Mechanism of gamma‐secretase modulators

Abstract

BackgroundAmyloid‐beta (Aβ) peptides are believed to be integral to Alzheimer’s disease (AD) pathogenesis through their role in the “amyloid hypothesis,” in which the accumulation of Aβ peptides initiates a cascade of pathological events leading to neurodegeneration and AD. γ‐Secretase is a transmembrane aspartyl protease which cleaves APP to generate Aβ peptides, making γ‐secretase an attractive drug target. However, inhibitors failed in clinical trials due to their unwanted side effects on other γ‐secretase substrates such as Notch. ɣ‐Secretase modulators (GSMs) selectively reduce levels of the pathogenic Aβ species without affecting Notch and overall APP processing. However, many questions on their mechanism still remain. Here we investigate the molecular mechanism of GSMs by (1) investigating the binding site of imidazole GSM E2012 using photoaffinity labeling and molecular docking and (2) investigating combination therapy with different GSMs on reducing Aβ production.MethodPart I: We incubated clickable GSM photoaffinity probes with cell membranes in the presence or absence of competitors, followed by UV irradiation to initiate photo‐crosslinking to nearby proteins and conjugation to biotin via click chemistry. Labeled proteins were isolated with affinity chromatography by streptavidin beads, eluted, and analyzed by western blot. Probes were docked onto γ‐secretase (PDB 7D8X) using tools from Schrodinger Suite 2021‐2. Part II: We treated N2a‐APP cells with GSM‐1 (acid GSM), E2012, or BPN‐15606 (imidazole GSMs) individually or in combination for 12h. Afterwards we collected cell culture media and measured secreted Aβ38, Aβ40, and Aβ42 levels using MesoScale Discovery.ResultWe demonstrate that GSM photoprobe E2012‐BPyne labels presenilin‐1 and nicastrin in the presence of transition state analog L‐685, 458, consistent with structural observations. From molecular docking studies we observe that GSM photoprobes differentially interacted with γ‐secretase and docking scores aligned with probe labeling efficiency. Furthermore, we demonstrate that acid and imidazole GSMs synergistically inhibit cellular Aβ production.ConclusionOur findings have revealed insight into the binding of GSMs and the effects of two GSM classes in combination. We are currently examining the effects of combined GSMs in primary neurons and on Notch production. These studies aim to better understand the mechanisms of GSMs to target γ‐secretase in AD.

Gamma-secretase modulators: a promising route for the treatment of Alzheimer's disease

“…The binding sites for the two classes of GSMs are likely different since they do not show competitive binding, and they affect the processing of APP differently, resulting in different Aβ profiles (Borgegård et al, 2012 ; Olsson et al, 2014 ). There is evidence suggesting that the carboxylic acid class of GSMs interacts with APP rather than GSEC (Kukar et al, 2008 ), a finding coherent with the fact that both classes of GSMs have synergistic properties in reducing longer forms of Aβ (Robertson et al, 2017 ; Luo et al, 2022 ).…”