Aminoquinolones and Their Benzoquinone Dimer Hybrids as Modulators of Prion Protein Conversion.

Prion Diseases or Transmissible Spongiform Encephalopathies are neurodegenerative conditions associated with a long incubation period and progressive clinical evolution, leading to death. Their pathogenesis is characterized by conformational changes of the cellular prion protein-PrPC-in its infectious isoform-PrPSc-which can form polymeric aggregates that precipitate in brain tissues. Currently, there are no effective treatments for these diseases. The 2,5-diamino-1,4-benzoquinone structure is associated with an anti-prion profile and, considering the biodynamic properties associated with 4-quinolones, in this work, 6-amino-4-quinolones derivatives and their respective benzoquinone dimeric hybrids were synthesized and had their bioactive profile evaluated through their ability to prevent prion conversion. Two hybrids, namely, 2,5-dichloro-3,6-bis((3-carboxy-1-pentyl-4-quinolone-6-yl)amino)-1,4-benzoquinone (8e) and 2,5-dichloro-3,6-bis((1-benzyl-3-carboxy-4-quinolone-6-yl)amino)-1,4-benzoquinone (8f), stood out for their prion conversion inhibition ability, affecting the fibrillation process in both the kinetics-with a shortening of the lag phase-and thermodynamics and their ability to inhibit the formation of protein aggregates without significant cytotoxicity at ten micromolar.

Evaluation of the effect of cafeteria diet on the kidney Na,K-ATPase activity, and oxidative stress.

In this study, renal tissue, subdivided into the cortex and medulla of Wistar rats subjected to a cafeteria diet (CAF) for 24 days or to normal diet, was used to analyze whether the renal enzyme Na,K-ATPase activity was modified by CAF diet, as well as to analyze the α1 subunit of renal Na,K-ATPase expression levels. The lipid profile of the renal plasma membrane and oxidative stress were verified. In the Na,K-ATPase activity evaluation, no alteration was found, but a significant decrease of 30% in the cortex was detected in the α1 subunit expression of the enzyme. There was a 24% decrease in phospholipids in the cortex of rats submitted to CAF, a 17% increase in cholesterol levels in the cortex, and a 23% decrease in the medulla. Lipid peroxidation was significantly increased in the groups submitted to CAF, both in the cortical region, 29%, and in the medulla, 35%. Also, a reduction of 45% in the glutathione levels was observed in the cortex and medulla with CAF. CAF showed a nearly two-fold increase in glutathione peroxidase (GPX) activity in relation to the control group in the cortex and a 59% increase in the GPx activity in the medulla. In conclusion, although the diet was administered for a short period of time, important results were found, especially those related to the lipid profile and oxidative stress, which may directly affect renal function.

Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain.

Inactivation of the tumor suppressor protein p53 by mutagenesis, chemical modification, protein-protein interaction, or aggregation has been associated with different human cancers. Although DNA is the typical substrate of p53, numerous studies have reported p53 interactions with RNA. Here, we have examined the effects of RNA of varied sequence, length, and origin on the mechanism of aggregation of the core domain of p53 (p53C) using light scattering, intrinsic fluorescence, transmission electron microscopy, thioflavin-T binding, seeding, and immunoblot assays. Our results are the first to demonstrate that RNA can modulate the aggregation of p53C and full-length p53. We found bimodal behavior of RNA in p53C aggregation. A low RNA:protein ratio (∼1:50) facilitates the accumulation of large amorphous aggregates of p53C. By contrast, at a high RNA:protein ratio (≥1:8), the amorphous aggregation of p53C is clearly suppressed. Instead, amyloid p53C oligomers are formed that can act as seeds nucleating de novo aggregation of p53C. We propose that structured RNAs prevent p53C aggregation through surface interaction and play a significant role in the regulation of the tumor suppressor protein.

Prion protein-coated magnetic beads: synthesis, characterization and development of a new ligands screening method.

Prion diseases are characterized by protein aggregation and neurodegeneration. Conversion of the native prion protein (PrP(C)) into the abnormal scrapie PrP isoform (PrP(Sc)), which undergoes aggregation and can eventually form amyloid fibrils, is a critical step leading to the characteristic path morphological hallmark of these diseases. However, the mechanism of conversion remains unclear. It is known that ligands can act as cofactors or inhibitors in the conversion mechanism of PrP(C) into PrP(Sc). Within this context, herein, we describe the immobilization of PrP(C) onto the surface of magnetic beads and the morphological characterization of PrP(C)-coated beads by fluorescence confocal microscopy. PrP(C)-coated magnetic beads were used to identify ligands from a mixture of compounds, which were monitored by UHPLC-ESI-MS/MS. This affinity-based method allowed the isolation of the anti-prion compound quinacrine, an inhibitor of PrP aggregation. The results indicate that this approach can be applied to not only "fish" for anti-prion compounds from complex matrixes, but also to screening for and identify possible cellular cofactors involved in the deflagration of prion diseases.

New structure-activity relationships of chalcone inhibitors of breast cancer resistance protein: polyspecificity toward inhibition and critical substitutions against cytotoxicity.

Adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) plays a major role in cancer cell multidrug resistance, which contributes to low efficacy of chemotherapy. Chalcones were recently found to be potent and specific inhibitors, but unfortunately display a significant cytotoxicity. A cellular screening against ABCG2-mediated mitoxantrone efflux was performed here by flow cytometry on 54 chalcone derivatives from three different series with a wide panel of substituents. The identified leads, with submicromolar IC50 (half maximal inhibitory concentration) values, showed that the previously identified 2'-OH-4',6'-dimethoxyphenyl, as A-ring, could be efficiently replaced by a 2'-naphthyl group, or a 3',4'-methylenedioxyphenyl with lower affinity. Such a structural variability indicates 3polyspecificity of the multidrug transporter for inhibitors. At least two methoxyl groups were necessary on B-ring for optimal inhibition, but substitution at positions 3, 4, and 5 induced cytotoxicity. The presence of a large O-benzyl substituent at position 4 and a 2'-naphthyl as A-ring markedly decreased the cytotoxicity, giving a high therapeutic ratio, which constitutes a critical requirement for future in-vivo assays in animal models.

Effect of different extracts from the Brazilian Atlantic Forest on the Pdr5p ATPase activity / Efeito de extratos de plantas da Floresta Atlântica Brasileira sobre a atividade da Pdr5p ATPase

In the current study, we tested the effect of 27 plant extracts and fractions from different botanical families on the activity of Pdr5p from yeast plasma membrane, responsible for the multidrug resistance phenotype in yeast cells. Some of the extracts were able to produce a good inhibition in the fixed concentration (200 µg/mL) and were selected for a deeper investigation. Dose-response curves were obtained for the crude ethanol extracts of Bathysa australis (A. St.-Hill.) Benth. & Hook f., Mabea fistulifera Mart. and Virola oleifera (Schott) A. C. Sm. with concentrations ranging up to 400 µg/mL. The lower IC50 value was obtained for Virola oleifera, 22.8 µg/mL, followed by Bathysa australis, 35.3 µg/mL, and Mabea fistulifera, 42.5 µg/mL. After fractionation of the crude extracts by liquid-liquid partition with different organic solvents and each fraction was tested again, only some of the fractions retained the ability to inhibit the enzymatic activity. When analyzed by HPLC/DAD, the active fractions showed the presence of flavonoid derivatives, already reported for their ability to inhibit Pdr5p ATPase activity, as well as other classes of secondary metabolites such as lignans and alkaloids.

No presente estudo, testamos o efeito de 27 extratos e frações de plantas de diferentes famílias botânicas sobre a atividade da proteína Pdr5p de membranas plasmáticas de leveduras, responsável pelo fenótipo de resistência a múltiplas drogas em leveduras. Alguns dos extratos foram capazes de produzir uma boa inibição na concentração fixa de 200 µg/mL e foram selecionados para uma investigação mais aprofundada. Curvas de dose-resposta foram obtidas para os extratos brutos etanólicos de Bathysa australis (A. St.-Hill.) Benth. & Hook f., Mabea fistulifera Mart. e Virola oleifera (Schott) A. C. Sm., com concentrações até 400 µg/mL. O menor valor de IC50 foi obtido para Virola oleifera, 22,8 µg/mL, seguido por Bathysa australis, 35,3 µg/mL e Mabea fistulifera, 42,5 µg/mL. Após o fracionamento dos extratos brutos por partição líquido-líquido com diferentes solventes orgânicos, cada fração foi novamente testada, sendo que apenas algumas das frações mantiveram a habilidade de inibir a atividade enzimática. Quando analisadas por HPLC/DAD, as frações ativas demonstraram a presença de derivados de flavonóides, que já demonstraram ter a habilidade de inibir a atividade ATPasica da Pdr5p, assim como outras classes de metabólitos secundários, tais como lignanas e alcalóides.