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Published on: May 5, 2023
Regulation of the NRF2 transcription factor by andrographolide and organic extracts from plant endophytes
Daphne Pei Wen Wong1, Mei Ying Ng1, Jia Yu Leung1
1Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Abstract:
The transcription factor NF-E2 Related Factor-2 (NRF2) is an important drug target. Activation of NRF2 has chemopreventive effects in cancer and exerts beneficial effects in a number of diseases, including neurodegenerative diseases, inflammatory diseases, hepatosteatosis, obesity and insulin resistance. Hence, there have been great efforts to discover and characterize novel NRF2 activators. One reported NRF2 activator is the labdane diterpenoid andrographolide. In this study, we identified the mechanism through which andrographolide activates NRF2. We showed that andrographolide inhibits the function of KEAP1, a protein that together with CUL3 and RBX1 forms an E3 ubiquitin ligase that polyubiquitinates NRF2. Andrographolide partially inhibits the interaction of KEAP1 with CUL3 in a manner dependent on Cys151 in KEAP1. This suggests that andrographolide forms Michael acceptor dependent adducts with Cys151 in KEAP1 in vivo, leading to inhibition of NRF2 ubiquitination and consequently accumulation of the transcription factor. Interestingly, we also showed that at higher concentrations andrographolide increases NRF2 protein expression in a Cys151 independent, but likely KEAP1 dependent manner, possibly through modification of other Cys residues in KEAP1. In this study we also screened secondary metabolites produced by endophytes isolated from non-flowering plants for NRF2-inducing properties. One of the extracts, ORX 41, increased both NRF2 protein expression and transcriptional activity markedly. These results suggest that endophytes isolated from non-flowering or other plants may be a good source of novel NRF2 inducing compounds.
Insights
Andrographolide activates the NRF2 pathway by inhibiting KEAP1, increasing NRF2 levels. Endophyte extracts also show potential for discovering new NRF2 activators.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Nuclear factor erythroid 2-related factor-2 (NRF2) is a crucial transcription factor with therapeutic potential in various diseases.
- Activating NRF2 offers chemopreventive benefits in cancer and therapeutic effects in neurodegenerative, inflammatory, and metabolic disorders.
- Discovering novel NRF2 activators is a significant area of pharmaceutical research.
Purpose of the Study:
- To elucidate the mechanism by which andrographolide activates NRF2.
- To investigate the role of KEAP1 cysteine residues in andrographolide-mediated NRF2 activation.
- To screen endophyte-derived secondary metabolites for NRF2-inducing properties.
Main Methods:
- Investigated andrographolide's effect on the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex.
- Assessed the role of KEAP1 Cys151 in andrographolide's interaction with KEAP1.
- Performed in vivo and in vitro assays to measure NRF2 ubiquitination and protein levels.
- Screened endophyte extracts for NRF2 activation using protein expression and transcriptional activity assays.
Main Results:
- Andrographolide inhibits KEAP1-CUL3 interaction, dependent on KEAP1 Cys151, leading to NRF2 stabilization.
- Higher andrographolide concentrations activate NRF2 via a Cys151-independent, KEAP1-dependent mechanism.
- Endophyte extract ORX 41 significantly enhanced both NRF2 protein expression and transcriptional activity.
Conclusions:
- Andrographolide activates NRF2 by directly inhibiting KEAP1's function through covalent modification of cysteine residues.
- Endophytes from non-flowering plants represent a promising source for novel NRF2-activating compounds.
- Understanding andrographolide's mechanism provides insights for developing NRF2-targeted therapeutics.
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