Mapping the Binding Site of BMS-708163 on γ-Secretase with Cleavable Photoprobes

gamma-Secretase, a four-subunit transmembrane aspartic proteinase, is a highly valued drug target in Alzheimer's disease and cancer. Despite significant progress in structural studies, the respective molecular mechanisms and binding modes of gamma-secretase inhibitors (GSIs) and modulators (GSMs) remain uncertain. Here, we developed biotinylated cleavable-linker photoprobes based on the BMS-708163 GSI to study its interaction with gamma-secretase. Comparison of four cleavable linkers indicated that the hydrazinelabile N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl (Dde) linker was cleaved most efficiently to release photolabeled and affinity-captured presenilin- 1 (PS1), the catalytic subunit of gamma-secretase. Peptide mapping showed that the BMS-708163-based probe photoinserted at L282 of PS1. This insertion site was consistent with the results of molecular dynamics simulations of the gamma-secretase complex with inhibitor. Taken together, this work reveals the binding site of a GSI and offers insights into the mechanism of action of this class of inhibitors.