Fig. 1.
In silico and ex vivo screening. (a) Residues undergoing global fluctuation displayed as a wire frame in red mapped on the mouse PrPC structure (residues 124–226) (12), and a binding pocket defined by those residues, colored green. S1, A, S2, B, and C indicate S1 strand, helix A, S2 strand, helix B, and helix C, respectively. The image was created by using PyMol (www.pymol.org). (b) 2-Pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8. (c) Western blotting of PrPSc in GT+FK cells after treatment with different compounds picked up by in silico screening. The cells treated with no. 8 compound showed significant reduction of PrPSc, which was better than that with 10 μg/ml pentosan polysulfate. DM, DMSO at 0.1%; PS, 10 μg/ml pentosan polysulfate. Molecular masses (37, 25, and 15 kDa) are indicated by bars on the right side of the panels. (d) PrPSc signals in serially diluted mock-treated samples and tested samples were scanned and quantified. The IC50 of no. 8 (72-h treatment), as determined by four repeated experiments, was 1.35 μM. (e) GN8 reduced PrPSc also in 22L-, Ch-, and BSE-infected cells. CR, Congo red at 10 μg/ml; PK, proteinase K digestion. (f) Kaplan–Meier's survival curves of FK-infected mice administered GN8 by intraventricular infusion. The control group (n = 6) was killed at 123.8 ± 7.4 days (black line). Average survival time of GN8-treated mice was 132.3 ± 9.2 days (42–70 d.p.i. group, n = 7, red line) and 141.5 ± 18.8 days (70–98 d.p.i. group, n = 6, green line). (g) Kaplan–Meier's survival curves of FK-infected mice administered GN8 subcutaneously. The control group (n = 9) was killed at 133.0 ± 4.9 days (black line). Average survival time of GN8-treated mice was 148.6 ± 10.3 days (67–95 d.p.i. group, n = 8, red line) and 151.4 ± 15.3 days (67–123 d.p.i. group, n = 10, green line).