Researchers from Gansu University of Chinese Medicine synthesized nitroxide radical–modified rhein derivatives to target the Keap1-Nrf2 antioxidant pathway. Among them, compound 4b displayed potent DPPH and ABTS free radical scavenging, protected L02 hepatocytes under oxidative stress, and extended Caenorhabditis elegans lifespan by over 40%, enhancing stress tolerance and antioxidant enzyme activity.
Key points
Design and synthesis of rhein nitroxide derivatives via DCC/DMAP coupling at the 3-carboxyl position.
Compound 4b exhibits IC₅₀ values of 0.51 mM (DPPH) and 0.12 mM (ABTS) and restores 95.4% viability in H₂O₂-challenged L02 hepatocytes.
In C. elegans, 300 µM 4b increases mean lifespan by 41%, enhances stress resistance, reduces ROS/MDA, and elevates GSH levels.
Why it matters:
This derivative offers a novel, targeted Keap1-Nrf2 modulation approach with strong anti-aging potential and improved antioxidant activity.
Q&A
What is the Keap1-Nrf2 pathway?
Why use C. elegans to test anti-aging compounds?
What are DPPH and ABTS assays measuring?
How does compound 4b compare with parent rhein?
Read full article
Academy
Keap1-Nrf2 Signaling Pathway
The Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid 2–related factor 2 (Nrf2) pathway is a central regulatory mechanism that protects cells from oxidative and electrophilic stress. Nrf2 is a transcription factor that, when activated, binds to antioxidant response elements (ARE) in the promoter regions of target genes and induces the expression of over 200 cytoprotective proteins. These include enzymes involved in antioxidant defense, detoxification, and stress resistance, such as glutathione S-transferases, NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), and key enzymes for glutathione biosynthesis.
Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, which serves as an adapter for a Cullin 3–based E3 ubiquitin ligase complex. Keap1 continually labels Nrf2 for ubiquitination and proteasomal degradation, keeping Nrf2 levels low. During oxidative or electrophilic stress, reactive species or modulators covalently modify critical cysteine residues on Keap1 (notably Cys151, Cys273, and Cys288). This modification hinders Keap1’s ability to target Nrf2 for degradation.
Activation Mechanism:
- Stress Sensing: Keap1 cysteines detect oxidants or electrophiles, triggering structural changes.
- Nrf2 Stabilization: Modified Keap1 releases Nrf2, allowing its accumulation.
- Nuclear Translocation: Stabilized Nrf2 moves into the nucleus.
- Transcriptional Upregulation: Nrf2 binds AREs and recruits coactivators, promoting transcription of antioxidant and cytoprotective genes.
Roles in Longevity: In model organisms from worms to mammals, Nrf2 activation correlates with extended lifespan and healthspan. Enhanced expression of antioxidant and detoxification enzymes prevents age-related accumulation of oxidative damage to DNA, proteins, and lipids, reducing the risk of neurodegenerative, cardiovascular, and metabolic disorders.
Pharmacological Modulation:
- Covalent Activators: Compounds like dimethyl fumarate and sulforaphane form Michael adducts with Keap1 cysteines, inactivating the Keap1–Cul3 complex.
- Protein–Protein Interaction Inhibitors: Non-electrophilic molecules block the interface between Keap1’s Kelch domain and the Neh2 domain of Nrf2, stabilizing Nrf2 in a redox-independent manner.
- Natural Product Derivatives: Plant-derived scaffolds, including rhein and its nitroxide-modified analogs, provide versatile templates for designing safer, potent Nrf2 activators.
Relevance to Anti-Aging Research: Targeting Keap1-Nrf2 represents a promising route to bolster intrinsic antioxidant defenses, slow cellular aging, and mitigate age-related pathologies. Studies like the development of nitroxide radical–modified rhein derivatives illustrate how chemical innovation can fine-tune this pathway, achieving robust antioxidant activity, enhanced cellular resilience, and extended organismal lifespan.
Future Directions: Ongoing research aims to optimize the specificity, potency, and safety profiles of Nrf2 modulators, balancing effective stress resistance with minimal off-target effects for potential translation into human anti-aging therapies.