Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity.

The prion protein, PrP, exists in several stable conformations, with the presence of one conformation, PrP(Sc), associated with transmissible neurodegenerative diseases. Targeting PrP by high-affinity ligands has been proven to be an effective way of preventing peripheral prion infections. Here, we have generated bacterially expressed single chain fragments of the variable domains (scFv) of a monoclonal antibody in Escherichia coli, originally raised against purified PrP(Sc) that recognizes both PrP(C) and PrP(Sc). This scFv fragment had a dissociation constant (K(D)) with recombinant PrP of 2 nM and cleared prions in ScN2a cells at 4 nM, as demonstrated by a mouse prion bioassay. A peptide corresponding to the complementarity determining region 3 of the heavy chain (CDR3H) selectively bound PrP(Sc) but had lost antiprion activity. However, synthesis and application of an improved peptide mimicking side chain topology of CDR3H while exhibiting increased protease resistance, a retro-inverso d-peptide of CDR3H, still bound PrP(Sc) and reinstated antiprion activity. We conclude that (1) scFvW226 is so far the smallest polypeptide with bioassay confirmed antiprion activity, and (2) differential conformation specificity and bioactivity can be regulated by orchestrating the participation of different CDRs.

Structure-activity relationship of tocopherol derivatives suggesting a novel non-antioxidant mechanism in antiprion potency.

Beneficial effects of tocopherols, or vitamin E, on degenerative brain conditions have been attributed mainly to their antioxidant effects. Non-antioxidant effects of the tocopherols have been shown to be mediated by inhibition of protein kinase C (PKC) signaling. Prion disease is a paradigmatic protein conformational disease characterized by the induced conversion of a normal host protein PrP(C) to adopt a pathogenic conformation PrP(Sc). The molecular regulation of prion replication is poorly understood. Here, we show that tocopherols inhibit prion replication by a structure-activity relationship for antiprion activity independent of antioxidant activity with tocopherol succinate (TS) posessing highest EC(50) at 7 microM. Only TS but not an equally antiprion active PKC inhibitor could be partially antagonized by substochiometric 1 nM rapamycin suggesting that there are pathways via mammalian target of rapamycin (mTOR) that interfere with tocopherol's biological effects. Interaction with the mTOR pathway is a yet undescribed characteristic of tocopherol derivatives, potentially significant for pathophysiological processes other than prion propagation.