O1–11–01: Gamma‐secretase Inhibitor (GSI) and Modulator (GSM) Photoaffinity Probes Reveal Distinct Binding Sites on Presenilin

g -Secretase modulators (GSMs) which reduce the production of A b 42 have emerged as a promising class of compounds for the treatment of Alzheimer's disease (AD). The target and mechanism of action of GSMs is a topic of much interest. Some evidence has suggested that GSMs function by binding the substrate, APP, while other evidence has suggested action through direct modulation of the enzyme, γ-secretase. We have developed several GSM and GSIphotoaffinity probes as tools to gain a better understanding of their binding sites and mechanism of modulation or inhibition. These include clickable photoaffinity probes based on acid and imidazole GSMs as well as sulfonamide GSIs that contain a photoreactive group for crosslinking the compound to cellular target proteins and an alkyne handles to attach a reporter tag by click chemistry for visualization and identification of the target(s). All 3 classes of photoprobes were found to specifically label the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling of PS1-NTF by the imidazole GSM photoprobe E2012-BPyne was competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, while labeling by the acid GSM photoprobe was competed in the presence of the parent acid GSM-1, but not with E2012. In both cases, the photolabeling was not competed by the allosteric GSI BMS-708163 or the substrate docking site peptide inhibitor pep11 providing evidence that all of these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active-site directed GSI L458. Taken together our results support the hypothesis that multiple binding sites within the g -secretase complex exist, each of which may contribute to different modes of modulatory or inhibitory action. In addition, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of g -secretase and the modulatory binding site of certain GSMs.