Schematic illustration of in-vitro assays, and dose response curves of an APP selective gamma-secretase inhibitor. (a) Recombinant E.coli expressed fusion protein substrates APP MBP-C125Sw (left side, numbering relative to Aβ peptide residues, red box) or mNotch100 substrate (right hand side, numbering relative to initiator methionine residue) were incubated in separate reactions with partially purified solubilized gamma-secretase enzyme from IMR-32 cells as detailed in methods. The reactions products were analyzed by ELISA using 2G3/6H9 ELISA for AβX-40 for GammaAPP assay product, or the 9F3/anti-HA ELISA for NICD, the GammaNotch assay product. MBP, N-terminal fusion with maltose binding protein (grey hatched box); TMD, transmembrane domain (blue hatched box); HA, hemagglutnin antigen epitope tag (green hatched box); HIS-6, hexahistidine residue epitope tag (yellow box). (b) Dose response curve demonstrating APP-selective inhibition from a representative screening hit ELN158762. The IC50 values for inhibition of APP and Notch substrate cleavage, and selectivity ratio in the biochemical assays with this compound are reported in Table 1.