Dissertation
Novel analogs of the natural product Fraxinellone protect against endogenous and exogenous toxicants
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2024
DOI: 10.25820/etd.007420
Abstract
Numerous insults, both endogenous (e.g., Glu) and exogenous (e.g., pesticides), compromisefunction of the nervous system and pose as risk factors for mediators of damage and/or later disease. For example, when extracellular levels of the excitatory neurotransmitter glutamate (Glu) rise due to pathological conditions (e.g., epilepsy, amyotrophic lateral sclerosis) or injury (e.g., stroke), aberrant synaptic signaling leads to excitotoxicity and subsequent oxidative stress, thought to contribute to neurodegeneration. Numerous pesticides, e.g., rotenone, are known to induce oxidative stress and cause injury to neurons. In previous reports, limonoids such as fraxinellone, showed significant neuroprotective activity against Glu excitotoxicity and reactive oxygen species (ROS) production in vitro. Given these findings, a library of novel fraxinellone analogs was synthesized with the goal of identifying analogs exhibiting neuroprotection against endogenous and exogenous insults. One analog was found to be protective against Glu-mediated toxicity with a measured EC50 of 44 nM and 39 nM in in vitro assays using PC12 and SH-SY5Y cells, respectively. To probe the mechanism of action, a series of experiments were performed demonstrating antioxidant activity following treatment with the fraxinellone-derived analog and rapid activation of the Nrf2 pathway. Pre-treatment with the agent yielded rapid induction of antioxidant genes, namely, GPX4, SOD1 and NQO1, as measured via qPCR. The analog mitigated Glu-mediated ROS as measured using two different fluorescent probes. Cytoprotection could be replicated using sulforaphane (SFN), a Nrf2 activator, and inhibited via ML-385, which binds Nrf2 and interferes with its binding to regulatory DNA sequences, thereby blocking downstream gene expression. Nrf2 DNA-binding activity of nuclear extracts was performed using a Nrf2 ELISA-based transcription factor assay kit. In addition, pre-treatment with the thiol N-acetyl Cys (NAC) completely mitigated SFN-mediated induction of antioxidant genes but had no effect on the activity of the fraxinellone analog, suggesting thiol modification is not critical for its mechanism of action. In summary, our data demonstrate a fraxinellone analog to be a novel, potent, and rapid activator of the Nrf2-mediated antioxidant defense system, providing robust protection against endogenous and exogenous insults. Given that, further development of this class of analogs may yield promising therapeutic strategies to address pathogenic or neurodegenerative conditions, such as those involving Glu excitotoxicity and excessive production of ROS.
Details
- Title: Subtitle
- Novel analogs of the natural product Fraxinellone protect against endogenous and exogenous toxicants
- Creators
- Anna Elizabeth Bartman
- Contributors
- Jonathan A Doorn (Advisor)Ethan J. Anderson (Committee Member)Marie E. Gaine (Committee Member)David B. Martin (Committee Member)Florence J. Williams (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Pharmacy (Medicinal and Natural Products Chemistry)
- Date degree season
- Spring 2024
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.007420
- Number of pages
- xvi, 104 pages
- Copyright
- Copyright 2024 Anna Elizabeth Bartman
- Language
- English
- Date submitted
- 01/03/2024
- Description illustrations
- illustrations, tables, graphs
- Description bibliographic
- Includes bibliographical references (pages 96-104).
- Public Abstract (ETD)
- Oxidative stress and oxidative-mediated cell death is implicated in many neurodegenerative diseases, including Alzheimer’s disease, (AD) Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Key causes of this oxidative-mediated cell death include several insults that come from within our own bodies or from environmental insults, like exposure to pesticides. Although numerous drugs evaluated as antioxidants or other mediators of oxidative stress have exhibited therapeutic potential in preclinical studies, many have been unsuccessful upon reaching clinical trials. A key group of natural products that are part of the limonoid class (e.g., fraxinellone) have shown great potential to protect against glutamate (Glu)- mediated oxidative cell death. However, many current drugs that mediate oxidative stress fail when they reach clinical trials because there is no understanding of the mechanism through which these natural products act and where they are most effective. My doctoral work has focused on assessing the activity of a library of novel analogs of the natural product fraxinellone and identifying their mechanism of protection through use of two different cell models. First, the goal of my work was to assess the activity of the various analogs against glutamate and then determine if the protection afforded by the fraxinellone analogs was generalizable to other oxidative insults. Surprisingly, I found several analogs better protected against each of these insults when compared to natural fraxinellone. Following identification of several active analogs, I was able to provide sound evidence suggesting they protect through Nrf2 activation, which is a defense mechanism within our bodies that induces an antioxidant response to protect against oxidative stress.
- Academic Unit
- Pharmacy
- Record Identifier
- 9984647356802771
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