Alzheimer’s disease (AD) is the most common type of neurodegenerative disorder. Amyloid-beta (Aβ) plaques are integral to the “amyloid hypothesis,” which states that the accumulation of Aβ peptides triggers a cascade of pathological events leading to neurodegeneration and ultimately AD. While the FDA approved aducanumab, the first Aβ-targeted therapy, multiple safe and effective treatments will be needed to target the complex pathologies of AD. γ-Secretase is an intramembrane aspartyl protease that is critical for the generation of Aβ peptides. Activity and specificity of γ-secretase are regulated by both obligatory subunits and modulatory proteins. Due to its complex structure and function and early clinical failures with pan inhibitors, γ-secretase has been a challenging drug target for AD. γ-secretase modulators, however, have dramatically shifted the approach to targeting γ-secretase. Here we review γ-secretase and small molecule modulators, from the initial characterization of a subset of NSAIDs to the most recent clinical candidates. We also discuss the chemical biology of γ-secretase, in which small molecule probes enabled structural and functional insights into γ-secretase before the emergence of high-resolution structural studies. Finally, we discuss the recent crystal structures of γ-secretase, which have provided valuable perspectives on substrate recognition and molecular mechanisms of small molecules. We conclude that modulation of γ-secretase will be part of a new wave of AD therapeutics.

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions.  Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread.  For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000.  Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase.  The 2.5 Å resolution x-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition.  Functional characterization of site-directed mutants targeting the substrate and NAD(H) binding sites identify key residues in the active site for ligand interactions, including those in the 'aromatic box' that define the aldehyde binding site.  Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.

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.