ABSTRACT
The search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrPC) conversion into protease-resistant forms (protease-resistant PrP [PrPRes] or the scrapie form of PrP [PrPSc]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identified in vitro to be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action. None of them changed PrPC mRNA levels in N2a cells, as shown by reverse transcription-quantitative real-time PCR. Among them, J8 and Y17 were effective in real-time quaking-induced conversion reactions using rodent recombinant PrP (rPrP) from residues 23 to 231 (rPrP23-231) as the substrate and PrPSc seeds from hamster and human brain. However, when rPrP from residues 90 to 231 (rPrP90-231), which lacks the N-terminal domain, was used as the substrate, only J8 remained effective, indicating that this region is important for Y17 activity, while J8 seems to interact with the PrPC globular domain. J8 also reduced the fibrillation of mouse rPrP23-231 seeded with in vitro-produced fibrils. Furthermore, most of the compounds decreased the amount of PrPC on the N2a cell surface by trapping this protein in the endoplasmic reticulum. On the basis of these results, we hypothesize that J8, a nontoxic compound previously shown to be a promising antiprion agent, may act by different mechanisms, since its efficacy is attributable not only to PrP conversion inhibition but also to a reduction of the PrPC content on the cell surface.
Subject(s)
Chalcones/pharmacology , Drugs, Investigational/pharmacology , Neurons/drug effects , Oxadiazoles/pharmacology , Prion Proteins/antagonists & inhibitors , Animals , Binding Sites , Cell Line, Tumor , Chalcones/chemical synthesis , Cloning, Molecular , Drugs, Investigational/chemical synthesis , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Kinetics , Mice , Molecular Docking Simulation , Neurons/metabolism , Neurons/pathology , Oxadiazoles/chemical synthesis , Prion Proteins/chemistry , Prion Proteins/genetics , Prion Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , ThermodynamicsABSTRACT
Eukaryotic cells have developed mechanisms to rapidly respond towards the environment by changing the expression of a series of genes. There is increasing evidence that reactive oxygen species (ROS), besides causing damage, may also fulfill an important role as second messengers involved in signal transduction. Recently, we have demonstrated that deletion of SOD1 is beneficial for the acquisition of tolerance towards heat and ethanol stresses. The present report demonstrates that a sod1 mutant was the only one capable of acquiring tolerance against a subsequent stress produced by menadione, although this mutant strain had exhibited high sensitivity to oxidative stress. By measuring the level of intracellular oxidation, lipid peroxidation as well as glutathione metabolism, we have shown that in the SOD1-deleted strain, an unbalance occurs in the cell redox status. These results indicated that the capacity of acquiring tolerance to oxidative stress is related to a signal given by one or all of the above factors.
