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

Abstract

SUMMARY γ-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 γ-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 γ-secretase. Comparison of four cleavable linkers indicated that the hydrazine-labile 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 γ-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 γ-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.

Computer-Aided Drug Design of β-Secretase, γ-Secretase and Anti-Tau Inhibitors for the Discovery of Novel Alzheimer’s Therapeutics

“…Multiscale MD simulations were employed to investigate substrate binding to γ-secretase and the role of substrate flexibility during the binding process [97]. MD simulations were combined with experimental techniques to map the binding site of BMS-708163 [98]. The molecular basis for Aβ peptides binding was studied with MD simulations and molecular docking indicating that the semi-open conformation of γ-secretase consistently shows the best binding mode for Aβ peptides, and it should be primarily targeted for the development of selective GSMs [99].…”

Mechanisms of γ-Secretase Activation and Substrate Processing

“…Computational modeling, especially molecular dynamics (MD) simulation, has proven useful in understanding the structural dynamics of γ -secretase. Previous studies have provided valuable insights into the conformational changes [15][16][17][18] , enzyme allosteric modulation 19 , substrate binding 15,18,[20][21][22] , water distribution [15][16] , lipid interactions 16 and ligand binding of γ -secretase [23][24][25] .…”

Small Molecule Interactome Mapping by Photo‐Affinity Labeling (SIM‐PAL) to Identify Binding Sites of Small Molecules on a Proteome‐Wide Scale

“…Comparison between samples can be achieved using quantification methods (e.g., label‐free, SILAC, tandem mass tag [TMT]). These approaches have been widely used to characterize the protein interactomes of metabolites, fragment libraries, bioactive small molecules, and drugs (Das, ; Flaxman & Woo, ; Gao, Mfuh, Amako, & Woo, ; Gertsik et al., ; MacKinnon, Garrison, Hegde, & Taunton, ; Parker et al., ). Cleavage of the enrichment handle by chemical or enzymatic means then allows for recovery of the small molecule–conjugated peptide for analysis of the binding site itself (Speers & Cravatt, ; Szychowski et al., ).…”