Identification of Presenilin 1-Selective γ-Secretase Inhibitors with Reconstituted γ-Secretase Complexes

Accumulation of the beta-amyloid (A beta) peptides is one of the major pathologic hallmarks in the brains of Alzheimer's disease (AD) patients. A beta is generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) catalyzed by beta- and gamma-secretases. Inhibition of A beta production by gamma-secretase inhibitors (GSIs) is thus being pursued as a target for treatment of AD. In addition to processing APP, gamma-secretase also catalyzes proteolytic cleavage of other transmembrane substrates, with the best characterized one being the cell surface receptor Notch. GSIs reduce A beta production in animals and humans but also cause significant side effects because of the inhibition of Notch processing. The development of GSIs that reduce A beta production and have less Notch-mediated side effect liability is therefore an important goal. gamma-Secretase is a large membrane protein complex with four components, two of which have multiple isoforms: presenilin (PS1 or PS2), aph-1 (aph-1a or aph-1b), nicastrin, and pen-2. Here we describe the reconstitution of four gamma-secretase complexes in Sf9 cells containing PS1-aph-1a, PS1-aph-1b, PS2-aph-1a, and PS2-aph-1b complexes. While PS1-aph-1a, PS1-aph-1b, and PS2-aph-1a complexes displayed robust gamma-secretase activity, the reconstituted PS2-aph-1b complex was devoid of detectable gamma-secretase activity. gamma-Secretase complexes containing PS1 produced a higher proportion of the toxic species A beta 42 than gamma-secretase complexes containing PS2. Using the reconstitution system, we identified MRK-560 and SCH 1500022 as highly selective inhibitors of PS1 gamma-secretase activity. These findings may provide important insights into developing a new generation of gamma-secretase inhibitors with improved side effect profiles.