RESUMO
The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.
Assuntos
Gentamicinas/farmacologia , Proteínas Priônicas/efeitos dos fármacos , Príons/antagonistas & inibidores , Aminoglicosídeos/metabolismo , Aminoglicosídeos/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Gentamicinas/metabolismo , Camundongos , Proteínas PrPC/efeitos dos fármacos , Proteínas PrPC/metabolismo , Proteínas PrPSc/efeitos dos fármacos , Proteínas PrPSc/metabolismo , Doenças Priônicas/prevenção & controle , Proteínas Priônicas/metabolismo , Príons/metabolismo , Inibidores da Síntese de ProteínasRESUMO
Prion diseases are fatal and infectious neurodegenerative diseases in humans and other mammals caused by templated misfolding of the endogenous prion protein (PrP). Although there is currently no vaccine or therapy against prion disease, several classes of small-molecule compounds have been shown to increase disease-free incubation time in prion-infected mice. An apparent obstacle to effective anti-prion therapy is the emergence of drug-resistant strains during static therapy with either single compounds or multi-drug combination regimens. Here, we treated scrapie-infected mice with dynamic regimens that alternate between different classes of anti-prion drugs. The results show that alternating regimens containing various combinations of Anle138b, IND24 and IND116135 reduce the incidence of combination drug resistance, but do not significantly increase long-term disease-free survival compared to monotherapy. Furthermore, the alternating regimens induced regional vacuolation profiles resembling those generated by a single component of the alternating regimen, suggesting the emergence of strain dominance.
Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Príons/antagonistas & inibidores , Scrapie/tratamento farmacológico , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Intervalo Livre de Doença , Quimioterapia Combinada , Período de Incubação de Doenças Infecciosas , Camundongos , Príons/efeitos dos fármacos , Scrapie/mortalidade , Scrapie/patologiaRESUMO
A multilayered computational workflow was designed to identify a druggable binding site on the surface of the E200K pathogenic mutant of the human prion protein, and to investigate the effect of the binding of small molecules in the inhibition of the early aggregation of this protein. At this purpose, we developed an efficient computational tool to scan the molecular interaction properties of a whole MD trajectory, thus leading to the characterization of plausible binding regions on the surface of PrP-E200K. These structural data were then employed to drive structure-based virtual screening and fragment-based approaches to the seeking of small molecular binders of the PrP-E200K. Six promising compounds were identified, and their binding stabilities were assessed by MD simulations. Therefore, analyses of the molecular electrostatic potential similarity between the bound complexes and unbound protein evidenced their potential activity as charged-based inhibitors of the PrP-E200K early aggregation.
Assuntos
Proteínas Mutantes/química , Príons/química , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Príons/antagonistas & inibidores , Príons/genética , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Yeast prions have become important models for the study of the basic mechanisms underlying human amyloid diseases. Yeast prions are pathogenic (unlike the [Het-s] prion of Podospora anserina), and most are amyloid-based with the same in-register parallel ß-sheet architecture as most of the disease-causing human amyloids studied. Normal yeast cells eliminate the large majority of prion variants arising, and several anti-prion/anti-amyloid systems that eliminate them have been identified. It is likely that mammalian cells also have anti-amyloid systems, which may be useful in the same way humoral, cellular, and innate immune systems are used to treat or prevent bacterial and viral infections.
Assuntos
Príons/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , HumanosRESUMO
The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.
Assuntos
Membrana Celular/química , Proteínas PrPC/análise , Príons/antagonistas & inibidores , Animais , Caseína Quinase II/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes , Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HEK293 , Harmalina/análogos & derivados , Harmalina/farmacologia , Hematoxilina/análogos & derivados , Hematoxilina/farmacologia , Humanos , Camundongos , Neuroblastoma , Proteínas PrPC/genética , Príons/biossíntese , Príons/toxicidade , Quinacrina/farmacologia , Tacrolimo/farmacologiaRESUMO
Incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD) are caused by transplantation of prion-contaminated hormones, cornea and dura mater as well as contact with prion- contaminated medical devices, such as stereotactic electrodes, used in neurosurgery. Because prions are highly resistant and difficult to inactivate, prion contamination is a severe risk when medical instruments are reused after surgical procedures involving suspicious and confirmed cases of patients with prion diseases. Therefore, when high-risk procedures such as cerebral surgery, craniotomy surgery, orthopaedic spinal surgery and ophthalmic surgery are performed for high-risk patients or individuals with prion diseases, it is neces- sary to appropriately treat the medical devices using scientifically proven prion inactivation methods. In this chapter, we introduce fundamental aspects of prion inactivation methods, looking specifically at the practical issues involved in their implementation.
Assuntos
Doenças Priônicas/prevenção & controle , Doenças Priônicas/transmissão , Príons/antagonistas & inibidores , Animais , Detergentes/química , Humanos , Doenças Priônicas/sangue , Doenças Priônicas/etiologia , Príons/sangue , Príons/efeitos dos fármacos , Fatores de Risco , Equipamentos CirúrgicosRESUMO
In a study aimed at identifying new anti-prion compounds we screened a library of 500 Australian marine invertebrate derived extracts using a yeast-based anti-prion assay. This resulted in an extract from the subtropical sponge Lamellodysidea cf. chlorea showing potent anti-prion activity. The bioassay-guided investigation of the sponge extract led to the isolation of three new bioactive polyoxygenated steroids, lamellosterols A-C (1-3). These sterols were all isolated in low yield, and their structures elucidated by extensive NMR and MS data analysis. Lamellosterols A-C displayed potent anti-prion activity against the [PSI+] yeast prion (EC50s of 12.7, 13.8, and 9.8 µM, respectively). Lamellosterol A (1) was further shown to bind to the Parkinson's disease implicated amyloid protein, α-synuclein, and to significantly inhibit its aggregation. Our findings indicate that these polyoxygenated sterol sulfates may be useful compounds to study mechanisms associated with neurodegenerative diseases.
Assuntos
Poríferos/metabolismo , Príons/antagonistas & inibidores , Esteróis/farmacologia , alfa-Sinucleína/antagonistas & inibidores , Animais , Estrutura Molecular , Príons/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Infectious proteins (prions) include an array of human (mammalian) and yeast amyloid diseases in which a protein or peptide forms a linear ß-sheet-rich filament, at least one functional amyloid prion, and two functional infectious proteins unrelated to amyloid. In Saccharomyces cerevisiae, at least eight anti-prion systems deal with pathogenic amyloid yeast prions by (1) blocking their generation (Ssb1,2, Ssz1, Zuo1), (2) curing most variants as they arise (Btn2, Cur1, Hsp104, Upf1,2,3, Siw14), and (3) limiting the pathogenicity of variants that do arise and propagate (Sis1, Lug1). Known mechanisms include facilitating proper folding of the prion protein (Ssb1,2, Ssz1, Zuo1), producing highly asymmetric segregation of prion filaments in mitosis (Btn2, Hsp104), competing with the amyloid filaments for prion protein monomers (Upf1,2,3), and regulation of levels of inositol polyphosphates (Siw14). It is hoped that the discovery of yeast anti-prion systems and elucidation of their mechanisms will facilitate finding analogous or homologous systems in humans, whose manipulation may be useful in treatment.
Assuntos
Príons/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Evolução Molecular , Genes Fúngicos , Variação Genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Príons/antagonistas & inibidores , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Prions are infectious proteins that cause fatal neurodegenerative disorders including Creutzfeldt-Jakob and bovine spongiform encephalopathy (mad cow) diseases. The yeast [PSI+] prion is formed by the translation-termination factor Sup35, is the best-studied prion, and provides a useful model system for studying such diseases. However, despite recent progress in the understanding of prion diseases, the cellular defense mechanism against prions has not been elucidated. Here, we report that proteolytic cleavage of Sup35 suppresses spontaneous de novo generation of the [PSI+] prion. We found that during yeast growth in glucose media, a maximum of 40% of Sup35 is cleaved at its N-terminal prion domain. This cleavage requires the vacuolar proteases PrA-PrB. Cleavage occurs in a manner dependent on translation but independently of autophagy between the glutamine/asparagine-rich (Q/N-rich) stretch critical for prion formation and the oligopeptide-repeat region required for prion maintenance, resulting in the removal of the Q/N-rich stretch from the Sup35 N terminus. The complete inhibition of Sup35 cleavage, by knocking out either PrA (pep4Δ) or PrB (prb1Δ), increased the rate of de novo formation of [PSI+] prion up to â¼5-fold, whereas the activation of Sup35 cleavage, by overproducing PrB, inhibited [PSI+] formation. On the other hand, activation of the PrB pathway neither cleaved the amyloid conformers of Sup35 in [PSI+] strains nor eliminated preexisting [PSI+]. These findings point to a mechanism antagonizing prion generation in yeast. Our results underscore the usefulness of the yeast [PSI+] prion as a model system to investigate defense mechanisms against prion diseases and other amyloidoses.
Assuntos
Proteínas Fúngicas/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Proteínas Priônicas/metabolismo , Príons/antagonistas & inibidores , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Peptídeo Hidrolases/metabolismo , Príons/metabolismo , Vacúolos/enzimologia , Leveduras/metabolismoRESUMO
The conversion of the native random coil amyloid beta (Aß) into amyloid fibers is thought to be a key event in the progression of Alzheimer's disease (AD). A significant body of evidence suggests that the highly dynamic Aß oligomers are the main causal agent associated with the onset of AD. Among many potential therapeutic approaches, one is the modulation of Aß conformation into off-pathway structures to avoid the formation of the putative neurotoxic Aß oligomers. A library of oligoquinolines was screened to identify antagonists of Aß oligomerization, amyloid formation, and cytotoxicity. A dianionic tetraquinoline, denoted as 5, was one of the most potent antagonists of Aß fibrillation. Biophysical assays including amyloid kinetics, dot blot, ELISA, and TEM show that 5 effectively inhibits both Aß oligomerization and fibrillation. The antagonist activity of 5 toward Aß aggregation diminishes with sequence and positional changes in the surface functionalities. 5 binds to the central discordant α-helical region and induces a unique α-helical conformation in Aß. Interestingly, 5 adjusts its conformation to optimize the antagonist activity against Aß. 5 effectively rescues neuroblastoma cells from Aß-mediated cytotoxicity and antagonizes fibrillation and cytotoxicity pathways of secondary nucleation induced by seeding. 5 is also equally effective in inhibiting preformed oligomer-mediated processes. Collectively, 5 induces strong secondary structure in Aß and inhibits its functions including oligomerization, fibrillation, and cytotoxicity.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Amiloide/química , Amiloide/toxicidade , Agregação Patológica de Proteínas/tratamento farmacológico , Doença de Alzheimer/metabolismo , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Humanos , Cinética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Príons/antagonistas & inibidores , Príons/química , Príons/metabolismo , Príons/toxicidade , Estrutura Secundária de Proteína/efeitos dos fármacosRESUMO
The discovery that the alteration of aging by reducing the activity of the insulin/IGF-1 signaling (IIS) cascade protects nematodes and mice from neurodegeneration-linked, toxic protein aggregation (proteotoxicity) raises the prospect that IIS inhibitors bear therapeutic potential to counter neurodegenerative diseases. Recently, we reported that NT219, a highly efficient IGF-1 signaling inhibitor, protects model worms from the aggregation of amyloid ß peptide and polyglutamine peptides that are linked to the manifestation of Alzheimer's and Huntington's diseases, respectively. Here, we employed cultured cell systems to investigate whether NT219 promotes protein homeostasis (proteostasis) in mammalian cells and to explore its underlying mechanisms. We found that NT219 enhances the aggregation of misfolded prion protein and promotes its deposition in quality control compartments known as "aggresomes." NT219 also elevates the levels of certain molecular chaperones but, surprisingly, reduces proteasome activity and impairs autophagy. Our findings show that IGF-1 signaling inhibitors in general and NT219 in particular can promote proteostasis in mammalian cells by hyperaggregating hazardous proteins, thereby bearing the potential to postpone the onset and slow the progression of neurodegenerative illnesses in the elderly.-Moll, L., Ben-Gedalya, T., Reuveni, H., Cohen, E. The inhibition of IGF-1 signaling promotes proteostasis by enhancing protein aggregation and deposition.
Assuntos
Homeostase/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Animais , Western Blotting , Células CHO , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Cricetinae , Cricetulus , Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Células NIH 3T3 , Doenças Neurodegenerativas/metabolismo , Compostos Orgânicos/farmacologia , Células PC12 , Príons/antagonistas & inibidores , Príons/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirogalol/análogos & derivados , Pirogalol/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Tioamidas/farmacologiaRESUMO
The first step in the conventional approach to self-assembled biomaterials is to develop well-defined nanostructures in vitro, which is followed by disruption of the preformed nanostructures at the inside of the cell to achieve bioactivity. Here, we propose an inverse strategy to develop in-cell gain-of-function self-assembled nanostructures. In this approach, the supramolecular building blocks exist in a unimolecular/unordered state in vitro or at the outside of the cell and assemble into well-defined nanostructures after cell internalization. We used block copolypeptides of an oligoarginine and a self-assembling peptide as building blocks and investigated correlations among the nanostructural state, antiprion bioactivity, and cytotoxicity. The optimal bioactivity (i.e., the highest antiprion activity and lowest cytotoxicity) was obtained when the building blocks existed in a unimolecular/unordered state in vitro and during the cell internalization process, exerting minimal cytotoxic damage to cell membranes, and were subsequently converted into high-charge-density vesicles in the low pH endosome/lysosomes in vivo, thus, resulting in the significantly enhanced antiprion activity. In particular, the in-cell self-assembly concept presents a feasible approach to developing therapeutics against protein misfolding diseases. In general, the in-cell self-assembly provides a novel inverse methodology to supramolecular bionanomaterials.
Assuntos
Ciclo Celular , Nanoestruturas/química , Peptídeos/química , Príons/antagonistas & inibidores , Materiais Biocompatíveis/química , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Peptídeos/antagonistas & inibidores , Príons/químicaRESUMO
Conformational conversion of the normal cellular prion protein, PrPC, into the misfolded isoform, PrPSc, is considered to be a central event in the development of fatal neurodegenerative diseases. Stabilization of prion protein at the normal cellular form (PrPC) with small molecules is a rational and efficient strategy for treatment of prion related diseases. However, few compounds have been identified as potent prion inhibitors by binding to the normal conformation of prion. In this work, to rational screening of inhibitors capable of stabilizing cellular form of prion protein, multiple approaches combining docking-based virtual screening, steady-state fluorescence quenching, surface plasmon resonance and thioflavin T fluorescence assay were used to discover new compounds interrupting PrPC to PrPSc conversion. Compound 3253-0207 that can bind to PrPC with micromolar affinity and inhibit prion fibrillation was identified from small molecule databases. Molecular dynamics simulation indicated that compound 3253-0207 can bind to the hotspot residues in the binding pocket composed by ß1, ß2 and α2, which are significant structure moieties in conversion from PrPC to PrPSc.
Assuntos
Príons , Avaliação Pré-Clínica de Medicamentos , Simulação de Dinâmica Molecular , Fármacos Neuroprotetores/química , Doenças Priônicas/tratamento farmacológico , Príons/antagonistas & inibidores , Príons/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
Neurodegenerative diseases are associated with accumulation of amyloid-type protein misfolding products. Prion protein (PrP) is known for its ability to aggregate into soluble oligomers that in turn associate into amyloid fibrils. Preventing the formation of these infective and neurotoxic entities represents a viable strategy to control prion diseases. Numerous attempts to find dietary compounds with anti-prion properties have been made; however, the most promising agent found so far was curcumin, which is poorly soluble and merely bioavailable. In the present work, we identify 3,4-dimethoxycinnamic acid (DMCA) which is a bioavailable coffee component as a perspective anti-prion compound. 3,4-Dimethoxycinnamic acid was found to bind potently to prion protein with a Kd of 405 nM. An in vitro study of DMCA effect on PrP oligomerization and fibrillization was undertaken using isothermal titration calorimetry (ITC), dynamic light scattering (DLS) and circular dichroism (CD) methodologies. We demonstrated that DMCA affects PrP oligomer formation reducing the oligomer content by 30-40%, and enhancing SH-SY5Y cell viability treated with prion oligomers. Molecular docking studies allowed to suggest a site where DMCA is able to bind stabilizing PrP tertiary structure. We suggest that DMCA is a perspective dietary compound for prophylaxis of neurodegenerative diseases that needs further research. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Cinamatos/química , Proteínas Priônicas/antagonistas & inibidores , Príons/antagonistas & inibidores , Sítios de Ligação , Simulação de Acoplamento Molecular , Doenças PriônicasRESUMO
To accelerate the logical drug design procedure, we created the program "NAGARA," a plugin for PyMOL, and applied it to the discovery of small compounds called medical chaperones (MCs) that stabilize the cellular form of a prion protein (PrP(C)). In NAGARA, we constructed a single platform to unify the docking simulation (DS), free energy calculation by molecular dynamics (MD) simulation, and interfragment interaction energy (IFIE) calculation by quantum chemistry (QC) calculation. NAGARA also enables large-scale parallel computing via a convenient graphical user interface. Here, we demonstrated its performance and its broad applicability from drug discovery to lead optimization with full compatibility with various experimental methods including Western blotting (WB) analysis, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) measurements. Combining DS and WB, we discovered anti-prion activities for two compounds and tegobuvir (TGV), a non-nucleoside non-structural protein NS5B polymerase inhibitor showing activity against hepatitis C virus genotype 1. Binding profiles predicted by MD and QC are consistent with those obtained by SPR and NMR. Free energy analyses showed that these compounds stabilize the PrP(C) conformation by decreasing the conformational fluctuation of the PrP(C). Because TGV has been already approved as a medicine, its extension to prion diseases is straightforward. Finally, we evaluated the affinities of the fragmented regions of TGV using QC and found a clue for its further optimization. By repeating WB, MD, and QC recursively, we were able to obtain the optimum lead structure.
Assuntos
Desenho de Fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Príons/antagonistas & inibidores , Purinas/química , Piridazinas/química , Software , Sítios de Ligação , Modelos Químicos , Ligação Proteica , Teoria QuânticaRESUMO
There is not a single pharmaceutical that halts or even slows any neurodegenerative disease. Mounting evidence shows that prions cause many neurodegenerative diseases, and arguably, scrapie and Creutzfeldt-Jakob disease prions represent the best therapeutic targets. We report here that the previously identified 2-aminothiazoles IND24 and IND81 doubled the survival times of scrapie-infected, wild-type mice. However, mice infected with Rocky Mountain Laboratory (RML) prions, a scrapie-derived strain, and treated with IND24 eventually exhibited neurological dysfunction and died. We serially passaged their brain homogenates in mice and cultured cells. We found that the prion strain isolated from IND24-treated mice, designated RML[IND24], emerged during a single passage in treated mice. Although RML prions infect both the N2a and CAD5 cell lines, RML[IND24] prions could only infect CAD5 cells. When passaged in CAD5 cells, the prions remained resistant to high concentrations of IND24. However, one passage of RML[IND24] prions in untreated mice restored susceptibility to IND24 in CAD5 cells. Although IND24 treatment extended the lives of mice propagating different prion strains, including RML, another scrapie-derived prion strain ME7, and chronic wasting disease, it was ineffective in slowing propagation of Creutzfeldt-Jakob disease prions in transgenic mice. Our studies demonstrate that prion strains can acquire resistance upon exposure to IND24 that is lost upon passage in mice in the absence of IND24. These data suggest that monotherapy can select for resistance, thus intermittent therapy with mixtures of antiprion compounds may be required to slow or stop neurodegeneration.
Assuntos
Resistência a Medicamentos/genética , Doenças Neurodegenerativas/tratamento farmacológico , Príons/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Encéfalo/patologia , Linhagem Celular , Primers do DNA/genética , Descoberta de Drogas , Feminino , Humanos , Immunoblotting , Medições Luminescentes , Camundongos , Príons/genéticaRESUMO
An altered form of the cellular prion protein, the PrPScor PrPRes, is implicated in the occurrence of the still untreatable transmissible spongiform encephalopathies. We have previously synthesized and characterized aromatic compounds that inhibit protease-resistant prion protein (PrPRes) accumulation in scrapie-infected cells. These compounds belong to different chemical classes, including acylhydrazones, chalcones and oxadiazoles. Some of the active compounds were non-toxic to neuroblastoma cells in culture and seem to possess drugable properties, since they are in agreement with the Lipinski´s rule of 5 and present desirable pharmacokinetic profiles as predicted in silico. Before the evaluation of the in vivo efficacy of the aromatic compounds in scrapie-infected mice, safety assessment in healthy mice is needed. Here we used Swiss mice to evaluate the acute toxicity profile of the six most promising anti-prionic compounds, the 2,4,5-trimethoxychalcones (J1, J8, J20 and J35) and the 1,3,4-oxadiazoles (Y13 and Y17). One single oral administration (300 mg/kg) of J1, J8, J20, J35, Y13 and Y17 or repeated intraperitoneal administration (10 mg/kg, 3 times a week, for 4 weeks) of J1, J8 and J35, did not elicit toxicity in mice. We strongly believe that the investigated trimethoxychalcones and oxadiazoles are interesting compounds to be further analyzed in vivo against prion diseases.
Assuntos
Chalconas/toxicidade , Oxidiazóis/toxicidade , Príons/antagonistas & inibidores , Scrapie/tratamento farmacológico , Animais , Relação Dose-Resposta a Droga , Feminino , Dose Letal Mediana , Masculino , CamundongosRESUMO
In prion diseases, a major issue in therapeutic research is the variability of the effect between strains. Stimulated by the report of an antiprion effect in a scrapie model and by ongoing international clinical trials using doxycycline, we studied the efficacy of cyclines against the propagation of human prions. First, we successfully propagated various Creutzfeldt-Jakob disease (CJD) isolates (sporadic, variant, and iatrogenic CJD) in neuronal cultures expressing the human prion protein. Then, we found that doxycycline was the most effective compound, with important variations between isolates. Isolates from sporadic CJD, the most common form of prion disease, showed the highest sensitivity.
Assuntos
Doxiciclina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Príons/antagonistas & inibidores , Príons/efeitos dos fármacos , Células Cultivadas , Doxiciclina/metabolismo , Doxiciclina/uso terapêutico , HumanosRESUMO
Structural information regarding normal prion protein (PrP(C)) and the scrapie isoform (PrP(Sc)) is of vital importance for elucidating the pathogenesis of prion diseases (PDs). Despite successful determination of the three-dimensional structures of PrP(C), the structural details of PrP(Sc) remain elusive. Nevertheless, accumulated evidence indicates that beta-sheets comprise the basic building blocks of PrP(Sc). Consensus has been reached about the beta-sheet constitution of the N-terminus of PrP, but the constitution of C-terminal beta-sheets is heavily debated. By evaluating the most recent observations regarding the dynamics and structures of PrP, we propose that helix 2 is more likely than helices 1 and 3 to participate in beta-sheet formation. This hypothesis also provides clues to explaining an intriguing phenomenon in prion biology-the lack of PDs in non-mammals.
Assuntos
Príons/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Doenças Priônicas/metabolismo , Príons/antagonistas & inibidores , Príons/genética , Príons/metabolismo , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance. It is well established that low concentrations of guanidinium chloride (GdmCl) inhibit the ATPase activity of Hsp104, leading to so called "prion curing," the loss of prion-related phenotypes. Here, we present mechanistic details about the Hsp100 chaperone inhibition by GdmCl using the Hsp104 homolog ClpB from Thermus thermophilus. Initially, we demonstrate that NBD1 of ClpB, which was previously considered inactive as a separately expressed construct, is a fully active ATPase on its own. Next, we show that only NBD1, but not NBD2, is affected by GdmCl. We present a crystal structure of ClpB NBD1 in complex with GdmCl and ADP, showing that the Gdm(+) ion binds specifically to the active site of NBD1. A conserved essential glutamate residue is involved in this interaction. Additionally, Gdm(+) interacts directly with the nucleotide, thereby increasing the nucleotide binding affinity of NBD1. We propose that both the interference with the essential glutamate and the modulation of nucleotide binding properties in NBD1 is responsible for the GdmCl-specific inhibition of Hsp100 chaperones.