RESUMO
Amphotericin B (AmB) is one of the rare drugs that affect the course of experimental prion diseases and modify the kinetics of abnormal prion protein accumulation in the central nervous system. Therefore, AmB could be used as a pharmacological tool to contribute to our understanding of the pathogenic mechanisms involved in these neurodegenerative disorders.
Assuntos
Anfotericina B/farmacologia , Antibacterianos/farmacologia , Doenças Priônicas/etiologia , Doenças Priônicas/metabolismo , Príons/efeitos dos fármacos , Príons/patogenicidade , Anfotericina B/efeitos adversos , Anfotericina B/química , Animais , Antibacterianos/efeitos adversos , Antibacterianos/química , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Bovinos , Cricetinae , Resistência Microbiana a Medicamentos , Humanos , Camundongos , Estrutura Molecular , Polienos/efeitos adversos , Polienos/química , Polienos/farmacologia , Proteínas PrPSc/efeitos dos fármacos , Príons/metabolismo , OvinosRESUMO
Five compounds related to Congo Red were found to inhibit generation of protease-resistant prion protein in a cell-free system. In this assay Trypan Blue, Evans Blue, Sirius Red F3B, Primuline and Thioflavin-S were all more inhibitory than Congo Red itself. In scrapie-infected mouse neuroblastoma cells one compound, Sirius Red F3B, was capable of blocking the formation of protease-resistant prion protein to a similar extent as Congo Red; however, the other four compounds were less effective. Some of these compounds should be considered for testing in TSE disease models in live animals.
Assuntos
Vermelho Congo/análogos & derivados , Vermelho Congo/farmacologia , Proteínas PrPC/efeitos dos fármacos , Proteínas PrPSc/química , Proteínas PrPSc/efeitos dos fármacos , Azul Tripano/farmacologia , Animais , Compostos Azo/farmacologia , Western Blotting , Sistema Livre de Células , Cricetinae , Camundongos , Neuroblastoma , Neurônios/química , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Scrapie/metabolismo , Tiazóis/farmacologia , Células Tumorais CultivadasAssuntos
Endopeptidases/metabolismo , Príons/análise , Animais , Encéfalo/metabolismo , Sistema Livre de Células , Células Cultivadas , Guanidina/química , Humanos , Immunoblotting/métodos , Metionina/metabolismo , Testes de Precipitina , Doenças Priônicas/metabolismo , Príons/metabolismo , Radioisótopos de EnxofreRESUMO
To date very few drugs have favorably influenced the course of transmissible spongiform encephalopathies. In previous studies, the polyene antibiotics amphotericin B (AmB) and MS-8209 prolonged the incubation time in Syrian hamsters of the 263K strain of scrapie, but AmB had no effect against other scrapie strains in Syrian hamsters. In the present experiments using transgenic mice expressing Syrian hamster PrP in neurons only, MS-8209 extended the life spans of animals infected with the 263K strain but not the DY strain. AmB was effective against both 263K and DY and prevented death in 18% of DY-infected animals. The AmB effect against strain 263K was more prominent in mice whose endogenous PrP gene had been inactivated by homologous recombination. It was unclear whether this difference was due to a change in the duration of the disease or to possible interactive effects between the mouse PrP gene and the drugs themselves. The effectiveness of treatment after intracerebral scrapie infection in transgenic mice expressing PrP only in neurons suggested that neurons are important sites of action for these drugs.
Assuntos
Anfotericina B/análogos & derivados , Anfotericina B/uso terapêutico , Antibacterianos/uso terapêutico , Antivirais/uso terapêutico , Neurônios/metabolismo , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/genética , Príons/genética , Animais , Cricetinae , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Príons/biossínteseRESUMO
To test the efficacy of a new amphotericin B derivative, MS-8209, in delaying scrapie, hamsters were infected intracerebrally with the 263K scrapie agent and treated with MS-8209 either early in the course of the disease or continuously. The results show that (i) all treatments lengthened the incubation period of hamster scrapie, (ii) continuous treatment with MS-8209 doubled the length of the incubation period compared with that observed in infected, untreated animals, and (iii) all treatments delayed the accumulation of a proteinase-resistant prion protein and glial fibrillary acidic protein in the brain. These findings suggest that MS-8209 is a powerful tool for investigating the pathogenesis of transmissible subacute spongiform encephalopathies.
Assuntos
Anfotericina B/análogos & derivados , Scrapie/tratamento farmacológico , Anfotericina B/uso terapêutico , Animais , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Cricetinae , Feminino , Proteína Glial Fibrilar Ácida/biossíntese , Mesocricetus , Proteínas PrPSc/biossíntese , Scrapie/metabolismoRESUMO
Congo red (CR) has been shown to inhibit the accumulation in scrapie-infected cells of prion protein (PrP) in the abnormal protease-resistant form (PrP-res). However, it was not clear if this effect was due to a direct interaction of CR with either PrP-res or its protease-sensitive precursor (PrP-sen) or to a less direct effect on living cells. Here we show that CR inhibits PrP-res formation in a simple cell-free reaction composed predominantly of purified PrP-res and PrP-sen. Structurally modified CR analogues were also compared in both the cell-free conversion reaction and scrapie-infected neuroblastoma cells. Methylation of the central phenyl groups at the 2,2' positions diminished the inhibitory potency by > or = 10-fold. In contrast, there was little effect of 3,3' methylation of the phenyls, deletion of one phenyl, or addition of an amido group between the phenyls. The relative activities of these compounds were well correlated in both cellular and acellular systems. Molecular modeling indicated that CR and 3,3'-methyl-CR have little rotational restriction about the biphenyl bond and can readily adopt a planar conformation, as can phenyl-CR and amido-CR. In contrast, 2,2'-methyl-CR is restricted to a nonplanar conformation of the biphenyl group. Thus, planarity and/or torsional mobility of the central phenyl rings of CR and its analogues is probably important for inhibition of PrP-res formation. On the other hand, variations in the intersulfonate distance in these molecules had little effect on PrP-res inhibition. These results indicated a high degree of structural specificity in the inhibition of PrP-res formation by CR and related compounds.
Assuntos
Corantes/química , Corantes/farmacologia , Vermelho Congo/química , Vermelho Congo/farmacologia , Endopeptidases/farmacologia , Príons/antagonistas & inibidores , Animais , Compostos de Bifenilo/metabolismo , Sistema Livre de Células/efeitos dos fármacos , Vermelho Congo/metabolismo , Cricetinae , Resistência a Medicamentos , Metilação , Príons/metabolismo , Precursores de Proteínas/metabolismo , Células Tumorais CultivadasRESUMO
Transmissible subacute spongiform encephalopathies (TSSE) are neurodegenerative diseases characterized by the presence of a modified, partially proteinase-resistant host protein, PrPSc, which accumulates in the brains of infected individuals. Recently it has been reported that amphotericin B (AmB) treatment of hamsters infected with scrapie strain 263K prolongs the incubation period of the disease, and dissociates in vivo replication of the scrapie agent from PrPSc accumulation. We report here on data obtained after treatment with AmB and one of its derivatives, MS-8209, in experimental scrapie of mouse and hamster. Treatment was carried out by the intraperitoneal route 6 days per week, at three different dosages initiated at the time of infection. Two regimens were used: during the early time of infection or throughout the experimental infection. Results indicate that MS-8209 was as efficient as AmB in prolonging the incubation time and decreasing PrPSc accumulation in the hamster scrapie model. A dose-dependent response was observed in mice treated early after experimental infection. At a dose of 2.5 mg/kg, MS-8209 significantly prolonged the incubation period (by 11.9%). In long-term treatment of mice, MS-8209 and AmB markedly reduced PrPSc levels in the preclinical stage of the disease. These data demonstrate that the effect of AmB is not restricted to one model (hamster-263K). This regimen leads to an inversion of the PrPSc to proteinase-sensitive protein (PrPSens) ratio, suggesting PrPSens (presumably cellular PrPC) accumulation occurs before its conversion into PrPSc. As it has been shown that AmB does not modify the infectivity titre, we conclude that the drugs could act by inhibiting either the interaction of the scrapie agent with PrPSens during the early times of infection or the conversion of PrPSens into PrPSc.
Assuntos
Anfotericina B/análogos & derivados , Anfotericina B/uso terapêutico , Antivirais/uso terapêutico , Doenças Priônicas/tratamento farmacológico , Príons/biossíntese , Príons/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Cricetinae , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPSc , Príons/efeitos dos fármacos , Príons/isolamento & purificação , Fatores de TempoRESUMO
Mice were infected intracerebrally with the bovine spongiform encephalopathy (BSE) or the scrapie agent and treated during 8 weeks postinfection to test the protective effect of a new amphotericin B (AmB) derivative, MS-8209, in experimental transmissible spongiform encephalopathies. The results show that (i) the treatment prolonged the incubation period of both BSE-infected and scrapie-infected mice, (ii) MS-8209 and AmB were much more efficient in delaying the onset of scrapie than that of BSE, and (iii) a delay in Prp-res (proteinase K-resistant prion protein) and GFAP (glial fibrillary acidic protein) accumulation was observed in the brains of scrapie-infected mice, but was not significant in BSE-infected mice. The analysis of the molecular and clinical results strongly suggests a common mechanism of action of this category of drugs on the different transmissible spongiform encephalopathy strains. This could be due to an interaction with the PrP transconformation process leading to the formation of PrP-res.
Assuntos
Anfotericina B/análogos & derivados , Antivirais/uso terapêutico , Encefalopatia Espongiforme Bovina/tratamento farmacológico , Scrapie/tratamento farmacológico , Anfotericina B/uso terapêutico , Animais , Antibacterianos , Bovinos , Relação Dose-Resposta a Droga , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Polienos , Proteínas PrPSc/metabolismoRESUMO
The development of transmissible spongiform encephalopathies in experimental models depends on two major factors: the intracerebral accumulation of an abnormal, protease-resistant isoform of PrP (PrPres), which is a host protein mainly expressed in neurons; and the existence of different strains of agent. In order to make a distinction between pathogenic mechanisms depending upon the accumulation of host-derived PrPres and the strain-specific effects, we quantified and compared the sequence of molecular [PrPres and glial fibrillary acidic protein (GFAP) accumulation] and pathological events in the brains of syngeneic mice throughout the course of infection with two different strains of agent. The bovine spongiform encephalopathy (BSE) agent exhibits properties different from any known scrapie source and has been studied in comparison with a classical scrapie strain. Convergent kinetic data in both models confirmed the cause-effect relationship between PrPres and pathological changes and showed that PrPres accumulation is directly responsible for astrocyte activation in vivo. Moreover, we observed a threshold level of PrPres for this effect on astroglial cells. However, despite similar infectivity titres, the BSE model produced less PrPres than scrapie, and the relative importance of gliosis was higher. The comparison of the molecular and pathological features after intracerebral or intraperitoneal inoculation also revealed differences between the models. Therefore, the mechanisms leading to the targeting and the fine regulation of the molecular events seem to be independent of the host PrP and to depend upon the agent. The possible involvement of a regulatory molecule accounting for these specificities has to be considered.
Assuntos
Encefalopatia Espongiforme Bovina/etiologia , Proteínas PrPSc/patogenicidade , Scrapie/etiologia , Animais , Astrócitos/metabolismo , Bovinos , Modelos Animais de Doenças , Encefalopatia Espongiforme Bovina/patologia , Encefalopatia Espongiforme Bovina/fisiopatologia , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Scrapie/patologia , Scrapie/fisiopatologia , Ovinos , Fatores de TempoRESUMO
Amphotericin B (AmB) is one of the few drugs able to prolong survival times in experimental scrapie and delays the accumulation of PrPres, a specific marker of this disease in the brain in vivo. Previous reports showed that the AmB effect is observed only if the drug is administered around the time of infection. In the present study, intracerebrally infected mice were treated with AmB or one of its derivatives, MS-8209, between 80 and 140 days postinoculation. We observed an increased incubation time and a delay in PrPres accumulation and glial fibrillary acidic protein gene expression. Treatment starting at 80 days postinoculation was as efficient as long-term treatment starting the day of inoculation. Our results indicate that polyene antibiotics may interfere, throughout the course of the experimental disease, with the propagation of the scrapie agent.
Assuntos
Anfotericina B/análogos & derivados , Polienos/farmacologia , Scrapie/tratamento farmacológico , Sequência de Aminoácidos , Anfotericina B/farmacologia , Animais , Antibacterianos/farmacologia , Esquema de Medicação , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas PrPSc , Coelhos , Scrapie/fisiopatologia , Fatores de TempoRESUMO
Using the severe combined immunodeficiency (SCID) mouse model, we investigated the requirement of the immune system for the development of scrapie after peripheral inoculation. A total of 33% of SCID mice, all but one immunologically reconstituted SCID mice (93%), and all CB17 control mice developed the disease. PrPres was detectable in the brains of all diseased animals and in the spleens of reconstituted SCID and CB17 control mice but not of the diseased non-immunologically reconstituted SCID mice. The immune system appears to be a primary target in the pathogenesis of scrapie, but direct spread to the central nervous system from the peritoneum via visceral nerve fibers can probably also occur.
Assuntos
Proteínas PrPSc/imunologia , Scrapie/imunologia , Animais , Encéfalo/imunologia , Sistema Imunitário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Proteínas PrPSc/biossíntese , Baço/imunologiaRESUMO
Amphotericin B (AmB) has been shown to delay hamster scrapie. Infectivity studies have been performed previously using AmB in order to understand the relationship between the accumulation of an abnormal isoform (PrPres) of the prion protein and 263K scrapie agent replication in the brain. The first study reported that AmB had no effect upon agent replication, although it delayed the development of both clinical signs and PrPres accumulation. However, subsequent experiments using the same model showed a significant effect both on agent replication and PrPres accumulation early in infection. This fundamental discrepancy was assumed to be linked to differences in experimental protocols. In order to unravel the issue, a new experiment has been performed encompassing different protocols and using an AmB derivative, MS-8209, that can be used at higher doses because of its lower toxicity. The findings of this study exclude the suspected differences in the protocols as the reason for previous conflicting results, and suggest strongly that these discrepancies were due to a low dose of AmB causing a 'threshold effect'. Overall, this study indicates that, in this model, PrPres cannot be dissociated from infectivity by polyene antibiotics.
Assuntos
Anfotericina B/análogos & derivados , Antivirais/farmacologia , Proteínas PrPSc/efeitos dos fármacos , Anfotericina B/farmacologia , Animais , Cricetinae , Relação Dose-Resposta a Droga , Feminino , Mesocricetus , Proteínas PrPSc/metabolismoRESUMO
The polyene antibiotic MS-8209 is currently one of the most effective drugs in the treatment of experimental scrapie. However, its mechanism of action and its site of intervention in the pathogenetical process of scrapie infection are largely unknown. It has been shown previously that the infection of immunodeficient SCID mice by the peripheral route provides a reliable model for direct scrapie neuroinvasion, bypassing the lymphoreticular system. Indeed, a proportion of SCID mice develop scrapie after a similar time to immunocompetent mice, despite their severe immune impairment. This model is now used to clarify the targets of MS-8209. In SCID mice, MS-8209 treatment protected against infection but did not prolong survival time. In SCID mice immunologically reconstituted prior to inoculation, the drug delayed the disease without an effect on the attack rate. These findings strongly suggest that MS-8209 acts by hampering the first step of the neuroinvasion process, i.e. the uptake of the infectious agent by peripheral nerve endings. The mechanism leading to the inhibition of agent propagation to nervous cells is discussed with regard to the properties of polyene antibiotics.