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First published online 11 October 2005
doi: 10.1242/jcs.02609

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Charged bipolar suramin derivatives induce aggregation of the prion protein at the cell surface and inhibit PrP^Sc replication

¹ Prion Research Group, Institute of Virology, Technical University of Munich, Biedersteiner-Str. 29, 80802 Munich, Germany
² Pharmaceutical Institute, Rheinische Friedrich-Wilhelms-Universität Bonn, An der Immenburg 4, 53121 Bonn, Germany
³ Institute for Novel and Emerging Infectious Diseases, Friedrich Loeffler Institute, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany

View larger version (22K): [in a new window]   Fig. 1. Chemical structures of suramin and its naphthalene and benzene derivatives with sulfonic, carbonic or phosphonic acid substitutions. Suramin, NF449, NF023, NF058, NF078, NF110, NF305, NF307, NF506 are characterised by symmetrical aromatic structure with sulfonic acid substitutions. Symmetrical compounds NF068, NF542, NF710 present phosphonic and NF043, carbonic acid groups. ANTS and NF007 have sulfonic acid substitutions but lack bipolar structure. NN is an uncharged aromatic compound with symmetrical structure.

View larger version (62K): [in a new window]   Fig. 2. Suramin and derivatives reduce the amount of PrPSc in prion-infected cells to undetectable levels and induce insoluble full-length PrP aggregates. (A,B,C) The effect of suramin derivatives on PrPSc was determined in prion-infected neuroblastoma cells (3F4-ScN2a) using a solubility assay and proteinase K (PK) treatment followed by immunoblot analysis. Cells were seeded on 60 mm plates and treated daily with suramin or with one of its derivatives, as indicated. All compounds were applied at a concentration of 200 µg/ml for 4 days. Mock-treated cells were used as a control. Cells were harvested and postnuclear lysates were either treated with 20 µg/ml PK for 30 minutes at 37°C or subjected to ultracentrifugation at 100,000 g in the presence of 1% sarcosyl, to separate soluble (S, supernatant) from insoluble (P, pellet) fractions, as indicated above the blots. PrP was visualised by immunoblotting using the monoclonal anti-PrP antibody 4H11. Molecular size markers are depicted on the left. No PK-resistant PrP is detected after treatment of the cells with NF058, NF078 (A, lanes 10,16); NF110, NF305, NF307, NF506 (B, lanes 1,4,7,10); suramin, NF023 or NF449 (C, lanes 4,7,10). NF542 and NF710 were highly cytotoxic. Actin loading controls are shown below each panel.

View larger version (41K): [in a new window]   Fig. 3. Suramin derivatives interfere with de novo PrPSc formation and reduce PrPc half-life. (A) 3F4-ScN2A cells were metabolically labelled with [35S]Met/Cys overnight in the presence of 200 µg/ml suramin or one of its derivatives or without addition of compounds, as a control. Cells were lysed after the pulse and subjected to digestion with 20 µg/ml PK for 30 minutes at 37°C. Lysates were ultracentrifuged in the presence of 1% sarcosyl and the pellets immunoprecipitated with the polyclonal anti-PrP antibody A7. Prion protein was deglycosylated with PNGase F and subjected to SDS-PAGE and autoradiography. De novo formation of PrPSc is inhibited when the cells are treated with suramin, NF023 or NF449 (lanes 2,3 and 4). NF007 and ANTS show no inhibitory effect on prion conversion (lanes 5 and 6). (B) Confluent wtN2a cells were incubated overnight with 200 µg/ml of the indicated compounds and then metabolically labelled with [35S]Met/Cys for 1 hour (in the presence of suramin derivatives) at 37°C. After the pulse, cells were incubated in culture medium without 35S (with compounds) at 37°C for the indicated chase time points, before harvesting. PrP was precipitated with polyclonal A7 antibody and deglycosylated with PNGase F to facilitate molecular size comparison and quantification. Samples were subjected to SDS-PAGE and autoradiography. Positions of molecular size markers in kDa are shown on the left. (C) Densitometric evaluation of autoradiograms. PrP harvested 1 hour after the radioactive pulse in the presence of suramin derivative (time estimated for proper uptake and activity of compounds) was set as the total PrP population (100 %) (lanes 1,6,11,16 in B; 1 hour chase time). Decrease in protein amounts at following chase points is expressed as a percentage of this total protein and plotted as a function of the chase times. The data points were fitted to an exponential curve using non-linear regression analysis. All the compounds tested reduce the PrPc half-life to different extents.

View larger version (58K): [in a new window]   Fig. 4. Treatment with suramin analogues does not downregulate cell surface expression of PrPc. (A) Expression levels of PrPc in 3F4-N2a cells treated for 3 days with 200 µg/ml NF023, NF449, NF023 or ANTS was measured by FACS analysis. The histograms depict the cell surface expression in non-permeabilised cells (upper panels) and the intracellular PrP expression in cells permeabilised with saponin buffer. FL1 represents the fluorescence intensity in treated cells (bold line) and in the mock-treated ones (dotted line), plotted against the number of cells (counts). For each cell population 10,000 events were measured. Living and dead cells were separated by staining with propidium iodide and gating. The monoclonal antibody 3F4 was used for detection of PrPc. No significant change in the intracellular or in the surface expression of PrPc was detected upon treatment of cells with any of the tested compounds. (B) PrPc localisation upon exposure to NF023 or NF449 was confirmed by confocal microscopy studies and compared to mock-treated cells in wtN2a cells, stably overexpressing PrPc. Tested compounds were added daily (48 hour treatment) to the culture medium at a concentration of 200 µg/ml, before fixation and permeabilisation. Staining of PrP and lysosomes was performed using the polyclonal anti-PrP antibody A7 (left-hand column) or a monoclonal antibody directed against the lysosomal protein LAMP-I (middle column), respectively. After treatment with NF023 or NF449, PrPc could still be detected on the cell surface (middle and lower left panels) at levels comparable to untreated cells (mock). Upon treatment with suramin derivatives, PrPc also shows increased localisation in intracellular compartments which partially correspond to lysosomes, as seen in an overlay of the two signals (merge). Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 5. Effective suramin analogues induce aggregation of PrPc independently of the mode of surface anchorage. (A,B,C) N2a cells were transiently transfected with 3F4-tagged wtPrP, CD4-PrP or Thy-1 and treated with 200 µg/ml of the indicated compounds (10 or 200 µg/ml for NN) for 3 days. Postnuclear lysates were subjected to ultracentrifugation at 100,000 g in the presence of 1% sarcosyl. Pellets and supernatants were run on SDS-PAGE and analysed by immunoblotting using the monoclonal antibody 3F4 or monoclonal anti-HA antibody to detect Thy-1. With the exception of ANTS and NN, Suramin and all the tested derivatives induce formation of insoluble PrP-aggregates (A, lanes 3,5,7,9,11,13) whereas PrPc remains soluble in cells treated with ANTS (A, lane 16) or NN (A, lanes 22 and 24). CD4-PrP also partitioned in the insoluble fraction upon incubation of cells with suramin derivatives (B, lanes 3, 5, 7, 9, 11), whereas the same treatment had no effect on Thy-1 solubility (C).

View larger version (51K): [in a new window]   Fig. 6. Suramin analogues induce aggregation of purified recombinant mouse prion protein. (A,B) Recombinant mouse PrP (amino acids 23-231) (50 ng/µl) was incubated overnight with 10 or 100 µg/ml of the suramin analogues indicated in sodium acetate buffer at pH 7. Samples were then subjected to ultracentrifugation in the presence of 1% sarcosyl. Pellets and supernatants were run on SDS-PAGE and analysed by immunoblotting using the mouse monoclonal antibody 4H11. Mock-treated recombinant PrP was detected in the soluble as well as in the insoluble fractions. Upon treatment with NF023, NF449, NF078, NF110, NF305, NF068 and suramin, a clear shift into pellet fractions was detected whereas ANTS (B, lanes 5-8) and NF043 (B, lanes 13-16) did not affect the solubility of PrP.

View larger version (29K): [in a new window]   Fig. 7. Treatment with effective compounds does not affect solubility of PrPc when transport of PrP along the secretory pathway is inhibited. 10 µg/ml Brefeldin A (BFA) were added to the culture medium of wtN2a cells. After 2 hours, cells were exposed to 200 µg/ml suramin, NF449 or NF023 in the presence of BFA and incubated at 37°C overnight. Cells were then harvested and subjected to a solubility assay. Soluble (S) and insoluble (P) fractions were analysed by immunoblotting, using the monoclonal anti-PrP antibody 4H11. Positions of molecular size markers are depicted on the left. Treatment of cells with BFA alone did not affect the solubility of PrPc (lane 3). Treatment with BFA in the presence of suramin, NF449 or NF023 significantly decreased the induction of PrPc aggregates (lanes 7,11 and 15).

View larger version (64K): [in a new window]   Fig. 8. Suramin derivatives, in contrast to suramin, do not exert their aggregation effect on lysosome-localised LAMP-PrP. (A) N2a cells transiently transfected with wild-type PrP or PrP-LAMP were treated for 48 hours with 200 µg/ml NF449, NF023 or suramin. Cells were then lysed and subjected to ultracentrifugation at 100,000 g in the presence of 1% sarcosyl. Supernatants (S) and pellets (P) were analysed by immunoblot analysis using the monoclonal antibody 3F4. Suramin (lane 11) but not NF449 or NF023 (lanes 7,9) significantly affected the solubility of lysosome-localised LAMP-PrP, confirming a different site of action of the tested compounds. (B) 3F4-N2a cells were biotinylated for 15 minutes on ice. Suramin, NF449 or NF023 were added to the culture medium and cells were placed at 37°C for 4 hours. Cells were then lysed and subjected to ultracentrifugation at 100,000 g in the presence of 1% sarcosyl. Supernatants (S) and pellets (P) were precipitated with the polyclonal antibody A7, analysed by immunoblotting, and biotinylated proteins were detected with streptavidin. Although surface-localised PrPc is soluble in mock-treated cells (lane 1), all tested compounds affect the solubility of this PrPc population (lanes 3,5 and 7).