Characterizing antiprion compounds based on their binding properties to prion proteins: implications as medical chaperones

Abstract

A variety of antiprion compounds have been reported that are effective in ex vivo and in vivo treatment experiments. However, the molecular mechanisms for most of these compounds remain unknown. Here we classified antiprion mechanisms into four categories: I, specific conformational stabilization; II, nonspecific stabilization; III, aggregation; and IV, interaction with molecules other than PrP(C). To characterize antiprion compounds based on this classification, we determined their binding affinities to PrP(C) using surface plasmon resonance and their binding sites on PrP(C) using NMR spectroscopy. GN8 and GJP49 bound specifically to the hot spot in PrP(C), and acted as "medical chaperones" to stabilize the native conformation. Thus, mechanisms I was predominant. In contrast, quinacrine and epigallocathechin bound to PrP(C) rather nonspecifically; these may stabilize the PrP(C) conformation nonspecifically including the interference with the intermolecular interaction following mechanism II. Congo red and pentosan polysulfate bound to PrP(C) and caused aggregation and precipitation of PrP(C), thus reducing the effective concentration of prion protein. Thus, mechanism III was appropriate. Finally, CP-60, an edarabone derivative, did not bind to PrP(C). Thus these were classified into mechanism IV. However, their antiprion activities were not confirmed in the GT + FK system, whose details remain to be elucidated. This proposed antiprion mechanisms of diverse antiprion compounds could help to elucidate their antiprion activities and facilitate effective antiprion drug discovery.

Figure 1

Characteristics of the binding sites for the antiprion compound GJP49. GJP49 was discovered by in silico screening using Autodock. (A) ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled recombinant prion protein, mouse PrP(121–231) (33 μM), with (red) or without (blue) GJP49 (500 μM). (B–D) Plots of chemical shift perturbations (Δδ = [(Δδ_1H)² + 0.17(Δδ_15N)²]^1/2) as a function of the residue number. (E) Mapping of the significantly perturbed residues on the three-dimensional structure of the prion (1AG2). The perturbed residues with Δδ values of >0.04 ppm are shown in red, and those with 0.4 < Δδ < 0.3 ppm are shown in orange. The binding pocket is overlaid in green. S1, HA, S2, HB, and HC indicate S1 strand, helix A, S2 strand, helix B, and helix C, respectively. The image was created using PyMol.

Figure 2

Surface plasmon resonance sensorgrams for various antiprion compounds. (A) Quinacrine; the concentrations from bottom to top are: 0.781, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μM. (B) Epigallocathechin gallate; the concentrations from bottom to top are: 3.91 7.81, 15.6, 31.3, 62.5, and 125 μM. (C) Congo red; the concentrations from bottom to top are: 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μM. (D) Pentosan polysulfate; the concentrations is: 1.95 μg/mL. (E–H) CP60, Edarabone derivative 13, D-PEN, and indole-3-glyoxylamide derivative 10; the concentrations from bottom to top are: 0.75, 1.25, 2.5, 5.0, 10, 20, and 40 μM. The symbol * indicates spike noise. Insets show the compound concentration dependence of the Biacore response. For quinacrine, Congo red, and pentosan polysulfate binding experiments, recombinant mouse PrP(121–231) was fixed on the surface of the sensor chip. For epigallocatechin gallate, CP60, edarabone derivative 13, D-PEN, and indole-3-glyoxylamide derivative 10 binding experiments, recombinant mouse PrP(23–231) was fixed on the surface of the sensor chip.

Figure 3

Chemical shift perturbations (Δδ) as a function of residue number and the mapping of Δδ onto the three dimensional structure of recombinant mouse PrP(121–231) (PDB ID: 1AG2). (A, B) GJP49, (C, D) quinacrine, and (E, F) epigallocatechin gallate (EGCG). Final concentrations of the protein and compounds were, respectively, 31 μM and 500 μM for GJP49 and EGCG binding experiments and 32 μM and 5.3 mM for quinacrine binding experiment.

Figure 4

Aggregation and precipitation of mouse PrP(121–231) by Congo red. (A) Precipitation of mouse PrP(121–231) by Congo red in a Shigemi tube. (B) 1D ¹H NMR spectra with (lower trace) and without (upper trace) Congo red.

Figure 5

Illustration of the pathogenic conversion process from PrP^C to PrP^Sc in the absence (A) and presence (B) of antiprion compounds and the classification of antiprion mechanisms based on interactions with PrP^C. Mechanism I molecules (c1), designated “medical chaperones,” stabilize the PrP^C conformation. Mechanism II molecules (c2) bind to PrP^C nonspecifically to stabilize the PrP^C and interfere with the interaction between PrP^C and PrP^Sc. Mechanism III molecules (c3) induce prion protein aggregation and cause precipitation, which reduces the amount of PrP^C. Mechanism IV molecules (c4) interact with molecules other than PrP^C, such as PrP^Sc, PrP*, or membrane proteins.