Brain Advance Access originally published online on July 1, 2006
Brain 2006 129(9):2241-2265; doi:10.1093/brain/awl150
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Review Article |
A systematic review of prion therapeutics in experimental models
MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London Queen Square, London, UK
Correspondence to: John Collinge, MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK E-mail: j.collinge{at}prion.ucl.ac.uk
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Summary |
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Prion diseases are transmissible, invariably fatal, neurodegenerative diseases which include Creutzfeldt–Jakob disease (CJD) in humans and bovine spongiform encephalopathy and scrapie in animals. A large number of putative treatments have been studied in experimental models over the past 30 years, with at best modest disease-modifying effects. The arrival of variant CJD in the UK in the 1990s has intensified the search for effective therapeutic agents, using an increasing number of animal, cellular and in vitro models with some recent promising proof of principle studies. Here, for the first time, we present a comprehensive systematic, rather than selective, review of published data on experimental approaches to prion therapeutics to provide a scientific resource for informing future therapeutics research, both in laboratory models and in clinical studies.
Key Words: prion; Creutzfeldt–Jakob disease; transmissible spongiform encephalopathy; experimental models; therapeutics
Abbreviations: CJD, Creutzfeldt–Jakob disease; CR, congo red; dpi, days post infection; DS, dextran sulphate; GAG, glycosaminoglycans; HM, heparan mimicking; HPA-23, heteropolyanion-23; IC50, half-maximal inhibition concentration in vitro; i.c., intracerebral; i.c.v., intracerebralventricle; i.p., intraperitoneal; iv, intravenous; LRS, lymphoreticular system; PPS, pentosan polysulphate; PrP, prion protein; PrPC, cellular prion protein; PrPSc, disease-associated prion protein; PrPsen, protease-sensitive prion protein; PrPres, protease-resistant prion protein (as monitored by proteinase-K resistant PrP immunoreactivity); Prnp, prion protein gene; SAF, scrapie-associated fibril; sc, subcutaneous (peripheral)
Received January 6, 2006. Revised May 4, 2006. Accepted May 8, 2006.
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Since the 1960s, there have been many attempts to treat prion diseases and prevent prion replication in a wide variety of experimental models. Here we present a systematic collation of these data from the published literature. Although the diversity of both the data and methodology precludes formal meta-analysis, this comprehensive overview allows a degree of qualitative analysis, which is particularly timely at the advent of controlled clinical trials in human prion diseases.
Prion diseases, or transmissible spongiform encephalopathies, include Creutzfeldt–Jakob disease (CJD) in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. They are incurable, fatal neurodegenerative diseases associated with neuronal cell death, characteristic ‘spongiform’ vacuolation of the brain tissue and accumulation of a disease-associated isoform of the endogenously expressed prion protein (for review see Collinge, 2001
). The human prion diseases have three distinct aetiologies: they may arise sporadically; be autosomal dominantly inherited conditions; or be acquired from exposure to environmental prions (via dietary exposure to human or bovine prions or accidental exposure to human prions during medical and surgical procedures) (Collinge, 2001). The recognition of a novel human prion disease, variant CJD (Will et al., 1996), and the experimental confirmation that it is caused by BSE-like prions (Collinge et al., 1996; Bruce et al., 1997; Hill et al., 1997), have led to fears of a vCJD epidemic in the UK. Fortunately, the number of recognized cases of vCJD has been relatively small to date (
160), but the number of asymptomatic infected individuals incubating or carrying the infection is unknown; human prion incubation periods may span decades (Collinge, 1999) and the recognition of secondary transmission by blood transfusion has raised new concerns (Llewelyn et al., 2004; Peden et al., 2004).
The central feature of prion diseases is the accumulation in the brain and some other tissues of the disease-associated PrPSc, which is derived from the host-encoded cellular PrPC. Although its function is unknown, PrPC is implicated in prion pathogenesis as coding mutations of the human prion protein gene (PRNP) result in inherited forms of prion disease (Collinge, 2001), and the presence of PrPC is required for prion propagation and development of prion pathology (Büeler et al., 1993). PrPSc is derived from PrPC by post-translational conformational change (Borchelt et al., 1990; Caughey and Raymond, 1991) and is extracted from diseased brain tissue as aggregated material, which is distinguishable from PrPC by its partial protease resistance and detergent insolubility. A wealth of evidence now supports the ‘protein-only’ hypothesis (Griffith, 1967; Prusiner, 1982), which states that PrPSc is the principal, and possibly the sole, constituent of the transmissible agent or prion (Bolton et al., 1982) and that it serves as a conformational template promoting the conversion of endogenous PrPC to PrPSc (for review see Prusiner, 2001). In vitro methods to produce protease-resistant material from PrPC have been developed (see Supplementary Material), but to date there has been no proof of the ‘protein-only’ hypothesis.
There is also increasing evidence to suggest that PrPSc in vivo is itself not directly neurotoxic and there is a lack of correlation between PrPSc deposition and disease severity (Hsiao et al., 1990; Medori et al., 1992; Büeler et al., 1994; Collinge et al., 1995; Lasmezas et al., 1997; Hill et al., 2000; Mallucci et al., 2003), suggesting that it is the process of conversion of PrPC to PrPSc that is the key event in prion pathogenesis, rather than the accumulation of PrPSc. The mechanism for the conversion and the structure of the infectious agent remain unclear.
On a molecular level, prion disease therapeutics can be targeted to PrPC, PrPSc or to the process of conversion between the two prion protein isoforms. Targeting PrPSc, the disease-associated isoform, may appear to be the most logical approach, but such targeting may have no effect of disease progression, or even enhance or prolong disease if PrPSc is a non-pathological end-point of a pathogenic conversion process, or if the rate of PrPSc deposition is critical to disease progression. Alternatively, targeting PrPC has the potential to remove the substrate for the pathogenesis and is applicable regardless of the disease aetiology.
The existence of distinct isolates or strains of prion has been recognized for many years. Strains were originally isolated by serial passage of natural scrapie samples in rodents and are distinguished by the production of different phenotypes in inbred mice (for review see Bruce et al., 1992 and Collinge, 2001) and by biochemical differences in the disease-associated prion protein isoform PrPSc (Bessen and Marsh, 1994; Collinge et al., 1996; Telling et al., 1996). It is important to consider prion strain type as well as experimental host with respect to model systems used to evaluate therapeutics; some therapeutic approaches may have different efficacy against different host/strain combinations. The pathogenesis may additionally be affected by the route of prion inoculation; following peripheral infection in some experimental animal models, prion replication is first detectable in the lymphoreticular tissues and spleen where levels plateau before detectable neuroinvasion, which occurs much later in the incubation period (for review see Aguzzi et al., 2003). Experimental models of prion propagation are reviewed in detail in the Supplementary Material.
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This review was carried out using methodology adapted from that for compiling systematic reviews of clinical studies of healthcare interventions (Egger et al., 2001
; Alderson et al., 2005). Bibliographic searches of the PubMed database were carried out using the criteria and search strategy detailed in the Supplementary Material, with the most recent search performed on March 10, 2006. Titles, abstracts or full references from the search results were screened by one individual (C.T.) to assess the eligibility for inclusion in the review. Essentially, experimental data describing the effect of therapeutic agents on prion propagation in animal, cell or cell-free models were eligible for inclusion. Further descriptions of the experimental models and data, both included and excluded from the collection, are given in the Supplementary Material. No unpublished studies were included.
Data from the selected references were extracted directly into the tables by one individual (C.T.). Separate tables were used for each experimental model (see Table 1 for summary of types of experimental model) with therapeutic agents organized as far as possible by structural class and/or pharmacological action (see Table 2 for summary of therapeutic approaches). For each individual agent, experimental data are arranged chronologically. The tables are presented in the Supplementary Material and are supported by descriptive text introducing the specific therapeutic agents and summarizing their effect in the experimental systems, with commentary kept to a minimum. No meta-analysis was performed owing to the heterogeneity of the methodology and data readout.
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The PubMed search, as detailed in the Supplementary Material, returned 1648 citations. Of these, 143 articles were selected according to the criteria outlined in the Supplementary Material. The data from these references are tabulated primarily by experimental model and secondarily by therapeutic agent type (as summarized in Tables 1 and 2), and they are presented in the Supplementary Material. Supplementary Table S1 lists experimental data from animal models of disease; Supplementary Table S2 lists the data from cell culture models; Supplementary Table S3 lists the data from cell-free propagation systems and Supplementary Tables S1a and S2a list the secondary infectivity bioassays performed on samples from primary experiments in either animal or cell models, respectively. The structures of many of the therapeutic agents are given in Supplementary Fig. S1. The accompanying text summarizing the data is arranged by therapeutic agent type.
Polyanionic compounds
Glycosaminoglycans (GAGs) are the polysaccharide side-chains of proteoglycans (PGs) and include heparan sulphate, heparin, dermatan sulphate, keratan sulphate, chrondroitin sulphate and carrageenans. PGs are components of the extracellular matrix and are involved in cell adhesion, migration and proliferation. Some cellular functions are also mediated by the free GAG chains. GAGs share a common architecture, being linear polymers of repeating disaccharide units, including an amino sugar and at least one negatively charged group (sulphate or carboxylate). This basic framework is also shared by the polyanionic glycans pentosan polysulphate (PPS) and dextran sulphate (DS), which are simplified semi-synthetic analogues of the endogenous GAGs (see Supplementary Fig. S1).
The endogenous heparan sulphate proteoglycan is associated with PrPSc deposits in prion disease (Snow et al., 1989; McBride et al., 1998) and also with the protein components of other amyloid deposits (for review see Diaz-Nido et al., 2002). GAGs can also associate with PrPC (Gabizon et al., 1993; Caughey et al., 1994), with the GAG analogues PPS and congo red (CR) showing higher affinity interactions than the endogenous heparin and chondroitin sulphate (Caughey et al., 1994). As described below, these polyanionic compounds are effective at decreasing detectable PrPSc and increasing prion disease incubation times in cellular and animal models, respectively, and their therapeutic efficacy is thought to be mediated by a competitive inhibition of the interaction between endogenous GAG molecules and PrPC and/or PrPSc.
Heteropolyanion-23
The antiviral compound heteropolyanion-23 (HPA-23, ammonium 5-tungsto-2-antimoniate) was initially tested against scrapie when the prevailing view was that the transmissible spongiform encephalopathies were caused by ‘slow viruses’. It was one of the first agents shown to have a beneficial effect on experimental prion disease, and the only effective one of a selection of antiviral agents tested (Kimberlin and Walker, 1979). HPA-23 delays the onset of scrapie following administration for over 9–12 days from the time of peripheral infection in mice and hamsters (Kimberlin and Walker, 1979, 1983, 1986), but it has no effect on the titre of the inoculum if pre-incubated with the inoculum (Kimberlin and Walker, 1986). The effects of HPA-23 treatment are prion strain dependent, and are less significant if administered after 12 days post infection, or if administered following intra-peritoneal rather than intravenous or subcutaneous inoculation with scrapie prions (Kimberlin and Walker, 1983, 1986).
Two other inorganic polyanions containing alternative or additional metal ions were ineffective against experimental rodent scrapie, whereas other organic polyanions, such as DS, tested alongside HPA-23 were found to be effective (Kimberlin and Walker, 1986). It is suggested that the efficacy of HPA-23 as an anti-prion agent is related to its polyanionic structure rather than its broad-range antiviral activity.
Dextran Sulphate (DS)
DS causes short-term impairment of the lymphoreticular system (LRS), and it was investigated as an anti-prion agent following demonstration of the involvement of the LRS in prion pathogenesis (Ehlers et al., 1984) in addition to the polyanion link (Kimberlin and Walker, 1986).
DS of molecular weight 500 kDa (DS500) causes a decrease in PrPSc levels in prion-infected cells (Caughey and Raymond, 1993; Barret et al., 2003) as well as a decrease in cell surface PrPC in uninfected cells (Shyng et al., 1995).
DS500 delays the disease onset following administration of a single dose around the time of i.p. infection in mice (Ehlers and Diringer, 1984; Ehlers et al., 1984; Farquhar and Dickinson, 1986), and both i.p. and i.c. infection in hamsters (Ladogana et al., 1992). There is a significant reduction in the prion titre in spleen following DS treatment (Ehlers and Diringer, 1984), but the effect of pre-incubation of the inoculum with DS500 is ambiguous, with no effect (Ehlers and Diringer, 1984) or a three log reduction in inoculum titre reported (Kimberlin and Walker, 1986) for mouse studies. Analyses of splenic PrPSc accumulation following DS500 treatment indicate a reduction in accumulation during the treatment period (Beringue et al., 2000a; Adjou et al., 2003). DS is most effective when administered at around the time of peripheral infection (Ehlers and Diringer, 1984), and in lymphotropic prion strains. DS500 treatment in mice is effective over a few weeks after peripheral inoculation in mice, during the period of replication in the LRS, but is ineffective if administered after the spleen accumulation plateau (Ehlers and Diringer, 1984; Kimberlin and Walker, 1986). In contrast to many mouse scrapie models, agent replication in the LRS prior to neuroinvasion is not necessary in hamsters peripherally infected with 263K scrapie. In hamsters, DS500 treatment is effective after both i.c. and i.p. inoculation but only if administered within 2 h of infection (Ladogana et al., 1992). Silica and trypan blue were tested along side DS as other agents causing impairment of the LRS, but neither shows any effect on scrapie incubation times (Ehlers et al., 1984).
DS500 is, however, toxic in mice [a 10% mortality is reported for animals treated with multiple doses of over a 4 week period (Adjou et al., 2003)], and other polyanions including PPS have been investigated as less toxic alternatives.
Pentosan Polysulphate (PPS)
PPS has anti-coagulant and anti-inflammatory activity, and has a veterinary license in the UK for treatment of osteoarthritis in the dog.
PPS has been reported to decrease PrPSc levels in prion-infected cells (Caughey and Raymond, 1993; Caughey et al., 1994; Birkett et al., 2001), but stimulates PrPSc formation in cell-free conversion system (Wong et al., 2001).
PPS causes a delay to the onset of disease following a single dose administered around the time of i.p. infection in mice (Ehlers and Diringer, 1984; Diringer and Ehlers, 1991; Farquhar et al., 1999), and both i.c. and i.p. infection in hamsters (Ladogana et al., 1992). PPS treatment by intra-ventricular infusion has also been shown to increase the incubation period of i.c. infected tg7 mice, even if the onset of the 4 week treatment period coincides with the onset of clinical symptoms (42 d.p.i.) (Doh-ura et al., 2004). Treatment was performed using an AZLET osmotic pump and intra-ventricular cannula inserted into the contralateral hemisphere from that used for scrapie prion inoculation. There are less severe pathological changes in the treated hemisphere, including reduced PrPSc deposition relative to the hemisphere inoculated. In animals treated late in the incubation period, pre-existing PrPSc deposition is not cleared by the treatment, but further PrPSc accumulation is inhibited. There is some evidence of dose response, and an inverse correlation between time of treatment and prolongation of incubation period, though the variability of the incubation time increases with increased PPS dosage. A control peripheral (subcutaneous) infusion experiment was ineffective in prolonging incubation times, and high doses (20 mg/kg/day) were found to cause haemorrhaging around the area of the pump insertion in 80% of animals (Doh-ura et al., 2004). The safety of intraventricular PPS treatment was assessed by continuous i.c.v. infusion of PPS at 110–460 µg/kg/day for 2 months into both rats and dogs. There were no adverse effects reported in rats, but doses >230 µg/kg/day caused severe adverse effects in dogs; the majority of animals suffered fatal seizures within 24 h of initiation of treatment and on pathological examination haematoma in the cerebral white matter surrounding the cannula insertion point was evident in many animals (Doh-ura et al., 2004).
PPS treatment was also found to inhibit PrPres accumulation in a CWD-infected deer-cell model of prion disease, with an IC50 of 10 ng/ml, equivalent to the IC50 for PPS treatment in RML-infected N2a cells (Raymond et al., 2006).
Endogenous GAGs
Heparan sulphate and heparin of various molecular weights decrease the accumulation of PrPSc in infected cell cultures (Gabizon et al., 1993; Caughey and Raymond, 1993; Caughey et al., 1994). Conversely, both heparan sulphate and PPS have been shown to stimulate the cell-free conversion of PrPC to protease-resistant forms (Wong et al., 2001). Other GAGs, including keratan sulphate and chondroitin sulphate, are less effective than heparan sulphate or DS at preventing PrPres accumulation in infected cells but also less effective at stimulating the in vitro conversion of PrPC to PrPres. These results suggest that sulphated glycans have a direct effect on the conversion of PrP, and that depending on the circumstances, they may be either cofactors or inhibitors of conversion. A model to explain the observed effects of GAGs in prion propagation systems proposes that sulphated glycans act as competitive inhibitors of endogenous GAGs in cells, whereas in the cell-free system the sulphated glycans are themselves cofactors for conversion (Wong et al., 2001).
Non-sulphated dextrans are not able to prevent PrPSc accumulation in cells (Caughey and Raymond, 1993), and inhibition of sulphation of GAGs by sodium chlorate also decreases the amount of PrPSc in scrapie-infected cell culture (Gabizon et al., 1993) indicating that the sulphation of these molecules is crucial to their involvement with prion conversion, whether as inhibitors or facilitators.
Heparan sulphate mimetics
Heparan mimicking (HM) molecules, developed initially for properties in wound healing (Desgranges et al., 1999), were investigated for activity as anti-prion drugs following observations of the efficacy of DS and other sulphated glycans. HMs are synthesized from dextran polymers by chemical modification of the hydroxyl groups with varying amounts of sulphate, carboxymethyl and benzylamide groups (Adjou et al., 2003; Schonberger et al., 2003); a representative structure is shown in Supplementary Fig. S1. There is a similar response of both HM2602 and HM5004 to DS500 in reducing PrPSc accumulation in chronically infected cells (Adjou et al., 2003; Schonberger et al., 2003), but only HM2606 is effective in vivo, offering a 14% increase in incubation time in scrapie prion-infected hamsters and a significant reduction in splenic PrPSc levels in scrapie prion-infected mice treated from the time of infection (Adjou et al., 2003).
Just as the non-sulphated dextrans are unable to prevent PrPSc accumulation in cells (Caughey and Raymond, 1993), the non-sulphated variants of the synthetic heparan mimetics are also ineffective (Adjou et al., 2003; Schonberger et al., 2003). An increased degree of both sulphate substitution and hydrophobic group substitution on the HM correlates with increased anti-prion efficacy in cells (Schonberger et al., 2003).
Phosphorothioate oligonucleotides
Four series of degenerate single-stranded oligonucleotides (DNA and analogues of natural nucleotides, see Supplementary Fig. S1) were investigated in a variety of prion propagation models (Kocisko et al., 2006b) following observation of the interaction of nucleic acids with PrP conversion and aggregation in vitro (Cordeiro et al., 2001; Deleault et al., 2005). Only the phosphorothioated oligonucleotides (randomers 1 and 2) are active in mouse-scrapie cell models, with IC50 values of 20 nM compared with 15–90 µM for related non-phosphorothioated nucleotides (DNA and randomer 3). The ability of the randomers to prevent PrPSc accumulation is strongly size-dependent (oligomers of 17 or more bases are much more effective than smaller oligomers) but largely independent of base-composition. The randomers active in preventing PrPSc accumulation in the cell system were found to bind recombinant mouse or hamster PrP competitively with DS5000 and PPS, and co-localized with internalized PrP in both infected and uninfected cells, suggesting that the effect is not mediated by interaction with PrPSc.
Randomer treatment of peripherally infected Tg7 mice leads to a dramatic increase in survival time; treatment with randomer 1 at 10 mg/kg more than doubles or triples the survival time for s.c. or i.p. inoculated mice, respectively. Pre-treatment of scrapie inocula with randomer also significantly decreases the effective titre of the inocula. Randomers showed a significantly lower anti-coagulation activity compared with PPS at equivalent molar doses, and may therefore have a clinical advantage over PPS.
Compounds related to GAGs/polysulphate polyanions
Congo Red (CR) and analogues
CR is a widely used histopathological stain for amyloid deposits including PrP. Since the earliest demonstration of CR efficacy in preventing prion accumulation in scrapie infected cells (Caughey and Race, 1992), there have been numerous studies of the effects of CR and its analogues as potential therapeutics for prion diseases.
Treatment of chronically infected cells with CR results in a decrease in PrPSc with IC50s reported between 1 nM and 1 µM (Caughey and Race, 1992; Caughey and Raymond, 1993; Caughey et al., 1993; Caspi et al., 1998; Demaimay et al., 1998; Demaimay et al., 2000; Milhavet et al., 2000; Mangé et al., 2000b; Rudyk et al., 2000; Poli et al., 2003). It is reported that both neuroblastoma and GT1 cells can be cured of prion infection by treatment with 1 µg/ml CR (Mangé et al., 2000b).
In cell-free conversion systems, CR causes a decrease in PrPres formation with IC50 of 8 µM (Demaimay et al., 1998, 2000; Kirby et al., 2003; Lucassen et al., 2003), although it has been observed that low concentrations of CR (<0.1 µM) actually stimulate PrPres formation under otherwise equivalent conditions (Demaimay et al., 1998, 2000; Kirby et al., 2003).
Two in vivo studies report that treatment with CR delays the disease onset in hamsters following ongoing administration starting around the time of i.p. or i.c. infection (Ingrosso et al., 1995; Poli et al., 2004), although the effect was not significant after i.c. infection with low titre scrapie (Poli et al., 2004) and it was noted that there was no effect on the progression of clinical disease (Ingrosso et al., 1995). A further study reports a transient increase in splenic PrPSc levels on CR treatment of peripherally infected mice (Beringue et al., 2000a).
Since CR is toxic, non-specific and does not cross the blood–brain barrier (Klunk et al., 1994, 1998), many analogues and derivatives of CR have been studied in both cell and cell-free systems in order to identify compounds with improved properties. There are three studies of small selections of compounds, either commercially available CR analogues and amyloid dyes (Demaimay et al., 1998, 2000) or synthetic naphthalene derivatives (Poli et al., 2003), which correspond to one of the terminal naphthalene groups on the symmetrical CR molecule (see Supplementary Fig. S1). Two further studies investigated large families of structurally related compounds for full structure–activity relationships (Rudyk et al., 2000; Sellarajah et al., 2004).
A general observation from these in vitro studies is that truncated molecules corresponding to one of the terminal moieties of CR are not effective at preventing PrPres propagation (Demaimay et al., 1998, 2000). One exception to this is the molecule CR-A (Poli et al., 2003), which in spite of the increased cytotoxicity compared with CR, was taken forward to tests in vivo (Poli et al., 2004) and was found to have similar efficacy to that of CR (see below).
Rudyk et al. (2000) observe that for some of the compounds, treatment with low concentrations of compound actually increases PrPres in SMB cells, whereas high concentrations lead to a decrease in PrPres. This is in accordance with the observed stimulation of cell-free conversion reactions at low concentrations of CR (Demaimay et al., 1998, 2000; Kirby et al., 2003). Many of the compounds tested show some activity, although only sirius red (see Supplementary Fig. S1) is as effective as CR in the ScN2a cell model (Demaimay et al., 2000). Conversely, in the SMB cells, sirius red is a better inhibitor than CR (Rudyk et al., 2000), and an in depth study of structure–activity relationships in SMB cells identified 10 compounds (from 54 tested) with improved activity compared with CR (Sellarajah et al., 2004). Although these 10 compounds represent 5 of the 7 different structural families tested, some structure–activity relationships have been described (Sellarajah et al., 2004).
A comparison of the cell-free conversion and cell culture experiments shows that there is some correlation between the results with SMB cells and the non-denaturing conversion system (Rudyk et al., 2000; Kirby et al., 2003), but otherwise no clear correlation between compound efficacy in cell culture and in cell-free systems (Demaimay et al., 1998, 2000). The amyloid specific dyes (evans red, trypan blue, sirius red, thioflavin S and primuline) are strongly inhibitory in the cell-free system but not in cells (Demaimay et al., 2000). Kirby et al. (2003) observe that CR is a less effective inhibitor of conversion under denaturing conversion conditions, which may account for some of the discrepancies between the different experimental systems.
In animals, CR-A (equivalent to the terminal moiety of CR) is more effective than CR after i.c. scrapie infection, but less effective after peripheral infection. CR-B (equivalent to the linker moiety of CR) is not effective in animals. In contrast to the equivalent concentration of CR, a combined treatment of two compounds corresponding to the terminal and linker moieties of CR in ratio 2:1 (CR-A:CR-B) did not increase the incubation time of infected hamsters (Poli et al., 2004).
Although CR is a small molecule (see Supplementary Fig. S1), it is able to stack extensively and mimic larger sulphated polyanions (Woody et al., 1981; Priola and Caughey, 1994) and hence may have some mechanistic similarity to the sulphated polyanionic compounds discussed above. Both CR and other GAG-type compounds cause a decrease in surface PrPC in uninfected cells (Shyng et al., 1995). CR competes with heparin binding to PrPsen (Caughey et al., 1994) and binds to and causes hyper-stabilization of PrPres, thereby preventing further amyloidogenesis both in cell culture systems and ex vivo brain homogenates (Caspi et al., 1998). The increase in splenic PrPSc after CR treatment (Beringue et al., 2000a) and the stimulation of in vitro conversion at low CR concentrations are consistent with the proposal that inhibition of PrPSc accumulation by CR is mediated by stabilization of PrPSc (Caspi et al., 1998).
Suramin
Suramin (polysulphonated naphthyl urea, see Supplementary Fig. S1) was initially developed to treat trypanosomiasis. It causes downregulation of surface proteins and interferes with the oligomerization state of proteins (Gilch et al., 2001) and has some structural homology to CR. A modest increase in incubation time is observed in hamsters treated with two or three doses of suramin at or around the time of i.p. inoculation (Ladogana et al., 1992), and in mice treated with a single dose around the time of i.p. infection (Gilch et al., 2001). Suramin has been shown to decrease PrPSc levels in infected cells (Gabizon et al., 1993; Doh-ura et al., 2000; Gilch et al., 2001), to decrease surface PrPC levels and cause intracellular PrP aggregation in uninfected cells and to cause aggregation of recombinant PrP (Gilch et al., 2001).
A variety of suramin derivatives and analogues were also tested in the ScN2a system; compounds effective at decreasing PrPSc accumulation are symmetrical aromatic structures with naphthalene- or benzene-sulphonic acid substitutions, whereas asymmetric or uncharged molecules and those with phosphonic or carbonic substitutions were less active (Nunziante et al., 2005). Unlike suramin, the analogues do not affect the cell surface expression of PrPC, but like suramin they do induce formation of detergent-insoluble PrP aggregates at the cell surface (Nunziante et al., 2005).
Polycationic compounds
In addition to the polyanionic compounds described above, various classes of cationic polyamine compounds, including components of lipid transfection media, have been identified as potential anti-prion agents (Supattapone et al., 1999, 2001; Winklhofer and Tatzelt, 2000; Solassol et al., 2004; Yudovin-Farber et al., 2005).
Dendritic polyamines
An incidental discovery showed that exposure of ScN2a cells to SuperFect decreased both pre-existing PrPSc and prevented the formation of de novo PrPSc (Supattapone et al., 1999). The component polyamine compounds of SuperFect that effected the decrease in PrPSc were identified as the dendritic polymers polypropyleneimine (PPI) generation 4.0, polyethyleneimine and polyamidoamide generation 4.0 (see Supplementary Fig. S1), which have IC50s for PrPSc decrease in the nanomolar range (Supattapone et al., 1999). Structure–activity relationships indicate that increased anti-prion efficacy correlates with increased branching and an increase in the surface density of primary amine groups. ScN2a cells treated with PPI generation 4.0 are cleared of PrPSc and are no longer infectious to mice (Supattapone et al., 2001). There is a good correlation between compound efficacy in cell culture and in vitro experiments in which purified mouse scrapie or scrapie-infected mouse brain homogenates are incubated with polyamines prior to detection of PrPSc. The in vitro exposure of RML prions to PPI at pH 4 caused disaggregation and a decrease in ß-sheet content of the preparation and an increase in proteolytic susceptibility of the protein that is both strain and sequence dependent (Supattapone et al., 1999, 2001). The proposed site of action of the polyamines is in lysosomal compartments, as demonstrated by their localization to lysosomes (Supattapone et al., 2001), their maximal efficacy in vitro at acidic pH (Supattapone et al., 1999), and the fact that the anti-prion effect of the polyamines can be blocked by the lysosome-rupturing agent chloroquine (Supattapone et al., 1999).
Phosphorus-containing dendrimers (pd) are a novel class of dendritic polyamines with improved bioavailability and decreased toxicity compared to the polyamines reported by Supattapone (Solassol et al., 2004). The phosphorus substituents of these dendrimers provide increased stability and tertiary amine groups render the molecule amphipathic. The efficacy of these compounds in the ScN2a cell culture system was found to increase with increasing generation (see Supplementary Fig. S1), but only up to generation 4 dendrimers (molecular weight 33 kDa), and cells cleared of PrPSc by pd-G4 treatment were no longer infectious to scrapie susceptible cells. Pd-G4 treatment of scrapie brain homogenate results in decreased PrPSc to varying degrees depending on the scrapie strain. In wild-type mice treated with pd-G4 after i.p. infection there was significant reduction in splenic PrPSc at 30 d.p.i. (Solassol et al., 2004), and although the effect on incubation time is not reported, there is a precedent for a correlation between depletion of splenic PrPSc in the early stages of i.p. infection and a prolongation of incubation time (Beringue et al., 2000b; Heppner et al., 2001; Barret et al., 2003; Mabbott et al., 2003). In addition to their anti-prion properties, these phosphorus dendrimers have the potential for targeting specific tissues and for use as soluble drug carriers (Solassol et al., 2004).
Other cationic polyamines
Following various lines of evidence that suggest an involvement of the cell membrane raft domains in the conversion of PrPC to PrPSc (Taraboulos et al., 1995; Kaneko et al., 1997a; Klein et al., 1998b), Winklhofer and Tatzelt (2000) screened a variety of lipid transfection reagents with membrane-association properties for their effect on PrPSc formation in ScN2a cells. Treatment with the polycationic lipo-polyamine DOSPA (see Supplementary Fig. S1) decreased the PrPSc levels in the cells by both clearance of pre-existing PrPSc and blocking de novo formation of PrPSc (Winklhofer and Tatzelt, 2000). The lipopolyamines tested are smaller than the dendritic molecules described above, and most were ineffective in clearing PrPSc from infected cells, including neutral and mono-cationic polyamines, as well as other poly-cationic lipids related to DOSPA with a spermine head-group (Winklhofer and Tatzelt, 2000). Similarly, Supattapone et al. (1999) reported the inability of a mono-cationic lipopolyamine (DOTAP) to decrease PrPSc levels in ScN2a cells. DOSPA treatment of cells showed no adverse effects on the integrity of the sphingolipid- cholesterol-rich membrane microdomains (rafts) (Winklhofer and Tatzelt, 2000). The membrane association of DOSPA is believed to be essential for its anti-prion activity.
Further polycationic compounds in which oligo-amines are conjugated to oxidized dextran or other sugar polymers have been tested for efficacy in the ScN2a cell model (Yudovin-Farber et al., 2005). These compounds are related to cationic polyamines and heparin mimetics and have been investigated for use as wound dressings and as gene transfection agents. Various combinations of polysaccharides and oligoamines were investigated, the most active components being dextran and spermine, respectively. Dextran–spermine was effective at completely clearing ScN2a cells of detectable PrPSc after 4 days of treatment at 31 ng/ml (3 nM); other combinations of saccharides and amines were effective at roughly one order of magnitude greater molarity. Dextran-spermine molecules were derivatized with the addition of methoxypoly-(ethylene glycol) (MPEG) or oleic acid substituents in order to produce compounds with improved bioavailability, but the derivatized compounds were less effective than the parent compound, with an inverse relationship between efficacy and degree of substitution.
Neither the branched (dendritic) polyamines nor the cationic polyamines affect PrPC production; it is suggested that their mechanism of action may be in part via stimulation of the normal cellular pathways of protein degradation to destroy PrPSc (Supattapone et al., 1999).
Tetrapyrrolic compounds
Tetrapyrrolic compounds are known effectors of protein conformational change, with structural similarities to CR (being both aromatic and sulphated) but with improved toxicological and solubility profiles. They also have the potential for extensive substitution and derivation. A variety of tetrapyrroles, including porphyrins and phthalocyanins, were investigated by Caughey and co-workers as potential anti-prion compounds using the ScN2a cell culture system and were found to decrease PrPres levels (Caughey et al., 1998), as well as to prevent propagation of PrPres in the cell-free conversion system with sub-micromolar IC50 (Caughey et al., 1998). Of those tested in the ScN2a model, the three most potent anti-prion compounds (shown in Supplementary Fig. S1) are phthalocyanin tetrasulphonate (PcTS), deuteroporphyrin IX 2,4-bis-(ethylene glycol) iron(III) (DPG2-Fe3+) and meso-tetra(2-N-methylpyridyl)porphine iron(III) (TMPP-Fe3+). In addition, the porphine complex indium (III) meso-tetra (4-sulphonatophenyl)porphine chloride (In-TSP) gives an IC50 of 0.3 µM in the CWD-infected deer-cell model (Raymond et al., 2006).
The most efficient of the cell culture compounds were examined using in vivo prion disease models—transgenic mice expressing hamster PrP and infected with hamster prion strains (tg7) (Priola et al., 2000), and subsequently in wild-type mice (Priola et al., 2003). A delay in the onset of disease follows multiple administrations over the first month after i.p. infection, but no significant effect is seen after i.c. infection, nor after treatment at a later stage in the disease progression after i.p. infection (Priola et al., 2000, 2003). Pre-treatment of the prion inocula with PcTS caused a significant increase in incubation period, but a greater increase in incubation time is observed following multiple treatments post inoculation. The suggested mode of action for the anti-prion efficacy of tetrapyrroles is in peripheral tissues at the initial stage of infection.
Further complexes of various divalent metals with two porphine tetrapyrroles, tetra (4-sulphonatophenyl)porphine (TSP) and tetra (4-N,N,N-trimethylanilinium)porphine (TAP), were investigated in the tg7 mouse model (Kocisko et al., 2006a). Prophylactic i.p. treatment with Fe-TAP in i.p. infected tg7 mice results in a 4-fold increase in survival time, much greater than that reported in previous tetrapyrrole studies. Significant increases in survival time of a lower magnitude are also seen for Fe-TSP and metal-free TSP and TAP treatments. Even Fe-TAP is ineffective, however, if administration is commenced more than 50 days after scrapie infection. Pre-incubation of the scrapie inoculum with metal–porphine complexes prior to i.c. inoculation results in modest increases in survival time for iron and nickel complexes, correlating to 3–4 log reduction in the effective titre of the inoculum. For i.c. porphine treatments commencing 2 weeks into the incubation period of i.c. infected mice, the most efficacious complex is Fe-TSP rather than Fe-TAP (the most effective in the i.p. treatment and i.p. infection model). Intracerebral Fe-TSP treatment is deemed by the authors to be as effective as similar regime of PPS, at 2-fold lower dose (mass per animal).
Polyene antibiotics
In the 1980s, Amyx reported on the treatment of experimental scrapie in hamsters and CJD in mice with 35 drugs encompassing antiviral, antibacterial, anti-parasitic, anti-fungal and anti-neoplastic drugs, hormonal agents and interferon. Only methotrexate and amphotericin B were found to significantly prolong the incubation time, following treatment throughout the incubation period, although none of the agents prevented disease. Amphotericin B treatment was also reported to significantly increase the incubation time of CJD-infected African green monkeys (Amyx et al., 1984).
Amphotericin B (AmB, see Supplementary Fig. S1) is a fungal antibiotic derived from Streptomyces nodosus, which acts by intercalation into and disruption of the cell membrane. AmB shows preferential binding for ergosterol over cholesterol, but in mammalian cells interacts with cholesterol and alters the membrane lipid composition by peroxidation and endocytic processes, resulting in modification of raft domain properties. Following the initial report from Amyx, there were many subsequent reports of treatment with amphotericin B and its derivatives in animal and cell culture models of prion propagation.
Amphotericin B treatment delays disease onset in hamsters infected both i.p. and i.c. with 263K scrapie (Pocchiari et al., 1987; Pocchiari et al., 1989; Xi et al., 1992; McKenzie et al., 1994; Adjou et al., 1995, 1999, 2000) and in C57BL/6 mice infected with C506M3 scrapie (Demaimay et al., 1994). In mice, treatment was effective even if administered at a late stage following i.c. infection, when there is already significant infectivity and PrPres accumulation in the brain (Demaimay et al., 1997), suggesting that AmB is an effective inhibitor of prion propagation within brain tissue. Effective late stage treatment in a hamster model has also been demonstrated (Adjou et al., 2000).
MS-8209 is a less toxic derivative of amphotericin B (Adjou et al., 1995), which can therefore be administered at higher doses and is generally more effective than amphotericin B. Treatment with AmB or MS-8209 shows strain-dependent efficacy; hamster scrapie strains DY and 139H show no effect or a less marked increase in incubation time compared with 263K scrapie (Xi et al., 1992; McKenzie et al., 1994), and treatment of mice with C506M3 scrapie is more successful than treatment of mice of the same strain with BSE infection (Adjou et al., 1996). In a transgenic mouse expressing hamster PrP under a neuron-specific enolase promoter (tg52NSE) infected with 263K hamster scrapie (Demaimay et al., 1999), both AmB and MS-8209 treatment was more effective in the absence of endogenous mouse PrP expression. Neither drug showed an equivalent efficacy in tg52NSE mice infected with the DY scrapie prion strain. MS-8209 treatment had no effect on the disease progression in SCID mice but decreased the proportion of mice succumbing to clinical disease (clinical attack rate), and conversely delayed disease onset without altering clinical attack rate in reconstituted SCID (R-SCID) mice (Beringue et al., 1999).
The amphotericin B analogue mepartricin is effective only against i.p. scrapie prion infection (Pocchiari et al., 1989) unlike AmB, which is effective against both i.c. and i.p. infection.
There are discrepancies in the findings from infectivity bioassays of AmB treated 263K-scrapie infected hamsters, which are ascribed to differences in experimental protocols; Xi et al. (1992) report no spleen infectivity and reduced brain infectivity to +50 d.p.i. in partially purified samples, whereas McKenzie et al. (1994) report merely a delay in accumulation of infectivity which reaches control levels at +70 d.p.i. Despite equivalent infectivity between treated and control hamsters, McKenzie et al. (1994) also report that at +70 p.d.i., the treated animals show no clinical sickness and 10-fold lower PrPres in brain tissue, compared to the clinically-sick untreated animals, again highlighting the lack of direct correlation between infectivity in the brain and clinical illness.
In cell culture systems, AmB decreases the PK-resistance of mutant PrP (Mangé et al., 2000a) and causes a decrease in level of PrPSc in infected neuronal cells which is not maintained on cessation of treatment (Mangé et al., 2000b). Filipin is a polyene antibiotic related to amphotericin B which is able to reduce PrPSc accumulation in ScN2a cells with an IC50 of 2 µM (Marella et al., 2002).
Tetracyclic compounds
The anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDOX) is an anticancer drug which was found to possess anti-amyloidogenic properties during a trial of cancer patients with immunoglobulin light-chain amyloidosis complications (Gianni et al., 1995). It was shown to encourage resorption of fibrils, and is also able to bind in vitro to a variety of natural amyloid fibres (Merlini et al., 1995). IDOX was tested in experimental hamster scrapie by ic administration of the scrapie inocula and drug together due to its high cytotoxicity and poor penetration of the blood–brain barrier; this treatment is effective in delaying the onset of clinical disease and there is an absence of key histopathological features of disease at onset (Tagliavini et al., 1997). Although not applicable as a therapeutic agent, IDOX is proposed as a prototype anti-prion compound (Tagliavini et al., 1997).
The tetracyclic antibiotics were investigated by the same group following the IDOX finding. In common with IDOX, tetrapyrrolic compounds and CR, the tetracyclic antibiotics tetracycline and doxycycline contain a hydrophobic core with hydrophilic substituents (see Supplementary Figure S1) and have improved cytotoxicity and pharmacokinetic properties compared with IDOX. The efficacy of tetracycline and doxycycline against experimental scrapie was demonstrated after incubation of the scrapie inoculum with drug prior to ic infection (Forloni et al., 2002), which results in a delayed onset of disease by reducing the titre of the initial inoculum. These results are presented as having potential for decontamination rather than for therapeutics (Forloni et al., 2002).
Tetracycline reduces protease-resistant PrP formation in the PMCA replication assay (Barret et al., 2003) and reduces detectable PrPres in treated brain homogenate (Tagliavini et al., 2000; Forloni et al., 2002). It is reported to bind to fibrillogenic synthetic PrP peptides, preventing acquisition of protease resistance (Tagliavini et al., 2000; Barret et al., 2003) and also to prevent PrP106-126 peptide-mediated cytotoxicity in primary cell culture (Tagliavini et al., 2000).
Tricyclic and related compounds
Given that the lysosome is a potential site of conversion of PrPC to PrPSc, Doh-ura et al. (2000) investigated the potential anti-prion effects of various lysosomotropic factors, including the anti-malarial drugs quinacrine and chloroquine (see Supplementary Figure S1), in the scrapie-infected cell system. Quinacrine was found to be a very efficient inhibitor or PrPSc propagation in infected cells, with an IC50 of 0.4 µM. Prusiner and colleagues later reported a cell culture system in which the efficacy of licensed tricyclic compounds were investigated (Korth et al., 2001). Compounds found to be effective in the ScN2a system included a variety of compounds in the acridine and phenothiazine classes, of which the most effective were quinacrine (IC50 0.3 µM) and chlorpromazine (IC50 3 µM), respectively. Ryou et al. (2003) also tested the relative efficacy of the different enantiomers of quinacrine in the cell system and report a 2- to 6-fold greater activity of (S)-quinacrine compared with (R)-quinacrine.
There are mixed reports of the efficacy of quinacrine in cell-free conversion systems; three groups reports that quinacrine has no effect on PrPSc propagation (Doh-ura et al., 2000; Kirby et al., 2003; Lucassen et al., 2003), whereas Barret et al. (2003) report that quinacrine decreases formation of protease-resistant PrP in the PMCA assay.
Despite its efficacy in cell culture, quinacrine treatment showed no effect on the incubation time of ic-infected animals, in either wild-type mice treated orally (Collins et al., 2002) or in tg7 mice treated by intra-ventricular infusion (Doh-ura et al., 2004). In fact a high dose of intra-ventricular quinacrine causes a decrease in the scrapie incubation time (Doh-ura et al., 2004), and a further study reports that ip quinacrine treatment of BSE-infected mice results in an increase in splenic PrPSc deposition at 30 days after infection (Barret et al., 2003).
Chlorpromazine is less effective than quinacrine in cell culture (Korth et al., 2001), but was reported to increase incubation time in mice after intracerebral but not intraperitoneal infection (Roikhel et al., 1984). These results were published using the compound name aminasine and have not been substantiated to date.
Following the success of acridine compounds in cells (Korth et al., 2001), a variety of bis-acridines were developed and investigated by the same group (May et al., 2003). Supplementary Figure S1 shows a representative bis-acridine compound. A total of 20 compounds of various linker types and lengths were investigated for bioactivity and cytotoxicity; three compounds were shown to have 10-fold increased efficacy compared with the parent quinacrine (i.e. IC50s 0.03 µM). There are no data reported for in vivo experiments with these compounds.
A study of further quinacrine-related (but not tri-cyclic) compounds has been reported by the same group of investigators who first observed the efficacy of quinacrine in cell systems (Murakami-Kubo et al., 2004). Two families of compounds containing a quinoline ring, typified by quinine and biquinoline (see Supplementary Fig. S1) were investigated. In the scrapie-infected N2a cell model, the effective compounds inhibited PrPSc accumulation with IC50s in the range 3 nM to 38 µM, without any effect on biosynthesis or turnover of PrPC. Other compounds were ineffective, and the authors describe structure–activity relationships for the compound families. In vitro biacore analyses show both quinine and biquinoline bind to recombinant PrPC. In vivo studies with transgenic mice expressing hamster PrP or overexpressing mouse PrP were treated for 1 month with an intra-ventricular infusion of quinine or biquinoline using the protocol established for intra-ventricular treatment with PPS. Treatment commenced either shortly after or 1 month after intracerebral scrapie inoculation, and in all cases the variance of the incubation times in the treatment groups was increased compared to the control groups. In spite of increases in the incubation times in many treatment groups, only some of these increases are statistically significant. There is no classical dose response to either compound. Administration of biquinoline at +10 d.p.i. by either intraperitoneal or intra-ventricular route gave a significant increase in incubation time, but neither route of biquinoline administration was significant when treatment was commenced late into the infection process (+35 d.p.i.). Pathological examination of the animals with prolonged incubation periods shows reduced PrPSc deposition on the brain hemisphere ipsilateral to the intra-ventricular cannula, but no difference in deposition and pathology on the contralateral side, the side of the scrapie inoculation.
Mefloquine is a quinoline anti-malarial effective against both RML and 22L infected N2a cells, but not effective as prophylaxis against peripherally inoculated scrapie in tg7 mice (Kocisko and Caughey, 2006). Other classes of anti-malarial compounds tested (i.e. not quinoline or acridine compounds) were generally toxic at the treatment concentrations (1–10 µM).
Beta-sheet breaker peptides
PrP residues 106–126 appear to be important in the conversion between prion protein isoforms (de Gioia et al., 1994), and an excess of peptides PrP106-136 or PrP109-141 (especially residues 119 and 120 from the hydrophobic sequence 113AGAAAAGA120) prevented the recruitment of PrPC in a cell-free conversion experiment (Chabry et al., 1998). Using prion protein peptides of both mouse and hamster sequences (residues 109–141, of which 119–136 are identical in mouse and hamster protein), Chabry et al. (1999) investigated the species specificity of conversion and report that both mouse and hamster peptides can inhibit conversion of proteins from either species. PrP119-136 is also effective at decreasing PrPSc in chronically infected MNB cells with an IC50 of 11 µM, whereas other peptides, including PrP119-128, did not show this effect (Chabry et al., 1999).
This result, and the successful employment of ß-sheet breaker peptides in models of Alzheimer's disease (Soto et al., 1998), spurred the design of a ß-sheet breaker peptide to specifically interact with prion protein conversion (Soto et al., 2000). A ß-sheet breaker peptide consists of a sequence from the target protein into which extra proline residues are inserted. Proline is an imino acid unable to take the conformation required by an ordered ß-sheet structure and its presence in a sequence of amino acid residues with otherwise high ß-propensity prevents the formation of ß-sheet by that peptide. The sequence of the prion protein ß-sheet peptide iPrP13 designed by Soto et al. is given in Supplementary Fig. S1. iPrP13 causes some reduction in the incubation time of scrapie infected mice if used to treat the inoculum prior to i.c. infection and decreases protease-resistant PrP in the CHO cell model expressing mutant PrP (Soto et al., 2000). A 1000-fold molar excess of iPrP13 over PrPSc is required for 90% reduction PrPSc in an in vitro experiment with scrapie infected brain homogenate (Soto et al., 2000). There was no decrease in PrPSc levels in chronically infected ScN2a cells treated with either monomeric or polymeric iPrP13 (Oishi et al., 2003).
Compounds identified by screening approaches
Cell culture screen
Chronically infected N2a cells were used to screen a library of 2000 commercially available drugs and natural products (The Spectrum Collection, MicroSource Discovery Inc.) for molecules capable of decreasing PrPres propagation (Kocisko et al., 2003). The initial screen conditions were 10 µM compound in RML-infected ScN2a cells for 5 days; compounds positive under these conditions were then screened at 1 µM in both 22L- and RML-infected ScN2a cells, and IC50 values were estimated from these two sets of treatment conditions. A total of 17 compounds were positive against both scrapie strains with sub-micromolar IC50s and without cytotoxicity (Supplementary Table S2). These compounds fall into five distinct classes: anti-histamines (astemizole, terfenadine) and polyphenolic compounds (tannic acid, katacine, bisepigallocatachin digallate), which define novel classes of anti-scrapie compounds, and anti-malarials (quinacrine, bebeerine, tetradrine, amodiaquine), phenothiazines (thiothixene, prochloroperazine, thioridazine, trifluoperazine) and steroid-type compounds (budesonide, clomiphene, lovastatin, chrysanthellinA). Compounds from these classes identified with anti-prion activity by other investigators include quinacrine (Doh-ura et al., 2000; Korth et al., 2001), chlorpromazine (Korth et al., 2001), prednisone (Outram et al., 1974) and lovastatin (Taraboulos et al., 1995; Bate et al., 2004b).
Some of the most promising compounds identified in this screen (Kocisko et al., 2003) were tested in the tg7 model of prion disease as both treatment for intracerebral scrapie infection and prophylaxis against peripheral scrapie infection. None showed any significant effect on prion disease incubation time following treatment throughout the incubation period (Kocisko et al., 2004).
Compounds from the initial screen in N2a cells (Kocisko et al., 2003) were subsequently tested in a Rov9 cell model of ovine scrapie, along with 32 novel inhibitors of PrPSc propagation identified by screening of both RML and 22L scrapie infected N2a cells (Kocisko et al., 2005). None of those tested in the Rov9 cells were as effective in the ovine model as in the murine models, with only 3 out of 32 showing IC50s 40 µM compared with submicromolar IC50s for RML-infected N2a and 1–10 µM for 22L-infected N2a.
Yeast-based screen
Further compounds were identified using an assay for prevention of yeast prion formation (Bach et al., 2003). A total of 2500 compounds, synthetic and natural products, were screened in a yeast assay. Two compounds were additionally shown to be effective in preventing prion propagation in the ScN2a model with IC50s of 5 µM. The kastellpaolitines constitute a new class of anti-prion compounds, and both these and the phenanthridines have some structural similarity to the tricyclic compounds.
Fluorescence screen
An assay for identification of potential anti-prion drugs from a library of 10 000 compounds was developed based on the scanning for intensely fluorescent targets (SIFT) technique (Bertsch et al., 2005). The assay involves the formation of a ternary complex between PrPSc, recombinant PrP and mAb which has both red and green fluorescence; in the presence of a compound that interferes with the binding of PrPSc to rPrP, a reduction in the green fluorescence of the complex is observed. Activities of test compounds were compared to that of positive control compound DOSPA (17 µM). A total of 80 promising compounds were tested in a secondary assay in ScN2a cells; 4 showed reproducible decreases in PrPSc signal in the absence of toxicity, with IC50s of 2 and 6 µM reported for two compounds.
Immunomodulation and immunotherapeutics
Although there is no obvious humoral immune response stimulated in prion disease (Porter et al., 1973), various elements of the immune system have been manipulated to probe prion pathogenesis, and these experiments in turn have suggested potential strategies for prion therapeutics; for a review of the involvement of the immune system in scrapie pathogenesis see Aguzzi et al. (2003). Early experiments assessed the efficacy of both immunostimulation and immunosuppression (see below), but since the experimental demonstration that antibodies can be raised to the prion protein, efforts have been focused on the therapeutic application of antibodies or of stimulating an antibody response. Initial attempts to produce anti-prion antibodies included immunization with purified prions (Bendheim et al., 1984) or scrapie-associated fibrils (SAFs) (Kascsak et al., 1987), with a greater response observed in prion knockout mice (Prusiner et al., 1993) compared to wild-type mice. With appropriate a