Gamma-secretase is a protease complex of four integral membrane proteins, with 
presenilin (PS) as the apparent catalytic component, and this enzyme processes 
the transmembrane domains of a variety of substrates, including the amyloid 
beta-protein precursor and the Notch receptor. Here we explore the mechanisms of 
structurally diverse gamma-secretase inhibitors by examining their ability to 
displace an active site-directed photoprobe from PS heterodimers. Most 
gamma-secretase inhibitors, including a potent inhibitor of the PS-like signal 
peptide peptidase, blocked the photoprobe from binding to PS1, indicating that 
these compounds either bind directly to the active site or alter it through an 
allosteric interaction. Conversely, some reported inhibitors failed to displace 
this interaction, demonstrating that these compounds do not interfere with the 
protease by affecting its active site. Differential effects of the inhibitors 
with respect to photoprobe displacement and in cell-based and cell-free assays 
suggest that these compounds are important mechanistic tools for deciphering the 
workings of this intramembrane-cleaving protease complex and its similarity to 
other polytopic aspartyl proteases.