Fig. 3. Suramin derivatives interfere with de novo PrP^Sc formation and reduce PrP^c 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 PrP^Sc 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 PrP^c half-life to different extents.