Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrP ^C ) to the scrapie form (PrP ^Sc ) is still unknown. Here we discovered a chemical chaperone to stabilize the PrP ^C conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a “pocket” created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl- N -[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrP ^Sc . Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrP ^C , will open the way to the development of novel anti-prion drugs.

Significance: On the basis of the amino acid sequence, a polypeptide folds into a unique structure. In contrast, aggregation-prone proteins often misfold into distinct aggregate conformations. Interestingly, each distinct aggregate conformation can dictate different phenotypic consequences, such as cellular toxicity. However, the underlying mechanism of the origin of such structural diversity of protein aggregates has been a critical open question. Using the intrinsically disordered domain of yeast prion protein Sup35, we revealed the structural diversification of prion aggregate is triggered by thermodynamic fluctuations within the monomeric protein prior to aggregation. Our findings not only provide a clue to explain previously puzzling observations in prion biology but also have important implications for therapeutic approaches for human diseases in which protein aggregation is involved.

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNPSc) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, ATG12–ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNPSc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNPSc in addition to attenuation of microgliosis and neuroprotection.