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1.
Int J Mol Sci ; 22(21)2021 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-34769172

RESUMEN

Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrPSc. PrPSc is produced through conformational conversion of the cellular isoform of prion protein, PrPC, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrPSc levels when cultured in advanced Dulbecco's modified eagle medium (DMEM) but not in classic DMEM. PrPC levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrPSc levels in N2aC24L1-3 cells, but not PrPC levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.


Asunto(s)
Encéfalo/metabolismo , Etanolamina/farmacología , Proteínas PrPSc , Enfermedades por Prión , Animales , Línea Celular Tumoral , Ratones , Ratones Endogámicos ICR , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo
2.
Expert Opin Ther Pat ; 31(12): 1097-1115, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34134584

RESUMEN

Introduction: Prion diseases are a class of rare and fatal neurodegenerative diseases for which no cure is currently available. They are characterized by conformational conversion of cellular prion protein (PrPC) into the disease-associated 'scrapie' isoform (PrPSc). Under an etiological point of view, prion diseases can be divided into acquired, genetic, and idiopathic form, the latter of which are the most frequent.Areas covered: Therapeutic approaches targeting prion diseases are based on the use of chemical and nature-based compounds, targeting either PrPC or PrPSc or other putative player in pathogenic mechanism. Other proposed anti-prion treatments include passive and active immunization strategies, peptides, aptamers, and PrPC-directed RNA interference techniques. The treatment efficacy has been mainly assessed in cell lines or animal models of the disease testing their ability to reduce prion accumulation.Expert opinion: The assessed strategies focussing on the identification of an efficient anti-prion therapy faced various issues, which go from permeation of the blood brain barrier to immunological tolerance of the host. Indeed, the use of combinatory approaches, which could boost a synergistic anti-prion effect and lower the potential side effects of single treatments and may represent an extreme powerful and feasible way to tackle prion disease.


Asunto(s)
Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPSc/antagonistas & inhibidores , Enfermedades por Prión/terapia , Animales , Humanos , Patentes como Asunto , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/fisiopatología
3.
Bioorg Med Chem ; 28(21): 115717, 2020 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-33065443

RESUMEN

Prions are misfolded proteins involved in neurodegenerative diseases of high interest in veterinary and public health. In this work, we report the chemical space exploration around the anti-prion compound BB 0300674 in order to gain an understanding of its Structure Activity Relationships (SARs). A series of 43 novel analogues, based on four different chemical clusters, were synthetized and tested against PrPSc and mutant PrP toxicity assays. From this biological screening, two compounds (59 and 65) emerged with a 10-fold improvement in anti-prion activity compared with the initial lead compound, presenting at the same time interesting cell viability.


Asunto(s)
Bencilaminas/química , Proteínas PrPSc/metabolismo , Animales , Bencilaminas/síntesis química , Bencilaminas/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Ratones , Mutagénesis , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/genética , Relación Estructura-Actividad
4.
Sci Rep ; 10(1): 4934, 2020 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-32188933

RESUMEN

Prion diseases comprise a fatal neuropathy caused by the conversion of prion protein from a cellular (PrPC) to a pathological (PrPSc) isoform. Previously, we obtained an RNA aptamer, r(GGAGGAGGAGGA) (R12), that folds into a unique G-quadruplex. The R12 homodimer binds to a PrPC molecule, inhibiting PrPC-to-PrPSc conversion. Here, we developed a new RNA aptamer, r(GGAGGAGGAGGAGGAGGAGGAGGA) (R24), where two R12s are tandemly connected. The 50% inhibitory concentration for the formation of PrPSc (IC50) of R24 in scrapie-infected cell lines was ca. 100 nM, i.e., much lower than that of R12 by two orders. Except for some antibodies, R24 exhibited the lowest recorded IC50 and the highest anti-prion activity. We also developed a related aptamer, r(GGAGGAGGAGGA-A-GGAGGAGGAGGA) (R12-A-R12), IC50 being ca. 500 nM. The structure of a single R12-A-R12 molecule determined by NMR resembled that of the R12 homodimer. The quadruplex structure of either R24 or R12-A-R12 is unimolecular, and therefore the structure could be stably formed when they are administered to a prion-infected cell culture. This may be the reason they can exert high anti-prion activity.


Asunto(s)
Aptámeros de Nucleótidos/química , Proteínas PrPSc/química , Técnica SELEX de Producción de Aptámeros , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/farmacología , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/genética , Proteínas Priónicas , Relación Estructura-Actividad
5.
Prion ; 13(1): 185-196, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-31578923

RESUMEN

Prion diseases are fatal transmissible neurodegenerative disorders that affect animals and humans. Prions are proteinaceous infectious particles consisting of a misfolded isoform of the cellular prion protein PrPC, termed PrPSc. PrPSc accumulates in infected neurons due to partial resistance to proteolytic digestion. Using compounds that interfere with the production of PrPSc or enhance its degradation cure prion infection in vitro, but most drugs failed when used to treat prion-infected rodents. In order to synergize the effect of anti-prion drugs, we combined drugs interfering with the generation of PrPSc with compounds inducing PrPSc degradation. Here, we tested autophagy stimulators (rapamycin or AR12) and cellulose ether compounds (TC-5RW or 60SH-50) either as single or combination treatment of mice infected with RML prions. Single drug treatments significantly extended the survival compared to the untreated group. As anticipated, also all the combination therapy groups showed extended survival compared to the untreated group, but no combination treatment showed superior effects to 60SH-50 or TC-5RW treatment alone. Unexpectedly, we later found that combining autophagy stimulator and cellulose ether treatment in cultured neuronal cells mitigated the pro-autophagic activity of AR12 and rapamycin, which can in part explain the in vivo results. Overall, we show that it is critical to exclude antagonizing drug effects when attempting combination therapy. In addition, we identified AR-12 as a pro-autophagic drug that significantly extends survival of prion-infected mice, has no adverse side effects on the animals used in this study, and can be useful in future studies.


Asunto(s)
Autofagia/efectos de los fármacos , Celulosa/uso terapéutico , Proteínas PrPSc/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Sirolimus/uso terapéutico , Animales , Celulosa/análogos & derivados , Sinergismo Farmacológico , Éteres/química , Éteres/uso terapéutico , Femenino , Ratones , Proteínas PrPSc/antagonistas & inhibidores , Enfermedades por Prión/metabolismo , Proteolisis/efectos de los fármacos
6.
Methods Mol Biol ; 1658: 83-94, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28861784

RESUMEN

During the course of prion infection, the normally soluble and protease-sensitive mammalian prion protein (PrPC) is refolded into an insoluble, partially protease-resistant, and infectious form called PrPSc. The conformational conversion of PrPC to PrPSc is a critical event during prion infection and is essential for the production of prion infectivity. This chapter briefly summarizes the ways in which cell biological approaches have enhanced our understanding of how PrP contributes to different aspects of prion pathogenesis.


Asunto(s)
Amiloide/química , Bioensayo , Ensayo de Immunospot Ligado a Enzimas/métodos , Proteínas PrPSc/genética , Agregado de Proteínas , Animales , Arvicolinae , Técnicas de Cultivo de Célula , Expresión Génica , Humanos , Ratones , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ovinos
7.
Methods Mol Biol ; 1658: 295-304, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28861797

RESUMEN

It is currently difficult to predict the number of asymptomatic prion carriers who will potentially go on to develop a prion disease or who will unknowingly transmit the prion agent to another individual. As prion therapeutic clinical trials have lacked success, there is a continuous need for novel therapeutics that have the potential to prevent, as for inherited prion disorders; slow, as for all prion disorders; and ultimately stop disease progression. Prion-infected cell models provide an ideal tool to search for new treatment avenues. This chapter describes the use of prion cell culture systems in the identification of prion therapeutics. It also deals with the methods required to validate the potential of an antiprion agent through cell viability and impact on cell growth rate.


Asunto(s)
Técnicas de Cultivo de Célula , Drogas en Investigación/farmacología , Ensayos Analíticos de Alto Rendimiento , Neuronas/efectos de los fármacos , Proteínas PrPSc/antagonistas & inhibidores , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Endopeptidasa K/química , Expresión Génica , Humanos , Neuronas/metabolismo , Neuronas/patología , Proteínas PrPSc/química , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Pliegue de Proteína
8.
ChemMedChem ; 12(16): 1286-1292, 2017 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-28722340

RESUMEN

Into the fold: Prion diseases are neurodegenerative disorders characterized by the accumulation in the brain of a self-replicating, misfolded isoform (PrPSc ) of the cellular prion protein (PrPC ). No therapies are available for these pathologies. We capitalized on previously described cell-based assays to screen a library of small molecules, and identified 55, a compound capable of counteracting both prion replication and toxicity. Compound 55 may represent the starting point for the development of a completely new class of therapeutics for prion diseases.


Asunto(s)
Proteínas Priónicas/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Mutagénesis , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Proteínas Priónicas/antagonistas & inhibidores , Proteínas Priónicas/genética , Unión Proteica , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/toxicidad
9.
PLoS One ; 12(1): e0170266, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28095474

RESUMEN

Prion propagation is mediated by the structural alteration of normal prion protein (PrPC) to generate pathogenic prion protein (PrPSc). To date, compounds for the inhibition of prion propagation have mainly been screened using PrPSc-infected cells. Real time-quaking-induced conversion (RT-QuIC) is one alternative screening method. In this study, we assessed the propagation inhibition effects of known anti-prion compounds using RT-QuIC and compared the results with those from a PrPSc-infected cell assay. Compounds were applied to RT-QuIC reactions at 0 h or 22 h after prion propagation to determine whether they inhibited propagation or reduced amplified aggregates. RT-QuIC reactions in presence of acridine, dextran sulfate sodium (DSS), and tannic acid inhibited seeded aggregation with sporadic Creutzfeldt-Jakob disease at 0 h. After treatment at 22 h, amplified fluorescence was decreased in wells treated with either acridine or tannic acid. Compound activities were verified by western blot of RT-QuIC products and in a dye-independent conversion assay, the Multimer Detection System. Protease K-resistant PrPSc fragments (PrPres) were reduced by DSS and tannic acid in the PrPSc-infected cell assay. Importantly, these inhibitory effects were similar despite different treatment times (0 h versus 3 days). Consequentially, RT-QuIC enabled the more specific classification of compounds according to action (i.e., inhibition of prion propagation versus reduction of amplified aggregates). RT-QuIC addresses the limitations of cell-based screening methods and can be used to further aid our understanding of the mechanisms of action of anti-prion compounds.


Asunto(s)
Acridinas/farmacología , Síndrome de Creutzfeldt-Jakob/metabolismo , Demencia/metabolismo , Dextranos/farmacología , Neuroblastoma/metabolismo , Proteínas PrPSc/antagonistas & inhibidores , Taninos/farmacología , Antiinfecciosos/farmacología , Anticoagulantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/tratamiento farmacológico , Síndrome de Creutzfeldt-Jakob/patología , Demencia/tratamiento farmacológico , Demencia/patología , Ensayos Analíticos de Alto Rendimiento , Humanos , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Proteínas PrPSc/metabolismo , Células Tumorales Cultivadas
10.
Expert Opin Drug Discov ; 12(3): 241-248, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28118747

RESUMEN

INTRODUCTION: To date, various therapeutic strategies identified numerous anti-prion compounds and antibodies that stabilize PrPC, block the conversion of PrPC-PrPSc and increased effect on PrPSc clearance. However, no suitable drug has been identified clinically so far due to the poor oral absorption, low blood-brain-barrier [BBB] penetration, and high toxicity. Although some of the drugs were proven to be effective in prion-infected cell culture and whole animal models, none of them increased the rate of survival compared to placebo. Areas covered: In this review, the authors highlight the importance of in silico approaches like molecular docking, virtual screening, pharmacophore analysis, molecular dynamics, QSAR, CoMFA and CoMSIA applied to detect molecular mechanisms of prion inhibition and conversion from PrPC-PrPSc. Expert opinion: Several in silico approaches combined with experimental studies have provided many structural and functional clues on the stability and physiological activity of prion mutants. Further, various studies of in silico and in vivo approaches were also shown to identify several new small organic anti-scrapie compounds to decrease the accumulation of PrPres in cell culture, inhibit the aggregation of a PrPC peptide, and possess pharmacokinetic characteristics that confirm the drug-likeness of these compounds.


Asunto(s)
Simulación por Computador , Diseño de Fármacos , Enfermedades por Prión/tratamiento farmacológico , Animales , Barrera Hematoencefálica/metabolismo , Modelos Animales de Enfermedad , Humanos , Simulación del Acoplamiento Molecular , Proteínas PrPC/efectos de los fármacos , Proteínas PrPC/metabolismo , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/efectos de los fármacos , Proteínas PrPSc/metabolismo , Tasa de Supervivencia , Distribución Tisular
11.
Mol Cell Biochem ; 428(1-2): 57-66, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28063003

RESUMEN

Biological effect of poly-L-arginine (PLR), the linear homopolymer comprised of L-arginine, was investigated to determine the activity of suppressing prions. PLR decreased the level of scrapie prion protein (PrPSc) in cultured cells permanently infected with prions in a concentration-dependent manner. The PrPSc inhibition efficacy of PLR was greater than that of another prion-suppressant poly-L-lysine (PLK) in a molecular mass-dependent fashion. The effective concentration of PLR to inhibit prions was achieved safely below the cytotoxic concentrations, and overall cytotoxicity of PLR was similar to that of PLK. PLR did not alter the cellular prion protein (PrPC) level and was unable to change the states of preformed recombinant PrP aggregates and PrPSc from prion-infected cells. These data eliminate the possibility that the action mechanism of PLR is through removal of PrPC and pre-existing PrPSc. However, PLR formed complexes with plasminogen that stimulates prion propagation via conversion of PrPC to the misfolded isoform, PrPSc. The plasminogen-PLR complex demonstrated the greater positive surface charge values than the similar complex with PLK, raising the possibility that PLR interferes with the role of cofactor for PrPSc generation better than PLK.


Asunto(s)
Péptidos/farmacología , Plasminógeno/metabolismo , Proteínas PrPSc/antagonistas & inhibidores , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Ratones , Polilisina/farmacología , Proteínas PrPC/metabolismo , Isoformas de Proteínas/metabolismo
12.
J Biol Chem ; 291(50): 26164-26176, 2016 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-27803163

RESUMEN

Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (Δ105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.


Asunto(s)
Piperidinas/química , Piperidinas/farmacología , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/metabolismo , Resonancia por Plasmón de Superficie/métodos , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas PrPSc/genética
13.
Antimicrob Agents Chemother ; 60(9): 5467-82, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27381401

RESUMEN

The transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrP(C)) into the accumulating, disease-associated isoform (PrP(Sc)). Despite extensive research into the inhibition of prion accumulation, no effective treatment exists. Previously, we demonstrated the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, against PrP(Sc) accumulation in sheep microglial cells. In an effort to determine the effect of structural substitutions on the antiprion activity of DB772, we employed an in vitro strategy to survey a library of structurally related, monothiophene- and furan-based compounds for improved inhibitory activity. Eighty-nine compounds were screened at 1 µM for effects on cell viability and prion accumulation in a persistently infected ovine microglia culture system. Eleven compounds with activity equivalent to or higher than that of DB772 were identified as preliminary hit compounds. For the preliminary hits, cytotoxicities and antiprion activities were compared to calculate the tissue culture selectivity index. A structure-activity relationship (SAR) analysis was performed to determine molecular components contributing to antiprion activity. To investigate potential mechanisms of inhibition, effects on PrP(C) and PrP(Sc) were examined. While inhibition of total PrP(C) was not observed, the results suggest that a potential target for inhibition at biologically relevant concentrations is through PrP(C) misfolding to PrP(Sc) Further, SAR analysis suggests that two structural elements were associated with micromolar antiprion activity. Taken together, the described data provide a foundation for deeper investigation into untested DB compounds and in the design of effective therapeutics.


Asunto(s)
Bencimidazoles/farmacología , Furanos/farmacología , Microglía/efectos de los fármacos , Proteínas PrPSc/antagonistas & inhibidores , Proteínas Priónicas/antagonistas & inhibidores , Animales , Ovinos , Relación Estructura-Actividad
14.
Sci Rep ; 5: 14944, 2015 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-26449325

RESUMEN

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrP(C)) to the pathogenic form, PrP(Sc). Compounds that inhibit this process by blocking conversion to the PrP(Sc) could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrP(C) interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrP(Sc) levels and one of these compounds also showed a high binding affinity for PrP(C). These results provide a promising starting point for the development of anti-prion compounds.


Asunto(s)
Simulación por Computador , Descubrimiento de Drogas/métodos , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPSc/antagonistas & inhibidores , Xenobióticos/farmacología , Animales , Línea Celular Tumoral , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Proteínas PrPC/química , Proteínas PrPSc/química , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Xenobióticos/química , Xenobióticos/clasificación
15.
Eur J Med Chem ; 91: 118-31, 2015 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-25042003

RESUMEN

To understand the pharmacophore properties of 2-aminothiazoles and design novel inhibitors against the prion protein, a highly predictive 3D quantitative structure-activity relationship (QSAR) has been developed by performing comparative molecular field analysis (CoMFA) and comparative similarity analysis (CoMSIA). Both CoMFA and CoMSIA maps reveal the presence of the oxymethyl groups in meta and para positions on the phenyl ring of compound 17 (N-[4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-yl]quinolin-2-amine), is necessary for activity while electro-negative nitrogen of quinoline is highly favorable to enhance activity. The blind docking results for these compounds show that the compound with quinoline binds with higher affinity than isoquinoline and naphthalene groups. Out of 150 novel compounds retrieved using finger print analysis by pharmacophoric model predicted based on five test sets of compounds, five compounds with diverse scaffolds were selected for biological evaluation as possible PrP inhibitors. Molecular docking combined with fluorescence quenching studies show that these compounds bind to pocket-D of SHaPrP near Trp145. The new antiprion compounds 3 and 6, which bind with the interaction energies of -12.1 and -13.2 kcal/mol, respectively, show fluorescence quenching with binding constant (Kd) values of 15.5 and 44.14 µM, respectively. Further fluorescence binding assays with compound 5, which is similar to 2-aminothiazole as a positive control, also show that the molecule binds to the pocket-D with the binding constant (Kd) value of 84.7 µM. Finally, both molecular docking and a fluorescence binding assay of noscapine as a negative control reveals the same binding site on the surface of pocket-A near a rigid loop between ß2 and α2 interacting with Arg164. This high level of correlation between molecular docking and fluorescence quenching studies confirm that these five compounds are likely to act as inhibitors for prion propagation while noscapine might act as a prion accelerator from PrP(C) to PrP(Sc).


Asunto(s)
Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores/química , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPSc/antagonistas & inhibidores , Quinolinas/química , Tiazoles/química , Animales , Sitios de Unión , Diseño de Fármacos , Humanos , Cinética , Ligandos , Simulación de Dinámica Molecular , Noscapina/química , Proteínas PrPC/química , Proteínas PrPSc/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad Cuantitativa , Espectrometría de Fluorescencia , Electricidad Estática , Homología Estructural de Proteína , Termodinámica
16.
PLoS One ; 9(9): e106516, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25181483

RESUMEN

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP(Sc)) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP(Sc) formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP(C)) and PrP(Sc) in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP(Sc) levels without altering intracellular distribution of PrP(Sc). PPS did not change the distribution and levels of PrP(C), whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP(C) to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP(Sc) formation. In contrast, CPZ and U18666A initiated the redistribution of PrP(Sc) from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP(C). The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP(Sc) redistribution by CPZ or U18666A partly antagonized PrP(Sc) degradation, suggesting that the transfer of PrP(Sc) to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP(Sc) degradation. This study revealed that precise analysis of the intracellular dynamics of PrP(C) and PrP(Sc) provides important information for understanding the mechanism of anti-prion agents.


Asunto(s)
Enfermedades por Prión/tratamiento farmacológico , Priones/antagonistas & inhibidores , Androstenos/farmacología , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Línea Celular Tumoral , Clorpromazina/farmacología , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Poliéster Pentosan Sulfúrico/farmacología , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPC/inmunología , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/inmunología , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades por Prión/terapia , Priones/inmunología , Priones/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo
17.
J Virol ; 88(8): 4083-99, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24453367

RESUMEN

UNLABELLED: A new type of antiprion compound, Gly-9, was found to inhibit abnormal prion protein formation in prion-infected neuroblastoma cells, in a prion strain-independent manner, when the cells were treated for more than 1 day. It reduced the intracellular prion protein level and significantly modified mRNA expression levels of genes of two types: interferon-stimulated genes were downregulated after more than 2 days of treatment, and the phosphodiesterase 4D-interacting protein gene, a gene involved in microtubule growth, was upregulated after more than 1 day of treatment. A supplement of interferon given to the cells partly restored the abnormal prion protein level but did not alter the normal prion protein level. This interferon action was independent of the Janus activated kinase-signal transducer and activator of transcription signaling pathway. Therefore, the changes in interferon-stimulated genes might be a secondary effect of Gly-9 treatment. However, gene knockdown of phosphodiesterase 4D-interacting protein restored or increased both the abnormal prion protein level and the normal prion protein level, without transcriptional alteration of the prion protein gene. It also altered the localization of abnormal prion protein accumulation in the cells, indicating that phosphodiesterase 4D-interacting protein might affect prion protein levels by altering the trafficking of prion protein-containing structures. Interferon and phosphodiesterase 4D-interacting protein had no direct mutual link, demonstrating that they regulate abnormal prion protein levels independently. Although the in vivo efficacy of Gly-9 was limited, the findings for Gly-9 provide insights into the regulation of abnormal prion protein in cells and suggest new targets for antiprion compounds. IMPORTANCE: This report describes our study of the efficacy and potential mechanism underlying the antiprion action of a new antiprion compound with a glycoside structure in prion-infected cells, as well as the efficacy of the compound in prion-infected animals. The study revealed involvements of two factors in the compound's mechanism of action: interferon and a microtubule nucleation activator, phosphodiesterase 4D-interacting protein. In particular, phosphodiesterase 4D-interacting protein was suggested to be important in regulating the trafficking or fusion of prion protein-containing vesicles or structures in cells. The findings of the study are expected to be useful not only for the elucidation of cellular regulatory mechanisms of prion protein but also for the implication of new targets for therapeutic development.


Asunto(s)
Proteínas Portadoras/metabolismo , Glicósidos/farmacología , Interferones/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/genética , Línea Celular , Proteínas del Citoesqueleto , Regulación hacia Abajo/efectos de los fármacos , Ratones , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/genética , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/genética , Transducción de Señal/efectos de los fármacos
18.
Curr Top Med Chem ; 13(19): 2484-90, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24059335

RESUMEN

An emerging common feature of the age-associated neurodegenerative disorders like Alzheimer's disease (AD) and Creutzfeldt-Jakob disease (CJD) is the ability of many disease-associated protein aggregates to induce conversion of a normal counterpart conformer leading to an acceleration of disease progression. Curative pharmacotherapy has not been achieved so far despite successes in elucidating pathomechanisms. Here, we review the pharmaceutical strategy of generating hybrid compounds, i.e. compounds consisting of several independently acting moieties with synergistic effects, on key molecular players in AD and CJD. For prion diseases, we review hybrid compounds consisting of two different heterocyclic compounds, their synergistic effects on prion replication in a cell culture model and their ability to prolong survival of experimentally prion-infected mice in vivo. While a combination therapy of several antiprion compounds including quinacrine, clomipramine, simvastatin and tocopherol prolonged survival time to 10-25%, administration of hybrid compound quinpramine alone, a chimera of acridine and iminodibenzyl scaffolds, led to 10% survival time extension. For AD, we review a hybrid compound consisting of an Aß recognizing D-peptide fused to a small molecule ß-sheet breaker, an aminopyrazole. This molecule was able to diminish Aß oligomers in cell culture and significantly decrease synaptotoxicity as measured by miniature excitatory postsynaptic responses in vitro. Hybrid compounds can dramatically increase potency of their single moieties and lead to novel functions when they act in a simultaneous or sequential manner thereby revealing synergistic properties. Their systematic generation combining different classes of compounds from peptides to small molecules has the potential to significantly accelerate drug discovery.


Asunto(s)
Compuestos Heterocíclicos/farmacología , Péptidos/farmacología , Proteínas PrPSc/antagonistas & inhibidores , Enfermedades por Prión/tratamiento farmacológico , Pliegue de Proteína/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Sinergismo Farmacológico , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/uso terapéutico , Humanos , Modelos Moleculares , Estructura Molecular , Péptidos/química , Péptidos/uso terapéutico , Proteínas PrPSc/metabolismo , Enfermedades por Prión/diagnóstico , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico
19.
PLoS One ; 8(1): e55282, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23383136

RESUMEN

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future.


Asunto(s)
Dendrímeros/farmacología , Polipropilenos/farmacología , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPC/metabolismo , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/metabolismo , Animales , Benzamidas/farmacología , Bioensayo/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Mesilato de Imatinib , Immunoblotting , Ratones , Microscopía Confocal , Piperazinas/farmacología , Proteína PrP 27-30/aislamiento & purificación , Pirimidinas/farmacología , Relación Estructura-Actividad , Suramina/farmacología
20.
Biochem Biophys Res Commun ; 432(1): 86-91, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23376069

RESUMEN

The bovine spongiform encephalopathy (BSE) agent is resistant to conventional microbial inactivation procedures and thus threatens the safety of cattle products and by-products. To obtain information necessary to assess BSE inactivation, we performed quantitative analysis of wet-heat inactivation of infectivity in BSE-infected cattle spinal cords. Using a highly sensitive bioassay, we found that infectivity in BSE cattle macerates fell with increase in temperatures from 133°C to 150°C and was not detected in the samples subjected to temperatures above 155°C. In dry cattle tissues, infectivity was detected even at 170°C. Thus, BSE infectivity reduces with increase in wet-heat temperatures but is less affected when tissues are dehydrated prior to the wet-heat treatment. The results of the quantitative protein misfolding cyclic amplification assay also demonstrated that the level of the protease-resistant prion protein fell below the bioassay detection limit by wet-heat at 155°C and higher and could help assess BSE inactivation. Our results show that BSE infectivity is strongly resistant to wet-heat inactivation and that it is necessary to pay attention to BSE decontamination in recycled cattle by-products.


Asunto(s)
Encefalopatía Espongiforme Bovina/transmisión , Calor , Proteínas PrPSc/antagonistas & inhibidores , Proteínas PrPSc/química , Médula Espinal , Animales , Bioensayo , Bovinos , Ratones , Ratones Transgénicos , Pliegue de Proteína
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