Identification of a Compound That Disrupts Binding of Amyloid-β to the Prion Protein Using a Novel Fluorescence-based Assay

Abstract

The prion protein (PrP) has been implicated both in prion diseases such as Creutzfeldt-Jakob disease, where its monomeric cellular isoform (PrP(C)) is recruited into pathogenic self-propagating polymers of misfolded protein, and in Alzheimer disease, where PrP(C) may act as a receptor for synaptotoxic oligomeric forms of amyloid-β (Aβ). There has been considerable interest in identification of compounds that bind to PrP(C), stabilizing its native fold and thereby acting as pharmacological chaperones to block prion propagation and pathogenesis. However, compounds binding PrP(C) could also inhibit the binding of toxic Aβ species and may have a role in treating Alzheimer disease, a highly prevalent dementia for which there are currently no disease-modifying treatments. However, the absence of a unitary, readily measurable, physiological function of PrP makes screening for ligands challenging, and the highly heterogeneous nature of Aβ oligomer preparations makes conventional competition binding assays difficult to interpret. We have therefore developed a high-throughput screen that utilizes site-specifically fluorescently labeled protein to identify compounds that bind to PrP and inhibit both Aβ binding and prion propagation. Following a screen of 1,200 approved drugs, we identified Chicago Sky Blue 6B as the first small molecule PrP ligand capable of inhibiting Aβ binding, demonstrating the feasibility of development of drugs to block this interaction. The interaction of Chicago Sky Blue 6B was characterized by isothermal titration calorimetry, and its ability to inhibit Aβ binding and reduce prion levels was established in cell-based assays.

Keywords: Alzheimer disease; amyloid-beta (AB); drug discovery; fluorescence anisotropy; high-throughput screening (HTS); isothermal titration calorimetry (ITC); neurodegenerative disease; prion.

FIGURE 1.

Representation of the structure of the human prion protein showing the sites where cysteine residues were introduced to permit labeling with IANBD.

FIGURE 2.

Characterization of the NBD-labeled proteins. A, Coomassie Blue-stained 16% polyacrylamide Tris-glycine gel showing NBD-labeled protein. Prior to loading, samples were heated to 95 °C for 5 min in SDS loading buffer in the absence of reducing agent. B, circular dichroism spectra of the PrP proteins labeled at the different sites. deg, degrees.

FIGURE 3.

Results for ELISA assays showing the relative binding of ICSM18 (A), ICSM35 (B), and Aβ (C) oligomers to the different NBD-labeled constructs. Curves showing binding to wild-type protein and 145 construct (the construct used in the screen) are shown as continuous lines, and binding curves for the other proteins are shown as dashed lines. Assays were performed in triplicate with the mean values ± S.E. shown.

FIGURE 4.

Interaction of biological ligands with the different prion protein reporter constructs. 120 nm labeled prion protein was incubated with 500 nm anti-prion antibodies ICSM18 and ICSM35, or with 1 μm synthetic amyloid β oligomers. Assays were performed in triplicate, with values expressed as mean ± S.E. All constructs demonstrated a statistically significant change in FP signal upon binding of all three ligands (one-way analysis of variance with Dunnett's multiple comparisons test). mP, millipolarization units.

FIGURE 5.

A, structure of Chicago Sky Blue 6B. B and C, dose-response curves for Chicago Sky Blue in fluorescence (B) and ELISA (C). Assays were performed in triplicate with the values expressed as the mean ± S.E. mP, millipolarization units; conc, concentration; AU, arbitrary units.

FIGURE 6.

Isothermal titration calorimetry isotherms for the interaction of Chicago Sky Blue with different length constructs of human prion protein 23–231 (A), 91–231 (B), and 119–231 (C).

FIGURE 7.

Activity of Chicago Sky Blue in cell-based assays. A, reduction in PrP^Sc in PK1 cells chronically infected with RML prions following 3 days of treatment with Chicago Sky Blue. conc, concentration. B, inhibition of the interaction of Aβ oligomers with COS-7 cells overexpressing mouse PrP. Data are the mean ± S.E. of four independent experiments. AU, arbitrary units.