Development of short-chain quinones
thesisposted on 2023-05-28, 00:19 authored by Feng, Z
Mitochondrial dysfunction leads to rare mitochondrial diseases but is also present in a vast number of common disorders such as glaucoma and diabetes. Despite the large number of affected patients, there is a striking unmet medical need for pharmacological approaches that directly target mitochondrial function. For this purpose, short-chain quinones (SCQs) have been widely investigated as potential therapeutic molecules for mitochondrial diseases and related disorders. To date, only the synthetic benzoquinone idebenone has been clinically approved in limited countries for a single mitochondrial disease, Leber's hereditary optic neuropathy (LHON). Although idebenone consistently shows very good safety and efficacy against vision loss in some LHON patients, its activity is limited by its rapid metabolic inactivation. To overcome these significant limitations, a library of novel SCQs was designed and synthesized at the University of Tasmania. From this panel, some compounds showed significantly improved cytoprotective activity in vitro compared to idebenone under conditions of mitochondrial dysfunction in the human hepatocarcinoma cell line HepG2. To identify the most promising SCQs from this novel library to aid its further development against mitochondrial diseases and related disorders, the current project aimed to characterize in vitro bioactivity, metabolic stability and toxicity profiles as well as in vivo therapeutic efficacy for selected candidate molecules. An in vitro bioactivity profile for 103 SCQs was generated that assessed metabolism related markers, redox activity, expression of cytoprotective proteins and oxidative damage in HepG2 cells. Cytoprotection by SCQs in the presence of rotenone was observed to be correlated with the NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent reduction of SCQs, which suggests an unexpected mode of action for SCQs that appears to involve a modification of NQO1-dependent signaling rather than a protective effect by the reduced quinone itself. Subsequently, a simple and efficient reverse-phase liquid chromatography (RP-LC)-based method was developed to determine the metabolic stability of the 16 most cytoprotective SCQs in HepG2 cells. In this assay, 15 SCQs, showed significantly higher metabolic stability than idebenone. Furthermore, detailed toxicity profiles were generated for the 11 best SCQs that displayed enhanced cytoprotection and improved metabolic stability. Compared to idebenone, lower metabolic toxicity, lower toxicity with regards to membrane integrity, lower long-term toxicity, as well as an absence of mitochondrial toxicity, pyknosis, DNA damage, or transformation potential were identified for different SCQs to different extents. Lastly, two novel SCQs identified with enhanced cytoprotection, improved metabolic stability, absence of mitochondrial toxicity or transformation potential were assessed for their protective activity in a rat model of diabetic retinopathy. Both compounds, given topically as eye drops, showed superior activity to restore vision in this pre-clinical model compared to the reference compounds idebenone and elamipretide. Unfortunately, due to aberrant animal housing conditions, the study was unable to demonstrate systemic therapeutic efficacy in this model. Overall, the results described in this thesis rationalize the selection of development candidates from a range of novel SCQs and inform their further development towards their clinical use.
Rights statementCopyright 2021 the author Chapter 2 is the following published article: Feng, Z., Nadikudi, M., Woolley, K. L., Hemasa, A. L., Chear, S., Smith, J. A., Gueven, N., 2021. Bioactivity profiles of cytoprotective short-chain quinones, Molecules, 26(5), 1382. Copyright: Copyright 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/). Chapter 3 is the following published article: Feng, Z., Smith, J. A., Gueven, N., Quirino, J. P., 2020. Metabolic stability of new mito-protective short-chain naphthoquinones, Pharmaceuticals, 13(2), 29. Copyright: Copyright 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/). Chapter 4 is the following published article: Feng, Z., Sedeeq, M., Daniel, A., Corban, M., Woolley, K. L., Condie, R., Azimi, I., Smith, J. A., Gueven, N., 2020. Comparative in vitro toxicology of novel cytoprotective short-chain naphthoquinones, Pharmaceuticals, 13(8), 184. Copyright: Copyright 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/).