Screening Congo Red and its analogues for their ability to prevent the formation of PrP-res in scrapie-infected cells

Hélène Rudyk1, Snezana Vasiljevic2, Ruth M. Hennion2, Christopher R. Birkett2, James Hope2 and Ian H. Gilbert1

Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK1
Institute for Animal Health, Compton Laboratories, Compton, Newbury, Berks RG20 7NN, UK2

Author for correspondence: James Hope. Fax +44 1635 577263. e-mail james.hope{at}bbsrc.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Transmissible spongiform encephalopathies (TSEs) are incurable, fatal diseases. The dye Congo Red (CR) can cure cells infected with agents of the sheep TSE, scrapie, but is not used as a therapeutic or prophylactic agent in vivo, as its effects are small, possibly due to low blood–brain barrier permeability, and complicated by its intrinsic carcinogenicity. In this paper, the development is described of a structure–activity profile for CR by testing a series of analogues of this dye for their ability to inhibit the formation of the protease-resistant prion protein, PrP-res, a molecular marker for the infectious agent, in the scrapie-infected, SMB cell line. It was found that the central benzidine unit in CR, which gives the molecule potential carcinogenicity, can be replaced by other, less toxic moieties and that the sulphonate groups on the core molecule can be replaced by carboxylic acids, which should improve the brain permeability of these compounds. However, detailed dose–response curves were generated for several derivatives and they revealed that, while some compounds showed inhibition of PrP-res accumulation at high concentrations, at low concentrations they actually stimulated levels of PrP-res above control values.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Transmissible spongiform encephalopathies (TSEs or prion diseases) are rare, progressive neurological disorders of humans and animals. They are invariably fatal, with a protracted, often asymptomatic time-course that can last months or even years. There is no cure for the TSEs and this is of particular concern in Europe, where a cross-species transmission of scrapie, the sheep TSE, to cattle has produced bovine spongiform encephalopathy (BSE) (Wells et al., 1987 ). This cattle epidemic has claimed nearly 200000 BSE-affected animals and led to the slaughter of large numbers of healthy but probably infected herds (Anderson et al., 1996 ). A novel human form, new variant Creutzfeldt–Jakob disease (vCJD) (Will et al., 1996 ), which is caused by the same (or similar) BSE strain of TSE agent (Bruce et al., 1997 ), has claimed 41 lives in the wake of BSE, presumably by the intake of infected food. This has stimulated an intense search for prophylactic or therapeutic drugs that are effective against oral exposure to these infectious disease agents.

The normal cellular prion protein (PrPC) is a 33–35 kDa, glycolipid-anchored membrane protein with two N-glycans that is relatively susceptible to proteolysis compared with an abnormal isoform, PrPSc (Basler et al., 1986 ; Hope et al., 1986 ). PrPSc can have variable N-glycosylation (Bolton et al., 1985 ; Stimson et al., 1999 ) and is degraded by proteinase K (PK) under mild denaturing conditions to a resistant core structure (PrP-res) with a molecular mass 6–7 kDa lower than that of PrPSc (Hope et al., 1988 ; Meyer et al., 1986 ). Conversion of PrPC to PrPSc is a biochemical correlate of agent replication in cell culture and animal models of TSEs. Sulphated polysaccharides and polyaromatic dyes, amphotericin B, deoxyrubicin, porphyrins and phthalocyanines can interfere with this biochemical conversion in vivo and/or in vitro (Gilbert & Rudyk, 1999 ). Some of these compounds, notably pentosan sulphate (Ehlers & Diringer, 1984 ; Ladogana et al., 1992 ) and Congo Red (CR) (Ingrosso et al., 1995 ), can also restrict the replication of the infectious agent and delay the onset of clinical disease when administered directly into the brain or peritoneum at or near the time of infection of mice or hamsters. These compounds are not effective when given much later than 1 month after infection in rodents and, in a single human case, one compound, amphotericin B, did not ameliorate the clinical signs of sporadic CJD (Masullo et al., 1992 ). In new variant but not sporadic CJD, PrPSc has been detected in spleen and tonsil (Hill et al., 1999 ), and it may be possible to block a peripheral phase of vCJD agent replication prior to its invasion of the CNS. Unfortunately, the current lead compounds have yet to be shown to be effective either when given orally or against oral infection (Farquhar et al., 1999 ). This may reflect the cellular complexity of oral pathogenesis. Recent work tracking the appearance of PrPSc by immunohistochemistry in orally infected hamsters has revealed at least two pathways of infection from alimentary canal to brain; either directly via the vagus nerve or indirectly via phagocytic, lymphoid cells of the gut (Beekes et al., 1998 ; McBride & Beekes, 1999 ). Clearly, different pharmacological agents may be needed to block each of these pathways and a multi-drug therapy may be required for an effective oral prophylaxis. We have focused our investigations on one type of infected cell, the non-neuronal SMB cell line, and one lead compound, CR.

In the past, cultured tumour cells of neuro-ectoderm origin, neuroblastoma (Caspi et al., 1998 ; Caughey, 1994 ; Caughey & Raymond, 1993 ; Priola et al., 1994 ; Race et al., 1987 ; Taraboulos et al., 1990 ; Tatzelt et al., 1996 ) or PC12 cells (Rubenstein et al., 1984 ), have been infected with rodent-passaged isolates of scrapie and used to screen potential drugs for their ability to inhibit replication of infectivity and production of PrPSc. However, these cells most likely do not represent those phagocytic cells involved in the initial uptake and replication of agent following peripheral infection. We have re-developed a persistently infected mouse cell line (SMB), cloned originally from a scrapie-infected mouse brain but of non-neuronal origin (Clarke, 1979 ; Clarke & Haig, 1970 ), which is highly phagocytic. We were interested to see whether a screening protocol could be set up to test for compounds that cure such phagocytes and if similar structure–activity relationships (SARs) were retained in comparison to their effects on infected cells of neuronal origin. We chose to investigate the SAR of CR on SMB cells because of its known molecular interactions with the normal and abnormal prion protein (Caughey et al., 1994 ; McBride et al., 1988 ; Wille et al., 1996 ), the availability of a limited SAR against scrapie-infected neuroblastoma cells (Demaimay et al., 1998 ) and its potential use as a lead compound for the development of therapeutics against other cerebro-vascular amyloidoses (Pollack et al., 1995a ; Shearman et al., 1994 ).

CR is a sulphonated azo dye commonly used in histopathology to stain the sparingly soluble, fibrillar deposits of protein (amyloid) found in many tissues as the by-product or cause of disease. CR-stained TSE amyloid was described in brain tissue of kuru-infected chimps (Beck et al., 1982 ) or scrapie-infected mice (Bruce & Fraser, 1975 ) long before the discovery of its molecular subunit, the abnormal prion protein (PrPSc). The crystal structure of a CR adduct of insulin fibrils has revealed a dye:peptide (1:2) complex (Turnell & Finch, 1992 ) that may be the structural basis of the fluorescent birefringence shown by this and other CR–amyloid complexes including CR–PrPSc under polarized light (Prusiner et al., 1983 ). The dye also interacts strongly with nucleotide-binding enzymes (Edwards & Woody, 1979 ) and is a very potent inhibitor of RNA polymerase (Liao et al., 1975 ) and the human immunodeficiency virus type 1 protease (Brinkworth & Fairlie, 1992 ). These factors may have originally suggested the use of CR as a TSE prophylactic. The mechanism by which CR inhibits replication of TSE agents and the accumulation of PrPSc in animals and infected neuroblastoma cell lines is not yet known, but may involve either an indirect perturbation of cellular levels or sites of PrPC or a direct interaction of CR with PrPSc. At high ionic strength or above 5 µM at physiological pH, the compound exists as a filamentous aggregate (Edwards & Woody, 1979 ), and it is in this form that it may stabilize the structure of PrPSc, preventing its involvement in further conversion of PrPC (Caspi et al., 1998 ; Demaimay et al., 1998 ). However, CR is unlikely to be a suitable drug candidate. It does not have sufficient selectivity to PrP, it cannot cross the blood–brain barrier and, when taken orally, it may be toxic, due to its breakdown to benzidine and its derivatives by microbes in the gastrointestinal tract (Bos et al., 1987 ; Gray & Ostby, 1993 ). We were interested to explore these SARs to see whether we could develop compounds based on CR that lacked these adverse properties. Therefore, we synthesized or purchased a number of analogues of CR and assayed them against persistently scrapie-infected SMB cells. Molecular modelling was also carried out to calculate the dihedral angle and the distance between the sulphonic acid groups of the analogues, and these data were incorporated into an SAR for their anti-scrapie effects.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Compounds.
Compounds III–VII, IX–XIII, XV, XVI and XIX–XXII (Figs 1 and 2 and Tables 1 and 2) were prepared by using the general methodology for synthesis of azo dyes (Zollinger, 1987 ). Details of their syntheses are to be published separately. Enquiries concerning the synthesis and structure of CR and analogues should be addressed to Ian Gilbert (fax +44 29 2087 4149; e-mail gilbertih@cardiff.ac.uk). The identity and purity of compounds was confirmed by NMR and mass spectrometry. CR and compounds I, II, VIII, XIV, XVII, XVIII and XXIII–XXVII were purchased from Sigma-Aldrich or Lancaster.



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Fig. 1. Structure of CR and its analogues. The generic structure can be seen as two identical terminator molecules attached via azo bonds to a linker moiety. Both the structure of the terminator and the linker geometry have been investigated in this study for their effects on inhibition of PrP-res formation in scrapie-infected cell cultures.

 


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Fig. 2. Polyaromatic polyanions unrelated to CR investigated for their anti-prion effects in cell culture.

 

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Table 1. Structures of azoic dyes and their fragments

 

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Table 2. Dihedral angles between phenyl groups and distances between sulphonate/carboxylate residues

 
{blacksquare} PrPSc assay.
The assay was adapted from the methods of Taraboulos et al. (1990) and Caughey & Raymond (1993) . SMB cells (Clarke & Haig, 1970 ) are persistently scrapie-infected non-neuronal cells. They were grown in tissue culture-treated plastic vessels (Becton-Dickinson) in Phenol Red-free medium 199 (Gibco BRL) containing 10% (v/v) newborn bovine serum and 5% (v/v) foetal calf serum (sera were heat-inactivated) at 33 °C under 5% CO2 in air at 95% relative humidity. To assess the effects of a drug, SMB cells were plated at 2x104 cells per well in 12-well cluster plates (~6x103 cells/cm2), 2 ml per well or, for Western blotting, they were plated at the same density in 60 mm Petri dishes, 4 ml per dish. After a minimum of 24 h (max. 72 h) to allow attachment to occur, the medium over the cells was replaced with fresh medium containing the drug. This medium was made by dilution from a 500-fold concentrate of the drug in DMSO; control wells always contained 0·2% (v/v) DMSO in standard growth medium. This concentration of DMSO had no effect on the synthesis of PrPSc (data not shown). Cells were extracted for assay 7 days after this first addition of drug; the growth medium (and drug or DMSO alone) was replaced on either day 3 or day 4.

For dot-blot analysis, cells growing in a 12-well cluster plate well were rinsed twice with PBS and then extracted with 0·2 ml lysis buffer (LyB) containing 10 mM Tris–HCl (pH 7·6), 100 mM NaCl, 10 mM EDTA, 0·5% (v/v) NP40 and 0·5% (w/v) sodium deoxycholate. The extract was centrifuged (1000 g, 5 min) and the post-nuclear supernatant was applied under vacuum to nitrocellulose membrane (0·45 µm pore; BA85, Schleicher & Schuell) via a 96-well dot-blot manifold. The membrane was removed from the apparatus and air-dried before processing in situ to discriminate PrPSc from PrPC. Briefly, the membranes were immersed in a 75 µg/ml solution of PK in 20 mM Tris–HCl-buffered saline (TBS) for 60 min at 37 °C. The proteolysis was stopped with 1 mM PMSF, and the membranes were then washed extensively with TBS and immersed in 3·0 M guanidine thiocyanate in TBS for 10 min at room temperature. This treatment denatures the residual PrPSc and exposes epitopes. After further extensive washing, the membrane was blocked in 5% (w/v) fat-free milk powder in PBS, processed with monoclonal anti-PrP antibody 6H4 (Prionics) at 0·8 µg/ml and developed by using ECL (Amersham Pharmacia Biotech). Relative quantification was made by normalizing loading of the blot to cellular protein in the post-nuclear extract, measured by detergent-compatible Lowry assay (Bio-Rad) with BSA as the standard, and densitometric analysis of multiple non-saturating films. The results were normalized to a fixed number of control SMB cells.

For Western blot analysis, cells growing in a 60 mm dish were rinsed twice with PBS and then scraped from the plate in 0·2 ml LyB. Nuclei and cytoskeletal remnants were removed by centrifugation at 2000 g for 5 min. To separate detergent-insoluble PrPSc complexes from the detergent-soluble PrPC fraction, the post-nuclear extract in LyB was incubated with 25 µg/ml PK for 60 min at 37 °C, which was then inhibited by adding PMSF to 1 mM. It was then diluted to 3·0 ml with LyB and centrifuged in a conical polyallomer tube (294000 g, 10 °C, 45 min) in an SW50.1 rotor (Beckman). The PrPSc-containing pellet was resuspended in LyB by cup-horn sonication, transferred to a clean tube and re-centrifuged under the same conditions. Pelleted proteins were dissolved by heating at 100 °C for 5 min in an electrophoresis sample buffer containing 4% (w/v) SDS and 5% (v/v) 2-mercaptoethanol (Laemmli, 1970 ). Proteins were separated on 15% polyacrylamide gels in Tris–glycine buffer and transferred electrophoretically to PVDF membranes (Immobilon P, Millipore) submerged in 25 mM Tris, 192 mM glycine and 0·05% SDS (Towbin et al., 1979 ). The membrane was blocked and processed to reveal PrPSc exactly as described for the dot-blot method (above).

{blacksquare} Molecular modelling.
Molecular modelling was carried out on O2 Silicon Graphics workstations using Macromodel 6.0. The structures were drawn in the ‘draw’ mode with the double bond in the trans configuration and sulphonate and carboxylate groups represented in the anionic form. Compounds were then minimized by using the Amber forcefield. The GB/SA solvation model for water as solvent was applied. Monte-Carlo minimization was then carried out. Either 500 or 1000 cycles were carried out for each compound, followed by minimization. The dihedral angles between the aromatic rings of the biphenyl group and the distances between the sulphonate or carboxylate groups were measured where appropriate. In some compounds, there were more than two sulphonate groups. In these cases, distances that diverged significantly from the commonly observed 20  were ignored.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
SAR studies
CR is a symmetrical molecule. Therefore, various fragments and analogues of just one half of the molecule were prepared (Fig. 1, Table 1: compounds I–V). None of these compounds showed activity at 1 µM (Fig. 3). However, compounds I, III, IV and V showed inhibition of PrP-res in SMB cells at 100 µM, albeit with much lower potency than CR. Compound I is a fragment, whilst compounds III, IV and V represent the half molecule or analogues of the half molecule of CR. This suggests that the whole molecule of CR, a linker and two terminator moieties (Fig. 1), is required for its anti-scrapie activity.



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Fig. 3. Relative potency of azoic dye fragments and CR in SMB cell assay. Compounds were tested at two concentrations; 1 µM (hatched bars) and 100 µM (solid bars). For the structures of compounds I–V, see Fig. 1 and Table 1. The measurements of PrP-res were made by dot-blot analysis and represent means±SD of at least two experiments, in which each concentration was tested in triplicate.

 
The central biphenyl group of CR was modified by substitutions at the 3,3' position of the biphenyl ring system (VI–VIII) (Figs 1 and 4, Table 1). These compounds had similar activity to CR at 100 µM. However, at 1 µM, the compounds were much less active, especially the dichloro derivative, compound VI, which paradoxically gave about twice the PrP-res level of the control. Some analogues were also tested in which the biphenyl spacer was replaced with a biphenylsulphone (compounds IX and X). These compounds showed similar activity to compounds VI–VII. Compound IX, the ‘para’-substituted biphenylsulphone compound, appeared to be slightly more active than the ‘meta’-substituted analogue, X, at 100 µM (Fig. 4). Removal of the naphthyl ring (Figs 1 and 4, Table 1: XI) gave reduced activity compared with the parent compound, IX, at 100 µM.



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Fig. 4. Relative potency of CR and analogues in SMB cell assay. Compounds were tested at two concentrations; 1 µM (hatched bars) and 100 µM (solid bars). For the structures of compounds VI–XXII, see Fig. 1 and Table 1. The measurements of PrP-res were made by dot-blot analysis and represent means±SD of at least two experiments, in which each concentration was tested in triplicate.

 
Several modifications to the naphthyl ring were undertaken (Figs 1 and 4, Table 2: XII–XVIII). Replacement of the naphthyl amino group with a hydroxyl group (XIII and XIV) or its acylation with trifluoroacetamide (XII) failed to elicit a significant increase in activity compared with the parent compounds at 100 µM (XII compared with CR; XIII with CR; XIV with VII). However, at 1 µM, there was again a paradoxical increase in PrP-res levels compared with control values. In compound XV, the position of the sulphonate group was adjusted slightly and an extra amino group was introduced. The activity remained very similar to that of the parent compounds XIV and CR. Similarly, increasing the level of sulphonation (compounds XVI–XVII) had little effect on the activity at 100 µM, although at 1 µM, these compounds appeared to stimulate PrP-res formation. The sulphonic acid group is mainly responsible for the highly ionic character of CR and for the low permeability of the cell membrane and blood–brain barrier to this compound. Structure–activity studies on this charged moiety should produce a compound with better pharmacokinetic properties. In particular, it has been found, in the case of Alzheimer’s disease models, that chrysamine G (XIX) shows similar or better anti-amyloid properties to CR, whilst showing improved uptake across the blood–brain barrier (Klunk et al., 1994 ). In chrysamine G, the sulphonic acid group is replaced by a carboxylic acid. Thus, compounds XIX, XX, XXI and XXII (Figs 1 and 4, Table 1) were tested as the chrysamine analogues of CR, VII, IX and X, respectively. Compound XIX showed similar activity to the parent at 100 µM but reduced activity at 1 µM. Compound XX showed greater activity than VII at 100 µM. In contrast, compounds XXI and XXII showed relatively ‘flat’ dose responses, with similar, low activities at both 1 and 100 µM.

We also included a series of more complex organic aromatic compounds in this analysis that were selected on the basis of their structures and their anti-amyloid activity in other disease models (Bandiera et al., 1997 ; Pollack et al., 1995b ) (Fig. 2). Compounds XXIII (Direct Red 75), XXIV (Sirius Red or Direct Red 80) and XXV (Calcion) showed good activity at 100 µM (Fig. 5). Compound XXIV (Sirius Red) was clearly the most active compound, even at a concentration of 1 µM (Fig. 5). This can also be seen on the dose–response curve (Fig. 6). The reason for this is unclear, but the extra degree of sulphonation of this compound is not the only factor contributing to its extra potency, as similarly sulphonated derivatives (e.g. XXV) were less active (Fig. 5). Compound XXVII (Mordant brown) has only one sulphonate group and is relatively inactive.



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Fig. 5. Relative potency of CR and analogues in SMB cell assay. Compounds were tested at two concentrations; 1 µM (hatched bars) and 100 µM (solid bars). For the structures of compounds XXIII–XXVII, see Fig. 1 and Table 1. The measurements of PrP-res were made by dot blot analysis and represent the means±SD of at least two experiments, in which each concentration was tested in triplicate.

 


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Fig. 6. (a) Dose–response curves of the effects of CR and related molecules on the formation of PrP-res in cultures of SMB cells. CR (•), VI ({circ}), IX ({square}), XII ({triangleup}), chrysamine G (XIX, {bigtriangledown}), XXIV ({lozenge}) were tested. The measurements of PrP-res were made by dot blot analysis and represent the means±SD of at least two experiments, in which each concentration was tested in triplicate. (b) The curves shown in (a) were additionally validated for two of these compounds, CR and IX, by Western blot analysis (n=2). Analysis of the effect of CR on SMB cell PrP-res content is shown. Lanes: 1, SMB cells alone; 2–7, SMB cells following treatment with CR at 10 µM (lane 2), 1 µM (3), 0·1 µM (4), 10 nM (5), 1 nM (6) or 0·1 nM (7). The symbols on the right indicate the positions of diglycosyl PrP-res ({blacksquare}), monoglycosyl PrP-res ({blacktriangleup}) and aglycosyl PrP-res (*).

 
Dose–response curves
Several of the compounds showed PrP-res levels higher than the control at 1 µM. Therefore, dose–response curves were generated for a number of compounds: CR, VI, IX, XII and XXIV (Fig. 6). Essentially, the data for CR, VI, XII and XXIV (Fig. 6) showed that, at high concentrations of inhibitor, PrP-res levels were depressed. As the concentration of compound was decreased, the levels of PrP-res increased and actually rose above the control level. The concentration of compound at which this occurred and the magnitude of the increase depended on the structure of the compound. As the concentration of compound was decreased further, the levels fell back to control. In contrast, a different dose–response curve was seen with compounds IX and XIX. In this case, as the concentration of compound was lowered, the PrP-res levels increased until they reached the control levels. They did not then increase further, but stayed at control levels as the concentration of compound was decreased further.

Modelling studies
Previous studies have correlated the inhibitory activity on PrP-res formation in cells and cell-free systems of a limited number of benzidine-based dyes with the dihedral angle between the two rings in the biphenyl ring of these compounds (Demaimay et al., 1998 ). Maximum potency was observed when this dihedral angle was about 35° (i.e. the biphenyl ring system should be as near to planar as possible) (Demaimay et al., 1998 ). CR, chrysamine G (XIX) and other azo dyes also bind to amyloid fibrils of the Alzheimer’s disease A{beta}(10-43) peptide. Modelling studies predicted a similar preferred conformation (Klunk et al., 1994 ). We therefore undertook modelling studies to determine the dihedral angle of our compounds and other structural parameters that might be important in determining activity, such as the intra-molecular distance between their sulphonic acid or carboxylate groups (Table 2). Most of the compounds analysed were measured with bond angles around 40° (Table 2). No significant structure–activity relationship could be determined from our data. Most of the distances measured between sulphonate or carboxylate groups were around 20 . However, for some, much shorter distances were measured: X, 8·3 ; XXII, 6·6 . These latter compounds showed slightly lower activity but, in general, among the other compounds there was no clear relationship between distance and activity.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
We have established the scrapie-infected SMB cell line as a tool for screening compounds for anti-scrapie activity and developed a structure–activity profile for CR, a compound with known effects in vivo on scrapie pathogenesis. This molecule is symmetrical and consists of two identical amino-substituted naphthalene sulphonates connected by azo linkages to a central biphenyl moiety. However, 1-amino-4-naphthalene sulphonic acid (I), 1-hydroxy-6-amino-3-naphthalene sulphonic acid (II) and the half molecule of CR (III) were ineffective as inhibitors of formation of PrP-res in SMB cells. The inhibitory effectiveness of this dye may therefore depend on the relative orientation and spacing of these naphthalene sulphonates in the complete molecule, as determined by the biphenyl spacer and its derivatives. However, modelling the dihedral angle between the phenyl groups and the distances between sulphonates/carboxylates on the naphthalate side-arms of our novel compounds did not provide further insights into a minimal structure for active molecules, but confirmed previous observations that planarity of the central biphenyl moiety or rotational freedom about the biphenyl bond were needed for inhibitory activity (Demaimay et al., 1998 ).

Dose–response curves for CR and compounds VI, XII and XXIV, the sulphonic acid derivatives, showed that, at high drug concentrations, the levels of PrP-res in SMB cells were suppressed compared with control levels. However, as the drug concentration was decreased, PrP-res levels rose above control levels. As the concentration of compound was decreased further, by definition, the PrP-res levels returned to control levels (Fig. 6). Carboxylate derivatives did not display these features (Fig. 6, compounds IX and XIX). This potentiation effect at low concentrations of compounds VI, XII and XXIV may be related to a phenomenon noted by Caspi et al. (1998) . They found that CR inhibited new PrPSc synthesis (or, at least, the incorporation of aglycosyl PrP into PrPSc) and old PrPSc degradation in scrapie-infected neuroblastoma cells. Alternatively, these compounds, as monomers at low concentrations, may stimulate PrPSc formation due to binding to PrPC at just one site, while at higher concentrations in physiological salt, they may form a supramolecular ligand, a liquid crystal, which sequesters PrPSc and/or PrPC, preventing their interaction (Skowronek et al., 1998 ; Stopa et al., 1998 ). The structure of PrPSc has been likened to the molten globule state of protein folding (Safar et al., 1994 ). This increases the complexity of possible CR–PrP interactions, as the liquid crystal form of this dye can bind to and stabilize intermediates in the transition of other proteins, such as the immunoglobulins, from native to molten globule state (Piekarska et al., 1996 ). It will be of interest to see whether the propensity of our different analogues to form liquid crystals under physiological conditions of pH and salt concentration matches their inhibitory activity on PrP-res accumulation in SMB cells. There is also a clear need to investigate the possible potentiation effects of these drugs in vivo because of the adverse implications such properties would have for their prophylactic benefit.

Our interest in CR was stimulated by its proven effects in vivo on extending the survival time of hamsters infected intraperitoneally with two different strains of scrapie (Ingrosso et al., 1995 ). If its effectiveness could be increased or widened to include oral prophylaxis, a major problem in the control and management of animal and human TSEs could be overcome. One obvious drawback to the use of CR is its toxicity. Embryopathic and teratogenic effects of CR have been observed in mice following oral or intraperitoneal administration of the dye (Gray et al., 1983 , 1992 ), and azo dyes based on benzidine have been implicated for over 100 years in the increased incidence of urinary bladder cancer amongst textile workers. Although the dyes are absorbed poorly by the gut, their metabolites, benzidene and other aromatic amines formed by reductive cleavage of the azo bonds by intestinal bacteria, are absorbed rapidly, and multiple processing steps in the liver, blood, kidney and bladder are implicated in its induction of bladder cancer (Zenser et al., 1998 ). We have shown that it is possible to replace the carcinogenic benzidine moiety in azo dyes and still retain activity against the conversion of cellular PrP to PrPSc. Similarly, carboxylate derivatives retained significant activity, as seen by comparison of CR and XIX (Fig. 4), and compounds VII and XX. This is encouraging, as this substitution is likely to improve the cellular and blood–brain barrier permeability of the compounds. Future work eliminating the azo bonds and disrupting the central biphenyl moiety will be aimed at further reducing the adverse effects of these dyes. The SMB cell line described here will provide a useful tool for screening the next generation of CR-based compounds for their ability to inhibit the conversion of PrPC to PrPSc.


   Acknowledgments
 
We would like to acknowledge the MRC for financial support (H.R., S.V.), the EPSRC Mass Spectrometry Service Centre at Swansea for mass spectra, Cardiff Centre for Molecular Modelling for use of the computing facilities and Dr Michael Knaggs for advice on carrying out the modelling.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 22 September 1999; accepted 30 November 1999.