern for the global population. The reports of the occurrence of various point mutations within the important therapeutic target protein of SARS-CoV-2 has elevated the problem. The SARS-CoV-2 main protease (Mpro) is a major therapeutic target for new antiviral designs. In this study, the efficacy of PF-00835231 was investigated (a Mpro inhibitor under clinical trials) against the Mpro and their reported mutants. Various in silico approaches were used to investigate and compare the efficacy of PF-00835231 and five drugs previously documented to inhibit the Mpro. Our study shows that PF-00835231 is not only effective against the wild type but demonstrates a high affinity against the studied mutants as well.


Assuntos
Antivirais/química , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Indóis/química , Indóis/farmacologia , Leucina/química , Leucina/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Sítios de Ligação , COVID-19/tratamento farmacológico , Simulação por Computador , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/genética , Bases de Dados de Proteínas , Diarilquinolinas/química , Diarilquinolinas/farmacologia , Di-Hidropiridinas/química , Di-Hidropiridinas/farmacologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrobenzenos/química , Nitrobenzenos/farmacologia , Nitrofenóis/química , Nitrofenóis/farmacologia , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Prolina/análogos & derivados , Prolina/química , Prolina/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética
4.
Theranostics ; 11(12): 5847-5862, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897885

RESUMO

Metastasis and chemoresistance are major causes of poor prognosis in patients with esophageal squamous cell carcinoma (ESCC), manipulated by multiple factors including deubiquitinating enzyme (DUB). DUB PSMD14 is reported to be a promising therapeutic target in various cancers. Here, we explored the antitumor activity of Thiolutin (THL), the PSMD14 inhibitor, as a new therapy strategy in ESCC. Methods: Through 4-NQO-induced murine ESCC model, we investigated the expression of PSMD14 in esophageal tumorigenesis. Ubiquitin-AMC assay was performed to evaluate DUB activity of PSMD14 with THL treatment. The effect of THL on epithelial-to-mesenchymal transition (EMT), invasion, stemness and chemosensitivity was detected by using in vitro and in vivo experiments. Immunoprecipitation and in vivo ubiquitination assay were conducted to examine whether THL could impair the deubiquitination and stability of SNAIL regulated by PSMD14. Results: Compared with normal esophageal epithelium, PSMD14 was upregulated in 4-NQO-induced murine esophageal epithelium dysplasia and ESCC tissues. THL could significantly weaken DUB activity of PSMD14. Furthermore, the results of in vitro and in vivo assays showed that THL efficiently suppressed motility and stemness and increased sensitivity to cisplatin in ESCC. Mechanically, THL impaired the interaction between PSMD14 and SNAIL, then promoted the ubiquitination and degradation of SNAIL to inhibit EMT which plays a crucial role in ESCC metastasis, stemness and chemosensitivity. TCGA database analysis revealed that high concomitant PSMD14/SNAIL expression predicted shorter overall survival in esophageal cancer. Conclusion: Our findings demonstrate for the first time that suppression of PSMD14/SNAIL axis by THL could be a novel and promising therapeutic approach for ESCC clinical therapy.


Assuntos
Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Transativadores/metabolismo , Animais , Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/patologia , Pirrolidinonas/farmacologia , Ubiquitinação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
5.
Theranostics ; 11(11): 5127-5142, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859738

RESUMO

Hypoxic microenvironment is a hallmark of solid tumors, especially glioblastoma. The strong reliance of glioma-propagating cells (GPCs) on hypoxia-induced survival advantages is potentially exploitable for drug development. Methods: To identify key signaling pathways for hypoxia adaptation by patient-derived GPCs, we performed a kinase inhibitor profiling by screening 188 small molecule inhibitors against 130 different kinases in normoxia and hypoxia. Potential kinase candidates were prioritized for in vitro and in vivo investigations using a ranking algorithm that integrated information from the kinome connectivity network and estimated patients' survival based on expression status. Results: Hypoxic drug screen highlighted extensive modifications of kinomic landscape and a crucial functionality of c-MET-PI3K. c-MET inhibitors diminished phosphorylation of c-MET and PI3K in GPCs subjected to hypoxia, suggesting its role in the hypoxic adaptation of GPCs. Mechanistically, the inhibition of c-MET and PI3K impaired antioxidant defense, leading to oxidative catastrophe and apoptosis. Repurposed c-MET inhibitors PF04217903 and tivantinib exhibited hypoxic-dependent drug synergism with temozolomide, resulting in reduced tumor load and growth of GPC xenografts. Detailed analysis of bulk and single-cell glioblastoma transcriptomes associates the cellular subpopulation over-expressing c-MET with inflamed, hypoxic, metastatic, and stem-like phenotypes. Conclusions: Thus, our "bench to bedside (the use of patient-derived GPCs and xenografts for basic research) and back (validation with independent glioblastoma transcriptome databases)" analysis unravels the novel therapeutic indications of c-MET and PI3K/Akt inhibitors for the treatment of glioblastoma, and potentially other cancers, in the hypoxic tumor microenvironment.


Assuntos
Glioma/genética , Hipóxia/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-met/genética , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioma/tratamento farmacológico , Humanos , Hipóxia/tratamento farmacológico , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Pirazinas/farmacologia , Pirrolidinonas/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Temozolomida/farmacologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Triazóis/farmacologia
6.
Molecules ; 26(5)2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33800024

RESUMO

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.


Assuntos
Moduladores de Receptores de Canabinoides/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/fisiologia , Receptores de Canabinoides/metabolismo , Animais , Canabinoides/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Corpo Estriado/citologia , Estrenos/farmacologia , Células-Tronco Neurais/fisiologia , Neurogênese/efeitos dos fármacos , Pirrolidinonas/farmacologia , Ratos , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/genética , Receptores de Canabinoides/genética , Fosfolipases Tipo C/antagonistas & inibidores
7.
Molecules ; 26(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924091

RESUMO

Ten benzoxazole clubbed 2-pyrrolidinones (11-20) as human monoacylglycerol lipase inhibitors were designed on the criteria fulfilling the structural requirements and on the basis of previously reported inhibitors. The designed, synthesized, and characterized compounds (11-20) were screened against monoacylglycerol lipase (MAGL) in order to find potential inhibitors. Compounds 19 (4-NO2 derivative) and 20 (4-SO2NH2 derivative), with an IC50 value of 8.4 and 7.6 nM, were found most active, respectively. Both of them showed micromolar potency (IC50 value above 50 µM) against a close analogue, fatty acid amide hydrolase (FAAH), therefore considered as selective inhibitors of MAGL. Molecular docking studies of compounds 19 and 20 revealed that carbonyl of 2-pyrrolidinone moiety sited at the oxyanion hole of catalytic site of the enzyme stabilized with three hydrogen bonds (~2 Å) with Ala51, Met123, and Ser122, the amino acid residues responsible for the catalytic function of the enzyme. Remarkably, the physiochemical and pharmacokinetic properties of compounds 19 and 20, computed by QikProp, were found to be in the qualifying range as per the proposed guideline for good orally bioactive CNS drugs. In formalin-induced nociception test, compound 20 reduced the pain response in acute and late stages in a dose-dependent manner. They significantly demonstrated the reduction in pain response, having better potency than the positive control gabapentin (GBP), at 30 mg/kg dose. Compounds 19 and 20 were submitted to NCI, USA, for anticancer activity screening. Compounds 19 (NSC: 778839) and 20 (NSC: 778842) were found to have good anticancer activity on SNB-75 cell line of CNS cancer, exhibiting 35.49 and 31.88% growth inhibition (% GI), respectively.


Assuntos
Antineoplásicos/química , Benzoxazóis/química , Monoacilglicerol Lipases/antagonistas & inibidores , Pirrolidinonas/farmacologia , Analgésicos/química , Analgésicos/farmacologia , Antineoplásicos/farmacologia , Benzoxazóis/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Humanos , Simulação de Acoplamento Molecular , Monoacilglicerol Lipases/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Pirrolidinonas/química , Relação Estrutura-Atividade
8.
J Physiol Biochem ; 77(2): 321-329, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33704695

RESUMO

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.


Assuntos
Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Fracionamento Celular , Membrana Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Estrenos/farmacologia , Regulação da Expressão Gênica , Transporte de Íons/efeitos dos fármacos , Isoxazóis/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Naftalenos/farmacologia , Fosforilação/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Cultura Primária de Células , Propionatos/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Pirrolidinonas/farmacologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Suínos , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
9.
Biomed Pharmacother ; 138: 111462, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33706129

RESUMO

The antiepileptic/anticonvulsive action of brivaracetam is considered to occur via modulation of synaptic vesicle protein 2A (SV2A); however, the pharmacological mechanisms of action have not been fully characterised. To explore the antiepileptic/anticonvulsive mechanism of brivaracetam associated with SV2A modulation, this study determined concentration-dependent effects of brivaracetam on astroglial L-glutamate release associated with connexin43 (Cx43), tumour-necrosis factor-α (TNFα) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/glutamate receptor of rat primary cultured astrocytes using ultra-high-performance liquid chromatography. Furthermore, interaction among TNFα, elevated extracellular K+ and brivaracetam on expression of SV2A and Cx43 was determined using capillary immunoblotting. TNFα and elevated extracellular K+ predominantly enhanced astroglial L-glutamate release associated with respective AMPA/glutamate receptor and hemichannel. These effects were enhanced by a synergistic effect of TNFα and elevated extracellular K+ in combination. The activation of astroglial L-glutamate release, and expression of SV2A and Cx43 in the plasma membrane was suppressed by subchronic brivaracetam administration but were unaffected by acute administration. These results suggest that migration of SV2A to the astroglial plasma membrane by hyperexcitability activates astroglial glutamatergic transmission, perhaps via hemichannel activation. Subchronic brivaracetam administration suppressed TNFα-induced activation of AMPA/glutamate receptor and hemichannel via inhibition of ectopic SV2A. These findings suggest that combined inhibition of vesicular and ectopic SV2A functions contribute to the antiepileptic/anticonvulsive mechanism of brivaracetam action.


Assuntos
Astrócitos/metabolismo , Ácido Glutâmico/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pirrolidinonas/farmacologia , Animais , Animais Recém-Nascidos , Anticonvulsivantes/farmacologia , Astrócitos/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Masculino , Proteínas do Tecido Nervoso/antagonistas & inibidores , Gravidez , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/metabolismo , Receptores de Glutamato/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
10.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669259

RESUMO

BACKGROUND: Pseurotins, a family of secondary metabolites of different fungi characterized by an unusual spirocyclic furanone-lactam core, are suggested to have different biological activities including the modulation of immune response. PURPOSE: Complex characterization of the effects of pseurotin D on human lymphocyte activation in order to understand the potential of pseurotin to modulate immune response in humans. METHODS: CD4+ and CD8+ T cells and CD19+ B cells isolated from human blood were activated by various activators simultaneously with pseurotin D treatment. The effects of pseurotin were tested on the basis of changes in cell viability, apoptosis, activation of signal transducers and activators of transcription (STAT) signaling pathways, production of tumor necrosis factor (TNF)-α by T cells, expression of activation markers CD69 and CD25 on T cells and Human Leukocyte Antigen-DR isotype (HLA-DR) on B cells, and the differentiation markers CD20, CD27, CD38, and immunoglobulin (Ig) D on B cells. RESULTS: Pseurotin D significantly inhibited the activation of both CD4+ and CD8+ human T cells complemented by the inhibition of TNF-α production without significant acute toxic effects. The Pseurotin D-mediated inhibition of T-cell activation was accompanied by the induction of the apoptosis of T cells. This corresponded with the inhibited phosphorylation of STAT3 and STAT5. In human B cells, pseurotin D did not significantly inhibit their activation; however, it affected their differentiation. CONCLUSIONS: Our results advance the current mechanistic understanding of the pseurotin-induced inhibition of lymphocytes and suggest pseurotins as new attractive chemotypes for future research in the context of immune-modulatory drugs.


Assuntos
Antígenos CD19/metabolismo , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Fatores Imunológicos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Pirrolidinonas/farmacologia , Adolescente , Adulto , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem
11.
Exp Eye Res ; 205: 108507, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33609510

RESUMO

Proliferative retinopathies, such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are major causes of visual impairment and blindness in industrialized countries. Prostaglandin E2 (PGE2) is implicated in cellular proliferation and migration via E-prostanoid receptor (EP4R). The aim of this study was to investigate the role of PGE2/EP4R signaling in the promotion of retinal neovascularisation. In a streptozotocin (STZ)-induced diabetic model and an oxygen-induced retinopathy (OIR) model, rats received an intravitreal injection of PGE2, cay10598 (an EP4R agonist) or AH23848 (an EP4R antagonist). Optical coherence tomography, retinal histology and biochemical markers were assessed. Treatment with PGE2 or cay10598 accelerated pathological retinal angiogenesis in STZ and OIR-induced rat retina, which was ameliorated in rats pretreated with AH23848. Serum VEGF-A was upregulated in the PGE2-treated diabetic rats vs non-treated diabetic rats and significantly downregulated in AH23848-treated diabetic rats. PGE2 or cay10598 treatment also significantly accelerated endothelial tip-cell formation in new-born rat retina. In addition, AH23848 treatment attenuated PGE2-or cay10598-induced proliferation and migration by repressing the EGF receptor (EGFR)/Growth factor receptor bound protein 2-associated binder protein 1 (Gab1)/Akt/NF-κB/VEGF-A signaling network in human retinal microvascular endothelial cells (hRMECs). PGE2/EP4R signaling network is thus a potential therapeutic target for pathological intraocular angiogenesis.


Assuntos
Dinoprostona/fisiologia , Receptores ErbB/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Neovascularização Retiniana/fisiopatologia , Animais , Animais Recém-Nascidos , Compostos de Bifenilo/farmacologia , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Endotélio Vascular/metabolismo , Injeções Intravítreas , Masculino , NF-kappa B/metabolismo , Oxigênio/toxicidade , Fosforilação , Pirrolidinonas/farmacologia , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Transdução de Sinais/fisiologia , Tetrazóis/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Biomolecules ; 11(2)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578683

RESUMO

A mimetic of the BDNF loop 4, bis (N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide, named GSB-106, was designed and synthesized in our scientific group. The compound activated TrkB, MAPK/ERK, PI3K/AKT, and PLCγ in in vitro experiments. In vivo experiments with rodents revealed its antidepressant-like activity in the forced swim and the tail suspension tests at the dose range of 0.1-5.0 mg/kg (i.p., p.o.). However, GSB-106 was not studied in depression models modulating major depression in humans. In the present study, the GSB-106 antidepressant-like activity was revealed in mice at the depression model induced by 28-day social defeat stress with 21-days oral administration (0.1 mg/kg) after stress. At the same time, GSB-106 restored reduced locomotor activity and completely eliminated the anhedonia manifestations. The compound also restored reduced levels of synaptophysin and CREB in the hippocampus. In addition, the Trk receptor antagonist K252A, and the PLC inhibitor U73122, were found to completely block the antidepressant-like activity of GSB-106 in the forced swimming test in mice. Thus, the present results demonstrate the dipeptide BDNF mimetic GSB-106 reversed depressive-like behavior and restored hippocampal neuroplasticity in a rodent depression model. These effects of GSB-106 are probably regulated by TrkB signaling.


Assuntos
Antidepressivos/uso terapêutico , Materiais Biomiméticos/uso terapêutico , Fator Neurotrófico Derivado do Encéfalo/química , Transtorno Depressivo/tratamento farmacológico , Dipeptídeos/uso terapêutico , Peptidomiméticos/uso terapêutico , Administração Oral , Animais , Antidepressivos/química , Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Carbazóis/farmacologia , Dipeptídeos/química , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Estrenos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Alcaloides Indólicos/farmacologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Proteínas Tirosina Quinases/metabolismo , Pirrolidinonas/farmacologia , Comportamento Social , Sinaptofisina/metabolismo
13.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477998

RESUMO

Stachybotrys microspora triprenyl phenol (SMTP) is a large family of small molecules derived from the fungus S. microspora. SMTP acts as a zymogen modulator (specifically, plasminogen modulator) that alters plasminogen conformation to enhance its binding to fibrin and subsequent fibrinolysis. Certain SMTP congeners exert anti-inflammatory effects by targeting soluble epoxide hydrolase. SMTP congeners with both plasminogen modulation activity and anti-inflammatory activity ameliorate various aspects of ischemic stroke in rodents and primates. A remarkable feature of SMTP efficacy is the suppression of hemorrhagic transformation, which is exacerbated by conventional thrombolytic treatments. No drug with such properties has been developed yet, and SMTP would be the first to promote thrombolysis but suppress disease-associated bleeding. On the basis of these findings, one SMTP congener is under clinical study and development. This review summarizes the discovery, mechanism of action, pharmacological activities, and development of SMTP.


Assuntos
Benzopiranos/farmacologia , Inflamação/tratamento farmacológico , AVC Isquêmico/tratamento farmacológico , Pirrolidinonas/farmacologia , Terapia Trombolítica/métodos , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Benzopiranos/uso terapêutico , Isquemia Encefálica/sangue , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Epóxido Hidrolases/efeitos dos fármacos , Epóxido Hidrolases/metabolismo , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Humanos , Inflamação/sangue , Inflamação/patologia , AVC Isquêmico/sangue , AVC Isquêmico/patologia , Plasminogênio/efeitos dos fármacos , Plasminogênio/metabolismo , Pirrolidinonas/uso terapêutico , Stachybotrys/química , Stachybotrys/metabolismo
14.
Theranostics ; 11(6): 2655-2669, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33456565

RESUMO

Increasing evidence reveals a close relationship between deubiquitinating enzymes (DUBs) and cancer progression. In this study, we attempted to identify the roles and mechanisms of critical DUBs in head and neck squamous cell carcinoma (HNSCC). Methods: Bioinformatics analysis was performed to screen differentially expressed novel DUBs in HNSCC. Immunohistochemistry assay was used to measure the expression of DUB PSMD14 in HNSCC specimens and adjacent normal tissues. The level of PSMD14 in HNSCC tumorigenesis was investigated using a 4-NQO-induced murine HNSCC model. The function of PSMD14 was determined through loss-of-function assays. Chromatin immunoprecipitation, immunoprecipitation and in vivo ubiquitination assay were conducted to explore the potential mechanism of PSMD14. The anti-tumor activity of PSMD14 inhibitor Thiolutin was assessed by in vitro and in vivo experiments. Results: We identified PSMD14 as one of significantly upregulated DUBs in HNSCC tissues. Aberrant expression of PSMD14 was associated with tumorigenesis and malignant progression of HNSCC and further indicated poor prognosis. The results of in vitro and in vivo experiments demonstrated PSMD14 depletion significantly undermined HNSCC growth, chemoresistance and stemness. Mechanically, PSMD14 inhibited the ubiquitination and degradation of E2F1 to improve the activation of Akt pathway and the transcription of SOX2. Furthermore, PSMD14 inhibitor Thiolutin exhibited a potent anti-tumor effect on HNSCC in vivo and in vitro by impairing DUB activity of PSMD14. Conclusion: Our findings demonstrate the role and mechanism of PSMD14 in HNSCC, and provide a novel and promising target for diagnosis and clinical therapy of HNSCC.


Assuntos
Enzimas Desubiquitinantes/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição E2F1/genética , Células-Tronco Neoplásicas/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas c-akt/genética , Fatores de Transcrição SOXB1/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Transativadores/genética , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Prognóstico , Pirrolidinonas/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
15.
Biomolecules ; 11(2)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33494474

RESUMO

Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: 1) BKca channels (Paxillin, 10-5 M); 2) L-type calcium channels (Verapamil, 10-5 M); 3) Ryanodine receptor, RyR (Ryanodine, 10-5 M); 4) inositol 1,4,5-triphosphate receptor, IP3R, (2-Aminoethyl diphenylborinate, 2-APB, 3 × 10-3 M); 5) phospholipase C, PLC, (U73122, 10-5 M), and 6) GPCR-Gαi signaling, (Pertussis Toxin, 10-5 M). EVOO phenols induced vasodilation of mesenteric arteries in a dose-dependent manner, and this effect was reduced by pre-incubation with Paxillin, Verapamil, Ryanodine, 2-APB, U73122, and Pertussis Toxin. Our data suggest that EVOO phenol-mediated vasodilation requires activation of BKca channels potentially through a local increase of subcellular calcium microdomains, a pivotal mechanism on the base of artery vasodilation. These findings provide novel mechanistic insights for understanding the vasodilatory properties of EVOO phenols on resistance arteries.


Assuntos
Microdomínios da Membrana/química , Artérias Mesentéricas/efeitos dos fármacos , Azeite de Oliva/química , Canais de Potássio/química , Fosfolipases Tipo C/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Compostos de Boro/farmacologia , Canais de Cálcio/química , Estrenos/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/química , Masculino , Paxilina/farmacologia , Toxina Pertussis/farmacologia , Fenol/química , Fenóis/farmacologia , Pirrolidinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Vasodilatação/efeitos dos fármacos , Verapamil/farmacologia
16.
ChemMedChem ; 16(6): 942-948, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33283984

RESUMO

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549/ACE2 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.


Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Células A549 , Alanina/análogos & derivados , Alanina/metabolismo , Alanina/farmacologia , Animais , Antivirais/síntese química , Antivirais/metabolismo , Domínio Catalítico , Chlorocebus aethiops , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Cisteína/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Ligação Proteica , Pirrolidinonas/síntese química , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacologia , SARS-CoV-2/enzimologia , Células Vero
17.
J Cell Mol Med ; 25(3): 1531-1545, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33372388

RESUMO

Previous studies identified the involvement of phosphoinositide-specific phospholipase C (PLC) γ1 in some events of chondrocytes. This study aims to investigate whether and how PLCγ1 modulates autophagy to execute its role in osteoarthritis (OA) progression. Rat normal or human OA chondrocytes were pretreated with IL-1ß for mimicking or sustaining OA pathological condition. Using Western blotting, immunoprecipitation, qPCR, immunofluorescence and Dimethylmethylene blue assays, and ELISA and transmission electron microscope techniques, we found that PLCγ1 inhibitor U73122 enhanced Collagen II, Aggrecan and GAG levels, accompanied with increased LC3B-II/I ratio and decreased P62 expression level, whereas autophagy inhibitor Chloroquine partially diminished its effect. Meanwhile, U73122 dissociated Beclin1 from Beclin1-IP3R-Bcl-2 complex and blocked mTOR/ULK1 axis, in which the crosstalk between PLCγ1, AMPK, Erk and Akt were involved. Additionally, by haematoxylin and eosin, Safranin O/Fast green, and immunohistochemistry staining, we observed that intra-articular injection of Ad-shPLCγ1-1/2 significantly enhanced Collagen and Aggrecan levels, accompanied with increased LC3B and decreased P62 levels in a rat OA model induced by anterior cruciate ligament transection and medial meniscus resection. Consequently, PLCγ1 inhibition-driven autophagy conferred cartilage protection against OA through promoting ECM synthesis in OA chondrocytes in vivo and in vitro, involving the crosstalk between PLCγ1, AMPK, Erk and Akt.


Assuntos
Autofagia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/etiologia , Osteoartrite/metabolismo , Fosfolipase C gama/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagia/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Células Cultivadas , Suscetibilidade a Doenças , Estrenos/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirrolidinonas/farmacologia , Ratos , Serina-Treonina Quinases TOR/metabolismo
18.
Gen Comp Endocrinol ; 300: 113637, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33017583

RESUMO

Allatotropin is a pleiotropic peptide originally characterized in insects. The existence of AT neuropeptide signaling was proposed in other invertebrates. In fact, we previously proposed the presence of an AT-like system regulating feeding behavior in Hydra sp. Even in insects, the information about the AT signaling pathway is incomplete. The aim of this study is to analyze the signaling cascade activated by AT in Hydra plagiodesmica using a pharmacological approach. The results show the involvement of Ca2+ and IP3 signaling in the transduction pathway of the peptide. Furthermore, we confirm the existence of a GPCR system involved in this pathway, that would be coupled to a Gq subfamily of Gα protein, which activates a PLC, inducing an increase in IP3 and cytosolic Ca2+. To the best of our knowledge, this work represents the first in vivo approach to study the overall signaling pathway and intracellular events involved in the myoregulatory effect of AT in Hydra sp.


Assuntos
Sinalização do Cálcio , Hydra/metabolismo , Hormônios de Inseto/metabolismo , Neuropeptídeos/metabolismo , Orexinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Maleimidas/farmacologia , Meliteno/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
19.
Cancer Res ; 80(24): 5491-5501, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33115803

RESUMO

Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of all renal cancers and around 30% of chRCC cases have mutations in TP53. chRCC is poorly supported by microvessels and has markably lower glucose uptake than clear cell RCC and papillary RCC. Currently, the metabolic status and mechanisms by which this tumor adapts to nutrient-poor microenvironments remain to be investigated. In this study, we performed proteome and metabolome profiling of chRCC tumors and adjacent kidney tissues and identified major metabolic alterations in chRCC tumors, including the classical Warburg effect, the downregulation of gluconeogenesis and amino acid metabolism, and the upregulation of protein degradation and endocytosis. chRCC cells depended on extracellular macromolecules as an amino acid source by activating endocytosis to sustain cell proliferation and survival. Inhibition of the phospholipase C gamma 2 (PLCG2)/inositol 1,4,5-trisphosphate (IP3)/Ca2+/protein kinase C (PKC) pathway significantly impaired the activation of endocytosis for amino acid uptakes into chRCC cells. In chRCC, whole-exome sequencing revealed that TP53 mutations were not related to expression of PLCG2 and activation of endocytosis. Our study provides novel perspectives on metabolic rewiring in chRCC and identifies the PLCG2/IP3/Ca2+/PKC axis as a potential therapeutic target in patients with chRCC. SIGNIFICANCE: This study reveals macropinocytosis as an important process utilized by chRCC to gain extracellular nutrients in a p53-independent manner.


Assuntos
Aminoácidos/metabolismo , Carcinoma de Células Renais/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Neoplasias Renais/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Compostos de Boro/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estrenos/farmacologia , Gluconeogênese , Humanos , Indóis/farmacologia , Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias Renais/patologia , Maleimidas/farmacologia , Metaboloma , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteoma , Pirrolidinonas/farmacologia
20.
PLoS One ; 15(10): e0240480, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33079945

RESUMO

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species.


Assuntos
Anfíbios/microbiologia , Antifúngicos/farmacologia , Batrachochytrium/efeitos dos fármacos , Batrachochytrium/crescimento & desenvolvimento , Micologia/métodos , Animais , Batrachochytrium/genética , Bleomicina/farmacologia , Cinamatos/farmacologia , Testes Diagnósticos de Rotina , Avaliação Pré-Clínica de Medicamentos , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Testes de Sensibilidade Microbiana , Neomicina/farmacologia , Puromicina/farmacologia , Pirrolidinonas/farmacologia , Seleção Genética
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