No abstract
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 ≥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.
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.
Transmissible subacute spongiform encephalopathies (TSSE) are neurodegenerative diseases characterized by the presence of a modified, partially proteinase-resistant host protein, PrP s~, 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 PrP s~ 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 PrP se 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 longterm treatment of mice, MS-8209 and AmB markedly reduced PrP se 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 PrP s~ to proteinasesensitive protein (PrP sens) ratio, suggesting PrP senS (presumably cellular PrP c) accumulation occurs before its conversion into PrP sc. 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 PrP sen~ during the early times of infection or the conversion of PrP sen~ into prp s~.
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 proteinaseresistant 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.Experimental scrapie in hamsters is a simple, reproducible model of transmissible spongiform encephalopathies (TSE) which have been described for many mammalian species. TSE include Creutzfeldt-Jakob disease in humans and scrapie in sheep and goats. These fatal diseases are characterized by a long incubation period and histopathological changes such as neuronal vacuolization and astrogliosis in the central nervous system. TSE are caused by as-yet-unknown infectious agents named transmissible spongiform agents, or prions, and characterized by the accumulation of an abnormal proteinase-resistant isoform (PrP-res) of the host-encoded prion protein (PrP), derived from PrP posttranslational modifications (2). The transcriptional accumulation of glial fibrillary acidic protein (GFAP), a specific marker of astrocytes, has also been observed (5).At present, no treatment is available for TSE. Many unsuccessful therapeutic trials have been performed in experimental animal models; the trials have included the use of hormones, antibiotics, antiviral drugs, and antifungal agents (3). Three groups of drugs exhibited relative efficacy against scrapie: polyanions (6), the amyloid-binding dye Congo red (7), and amphotericin B (AmB) (1,12). AmB belongs to the group of polyene antibiotics with a ring structure that includes a hydrophobic region of conjugated double bonds and a hydrophilic region of hydroxyl groups with a mycosamine sugar moiety. AmB is an effective antifungal agent that is widely used for the treatment of systemic fungal infections such as candidiasis, histoplasmosis, and aspergillosis (9). It has been reported to delay the appearance of experimental scrapie in hamsters infected intracerebrally or intraperitoneally with the 263K scrapie strain (11). AmB also delayed PrP-res accumulation in the brain (15), but its effect on scrapie agent replication remains unclear (8, 15). The efficacy of AmB is limited by the absolute need to start the treatment in the early stages of scrapie infection and by its acute toxicity. MS-8209 is a polyene macrolide antibiotic derived from AmB which exhibits identical antifungal properties but in addition possesses greater water solubility and is at least five times less toxic (14). MS-8209 is thus an antiviral drug that might be useful for ex...
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