γ-Secretase Modulator (GSM) Photoaffinity Probes Reveal Distinct Allosteric Binding Sites on Presenilin

gamma-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic beta-amyloid peptides (i.e. A beta 42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule gamma-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of A beta 42 while not blocking the processing of gamma-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that 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 L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active gamma-secretase) but not full-length PS1 (inactive gamma-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the gamma-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of gamma-secretase and the modulatory binding site of certain GSMs.