The prion protein (PrP) has been implicated both in prion diseases

The prion protein (PrP) has been implicated both in prion diseases such as Creutzfeldt-Jakob disease where its monomeric cellular isoform (PrPC) is recruited into pathogenic self-propagating polymers of misfolded protein and in Alzheimer disease where PrPC may act as a receptor for synaptotoxic oligomeric forms of amyloid-β (Aβ). 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 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 Firategrast (SB 683699) binding and reduce prion levels was established in cell-based assays. either target aggregated prion protein (37) a downstream target or have an unknown molecular target (38). We therefore sought to develop an assay capable of screening large numbers of compounds to identify ones that bind to PrPC. A fluorescence polarization (FP)-based assay to detect ligand binding was first established using several known biological ligands for PrP including Aβ oligomers. Rather than attempting to directly detect small molecule-induced changes in the FP signal we measured the ability of compounds to inhibit the binding of the Aβ oligomers. This assay was then used to screen a small collection of pharmacologically active compounds with rapid confirmation of binding using an orthologous assay for Aβ binding in Firategrast (SB 683699) an ELISA format. The binding of the only compound to show Rabbit Polyclonal to MEF2C (phospho-Ser396). robust activity in both assays Chicago Sky Blue 6B was then characterized using isothermal titration calorimetry (ITC) and its activity in a cellular context was confirmed by measuring the ability of the compound to lower prion levels in a chronically prion-infected mouse neuroblastoma cell line (14). Experimental Procedures Aβ1-42 and Aβ1-42 with biotin attached to Asp-1 using a 6-carbon linker (bAβ1-42) were synthesized and purified by Dr. James I. Elliott at Yale University (New Haven CT). Aβ oligomers were produced as described previously (17) and contained a mixture of monomer spherical oligomers and protofibrils. Anti-PrP antibodies ICSM35 and ICSM18 were from D-Gen Ltd. (London UK). The Prestwick Chemical Library compound collection of 1 200 compounds was purchased as 10 mm stock in dimethyl sulfoxide (DMSO) from Prestwick Chemical (Illkirch-Graffenstaden France). Chicago Sky Blue 6B solid was purchased from Sigma and dissolved in DMSO 24 h prior to use. Mutations to the wild-type human prion protein Firategrast (SB 683699) (amino acids 23-231) were generated by QuikChange mutagenesis and verified by sequencing. Prion protein with an N-terminal hexahistidine tag followed by a 23-amino acid linker Firategrast (SB 683699) and a thrombin cleavage site was expressed in (39) solubilized in 6 m guanidine hydrochloride and purified by affinity chromatography on a nickel-nitrilotriacetic acid column prior to undergoing stepwise oxidation and elution (40). Following elution proteins were dialyzed for 1 h against 10 mm Tris-HCl 100 mm sodium phosphate pH 5.8 prior to being labeled with IANBD ((39) and purified (40) as described previously prior to being labeled with IANBD ester. Despite the incorporation of an additional cysteine into a protein that already has two cysteine residues slow oxidation of the protein encouraged formation of the most thermodynamically stable disulfide whereas elution at a relatively low pH prevented the formation of intermolecular dimers such that following oxidative refolding on a nickel-nitrilotriacetic acid column and labeling with IANBD > 70% of the protein was monomeric as judged by SDS-PAGE in the absence of reducing agent (Fig. 2and = 2.80 ± 0.09 ?? ?13.1 ± 0.3 kcal mol?1 = ?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. 61.43 μm ± 0.24 μm = 2.89 ± 0.18 Δ= ?4.8 ± 0.2 kcal mol?1 = 3.2 ± 0.3 kcal mol?1) but shows no binding to a construct that contains only residues 119-231 (Fig. 6of 0.55 μm as determined by ITC. The utility of the fluorescence assay to screen large numbers of compounds followed by the ELISA assay to confirm the interaction was demonstrated by the observation that the remaining hit compound Chicago Sky Blue.