Novel short-chain quinones against mitochondrial dysfunction
Creators: name: Nadikudi, M
Publisher: name: University Of Tasmania
Year: 2023

description: Mitochondria regulate crucial cellular processes such as energy production in the form of adenosine triphosphate (ATP), Ca\\(^{2+}\\) homeostasis, cellular redox status and cell death. Consequently, cells and tissues that depend on these functions are susceptible to mitochondrial dysfunction. Any insult or genetic predisposition that leads to mitochondrial dysfunction can lead to a range of disorders that can manifest in many different tissues. Mitochondrial diseases, caused by mutations in mitochondrial DNA typically demonstrate severe neurological pathologies. These disorders are usually associated with defects in oxidative phosphorylation, reduced ATP levels, increased oxidative stress and decreased cellular viability. A class of compounds known as quinones are reported to protect against mitochondrial dysfunction. Currently, only one benzoquinone, idebenone, is approved in Europe to specifically treat a mitochondrial disease, while a few other drug candidates are in clinical development. Idebenone is reported to rescue cellular ATP levels and act as an antioxidant. Despite reported preclinical and clinical efficacy, no causative mode of action has been confirmed for this class of molecules that goes beyond pure associations. It is thought that quinones need to be bio-activated by cellular reductases such as NAD(P)H quinone oxidoreductase 1 (NQO1) for their therapeutic activity. Due to a rapid first pass metabolism and dependence on a single enzyme (NQO1) for its bio-activation, the clinical efficacy of idebenone is limited. Naphthoquinones, structural analogues of vitamin K, have also been investigated as potential therapeutic molecules to treat mitochondrial dysfunction-induced neurological disorders. In fact, growing evidence suggests that naphthoquinones could be beneficial to counteract mitochondrial dysfunction that is associated with neurological disorders such as epilepsy and Parkinson's disease. In this study, I characterised more than 110 novel short-chain naphthoquinones (SCQs) to protect against mitochondrial dysfunction while working towards a better understanding of their mechanism of action. These SCQs were characterized for their ability to protect against mitochondrial dysfunction in vitro. Consistent with previous data from benzoquinones, most novel SCQs were mainly bioactivated by NQO1, while some were also bio-activated by other unidentified cellular reductases. Overall, more than 20 of our SCQs showed significantly better in vitro cytoprotection against the mitochondrial complex-I (C-I) inhibitor, rotenone, compared to idebenone. For the first time, together with my collaborators from Department of Chemistry, we established a clear structure-activity relationship and identified optimal chemical characteristics for naphthoquinone-based SCQs. This included a specific solubility range with a defined balance between side-chain polarity, fattiness and the presence of specific functional groups attached to the quinone core. Interestingly, more than half of the novel SCQs significantly increased basal ketone levels in cellular supernatant suggesting possible upregulation of fatty acid metabolism. In contrast to what is currently believed as the mechanism of action of quinones, a striking lack of correlation between cytoprotection by our SCQs and their effects on ATP levels, lipid peroxidation, lactate or ketone levels suggests that the mechanism of action of these compounds is likely much more complex than previously anticipated. One of the most common, maternally-inherited mitochondrial disorders, Leber's Hereditary Optic Neuropathy (LHON) is caused by mutations in the mitochondrial DNA that encode subunits of mitochondrial C-I. These LHON mutations result in mitochondrial C-I deficiency, decreased ATP generation and increased oxidative stress which cause reduced retinal thickness and ultimately loss of retinal ganglion cells (RGC). LHON patients show a rapid and progressive loss of visual acuity. However, a rare possibility of partial recovery of vision was reported for some patients, sometimes even years after disease onset. Currently, idebenone is the only drug approved in the Europe for the treatment of LHON. In order to translate my in vitro findings to a disease model, two of the most promising cytoprotective novel SCQs that also significantly rescued ATP levels in vitro in the presence of the C-I inhibitor, rotenone, were tested in an in vivo mouse model of LHON. Intraocular injection of rotenone in wild-type C57Bl/6 mice significantly (p&lt;0.05) reduced both retinal RGC counts and the retinal thickness, which was associated with rapid loss of visual acuity. After 70 days of oral treatment with the novel SCQs, visual acuity was restored significantly (p&lt;0.05) better than sham or idebenone-treated mice, with improvements evident from the first week of treatment. Both SCQs significantly (p&lt;0.05) protected against RGC loss and reduced retinal thickness under conditions where idebenone showed no efficacy. In addition, no overt signs of SCQ-induced toxicity were observed in any of the animals. Overall, my in vitro and in vivo data suggest that these novel SCQs can be developed into superior, effective and safe drugs to treat disorders associated with mitochondrial dysfunction.