RESUMO
Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Doenças Priônicas/tratamento farmacológico , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Dobramento de Proteína , Animais , Sítios de Ligação , Simulação por Computador , Retículo Endoplasmático/metabolismo , Fibroblastos , Células HEK293 , Humanos , Ligantes , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos TestesRESUMO
Heterozygous mutations of the lysosomal enzyme glucocerebrosidase (GBA1) represent the major genetic risk for Parkinson's disease (PD), while homozygous GBA1 mutations cause Gaucher disease, a lysosomal storage disorder, which may involve severe neurodegeneration. We have previously demonstrated impaired autophagy and proteasomal degradation pathways and mitochondrial dysfunction in neurons from GBA1 knockout (gba1-/-) mice. We now show that stimulation with physiological glutamate concentrations causes pathological [Ca2+]c responses and delayed calcium deregulation, collapse of mitochondrial membrane potential and an irreversible fall in the ATP/ADP ratio. Mitochondrial Ca2+ uptake was reduced in gba1-/- cells as was expression of the mitochondrial calcium uniporter. The rate of free radical generation was increased in gba1-/- neurons. Behavior of gba1+/- neurons was similar to gba1-/- in terms of all variables, consistent with a contribution of these mechanisms to the pathogenesis of PD. These data signpost reduced bioenergetic capacity and [Ca2+]c dysregulation as mechanisms driving neurodegeneration.
Assuntos
Cálcio/metabolismo , Metabolismo Energético , Glucosilceramidase/deficiência , Neurônios/patologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/patologia , Radicais Livres/metabolismo , Glucosilceramidase/metabolismo , Ácido Glutâmico/toxicidade , Homeostase/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Receptores de Glutamato/metabolismoRESUMO
Glycosides are ubiquitous plant secondary metabolites consisting of a non-sugar component called an aglycone, attached to one or more sugars. One of the most interesting aglycones in grapes and wine is methyl salicylate (MeSA), an organic ester naturally produced by many plants, particularly wintergreens. To date, nine different MeSA glycosides from plants have been reported, mainly spread over the genera Gaultheria, Camellia, Polygala, Filipendula, and Passiflora. From a sensorial point of view, MeSA has a balsamic-sweet odor, known as Wintergreen. MeSA was found in Vitis riparia grapes, in Vitis vinifera sp. and in the Frontenac interspecific hybrid. We found that the MeSA glycosides content in Verdicchio wines and in some genetically related varieties (Trebbiano di Soave and Trebbiano di Lugana) was very high. In order to understand which glycosides were present in wine, the methanolic extract of Verdicchio wine was injected into a UPLC-Q-TOF-HDMS and compared to the extracts of different plants rich in such glycosides. Using pure standards, we confirmed the existence of two glycosides in wine: MeSA 2-O-ï¢-d-glucoside and MeSA 2-O-ï¢-d-xylopyranosyl (1-6) ï¢-d-glucopyranoside (gaultherin). For the first time, we also tentatively identified other diglycosides in wine: MeSA 2-O-ï¡-l-arabinopyranosyl (1-6)-ï¢-d-glucopyranoside (violutoside) and MeSA 2-O-ï¢-d-apiofuranosyl (1-6)-ï¢-d-glucopyranoside (canthoside A), MeSA 2-O-ï¢-d-glucopyranosyl (1-6)-O-ï¢-d-glucopyranoside (gentiobioside) and MeSA 2-O-ï¡-l-rhamnopyranosyl (1-6)-ï¢-d-glucopyranoside (rutinoside). Some of these glycosides have been isolated from Gaultheria procumbens leaves by preparative liquid chromatography and structurally annotated by 1H- and 13C-NMR analysis. Two of the peaks isolated from Gaultheria procumbens leaves, namely MeSA sambubioside and MeSA sophoroside, were herein observed for the first time. Six MeSA glycosides were quantified in 64 Italian white wines, highlighting the peculiar content and pattern in Verdicchio wines and related cultivars. The total concentration in bound and free MeSA in Verdicchio wines varied in the range of 456-9796 ïg/L and 5.5-143 ïg/L, respectively, while in the other wines the bound and free MeSA was below 363 ïg/L and 12 ïg/L, respectively. As this compound's olfactory threshold is between 50 and 100 ïg/L, our data support the hypothesis that methyl salicylate can contribute to the balsamic scent, especially in old Verdicchio wines.
Assuntos
Glicosídeos/química , Salicilatos/química , Vitis/química , Vinho/análise , Cromatografia Líquida , Dissacarídeos/química , Dissacarídeos/isolamento & purificação , Glicosídeos/classificação , Glicosídeos/isolamento & purificação , Humanos , Extratos Vegetais/química , Folhas de Planta/química , Salicilatos/classificação , Salicilatos/isolamento & purificaçãoRESUMO
Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest. Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus, was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action. EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells). A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed. At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown. EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner. A role of RyR as a direct target of EC was also suggested by molecular modelling studies. In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.
Assuntos
Organismos Aquáticos/metabolismo , Euplotes/metabolismo , Melanoma/tratamento farmacológico , Sesquiterpenos/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Agonistas dos Canais de Cálcio/isolamento & purificação , Agonistas dos Canais de Cálcio/farmacologia , Agonistas dos Canais de Cálcio/uso terapêutico , Linhagem Celular Tumoral , Dantroleno/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Oncogênica v-akt/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/uso terapêuticoRESUMO
Although the composition of strawberry fruit has been extensively studied, especially for the most abundant phenolic compounds, agrimoniin has never been univocally identified as one of the most abundant phenolic compounds in the fruit. In this study agrimoniin was isolated in the fruit of Fragaria vesca and its structure characterized. Furthermore, its presence was definitively established to be the main ellagitannin in both F. vesca and Fragaria ananassa D. fruit. The presence of sanguiin H-6 and lambertianin C as minor compounds was confirmed in both F. vesca and F. ananassa D. samples. For the first time here is reported the full NMR assignments for agrimoniin. These data should represent a point of reference for NMR analysis of this and other structurally related ellagitannins. Finally, the establishment of an HPLC protocol for separation provided information making it possible to avoid confusion with sanguiin H-6, the main ellagitannin in Rubus species, which is also present in strawberries but at a much lower concentration.