Turning the tide on Alzheimer’s disease: modulation of γ-secretase

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.

Targeted Activation in Localized Protein Environments via Deep Red Photoredox Catalysis

State-of-the art photoactivation strategies in chemical biology provide spatiotemporal control and visualization of biological processes. However, using high energy light (λ < 500 nm) for substrate or photocatalyst sensitization can lead to background activation of photoactive small molecule probes and reduce its efficacy in complex biological environments. Here we describe the development of targeted aryl azide activation via deep red light (λ = 660 nm) photoredox catalysis and its use in photocatalyzed proximity labeling. We demonstrate that aryl azides are converted to triplet nitrenes via a novel redox-centric mechanism and show that its spatially localized-formation requires both red light and a photocatalyst-targeting modality. This technology was applied in different colon cancer cell systems for targeted protein environment labeling of epithelial cell adhesion molecule (EpCAM). We identified a small subset of proteins with previously known and unknown association to EpCAM, including CDH3, a clinically relevant protein that shares high tumor selective expression with EpCAM.

Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes

ADP-ribosylation is a key post-translational modification that regulates a wide variety of cellular stress responses. The ADP-ribosylation cycle is maintained by writers and erasers. For example, poly(ADP-ribosyl)ation cycles consist of two predominant enzymes, poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG). However, historically, mechanisms of erasers of ADP-ribosylations have been understudied, primarily due to the lack of quantitative tools to selectively monitor specific activities of different ADP-ribosylation reversal enzymes. Here, we developed a new NUDT5-coupled AMP-Glo (NCAG) assay to specifically monitor the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes. We found that NUDT5 selectively cleaves protein-free ADP-ribose, but not protein-bound poly- and mono-ADP-ribosylations, protein-free poly(ADP-ribose) chains, or NAD+. As a proof-of-concept, we successfully measured the kinetic parameters for the exo-glycohydrolase activity of PARG, which releases monomeric ADP-ribose, and monitored activities of site-specific mono-ADP-ribosyl-acceptor hydrolases, such as ARH3 and TARG1. This NCAG assay can be used as a general platform to study the mechanisms of diverse ADP-ribosylation reversal enzymes that release protein-free ADP-ribose as a product. Furthermore, this assay provides a useful tool to identify small-molecule probes targeting ADP-ribosylation metabolism and to quantify ADP-ribose concentrations in cells.

Ferropsis cell death can cause complications that may be difficult to detect and quantify: autophagy role and possible therapeutics

It is possible to understand both the processes of ferroptosis and how this type of cell death will be harnessed in the near future. The novel ferroposis-based therapies will also be created and evaluated using various biomarkers. Selenium and vitamin E have been shown to support some types of cancer in several studies. Such environmental factors should be accounted for while exploring ferroaptosis's duties. The effect of these modifications will be dictated by the cellular and environmental context in which they are created. It is critical to determine whether the inhibitor can act as a lipophilic radical-trapping antioxidant and prevent ferroPTosis independently of the enzyme under investigation, as several studies with lipoxygenases have shown.Using pharmacological approaches to evaluate ferropsosis can have potential side effects that are difficult to measure. Using small-molecule probes may be investigated using small-molecules probes. It's essential to utilize a biochemical test or a pharmacodynamic marker of target inhibition when employing RSL3 to inhibit GPX4, or when using erastin to inhibit the system xc–cystine/glutamate antiporter, or by using an Xc–Cystine/Glutamate/Glutamic antiporter inhibitor, or an erast inhibitor, to inhibit Gpx4, GSH, and other types of protein thiols, such as GSH and protein thiola. Many tiny molecules that are potent and selective probes in cellular assays have limited applicability in animal studies. RSL3, a poor solubility, is useful only in instances when an injection is made directly into tissues or tumors. Interfering with iron to modulate ferroptosis has far-reaching ramifications. Findings obtained with iron chelators only should be interpreted with caution as depleting iron can have other side effects other than ferroPTosis suppression.

Visualizing Borrelia burgdorferi infection using a small molecule imaging probe.

In vivo diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here we establish HS-198 as a small molecule-fluorescent conjugate that selectively targets the highly conserved bacterial protein, HtpG (High temperature protein G), within B. burgdorferi, the bacterium responsible for Lyme Disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase as well as in inactivated spirochetes. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in post necropsy tissue sections. These findings demonstrate how small molecule probes directed at conserved bacterial protein targets can function to identify the microbe using non-invasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen.

Two-photon small-molecule fluorescence-based agents for sensing, imaging, and therapy within biological systems

In this tutorial review, we will explore recent advances for the design, construction and application of two-photon excited fluorescence (TPEF)-based small-molecule probes.

Recent advances in stimuli-responsive in situ self-assembly of small molecule probes for in vivo imaging of enzymatic activity

Stimuli-responsive in situ self-assembly of small molecule probes into nanostructures has been promising for the construction of molecular probes for in vivo imaging.