Analyzing Amyloid-beta Peptide Modulation Profiles and Binding Sites of gamma-Secretase Modulators

gamma-Secretase is a key player in the pathogenesis of Alzheimer's disease (AD). The intramembrane-cleaving enzyme initially cleaves a C-terminal fragment of the amyloid precursor protein (APP) at the e-site within its transmembrane domain to release the APP intracellular domain. Subsequent stepwise carboxy-terminal trimming cleavages eventually release amyloid-beta (A beta) peptides of 37-43 amino acids into the extracellular space. A beta(42) as well as the much less abundant A beta beta(43) species are highly aggregation prone and can deposit as plaques in the brains of affected patients, which are widely believed to be causative of AD. Disappointingly, due to lack of efficacy and side effects likely attributable to the inhibition of the crucial substrate Notch, inhibitors of gamma-secretase that lower A beta generation failed in clinical trials of AD. There is hope, however, that recently developed potent gamma-secretase modulators (GSMs) provide a safer approach for disease modification. These compounds have the unique property of primarily shifting the generation of A beta(42) toward that of shorter peptides without affecting the e-site cleavage of Notch and other substrates. In this chapter, we describe methods to investigate how GSMs affect the activity of the enzyme as well as how their molecular targets are identified.