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1.
Molecules ; 26(6)2021 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-33802860

RESUMEN

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.


Asunto(s)
Antivirales/química , Antivirales/farmacología , Indoles/química , Indoles/farmacología , Leucina/química , Leucina/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Pirrolidinonas/química , Pirrolidinonas/farmacología , Sitios de Unión , Simulación por Computador , /genética , Bases de Datos de Proteínas , Diarilquinolinas/química , Diarilquinolinas/farmacología , Dihidropiridinas/química , Dihidropiridinas/farmacología , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Nitrobencenos/química , Nitrobencenos/farmacología , Nitrofenoles/química , Nitrofenoles/farmacología , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Piperazinas/química , Piperazinas/farmacología , Prolina/análogos & derivados , Prolina/química , Prolina/farmacología , /genética
2.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-33669259

RESUMEN

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.


Asunto(s)
Antígenos CD19/metabolismo , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Factores Inmunológicos/farmacología , Activación de Linfocitos/efectos de los fármacos , Pirrolidinonas/farmacología , Adolescente , Adulto , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Adulto Joven
3.
PLoS One ; 15(8): e0238155, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32841278

RESUMEN

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.


Asunto(s)
Antineoplásicos/farmacología , Señalización del Calcio , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Gefitinib/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Estrenos/farmacología , Humanos , Factores de Transcripción NFATC/biosíntesis , Pirrolidinonas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores
4.
PLoS Pathog ; 16(7): e1008595, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32628727

RESUMEN

Sarocladium zeae is a fungal endophyte of maize and can be found co-inhabiting a single seed with Fusarium verticillioides, a major mycotoxigenic food safety threat. S. zeae produces pyrrocidines A and B that inhibit the growth of F. verticillioides and may limit its spread within the seed to locations lacking S. zeae. Although coinhabiting single seeds, the fungi are generally segregated in separate tissues. To understand F. verticillioides' protective physiological response to pyrrocidines we sequenced the F. verticillioides transcriptome upon exposure to purified pyrrocidine A or B at sub-inhibitory concentrations. Through this work we identified a F. verticillioides locus FvABC3 (FVEG_11089) encoding a transporter critical for resistance to pyrrocidine. We also identified FvZBD1 (FVEG_00314), a gene directly adjacent to the fumonisin biosynthetic gene cluster that was induced several thousand-fold in response to pyrrocidines. FvZBD1 is postulated to act as a genetic repressor of fumonisin production since deletion of the gene resulted in orders of magnitude increase in fumonisin. Further, pyrrocidine acts, likely through FvZBD1, to shut off fumonisin biosynthesis. This suggests that S. zeae is able to hack the secondary metabolic program of a competitor fungus, perhaps as preemptive self-protection, in this case impacting a mycotoxin of central concern for food safety.


Asunto(s)
Acremonium , Fumonisinas/metabolismo , Fusarium/genética , Micosis/microbiología , Enfermedades de las Plantas/microbiología , Zea mays/microbiología , Hidrocarburos Aromáticos con Puentes/metabolismo , Hidrocarburos Aromáticos con Puentes/farmacología , Coinfección , Resistencia a la Enfermedad/genética , Genes Fúngicos , Micosis/metabolismo , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacología
5.
Psychopharmacology (Berl) ; 237(8): 2317-2326, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32382782

RESUMEN

RATIONALE: Prolinol aryl ethers and their rigidified analogues pyrrolidinyl benzodioxanes have a high affinity for mammalian α4ß2 nicotinic acetylcholine receptors (nAChRs). Electrophysiological studies have shown that the former are full agonists and the latter partial agonists or antagonists of human α4ß2 receptors, but their in vivo effects are unknown. OBJECTIVES AND METHODS: As α4ß2 nAChRs play an important role in the cognition and the rewarding effects of nicotine, we tested the effects of two full agonists and one antagonist on spatial learning, memory and attention in zebrafish using a T-maze task and virtual object recognition test (VORT). The effect of a partial agonist in reducing nicotine-induced conditioned place preference (CPP) was also investigated. RESULTS: In comparison with the vehicle alone, the full agonists MCL-11 and MCL-28 induced a significant cognitive enhancement as measured by the reduced running time in the T-maze and increased attention as measured by the increased discrimination index in the VORT. MCL-11 was 882 times more potent than nicotine. The two compounds were characterised by an inverted U-shaped dose-response curve, and their effects were blocked by the co-administration of the antagonist MCL-117, which alone had no effect. The partial agonist MCL-54 induced CPP and had an inverted U-shaped dose-response curve similar to that of nicotine but blocked the reinforcing effect of co-administered nicotine. Binding studies showed that all of the compounds have a higher affinity for heteromeric [3H]-epibatidine receptors than [125I]-αBungarotoxin receptors. MCL-11 was the most selective of heteromeric receptors. CONCLUSIONS: These behavioural studies indicate that full agonist prolinol aryl ethers are very active in increasing spatial learning, memory and attention in zebrafish. The benzodioxane partial agonist MCL-54 reduced nicotine-induced CPP, and the benzodioxane antagonist MCL-117 blocked all agonist-induced activities.


Asunto(s)
Aprendizaje por Laberinto/efectos de los fármacos , Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/metabolismo , Pirrolidinas/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Éteres/metabolismo , Éteres/farmacología , Femenino , Masculino , Aprendizaje por Laberinto/fisiología , Morfinanos/metabolismo , Morfinanos/farmacología , Nicotina/metabolismo , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Pirrolidinas/farmacología , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacología , Pez Cebra
6.
PLoS One ; 15(5): e0233485, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32470050

RESUMEN

Antimicrobial resistance is a growing global health and economic concern. Current antimicrobial agents are becoming less effective against common bacterial infections. We previously identified pyrrolocins A and C, which showed activity against a variety of Gram-positive bacteria. Structurally similar compounds, known as pyrrolidinediones (e.g., TA-289, equisetin), also display antibacterial activity. However, the mechanism of action of these compounds against bacteria was undetermined. Here, we show that pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase (ACC), the first step in fatty acid synthesis. We used transcriptomic data, metabolomic analysis, fatty acid rescue and acetate incorporation experiments to show that a major mechanism of action of the pyrrolidinediones is inhibition of fatty acid biosynthesis, identifying ACC as the probable molecular target. This hypothesis was further supported using purified proteins, demonstrating that biotin carboxylase is the inhibited component of ACC. There are few known antibiotics that target this pathway and, therefore, we believe that these compounds may provide the basis for alternatives to current antimicrobial therapy.


Asunto(s)
Acetil-CoA Carboxilasa/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/metabolismo , Pirrolidinonas/farmacología , Tetrahidronaftalenos/farmacología , Acetil-CoA Carboxilasa/química , Antibacterianos/farmacología , Proteínas Bacterianas/química , Dominio Catalítico/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Ácidos Grasos/biosíntesis , Perfilación de la Expresión Génica , Bacterias Grampositivas/crecimiento & desarrollo , Humanos , Metabolómica , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismo
7.
Epilepsia ; 61(5): 914-923, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32297665

RESUMEN

OBJECTIVE: The antiepileptic drug candidate, padsevonil, is the first in a novel class of drugs designed to interact with both presynaptic and postsynaptic therapeutic targets: synaptic vesicle 2 proteins and γ-aminobutyric acid type A receptors (GABAA Rs), respectively. Functional aspects of padsevonil at the postsynaptic target, GABAA Rs, were characterized in experiments reported here. METHODS: The effect of padsevonil on GABA-mediated Cl- currents was determined by patch clamp on recombinant human GABAA Rs (α1ß2γ2) stably expressed in a CHO-K1 cell line and on native GABAA Rs in cultured rat primary cortical neurons. Padsevonil selectivity for GABAA R subtypes was evaluated using a two-electrode voltage clamp on recombinant human GABAA Rs (α1-5/ß2/γ2) in Xenopus oocytes. RESULTS: In recombinant GABAA Rs, padsevonil did not evoke Cl- currents in the absence of the agonist GABA. However, when co-administered with GABA at effective concentration (EC)20 , padsevonil potentiated GABA responses by 167% (EC50 138 nmol/L) and demonstrated a relative efficacy of 41% compared with zolpidem, a reference benzodiazepine site agonist. Similarly, padsevonil demonstrated GABA-potentiating activity at native GABAA Rs (EC50 208 nmol/L) in cultured rat cortical neurons. Padsevonil also potentiated GABA (EC20 ) responses in GABAA Rs expressed in oocytes, with higher potency at α1- and α5-containing receptors (EC50 295 and 281 nmol/L) than at α2- and α3-containing receptors (EC50 1737 and 2089 nmol/L). Compared with chlordiazepoxide-a nonselective, full GABAA R agonist-the relative efficacy of padsevonil was 60% for α1ß2γ2, 26% for α2ß2γ2, 56% for α3ß2γ2, and 41% for α5ß2γ2; no activity was observed at benzodiazepine-insensitive α4ß2γ2 receptors. SIGNIFICANCE: Results of functional investigations on recombinant and native neuronal GABAA Rs show that padsevonil acts as a positive allosteric modulator of these receptors, with a partial agonist profile at the benzodiazepine site. These properties may confer better tolerability and lower potential for tolerance development compared with classic benzodiazepines currently used in the clinic.


Asunto(s)
Anticonvulsivantes/farmacología , Imidazoles/farmacología , Pirrolidinonas/farmacología , Receptores de GABA-A/efectos de los fármacos , Tiadiazoles/farmacología , Animales , Células CHO , Cricetulus , Femenino , Humanos , Neuronas/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Ratas Wistar , Receptores Presinapticos/efectos de los fármacos , Proteínas Recombinantes , Potenciales Sinápticos/efectos de los fármacos , Xenopus laevis
8.
J Appl Oral Sci ; 28: e20190516, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32236357

RESUMEN

INTRODUCTION: This study investigated the effect of a calcium hydroxide (CH) paste (CleaniCal®) containing N-2-methyl pyrrolidone (NMP) as a vehicle on Enterococcus faecalis (E. faecalis) biofilms compared with other products containing saline (Calasept Plus™) or propylene glycol (PG) (Calcipex II®). METHODOLOGY: Standardized bovine root canal specimens were used. The antibacterial effects were measured by colony-forming unit counting. The thickness of bacterial microcolonies and exopolysaccharides was assessed using confocal laser scanning microscopy. Morphological features of the biofilms were observed using field-emission scanning electron microscopy (FE-SEM). Bovine tooth blocks covered with nail polish were immersed into the vehicles and dispelling was observed. The data were analyzed using one-way analysis of variance and Tukey tests (p<0.05). RESULTS: CleaniCal® showed the highest antibacterial activity, followed by Calcipex II® (p<0.05). Moreover, NMP showed a higher antibacterial effect compared with PG (p<0.05). The thickness of bacteria and EPS in the CleaniCal® group was significantly lower than that of other materials tested (p<0.05). FE-SEM images showed the specimens treated with Calasept Plus™ were covered with biofilms, whereas the specimens treated with other medicaments were not. Notably, the specimen treated with CleaniCal® was cleaner than the one treated with Calcipex II®. Furthermore, the nail polish on the bovine tooth block immersed in NMP was completely dispelled. CONCLUSIONS: CleaniCal® performed better than Calasept Plus™ and Calcipex II® in the removal efficacy of E. faecalis biofilms. The results suggest the effect might be due to the potent dissolving effect of NMP on organic substances.


Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Hidróxido de Calcio/farmacología , Enterococcus faecalis/efectos de los fármacos , Pirrolidinonas/farmacología , Irrigantes del Conducto Radicular/farmacología , Análisis de Varianza , Animales , Cloruro de Calcio/química , Cloruro de Calcio/farmacología , Hidróxido de Calcio/química , Bovinos , Recuento de Colonia Microbiana , Combinación de Medicamentos , Ensayo de Materiales , Microscopía Confocal , Microscopía Electrónica de Rastreo , Cloruro de Potasio/química , Cloruro de Potasio/farmacología , Pirrolidinonas/química , Reproducibilidad de los Resultados , Irrigantes del Conducto Radicular/química , Bicarbonato de Sodio/química , Bicarbonato de Sodio/farmacología , Cloruro de Sodio/química , Cloruro de Sodio/farmacología , Estadísticas no Paramétricas
9.
Eur J Med Chem ; 191: 112120, 2020 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-32120339

RESUMEN

N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4.


Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Diseño de Fármacos , Pirrolidinonas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Transferencia Resonante de Energía de Fluorescencia , Humanos , Modelos Moleculares , Estructura Molecular , Pirrolidinonas/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismo
10.
Cell Prolif ; 53(4): e12749, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32167212

RESUMEN

OBJECTIVES: Given that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. Here, we investigated ability of TN-16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death. MATERIALS AND METHODS: The effect of TN-16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flowcytometric assays. In vivo anti-tumour efficacy of TN-16 was investigated in syngeneic mouse model of breast cancer. RESULTS: TN-16 inhibited cancer cell proliferation by impairing late-stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy-specific substrate p62 and lack of additional LC3-II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome-lysosome fusion. Further studies revealed that TN-16-mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of our in vivo study revealed that TN-16-mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis. CONCLUSIONS: Our data signify that TN-16 is a potent autophagy flux inhibitor and might be suitable for (pre-) clinical use as standard inhibitor of autophagy with anti-cancer activity.


Asunto(s)
Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Pirrolidinonas/uso terapéutico , Moduladores de Tubulina/uso terapéutico , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/patología , Neoplasias/metabolismo , Neoplasias/patología , Pirrolidinonas/farmacología , Moduladores de Tubulina/farmacología
11.
Phytomedicine ; 69: 153194, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32146299

RESUMEN

BACKGROUND: The frequency of allergic diseases is constantly rising. Dysregulated production of isotype E immunoglobulins is one of the key factors behind allergic reactions and its modulation is therefore an important target for pharmacological intervention. Natural products of the pseurotin family were reported to be inhibitors of IgE production in B-cells. Mechanistic details underlying these effects are however not well understood. PURPOSE: In the present study, we synthesized new analogs of natural pseurotins and extensively investigated their inhibitory effects on activation, proliferation and differentiation of B-cells, as well as on the production of IgE. STUDY DESIGN: Effects of two natural pseurotins (pseurotins A and D) and a collection of fully synthetic pseurotin analogs were studied on mouse B-cells stimulated by the combination of IL-4 and E. coli lipopolysaccharide. The IgE production was determined along with cell viability and cell proliferation. The phosphorylation of selected members of the STAT transcription factor family was subsequently investigated. Finally, the in vivo effect of pseurotin D on the ovalbumin-induced delayed type hypersensitivity response was tested in mice. RESULTS: We discovered that several fully synthetic pseurotin analogs were able to decrease the production of IgE in stimulated B-cells with potency comparable to that of pseurotins A and D. We found that the two natural pseurotins and the active synthetic analogs inhibited the phosphorylation of STAT3, STAT5 and STAT6 proteins in stimulated B-cells, resulting in the inhibition of B-cell proliferation and differentiation into the plasma cells. In vivo, pseurotin D decreased ovalbumin-induced foot pad edema. CONCLUSION: Our results advance the current mechanistic understanding of the pseurotin-induced inhibition of IgE production in B-cells by linking the effect to STAT signaling, and associated modulation of B-cell proliferation and differentiation. Together with our finding that structurally simpler pseurotin analogs were able to reproduce the effects of natural pseurotins, the presented work has implications for the future research on these secondary metabolites in the context of allergic diseases.


Asunto(s)
Linfocitos B/efectos de los fármacos , Inmunoglobulina E/metabolismo , Células Plasmáticas/citología , Pirrolidinonas/química , Pirrolidinonas/farmacología , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Diferenciación Celular/efectos de los fármacos , Edema/inducido químicamente , Edema/tratamiento farmacológico , Escherichia coli/química , Inmunoglobulina E/sangre , Inmunoglobulina M/sangre , Inmunoglobulina M/metabolismo , Lipopolisacáridos/farmacología , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ovalbúmina/toxicidad , Fosforilación/efectos de los fármacos , Células Plasmáticas/fisiología , Factores de Transcripción STAT/metabolismo
12.
Am J Physiol Cell Physiol ; 318(5): C954-C968, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32186932

RESUMEN

The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K+ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.


Asunto(s)
Canal de Potasio Kv.1.2/genética , Canal de Potasio Kv1.5/genética , Hipertensión Arterial Pulmonar/genética , Receptores Sensibles al Calcio/genética , Molécula de Interacción Estromal 2/genética , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Señalización del Calcio/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Estrenos/farmacología , Células HEK293 , Humanos , Indoles/farmacología , Proteína Jagged-1/genética , Potenciales de la Membrana/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Pirrolidinonas/farmacología , Receptores Sensibles al Calcio/efectos de los fármacos , Receptores Notch/genética , Análisis de la Célula Individual
13.
Oxid Med Cell Longev ; 2020: 9369524, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32190179

RESUMEN

Aging has been characterized with the accumulation of oxidized proteins, as a consequence of progressive decline in proteostasis capacity. Among others, proteasomal system is an efficient protein turnover complex to avoid aggregation of oxidized proteins. Heat shock protein 70 (HSP70) is another critical player that is involved in some key processes including the correct folding of misfolded proteins and targeting aggregated proteins to the proteasome for rapid degradation. The aim of this study was to determine the role of proteasomal system and heat shock proteins to maintain proteome balance during replicative senescence in mild hyperthermia conditions. Our results demonstrated that HSP40/70 machinery is induced by mild hyperthermia conditions independent from senescence conditions. Since HSP70 is largely responsible for the rapidly inducible cell protection following hyperthermia, the activation of "heat shock response" resulted in the elevation of HSP40/70 expressions as well as the proteasome activity. Interestingly, when HSP70 expression was inhibited, increased proteasomal activation was shown to be responsive to mild hyperthermia. Since HSP70 is involved in various stress-related pathways such as oxidative and endoplasmic reticulum stress, depletion of HSP70 expression may induce proteasomal degradation to maintain proteome balance of the cell. Thus, our data suggests that in mild heat stress conditions, molecular chaperone HSP70 plays an important role to avoid protein oxidation and aggregation; however, activities of proteasomal system are induced when HSP70 expression is depleted.


Asunto(s)
Senescencia Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Hipertermia Inducida , Complejo de la Endopetidasa Proteasomal/metabolismo , Compuestos de Bencidrilo/farmacología , Senescencia Celular/genética , Silenciador del Gen , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Humanos , Masculino , Proteostasis , Pirrolidinonas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo
14.
Eur J Med Chem ; 191: 112150, 2020 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-32105981

RESUMEN

Since the discovery and early characterization of the histamine H3 receptor (H3R) in the 1980's, predominantly imidazole-based agonists were presented to the scientific community such as Nα-methylhistamine (Nα-MeHA) or (R)-α-methylhistamine ((R)α-MeHA). Whereas therapeutic applications have been prompted for H3R agonists such as treatment of pain, asthma and obesity, several drawbacks associated with imidazole-containing ligands makes the search for new agonists for this receptor demanding. Accordingly, high interest arose after publication of several pyrrolidindione-based, highly affine H3R agonists within this journal that avoid the imidazole moiety and thus, presenting a novel type of potential pharmacophores (Ghoshal, Anirban et al., 2018). In our present study performed in two independent laboratories, we further evaluated the exposed lead-compound (EC50 = 0.1 nM) of the previous research project with regards to pharmacological behavior at H3R. Thereby, no binding affinity was observed in neither [3H]Nα-MeHA nor bodilisant displacement assays that contradicts the previously published activity. Additional functional exploration employing GTPγ[35S], cAMP-accumulation assay and cAMP response element (CRE)-driven reporter gene assays exhibited slight partial agonist properties of such pyrrolidindiones but acting apart from the reported concentration range. We conclude, that the previously reported actions of such pyrrolidindiones result from an overestimation based on the method of measurement and thus, we cast doubt on the new pharmacophores with H3R agonist activity.


Asunto(s)
Pirrolidinonas/farmacología , Receptores Histamínicos H3/metabolismo , Relación Dosis-Respuesta a Droga , Polarización de Fluorescencia , Células HEK293 , Humanos , Estructura Molecular , Unión Proteica/efectos de los fármacos , Pirrolidinonas/síntesis química , Pirrolidinonas/química , Relación Estructura-Actividad
15.
Behav Pharmacol ; 31(2&3): 179-185, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31770112

RESUMEN

The clinical use of opioid analgesics, such as morphine, is limited by analgesic tolerance, molecular mechanism of which is not well understood. Recently, molecular chaperone heat shock protein 70 (Hsp70) has been demonstrated to play important roles in morphine-induced neuroadaptation. Here, we focused on the involvement of Hsp70 in the development of analgesic tolerance to morphine. Rats were treated with morphine (5, 10, 20 mg/kg, subcutaneously) or saline once daily for 10 consecutive days. Hsp70 modulator N-formyl-3, 4-methylenedioxybenzylidine-γ-butyrolactam [KNK437, 100 mg/kg, intraperitoneally (i.p.)], geranylgeranylacetone (500 mg/kg, i.p.) or pifithrin-µ (20 mg/kg, i.p.) was administered before morphine (10 mg/kg, subcutaneously)/saline treatment. Analgesic effect of morphine was measured using the tail-flick latency test, and Hsp70 protein expression was examined by western blot. Analgesic effect of morphine decreased gradually with the increase in the number of days of morphine injection, indicating development of analgesic tolerance. A significant increase of Hsp70 expression in the periaqueductal gray was observed during the development of analgesic tolerance after repeated morphine injections. The development of morphine analgesic tolerance was suppressed by pre-treatment with Hsp70 transcriptional inhibitor KNK437 or functional antagonist pifithrin-µ, while promoted by pre-treatment with Hsp70 transcriptional inducer geranylgeranylacetone. Our results demonstrated that the development of morphine analgesic tolerance was dual regulated by Hsp70 modulators, suggesting Hsp70 as an interesting and new target for preventing the development of opioid analgesic tolerance.


Asunto(s)
Compuestos de Bencidrilo/farmacología , Tolerancia a Medicamentos/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Pirrolidinonas/farmacología , Analgésicos/farmacología , Analgésicos Opioides/farmacología , Animales , Compuestos de Bencidrilo/metabolismo , Encéfalo/metabolismo , Femenino , Masculino , Morfina/farmacología , Sustancia Gris Periacueductal/metabolismo , Pirrolidinonas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/metabolismo
16.
J Pharmacol Exp Ther ; 372(1): 11-20, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31619464

RESUMEN

The antiepileptic drug (AED) candidate, (4R)-4-(2-chloro-2,2-difluoroethyl)-1-{[2-(methoxymethyl)-6-(trifluoromethyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl]methyl}pyrrolidin-2-one (padsevonil), is the first in a novel class of drugs that bind to synaptic vesicle protein 2 (SV2) proteins and the GABAA receptor benzodiazepine site, allowing for pre- and postsynaptic activity, respectively. In acute seizure models, padsevonil provided potent, dose-dependent protection against seizures induced by administration of pilocarpine or 11-deoxycortisol, and those induced acoustically or through 6 Hz stimulation; it was less potent in the pentylenetetrazol, bicuculline, and maximal electroshock models. Padsevonil displayed dose-dependent protective effects in chronic epilepsy models, including the intrahippocampal kainate and Genetic Absence Epilepsy Rats from Strasbourg models, which represent human mesial temporal lobe and absence epilepsy, respectively. In the amygdala kindling model, which is predictive of efficacy against focal to bilateral tonic-clonic seizures, padsevonil provided significant protection in kindled rodents; in mice specifically, it was the most potent AED compared with nine others with different mechanisms of action. Its therapeutic index was also the highest, potentially translating into a favorable efficacy and tolerability profile in humans. Importantly, in contrast to diazepam, tolerance to padsevonil's antiseizure effects was not observed in the pentylenetetrazol-induced clonic seizure threshold test. Further results in the 6 Hz model showed that padsevonil provided significantly greater protection than the combination of diazepam with either 2S-(2-oxo-1-pyrrolidinyl)butanamide (levetiracetam) or 2S-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl] butanamide (brivaracetam), both selective SV2A ligands. This observation suggests that padsevonil's unique mechanism of action confers antiseizure properties beyond the combination of compounds targeting SV2A and the benzodiazepine site. Overall, padsevonil displayed robust efficacy across validated seizure and epilepsy models, including those considered to represent drug-resistant epilepsy. SIGNIFICANCE STATEMENT: Padsevonil, a first-in-class antiepileptic drug candidate, targets SV2 proteins and the benzodiazepine site of GABAA receptors. It demonstrated robust efficacy across a broad range of rodent seizure and epilepsy models, several representing drug-resistant epilepsy. Furthermore, in one rodent model, its efficacy extended beyond the combination of drugs interacting separately with SV2 or the benzodiazepine site. Padsevonil displayed a high therapeutic index, potentially translating into a favorable safety profile in humans; tolerance to antiseizure effects was not observed.


Asunto(s)
Anticonvulsivantes/uso terapéutico , Epilepsia/tratamiento farmacológico , Imidazoles/uso terapéutico , Pirrolidinonas/uso terapéutico , Convulsiones/tratamiento farmacológico , Tiadiazoles/uso terapéutico , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/fisiopatología , Animales , Anticonvulsivantes/efectos adversos , Anticonvulsivantes/farmacología , Evaluación Preclínica de Medicamentos , Femenino , Imidazoles/efectos adversos , Imidazoles/farmacología , Excitación Neurológica , Masculino , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Pirrolidinonas/efectos adversos , Pirrolidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Tiadiazoles/efectos adversos , Tiadiazoles/farmacología
17.
Am J Physiol Regul Integr Comp Physiol ; 318(1): R38-R48, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31596114

RESUMEN

Astrocytes generate robust cytoplasmic calcium signals in response to reductions in extracellular glucose. This calcium signal, in turn, drives purinergic gliotransmission, which controls the activity of catecholaminergic (CA) neurons in the hindbrain. These CA neurons are critical to triggering glucose counter-regulatory responses (CRRs) that, ultimately, restore glucose homeostasis via endocrine and behavioral means. Although the astrocyte low-glucose sensor involvement in CRR has been accepted, it is not clear how astrocytes produce an increase in intracellular calcium in response to a decrease in glucose. Our ex vivo calcium imaging studies of hindbrain astrocytes show that the glucose type 2 transporter (GLUT2) is an essential feature of the astrocyte glucosensor mechanism. Coimmunoprecipitation assays reveal that the recombinant GLUT2 binds directly with the recombinant Gq protein subunit that activates phospholipase C (PLC). Additional calcium imaging studies suggest that GLUT2 may be connected to a PLC-endoplasmic reticular-calcium release mechanism, which is amplified by calcium-induced calcium release (CICR). Collectively, these data help outline a potential mechanism used by astrocytes to convert information regarding low-glucose levels into intracellular changes that ultimately regulate the CRR.


Asunto(s)
Astrocitos/fisiología , Calcio/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glucosa/metabolismo , Rombencéfalo/citología , Fosfolipasas de Tipo C/metabolismo , Anilidas/farmacología , Animales , Antioxidantes/farmacología , Compuestos de Boro/farmacología , Calcio/farmacología , Dantroleno/farmacología , Estrenos/farmacología , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Florizina/farmacología , Profármacos , Pirrolidinonas/farmacología , Quercetina/farmacología , Ratas , Ratas Long-Evans , Fosfolipasas de Tipo C/antagonistas & inhibidores
18.
Oncogene ; 39(2): 249-261, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31477839

RESUMEN

As an inhibitor of heat shock proteins (HSPs), KNK437 has been reported to play an anti-tumor role in several cancers. But its therapeutic effect and mechanisms in colorectal cancer (CRC) remain unclear. Here, KNK437 sharply inhibited the level of DnaJ heat shock protein family (Hsp40) member A1 (DNAJA1), followed by DNAJB1, but had little effect on the levels of HSP27, HSP105, HSP90, and HSP70 in CRC cells. DNAJA1 promoted CRC cell proliferation in vitro and tumor growth and metastasis in vivo. Mechanistically, DNAJA1 was activated by E2F transcription factor 1 (E2F1) and then promoted cell cycle by stabilizing cell division cycle protein 45 (CDC45), which could be reversed by KNK437. DNAJA1 was significantly upregulated in CRC tissues and positively correlated with serosa invasion, lymph node metastasis. High level of DNAJA1 predicted poor prognosis for CRC patients. Its expression was highly linked with E2F1 and CDC45 in CRC tissues. More importantly, KNK437 significantly suppressed the growth of DNAJA1 expressing tumor in vivo. The combined treatment of KNK437 with 5-FU/L-OHP chemotherapy reduced liver metastasis of CRC. These data reveal a novel mechanism of KNK437 in anti-tumor therapy of CRC and provides a newly therapeutic strategy with potential translation to the CRC patients.


Asunto(s)
Compuestos de Bencidrilo/farmacología , Proteínas de Ciclo Celular/genética , Neoplasias Colorrectales/tratamiento farmacológico , Factor de Transcripción E2F1/genética , Proteínas del Choque Térmico HSP40/genética , Pirrolidinonas/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/antagonistas & inhibidores , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
19.
RNA ; 26(1): 10-18, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31601735

RESUMEN

Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pathways, which affect mRNA stability, may themselves be perturbed by the approaches used to measure mRNA stability, leading to artifactual results. Here, we have focused on common strategies to measure mRNA half-lives in yeast and determined that commonly used transcription inhibitors thiolutin and 1,10 phenanthroline inhibit TORC1 signaling, PKC signaling, and partially activate HOG signaling. Additionally, 4-thiouracil (4tU), a uracil analog used in mRNA pulse-labeling approaches, modestly induces P-bodies, mRNA-protein granules implicated in storage and decay of nontranslating mRNA. Thiolutin also induces P-bodies, whereas phenanthroline has no effect. Doxycycline, which controls "Tet On/Tet Off" regulatable promoters, shows no impact on the above signaling pathways or P-bodies. In summary, our data argues that broad-acting transcriptional inhibitors are problematic for determining mRNA half-life, particularly if studying the impacts of the TORC1, HOG, or PKC pathway on mRNA stability. Regulatable promoter systems are a preferred approach for individual mRNA half-life studies, with 4tU labeling representing a good approach to global mRNA half-life analysis, despite modestly inducing P-bodies.


Asunto(s)
Estabilidad del ARN/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Citoplasma/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Semivida , Fenantrolinas/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Pirrolidinonas/farmacología , ARN de Hongos/química , ARN de Hongos/efectos de los fármacos , ARN Mensajero/química , ARN Mensajero/efectos de los fármacos , Saccharomyces cerevisiae/fisiología , Estrés Fisiológico
20.
Prostaglandins Other Lipid Mediat ; 146: 106389, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31689497

RESUMEN

There is considerable controversy regarding the vasoactive action of prostaglandin E2 (PGE2). On the one hand, indirect evidence implicates that astrocytic release of PGE2 contributes to neurovascular coupling responses mediating functional hyperemia in the brain. On the other hand, overproduction of PGE2 was also reported to contribute to cerebral vasospasm associated with subarachnoid hemorrhage. The present study was conducted to resolve this controversy by determining the direct vasoactive effects of PGE2 in resistance-sized human cerebral parenchymal arterioles. To achieve this goal PGE2-induced isotonic vasomotor responses were assessed in parenchymal arterioles isolated from fronto-temporo-parietal cortical tissues surgically removed from patients and expression of PGE2 receptors were examined. In functionally intact parenchymal arterioles lower concentrations of PGE2 (from 10-8 to 10-6 mol/l) caused significant, endothelium-independent vasorelaxation, which was inhibited by the EP4 receptor blocker BGC201531. In contrast, higher concentrations of PGE2 evoked significant EP1-dependent vasoconstriction, which could not be reversed by the EP4 receptor agonist CAY10598. We also confirmed previous observations that PGE2 primarily evokes constriction in intracerebral arterioles isolated from R. norvegicus. Importantly, vascular mRNA and protein expression of vasodilator EP4 receptors was significantly higher than that of vasoconstrictor EP1 receptors in human cerebral arterioles. PGE2 at low concentrations dilates whereas at higher concentrations constricts human cerebral parenchymal arterioles. This bimodal vasomotor response is consistent with both the proposed vasodilator role of PGE2 during functional hyperemia and its putative role in cerebral vasospasm associated with subarachnoid hemorrhage in human patients.


Asunto(s)
Encéfalo , Dinoprostona/farmacología , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Arteriolas/metabolismo , Arteriolas/fisiopatología , Encéfalo/irrigación sanguínea , Encéfalo/fisiopatología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Persona de Mediana Edad , Piridinas/farmacología , Pirrolidinonas/farmacología , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Hemorragia Subaracnoidea/metabolismo , Sulfonamidas/farmacología , Tetrazoles/farmacología
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