Identification of a Compound That Disrupts Binding of Amyloid-β to the Prion Protein Using a Novel Fluorescence-based Assay.
Bottom Line: 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.
Affiliation: From the Medical Research Council (MRC) Prion Unit and Department of Neurodegenerative Disease, University College London (UCL) Institute of Neurology, London WC1N 3BG, United Kingdom.Show MeSH
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Mentions: Because both the ELISA and the fluorescence assays involve incubating the compound in the presence of PrP and Aβ oligomers, it is possible that the compounds are binding to, and disrupting the Aβ oligomers rather than interacting with PrP. We therefore used ITC to confirm and further characterize the nature of the interaction between the compound and PrP. These experiments showed that Chicago Sky Blue binds the 23–231 construct (Fig. 6A) with a 3:1 ratio with a dissociation constant of 0.55 μm ± 0.04 μm (n = 2.80 ± 0.09, ΔH = −13.1 ± 0.3 kcal mol−1, TΔS = −4.6 ± 0.3 kcal mol−1). The compound binds with similar, but slightly reduced affinity to a shorter construct containing residues 91–231 (Fig. 6B), which still encompasses the primary Aβ oligomer binding region of the prion protein (KD 1.43 μm ± 0.24 μm, n = 2.89 ± 0.18, ΔH = −4.8 ± 0.2 kcal mol−1, TΔS = 3.2 ± 0.3 kcal mol−1), but shows no binding to a construct that contains only residues 119–231 (Fig. 6C), suggesting that the Chicago Sky Blue binding site overlaps that of the Aβ oligomers.
Affiliation: From the Medical Research Council (MRC) Prion Unit and Department of Neurodegenerative Disease, University College London (UCL) Institute of Neurology, London WC1N 3BG, United Kingdom.