In silico and ex vivo screening. (a) Residues undergoing global fluctuation displayed as a wire frame in red mapped on the mouse PrP^C 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 PrP^Sc 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 PrP^Sc, 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) PrP^Sc 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 PrP^Sc 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).

Interaction of an anti-prion compound, GN8, and a recombinant mouse PrP^C. (a) Scatchard plot (Req vs. Req/C, where Req and C are the equilibrium response of SPR and the concentration of GN8, respectively) of the specific binding of GN8 with the PrP obtained by a surface plasmon resonance sensorgram. From the slope of the line, K_d was estimated to be 3.9 μM. (Details are shown in SI Fig. 5 and SI Methods.) (b) An overlay of the ¹H-¹⁵N heteronuclear single quantum coherence NMR spectra of PrP in the absence and presence of GN8. Blue contours show the spectrum of PrP^C without GN8, and red contours show the spectrum in the presence of 1.0 mM GN8 at pH 4.5. (c) Plot of the weighted averages of the ¹H and ¹⁵N chemical shift changes, calculated by using the function Δδ = [(Δδ_1H)² + 0.17(Δδ_15N)²]^1/2 against the residue number. The absence of bars in the plot indicates unassigned residues, proline residues, or unmeasured shifts due to resonance overlaps. Perturbed residues with Δδ values of >0.9 ppm are shown in red, and those with 0.9 > Δδ > 0.5 ppm are in orange. (d) Mapping of the perturbed residues on the structure of mPrP(121–231) (PDB entry 1AG2). The perturbed residues with Δδ values of >0.9 ppm are shown in red, and those with 0.9 > Δδ > 0.5 ppm are in orange. Binding pocket is overlaid in 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. (e) Thermal unfolding profiles of recombinant mouse PrP (amino acids 23–231, 5 μM) without (blue) or with (red) GN8 (10 μM). CD intensities of PrP in the presence of GN8 were normalized to those of PrP without GN8, and fitted curves (see SI Methods) are also shown. Binding with GN8 stabilizes the conformation of PrP^C. (Inset) The difference in extinction coefficients at 222 nm of PrP without GN8 and those in the presence of GN8, as a function of temperature.

Inhibitory mechanism of GN8 for pathogenic conversion. (a) Stereoview of the optimized complex structure of the GN8 and mouse PrP^C, with putative hydrogen bonds, calculated by using ICM version 3.0. Presumed hydrogen bonds between GN8 (green) and E196 (red) and between GN8 (green) and N159 (blue) are shown in orange (dotted lines). The images were created with VMD (52). (b) Illustration of the Gibbs free energy as a function of the conformational space to explain the inhibitory mechanism of a chemical chaperone, GN8. GN8 stabilizes the PrP^C conformation and reduces the population of PrP*, PrP^Sc, and PrP^U. PrP* may not interact with GN8, because the specific conformation around the binding sites would be lost (14). Thus, the free energy level of PrP*, PrP^U, and PrP^Sc would not change much in the presence of GN8.