| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA
2 Department of Neurology, University of California San Francisco, San Francisco, CA, USA
3 Institute for Neurodegenerative Diseases, University of California San Francisco, San Francisco, CA, USA
4 Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA
Correspondence
Barnaby C. H. May
bmay{at}ind.ucsf.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Supplementary material is available in JGV Online.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Whilst PrPC is soluble, largely 
-helical and monomeric, PrPSc is insoluble, 
-rich and oligomeric. Prion diseases can arise through (i) transmission of infectious PrPSc, (ii) inheritance of some mutations in the prion protein gene (Prnp) or (iii) sporadic, spontaneous molecular events. Drug development for prion disease has been hampered by a lack of structural resolution of PrPSc and an understanding of the cellular mechanisms involved in the misfolding pathway. However, drug-discovery efforts have moved forward by using biochemical, cellular and animal models of prion replication. Several classes of compound have been identified that reduce the level of PrPSc in cultured cells (Trevitt & Collinge, 2006). The majority of prototypic anti-prion compounds described to date have come from phenotypic-screening efforts and, in some cases, the mode of action of these compounds is poorly understood.
Given the current understanding of the mechanisms involved in prion replication, several approaches to intervention could be imagined and exploited for drug discovery (Fig. 1). Strategies include identifying compounds that: (1.) increase the stability of PrPC or reduce the availability of the substrate, either at the level of PrP gene expression or through changes in cellular localization and/or trafficking; (2.) reduce the availability or involvement of cellular factors that chaperone conversion of PrPC; (3.) act on PrPSc to occlude available sites for templating PrPC; (4.) stabilize higher-order PrPSc oligomers and thus inhibit the necessary generation of lower-order oligomeric templates; and (5.) accelerate PrPSc clearance by decreasing the stability of the infectious isoform or stimulating natural clearance pathways.
|
) was described previously as being effective at reducing PrPSc in a cell-based model of prion replication. The concentration of quinacrine required to reduce the amount of PrPSc by 50 % (EC50), relative to untreated control ScN2a cells, was 300 nM (Doh-Ura et al., 2000). Structure–activity relationship (SAR) studies helped to define quinacrine as a prototype for a family of 9-aminoacridine anti-prion compounds (Korth et al., 2001; Ryou et al., 2003; May et al., 2006a). Subsequently, dimeric bis-acridine compounds (Fig. 2a) were identified as a potent class of 9-aminoacridine compound (May et al., 2003). The protective effect of quinacrine administration to animals inoculated experimentally with prions has yet to be clearly demonstrated (Collins et al., 2002; Barret et al., 2003; Doh-ura et al., 2004; Ryou et al., 2004). The clinical use of quinacrine for over half a century for the treatment of malaria and giardiasis prompted an examination of its effectiveness in the treatment of human prion disease.
|
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) was prepared in house and goat anti-human IgG Fab horseradish peroxidase conjugate was purchased from Fisher Scientific.
Compound synthesis.
The required spermidine-linked bis-acridine compound 1,4-butanediamine-N-(6-chloro-2-methoxy-9-acridinyl)-N'-{3-[(6-chloro-2-methoxy-9-acridinyl)amino]propyl}-(9-chloro) was prepared as described previously by reacting spermidine with 9,6-dicholoro-3-methoxyacridine (Girault et al., 2000). The purified compound was characterized by 1H NMR and LCMS prior to use.
Affinity-matrix synthesis.
Freeze-dried, epoxy-activated Sepharose 6B beads (100 mg, 10 µmol reactive epoxide) were suspended in deionized H2O for 30 min to swell the matrix to a bead-bed volume of approximately 350 µl. The resulting slurry was washed for 30 min with deionized H2O (3x200 ml) and filtered. The bis-acridine (40 mg, 6.3 µmol) was dissolved in 50 % N,N-dimethylformamide/0.1 M Na2CO3 (40 ml) and then added to the swollen beads (350 µl). Coupling proceeded overnight at 50 °C with vigorous shaking. The bead bed was drained and washed with 50 % N,N-dimethylformamide/0.1 M Na2CO3 (3x50 ml). An aqueous solution of 1 M ethanolamine (10 ml, pH 8.0) was added to the drained beads and reacted at 45 °C for 16 h to cap unreacted epoxy functional groups. The bead bed was drained and washed with H2O (3x50 ml) to yield the bis-acridine functionalized bead BA (Fig. 2b
) that was stored in 0.1 M NaCl buffer at 4 °C.
To generate the control matrix, Co, (Fig. 2b), epoxy-activated Sepharose 6B beads (100 mg, 10 µmol) were swollen, rinsed, then incubated with 1 M aqueous ethanolamine (10 ml, pH 8.0) for 16 h at 45 °C, washed with H2O (3x50 ml) and stored in 0.1 M NaCl at 4 °C until use.
Preparation of scrapie-infected cell lysate.
Neuroblastoma (N2a) cells were infected with the Rocky Mountain Laboratory (RML) strain of mouse-adapted scrapie prions and subcloned as described previously (Butler et al., 1988). Both scrapie-infected (ScN2a) and non-infected (N2a) cells were maintained at 37 °C in MEM supplemented with 10 % FBS and 1 % GlutaMAX. A 100 mmM plate of ScN2a or N2a cells was grown to 95 % confluence and 1 ml cold lysis buffer (10 mM Tris/HCl, pH 8.0; 150 mM NaCl; 0.5 % Nonidet P-40; 0.5 % sodium deoxycholate) was added. After 5 min, the nuclear pellet was removed and the cell lysate was transferred. The protein concentration was determined by using a Bio-Rad BCA protein assay kit prior to use. Cell lysates were adjusted to 5 mM dithiothreitol (DTT) and a 1 : 500 dilution of protease inhibitors was added [leupeptin (1 mg ml–1), aprotinin (1 mg ml–1), PMSF (100 mM)]. The final NaCl concentration was adjusted to 1 M by the addition of solid NaCl [50 mg (ml cell lysate)–1].
Preparation of prion-infected brain homogenate.
Animals (n=4 or more) were inoculated with prions as described by Chandler (1961). Animals were monitored for clinical signs of neurological dysfunction as described previously (Carlson et al., 1986). Terminal animals were sacrificed and whole brains were collected. Brain homogenates (10 %, w/v) were prepared at 4 °C in lysis buffer (10 mM Tris/HCl, pH 8.0; 150 mM NaCl; 0.5 % Nonidet P-40; 0.5 % sodium deoxycholate) in a FastPrep FP120 (BIO 101) tissue disruptor (3x45 s). The suspension was clarified by centrifugation at 3000 g for 10 min at 4 °C. The supernatant was removed and adjusted to 5 mM DTT, and a 1 : 500 dilution of protease inhibitors was added [leupeptin (1 mg ml–1), aprotinin (1 mg ml–1), PMSF (100 mM)]. The final NaCl concentration was adjusted to 1 M by the addition of solid NaCl [50 mg (ml brain homogenate)–1].
Preparation of PTA-precipitated Syrian hamster brain homogenate.
wt Syrian hamsters were inoculated with Sc237 hamster prions and sacrificed at the onset of disease. Whole brains were collected (approx. 1 g) and homogenized in PBS (9 ml) in a FastPrep FP120 (BIO 101) tissue disruptor (3x45 s) to give 10 % (w/v) brain homogenates. The sample was diluted with 4 % Sarkosyl in 10 ml PBS to give a 5 % (w/v) brain homogenate and then treated with 25 mg PK ml–1 at 37 °C for 1 h with shaking (350 r.p.m.). PK was inhibited by the addition of 100 mM PMSF (1 : 500 dilution). An aliquot (1.8 ml) of the PK-digested sample was transferred and clarified (500 g, 5 min, room temperature). The supernatant (1.2 ml) was transferred and sodium PTA (38 ml of a 10 % aqueous PTA solution, pH 7.4; 85 mM MgCl2) was added. The sample was incubated at 37 °C for 16 h with shaking (350 r.p.m.). PK-digested PrPSc was pelleted from the sample (14 000 g, 30 min, room temperature), the supernatant was discarded and the pellet was resuspended in 0.2 % Sarkosyl in PBS (40 ml). Several aliquots were prepared and pooled prior to dialysis to provide sufficient material (approx. 400 µl). The samples were dialysed against 500 ml dialysis buffer (10 mM sodium EDTA, pH 7.4; 0.1 % Sarkosyl; 1 mM NaN3) for 48 h, with a change of dialysis buffer after 24 h. Dialysis cartridges (Pierce) with a 3500 Da molecular mass cut-off were used. Following dialysis, the sample was diluted to 1 ml with 0.2 % Sarkosyl in PBS. Protease inhibitors were added [1 : 500, leupeptin (1 mg ml–1), aprotinin (1 mg ml–1), PMSF (100 mM)] and the final NaCl concentration was adjusted to 1 M by the addition of solid NaCl.
Affinity-purification assay.
In a typical experiment, ligated matrices (20 µl bead-bed volume) were incubated with either cell lysate or 10 % brain homogenate (1 ml) and mixed at 4 °C for 16 h. The sample was transferred onto a mini-spin column (Pierce Handee Mini-Spin Column) and beads were collected by centrifugation. The beads were washed successively with lysis buffer (1x400 µl), 1 M aqueous NaCl (3x400 µl) and lysis buffer (1x400 µl). Washing was aided by the use of a mini-spin column, whereby wash buffer was added, the sample was vortexed for 5 s, then beads were collected by centrifugation.
Immunoblotting.
To the matrices (BA or Co, 20 µl), 20 µl SDS loading buffer (0.125 M Tris/HCl, pH 6.8; 10 % 2-mercaptoethanol; 20 % glycerol; 4 % SDS) was added and the mixture was heated at 100 °C for 5 min. Matrix beads and loading buffer were loaded on a precast Bio-Rad Criterion gel (12.5 % acrylamide) and proteins were resolved and transferred to an Immobulin-P membrane (Millipore) as described previously (May et al., 2003). Proteins were visualized by using the recombinant Fab fragments D13, secondary horseradish peroxidase-labelled antibody and an enhanced chemiluminescence (ECL) development system (Amersham Biosciences). Densitometry was performed with the National Institutes of Health ImageJ software, computing at least three independent experiments. Total proteins were visualized by silver staining the intact gel using a Bio-Rad Silver Stain Plus kit according to the manufacturer’s instructions.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
), was prepared by reacting a spermidine-linked bis-acridine compound with epoxy-activated Sepharose 6B solid-phase beads under basic conditions. The bis-acridine parent compound, 1,4-butanediamine-N-(6-chloro-2-methoxy-9-acridinyl)-N'-{3-[(6-chloro-2-methoxy-9-acridinyl)amino]propyl}-(9-chloro), was bioactive at nanomolar concentrations in the ScN2a cell model of prion replication (May et al., 2003). A second matrix was prepared similarly by reacting ethanolamine with epoxide-activated Sepharose 6B beads to yield a ‘non-reactive’ control matrix, Co (Fig. 2b).
Cell lysates were prepared from ScN2a cells and incubated with either the BA or Co matrix. Matrices were washed extensively and captured proteins were resolved by gel electrophoresis. Probing a Western blot of proteins bound by the BA matrix with the anti-prion antibody D13 (Williamson et al., 1998) revealed PrP-immunoreactive proteins of the appropriate molecular mass (27–30 kDa) (Fig. 2c, lane 3). The Co matrix failed to bind PrP-immunoreactive material under the conditions employed (Fig. 2c, lane 2). Whilst protease resistance is not required for prion disease (Nazor et al., 2005), it is a hallmark of the infectious isoform, PrPSc, derived from ScN2a cells (Butler et al., 1988). Limited PK digestion of an ScN2a lysate truncates PrPSc to yield a protease-resistant core, denoted PrP 27–30 (Oesch et al., 1985). Proteins bound by the 9-aminoacridine ligands were digested with PK under standard conditions while remaining bound to the BA matrix. Following the ‘on-bead’ PK digestion and extensive washing of the BA matrix, PK-resistant PrP 27–30 was detected (Fig. 2c, lane 6). To investigate the isoform selectivity of 9-aminoacridine binding, BA and Co matrices were incubated with a cell lysate derived from non-infected neuroblastoma cells (N2a). PrPC was not detected bound to the BA matrix (Fig. 2c, lane 9), even when a large excess of total cellular proteins was used (Fig. 2c, lane 10).
We correlated the bioactivity of various structurally related compounds in ScN2a cells with the efficiency of affinity purification of PrPSc using these same compounds. Quinacrine and the related compounds azacrine, chlorpromazine and chloroquine were assayed for bioactivity in ScN2a cells by using a procedure established by one of us (May et al., 2006b). The bioactivity of the 9-aminoacridine compound quinacrine (EC50=0.9±0.1 µM) was approximately four- to eightfold higher those of the structurally related compounds (EC50=3.5–7.8 µM) (see Supplementary Table S1, available in JGV Online). Separately, we prepared Sepharose matrices coupled to synthetic analogues of the parent compounds quinacrine, azacrine, chlorpromazine and chloroquine (see Supplementary Scheme S1, available in JGV Online). The matrices (QC, AC, CP and CQ; Supplementary Table S1) were incubated with ScN2a lysates, and bound PrPSc was quantified by Western blotting and densitometry. The relative amount of PrPSc captured by the quinacrine-ligated matrix was approximately fivefold greater than for the other ligated matrices (see Supplementary Table S1, available in JGV Online). Thus, the affinity of the various related heterocyclic ligands for PrPSc correlates well with bioactivity in ScN2a cells. If the mechanism of action of this class of related compounds is via binding to PrPSc, it would be expected that ScN2a cell bioactivity and ligand affinity for PrPSc would correlate.
9-Aminoacridine compounds bind misfolded PrP from prion-diseased animals
We sought to determine whether the acridine-functionalized matrix could selectively bind PrPSc from prion-infected animals. wt CD1 mice were inoculated with the mouse-adapted prion strain RML and sacrificed at the onset of prion disease. Brain homogenates were prepared from these ill animals (Fig. 3a, b, lanes 4–6), whilst brain homogenates from uninoculated animals served as controls (Fig. 3a, b, lanes 1–3). Both Co and BA matrices failed to precipitate PrPC from non-infected animals (Fig. 3a, lanes 2 and 3). In contrast, the BA matrix precipitated PrPSc from prion-infected brain homogenates at approximately fourfold the level found in the Co matrix (Fig. 3a, lane 5), as determined by densitometry. Silver staining of SDS-PAGE-resolved proteins revealed that the BA matrix also enriches for other cellular proteins compared with the Co matrix (Fig. 3b, compare lanes 2 and 3), demonstrating that 9-aminoacridine binding is not wholly selective for PrPSc. The Co matrix was essentially free of non-specifically bound proteins, as judged by silver stain (Fig. 3b, lanes 2 and 5).
|
). Affinity purification of a Tg4053 homogenate using either the Co or the BA matrix did not isolate substantial amounts of PrPC (Fig. 4a, lanes 2 and 3, respectively), even in the presence of a large excess of PrPC (approx. 900 µg PrPC per 20 µl BA matrix; data not shown) derived from the Tg overexpression.
|
23–88,141–176) (Supattapone et al., 1999) or MoPrP(23–88) (Supattapone et al., 2001)] after RML inoculation (Fig. 4b, lanes 3 and 9). We also prepared brain homogenates from B6.I mice (Carlson et al., 1994) inoculated with mouse-passaged bovine spongiform encephalopathy prions (301V) (Farquhar et al., 1996) (Fig. 4b, lane 6), spontaneously ill Tg mice bearing the Gerstmann–Sträussler–Scheinker mutation [Tg(MoPrP,P101L)2866] (Telling et al., 1996) (Fig. 4b, lane 15) and Syrian hamsters inoculated with Sc237 hamster prions (Marsh & Kimberlin, 1975) (Fig. 4b, lane 12). The BA matrix readily precipitated PrPSc from all samples. The PrP-immunoreactive fraction precipitated from Syrian hamster brain homogenate included higher-molecular-mass and presumably oligomeric PrPSc (Fig. 4b, lane 12).
9-Aminoacridine–PrP binding is unlikely to be mediated by additional proteins or polynucleic acids
We sought to investigate the involvement of other proteins in the observed 9-aminoacridine–PrPSc interaction. If additional proteins mediate the observed acridine–PrPSc binding, it would be expected that protease digestion of a scrapie-infected lysate would result in loss of the observed binding between the 9-aminoacridine matrix and PrPSc. Thus, a brain homogenate prepared from an ill Syrian hamster was digested with PK under standard conditions and incubated with sodium PTA to selectively precipitate PrP 27–30 (Lee et al., 2005). The sample was dialysed to remove PTA and peptide fragments, and incubated with BA and Co matrices. Only the BA matrix precipitated PrP 27–30 (Fig. 5a, lane 3). This result suggests that additional proteins are not involved in the 9-aminoacridine–PrPSc interaction. However, we cannot rule out the involvement of low-abundance proteins that are protected from protease digestion by their close association with PrPSc.
|
, compare lanes 2 and 3). Subsequently, benzonase-treated ScN2a cell lysates were incubated with BA and Co matrices. In the absence of polynucleic acids, the BA matrix was able to precipitate PrP (Fig. 5c, lane 5). It was also possible to perform an ‘on-bead’ digestion by treating BA matrix-bound proteins with benzonase at 20 U ml–1. No loss in PrP immunoreactivity was observed by Western blot of the precipitated proteins (Fig. 5c, lane 6). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Given the existing SAR of this class (Korth et al., 2001; May et al., 2003, 2006a), we believed that this attachment strategy would permit binding to one or more cellular 9-aminoacridine receptors and subsequent purification of the receptor(s).
A major hurdle to successful target identification by affinity purification is the need to overcome non-specific binding of cellular macromolecules to the solid-phase matrix (Burdine & Kodadek, 2004). By using the control matrix (Co), we identified conditions that gave negligible non-specific binding of proteins to the Co matrix, as judged by silver staining after SDS-PAGE (Fig. 3b, lanes 2 and 5). The isotonic strengths of the cell lysate and lysis detergent were found to be important determinants of non-specific binding to the Co matrix (see Supplementary Fig. S1, available in JGV Online).
Given the amenability of the ScN2a cell model to the study of prion replication and the fact that 9-aminoacridine compounds are effective at reducing PrPSc in ScN2a cells, we chose to use this system initially for our mechanistic studies. Additionally, given their central involvement with prion disease, we initially focused on the affinity purification of the PrP isoforms by the BA matrix. Our results suggested that 9-aminoacridine compounds presented on a multivalent scaffold have a higher affinity for PrPSc than for PrPC. These findings were extended to the affinity purification of PrPSc from brain homogenates prepared from prion-diseased animals. Affinity purification of PrPSc from brain homogenates prepared from Tg(MoPrP,23–88,141–176) and Tg(MoPrP,23–88) mice expressing truncated PrP ruled out the existence of relevant acridine-binding epitopes at the N terminus (residues 23–88) and between residues 141 and 176 (Fig. 4b, lanes 3 and 9).
PrP is known to interact with a number of cellular factors, making it possible for 9-aminoacridine–PrP binding to be either direct (9-aminoacridine–PrP) or indirect (9-aminoacridine–X–PrP), where binding is mediated by a cellular component (X). Identifying additional binding components is of importance, as these molecules would be of therapeutic and biological relevance to prion disease. Macromolecules are known to bind PrP, including neural cell-adhesion molecules, glucosaminoglycans, nucleic acids, plasminogen and the laminin receptor (Lee et al., 2003). Whilst the role of these molecules in prion replication and disease is not clearly defined, the possibility exists that one or more of these molecules mediates the observed binding interaction between 9-aminoacridine ligands and PrPSc (9-aminoacridine–X–PrPSc). Additionally, 9-aminoacridine compounds are relatively promiscuous ligands. Receptors of quinacrine or related 9-aminoacridine compounds include nicotinic acetylcholine receptor (Spitzmaul et al., 2001), DNA (Gaugain et al., 1981), diamine oxidase (Ma & Sourkes, 1980) and phospholipase A2 (Mustonen et al., 1998). Other proteins, in addition to PrP, were shown to bind the 9-aminoacridine matrix (Fig. 3b). Future studies aim to identify these proteins by using suitable mass-spectroscopy techniques.
To investigate the possible involvement of proteins in the observed 9-aminoacridine–PrPSc binding, we sought to eliminate additional proteins during affinity purification of PrPSc. An ‘on-bead’ digestion of 9-aminoacridine-captured proteins failed to reduce the amount of bound PrPSc significantly (Fig. 2c). However, because it is possible that the solid-phase affinity matrix, or bound PrPSc, could provide protection to coordinated proteins from protease digestion, we attempted to digest a scrapie-infected cell lysate with PK prior to affinity purification. Protease pretreatment of the lysate prior to affinity purification resulted in substantial non-specific binding to the Co matrix, presumably resulting from the presence of low-molecular-mass peptide fragments (data not shown). To circumvent the non-specific binding, we selectively precipitated PrP 27–30 following PK digestion (Fig. 5a). Binding of the 9-aminoacridine matrix to PrPSc in the absence of additional cellular proteins was not reduced, suggesting that additional proteins did not mediate 9-aminoacridine–PrPSc binding.
Although not required for prion conversion or disease (Safar et al., 2005a), nucleic acids are known to associate with PrP (Deleault et al., 2003). Anti-DNA antibodies have been used to immunoprecipitate PrPSc from prion-infected brain homogenates, presumably as they immunoreact with nucleic acids present in PrP complexes (Zou et al., 2004). The binding of 9-aminoacridine and bis-acridine compounds to polynucleotides is well characterized and responsible for the observed cytotoxicity of certain acridine compounds (Le Pecq et al., 1975). Thus, we sought to address whether the observed binding of the BA matrix to PrPSc was mediated by polynucleotides. As benzonase digestion did not reduce the amount of PrPSc precipitated by the acridine-ligated matrix, we conclude that nucleic acids do not participate in the 9-aminoacridine–PrPSc complex.
PrPs are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) moiety (Stahl et al., 1987) and reside in caveolae-like domains of lipid rafts (Prusiner, 2004). Quinacrine and related acridine compounds are lipidophilic and are known to associate with lipid membranes (Dise et al., 1982; Pajeva et al., 1996). This complementary lipid binding allows for the possibility that the observed binding of PrPSc to the BA matrix was lipid-mediated. The use of NP-40-based cell lysates reduces the likelihood that intact lipid membranes mediate the observed 9-aminoacridine–PrPSc binding. In addition, the failure of the BA matrix to capture the GPI-anchored protein Thy-1 (Madore et al., 1999) provides further evidence against lipid-mediated 9-aminoacridine–PrPSc binding (see Supplementary Fig. S2, available in JGV Online).
Considerable effort has been devoted to developing diagnostic reagents for the detection of PrPSc isoforms (Castilla et al., 2005; Safar et al., 2005b). These reagents have immediate application in protecting the blood supply from prion contamination and for early diagnosis of prion diseases. Whilst 9-aminoacridine compounds precipitate PrPSc selectively, the low efficiency of this process precludes their current application in prion detection. We found that our current bis-acridine-ligated matrix recovers only approximately 5 % of the PrPSc from scrapie-infected cell lysates (Fig. 6). This low recovery of PrPSc by the BA matrix might be attributed either to low PrPSc–BA matrix binding efficiency or to the fact that only a small fraction of the total PrPSc possesses conformations that are compatible with binding to the BA matrix. It is possible that, after further structural optimization of the acridine class or optimization of acridine-ligated matrices, increased binding efficiency may result.
|
). However, 9-aminoacridine compounds do not reduce PrPC expression levels, thus ruling out PrPC depletion as a possible mechanism of action (Korth et al., 2001). It has been suggested that quinacrine acts on PrPSc formation by destabilizing cellular lipid rafts (Klingenstein et al., 2006) that are important for prion conversion (Taraboulos et al., 1995). Others have suggested that 9-aminoacridine compounds may modify lysine residues of PrP, leading to inhibition of prion replication (Sebestik et al., 2006). NMR studies mapped a quinacrine-binding epitope on the surface of recombinant human PrPC (residues 121–230), with a dissociation constant for the quinacrine–PrPC complex of 4.6 mM (Zahn et al., 2000). A weak quinacrine–PrPC binding interaction has also been characterized by surface plasma resonance using recombinant PrPs (Touil et al., 2006). These findings are consistent with our own, as we have been unable to affinity-purify PrPC by using a 9-aminoacridine-ligated matrix. For a protein to be isolated by affinity purification, we would expect a binding affinity between the immobilized ligand and protein receptor to be in the nanomolar concentration range.
The selectivity of the 9-aminoacridine matrix for PrPSc must be enciphered in the conformational and/or oligomeric differences between PrPC and PrPSc. Binding between 9-aminoacridine compounds and -rich peptides has been reported previously. For example, quinacrine is known to bind fibrillogenic PrP peptides and to inhibit PrP peptide fibrillization (Barret et al., 2003). Quinacrine mustard, a closely related 9-aminoacridine compound, inhibits the fibrillization of tau, a protein that adopts a -rich oligomeric state associated with the pathogenesis of Alzheimer’s disease (Taniguchi et al., 2005). These studies and our own evidence for direct 9-aminoacridine–PrPSc binding suggest a possible mechanism of action of anti-prion 9-aminoacridine compounds. In binding PrPSc, 9-aminoacridine compounds may inhibit PrPSc replication by occluding necessary epitopes for templating PrPC conversion or by altering the stability of PrPSc oligomers.
In conclusion, we have taken a chemical proteomics approach toward identifying a relevant 9-aminoacridine receptor involved in prion disease. We have shown that a 9-aminoacridine-functionalized matrix is conformationally selective for PrPSc. Whilst the recovery of PrPSc by our 9-aminoacridine matrix is low, and hence not currently applicable as a diagnostic reagent, insights gained from our studies may be useful in future drug discovery.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Burdine, L. & Kodadek, T. (2004). Target identification in chemical genetics: the (often) missing link. Chem Biol 11, 593–597.[CrossRef][Medline]
Butler, D. A., Scott, M. R., Bockman, J. M., Borchelt, D. R., Taraboulos, A., Hsiao, K. K., Kingsbury, D. T. & Prusiner, S. B. (1988). Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J Virol 62, 1558–1564.
Carlson, G. A., Kingsbury, D. T., Goodman, P. A., Coleman, S., Marshall, S. T., DeArmond, S., Westaway, D. & Prusiner, S. B. (1986). Linkage of prion protein and scrapie incubation time genes. Cell 46, 503–511.[CrossRef][Medline]
Carlson, G. A., Ebeling, C., Yang, S. L., Telling, G., Torchia, M., Groth, D., Westaway, D., DeArmond, S. J. & Prusiner, S. B. (1994). Prion isolate specified allotypic interactions between the cellular and scrapie prion proteins in congenic and transgenic mice. Proc Natl Acad Sci U S A 91, 5690–5694.
Castilla, J., Saa, P. & Soto, C. (2005). Detection of prions in blood. Nat Med 11, 982–985.[Medline]
Chandler, R. L. (1961). Encephalopathy in mice produced by inoculation with scrapie brain material. Lancet i, 1378–1379.
Collins, S. J., Lewis, V., Brazier, M., Hill, A. F., Fletcher, A. & Masters, C. L. (2002). Quinacrine does not prolong survival in a murine Creutzfeldt-Jakob disease model. Ann Neurol 52, 503–506.[CrossRef][Medline]
Deleault, N. R., Lucassen, R. W. & Supattapone, S. (2003). RNA molecules stimulate prion protein conversion. Nature 425, 717–720.[CrossRef][Medline]
Dise, C. A., Burch, J. W. & Goodman, D. B. (1982). Direct interaction of mepacrine with erythrocyte and platelet membrane phospholipid. J Biol Chem 257, 4701–4704.
Doh-Ura, K., Iwaki, T. & Caughey, B. (2000). Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation. J Virol 74, 4894–4897.
Doh-ura, K., Ishikawa, K., Murakami-Kubo, I., Sasaki, K., Mohri, S., Race, R. & Iwaki, T. (2004). Treatment of transmissible spongiform encephalopathy by intraventricular drug infusion in animal models. J Virol 78, 4999–5006.
Farquhar, C. F., Dornan, J., Moore, R. C., Somerville, R. A., Tunstall, A. M. & Hope, J. (1996). Protease-resistant PrP deposition in brain and non-central nervous system tissues of a murine model of bovine spongiform encephalopathy. J Gen Virol 77, 1941–1946.
Gaugain, B., Markovits, J., Le Pecq, J. B. & Roques, B. P. (1981). Hydrogen bonding in deoxyribonucleic acid base recognition. I. Proton nuclear magnetic resonance studies of dinucleotide-acridine alkylamide complexes. Biochemistry 20, 3035–3042.[CrossRef][Medline]
Girault, S., Grellier, P., Berecibar, A., Maes, L., Mouray, E., Lemiere, P., Debreu, M. A., Davioud-Charvet, E. & Sergheraert, C. (2000). Antimalarial, antitrypanosomal, and antileishmanial activities and cytotoxicity of bis(9-amino-6-chloro-2-methoxyacridines): influence of the linker. J Med Chem 43, 2646–2654.[CrossRef][Medline]
Klingenstein, R., Lober, S., Kujala, P., Godsave, S., Leliveld, S. R., Gmeiner, P., Peters, P. J. & Korth, C. (2006). Tricyclic antidepressants, quinacrine and a novel, synthetic chimera thereof clear prions by destabilizing detergent-resistant membrane compartments. J Neurochem 98, 748[CrossRef][Medline]
Korth, C., May, B. C., Cohen, F. E. & Prusiner, S. B. (2001). Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease. Proc Natl Acad Sci U S A 98, 9836–9841.
Lee, K. S., Linden, R., Prado, M. A., Brentani, R. R. & Martins, V. R. (2003). Towards cellular receptors for prions. Rev Med Virol 13, 399–408.[CrossRef][Medline]
Lee, I. S., Long, J. R., Prusiner, S. B. & Safar, J. G. (2005). Selective precipitation of prions by polyoxometalate complexes. J Am Chem Soc 127, 13802–13803.[CrossRef][Medline]
Le Pecq, J. B., Le Bret, M., Barbet, J. & Roques, B. (1975). DNA polyintercalating drugs: DNA binding of diacridine derivatives. Proc Natl Acad Sci U S A 72, 2915–2919.
Ma, K. & Sourkes, T. L. (1980). Inhibition of diamine oxidase by antimalarial drugs. Agents Actions 10, 395–398.[CrossRef][Medline]
Madore, N., Smith, K. L., Graham, C. H., Jen, A., Brady, K., Hall, S. & Morris, R. (1999). Functionally different GPI proteins are organized in different domains on the neuronal surface. EMBO J 18, 6917–6926.[CrossRef][Medline]
Marsh, R. F. & Kimberlin, R. H. (1975). Comparison of scrapie and transmissible mink encephalopathy in hamsters. II. Clinical signs, pathology, and pathogenesis. J Infect Dis 131, 104–110.[Medline]
May, B. C., Fafarman, A. T., Hong, S. B., Rogers, M., Deady, L. W., Prusiner, S. B. & Cohen, F. E. (2003). Potent inhibition of scrapie prion replication in cultured cells by bis-acridines. Proc Natl Acad Sci U S A 100, 3416–3421.
May, B. C., Witkop, J., Sherrill, J., Anderson, M. O., Madrid, P. B., Zorn, J. A., Prusiner, S. B., Cohen, F. E. & Guy, R. K. (2006a). Structure-activity relationship study of 9-aminoacridine compounds in scrapie-infected neuroblastoma cells. Bioorg Med Chem Lett 16, 4913–4916.[CrossRef][Medline]
May, B. C., Zorn, J. A., Witkop, J., Sherrill, J., Wallace, A. C., Legname, G., Prusiner, S. B. & Cohen, F. E. (2006b). Structure–activity relationship study of prion inhibition by 2-aminopyridine-3,5-dicarbonitrile-based compounds: parallel synthesis, bioactivity, and in vitro pharmacokinetics. J Med Chem 50, 65–73.
Mustonen, P., Lehtonen, J. Y. & Kinnunen, P. K. (1998). Binding of quinacrine to acidic phospholipids and pancreatic phospholipase A2. Effects on the catalytic activity of the enzyme. Biochemistry 37, 12051–12057.[CrossRef][Medline]
Nazor, K. E., Kuhn, F., Seward, T., Green, M., Zwald, D., Purro, M., Schmid, J., Biffiger, K., Power, A. M. & other authors (2005). Immunodetection of disease-associated mutant PrP, which accelerates disease in GSS transgenic mice. EMBO J 24, 2472–2480.[CrossRef][Medline]
Oesch, B., Westaway, D., Walchli, M., McKinley, M. P., Kent, S. B., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B. & other authors (1985). A cellular gene encodes scrapie PrP 27-30 protein. Cell 40, 735–746.[CrossRef][Medline]
Pajeva, I. K., Wiese, M., Cordes, H. P. & Seydel, J. K. (1996). Membrane interactions of some catamphiphilic drugs and relation to their multidrug-resistance-reversing ability. J Cancer Res Clin Oncol 122, 27–40.[CrossRef][Medline]
Prusiner, S. B. (2004). Prion Biology and Diseases, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Ryou, C., Legname, G., Peretz, D., Craig, J. C., Baldwin, M. A. & Prusiner, S. B. (2003). Differential inhibition of prion propagation by enantiomers of quinacrine. Lab Invest 83, 837–843.[Medline]
Ryou, C., Lessard, P., Freyman, Y., Guglielmo, B. J., Yung, L., Baldwin, M. A., Craig, J. C., DeArmond, S., May, B. C. H. & other authors (2004). In vivo efficacy of quinacrine in animal models of prion disease. In The First International Conference of the Network of Excellence NeuroPrion, Paris, p. 169. http://www.neuroprion.com/pdf_docs/conferences/prion2004/abstract_book.pdf.
Safar, J. G., Kellings, K., Serban, A., Groth, D., Cleaver, J. E., Prusiner, S. B. & Riesner, D. (2005a). Search for a prion-specific nucleic acid. J Virol 79, 10796–10806.
Safar, J. G., Geschwind, M. D., Deering, C., Didorenko, S., Sattavat, M., Sanchez, H., Serban, A., Vey, M., Baron, H. & other authors (2005b). Diagnosis of human prion disease. Proc Natl Acad Sci U S A 102, 3501–3506.
Sebestik, J., Safarik, M., Stibor, I. & Hlavacek, J. (2006). Acridin-9-yl exchange: a proposal for the action of some 9-aminoacridine drugs. Biopolymers 84, 605–614.[CrossRef][Medline]
Spitzmaul, G., Dilger, J. P. & Bouzat, C. (2001). The noncompetitive inhibitor quinacrine modifies the desensitization kinetics of muscle acetylcholine receptors. Mol Pharmacol 60, 235–243.
Stahl, N., Borchelt, D. R., Hsiao, K. & Prusiner, S. B. (1987). Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51, 229–240.[CrossRef][Medline]
Stuhlmeier, K. M. (2003). Mepacrine inhibits matrix metalloproteinases-1 (MMP-1) and MMP-9 activation in human fibroblast-like synoviocytes. J Rheumatol 30, 2330–2337.
Supattapone, S., Bosque, P., Muramoto, T., Wille, H., Aagaard, C., Peretz, D., Nguyen, H. O., Heinrich, C., Torchia, M. & other authors (1999). Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice. Cell 96, 869–878.[CrossRef][Medline]
Supattapone, S., Muramoto, T., Legname, G., Mehlhorn, I., Cohen, F. E., DeArmond, S. J., Prusiner, S. B. & Scott, M. R. (2001). Identification of two prion protein regions that modify scrapie incubation time. J Virol 75, 1408–1413.
Taniguchi, S., Suzuki, N., Masuda, M., Hisanaga, S., Iwatsubo, T., Goedert, M. & Hasegawa, M. (2005). Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J Biol Chem 280, 7614–7623.
Taraboulos, A., Scott, M., Semenov, A., Avrahami, D., Laszlo, L., Prusiner, S. B. & Avraham, D. (1995). Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibit formation of the scrapie isoform. J Cell Biol 129, 121–132.
Telling, G. C., Haga, T., Torchia, M., Tremblay, P., DeArmond, S. J. & Prusiner, S. B. (1996). Interactions between wild-type and mutant prion proteins modulate neurodegeneration in transgenic mice. Genes Dev 10, 1736–1750.
Touil, F., Pratt, S., Mutter, R. & Chen, B. (2006). Screening a library of potential prion therapeutics against cellular prion proteins and insights into their mode of biological activities by surface plasmon resonance. J Pharm Biomed Anal 40, 822–832.[CrossRef][Medline]
Trevitt, C. R. & Collinge, J. (2006). A systematic review of prion therapeutics in experimental models. Brain 129, 2241–2265.
Williamson, R. A., Peretz, D., Pinilla, C., Ball, H., Bastidas, R. B., Rozenshteyn, R., Houghten, R. A., Prusiner, S. B. & Burton, D. R. (1998). Mapping the prion protein using recombinant antibodies. J Virol 72, 9413–9418.
Zahn, R., Liu, A., Luhrs, T., Riek, R., von Schroetter, C., Lopez Garcia, F., Billeter, M., Calzolai, L., Wider, G. & Wuthrich, K. (2000). NMR solution structure of the human prion protein. Proc Natl Acad Sci U S A 97, 145–150.
Zou, W. Q., Zheng, J., Gray, D. M., Gambetti, P. & Chen, S. G. (2004). Antibody to DNA detects scrapie but not normal prion protein. Proc Natl Acad Sci U S A 101, 1380–1385.
Received 28 September 2006;
accepted 13 December 2006.
This article has been cited by other articles:
|
|
 |
|
  P. Spilman, P. Lessard, M. Sattavat, C. Bush, T. Tousseyn, E. J. Huang, K. Giles, T. Golde, P. Das, A. Fauq, et al. A {gamma}-secretase inhibitor and quinacrine reduce prions and prevent dendritic degeneration in murine brains PNAS, July 29, 2008; 105(30): 10595 - 10600. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
