Design of Substrate Transmembrane Mimetics as Structural Probes for γ-Secretase

Abstract

γ-Secretase is a membrane-embedded aspartyl protease complex central in biology and medicine. How this enzyme recognizes transmembrane substrates and catalyzes hydrolysis in the lipid bilayer is unclear. Inhibitors that mimic the entire substrate transmembrane domain and engage the active site should provide important tools for structural biology, yielding insight into substrate gating and trapping the protease in the active state. Here we report transmembrane peptidomimetic inhibitors of the γ-secretase complex that contain an N-terminal helical peptide region that engages a substrate docking exosite and a C-terminal transition-state analog moiety targeted to the active site. Both regions are required for stoichiometric inhibition of γ-secretase. Moreover, enzyme inhibition kinetics and photoaffinity probe displacement experiments demonstrate that both the docking exosite and the active site are engaged by the bipartite inhibitors. The solution conformations of these potent transmembranemimetic inhibitors are similar to those of bound natural substrates, suggesting these probes are preorganized for high-affinity binding and should allow visualization of the active γ-secretase complex, poised for intramembrane proteolysis, by cryo-electron microscopy.

Probing Mechanisms and Therapeutic Potential of γ-Secretase in Alzheimer’s Disease

“…More recently, HPI and TSA inhibitors have been combined to create substrate-based structural probes for the γ-secretase complex [ 62 ]. The structure of the γ-secretase complex was elucidated by cryo-electron microscopy (cryo-EM) in 2015 [ 63 ].…”

“…Thus, the enzyme was catalytically inactive, and the Cys mutations with crosslinking raised the possibility of artifacts. To trap the active enzyme without crosslinking, full TMD substrate-based mimetics were recently developed as tight-binding inhibitors of γ-secretase [ 62 ]. In the design of these structural probes, linking 10-residue helical peptide inhibitors (HPIs) to transition-state analog inhibitors (TSA) was envisioned to provide potent inhibitors that would simultaneously bind to both the docking site and active site.…”

“…TSA 10 and HPI 11 were selected for these studies ( Figure 7 ) [ 62 ]. TSA 10 , a pentapeptide analog spanning residues P2 through P3′, was among the most potent compounds to emerge from systematic variation of hydroxyethylurea peptidomimetics [ 54 ].…”

“…Enzyme inhibition kinetics demonstrated that 15 had a Ki of 0.42 nM toward γ-secretase [ 62 ]. Moreover, cross-competition kinetic experiments revealed that while TSA and HPI compounds were noncompetitive with respect to each other, 15 was competitive with both TSA and HPI, consistent with interaction of 15 with both docking site and active site.…”

Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction

“…For cross-competition studies between two inhibitors, enzyme reactions were run as activity assays, where varying concentration of two inhibitors were titrated against each-other, keeping the concentrations of C100 (0.50 μM) constant. 31,41 Reciprocal plots were generated, and data were fit using GraphPad Prism 9.0 into the following equation:…”