Thiol-reactive small molecules to accentuate oxidative stress in mitochondria

Cardwell, Patrick A. (2025) Thiol-reactive small molecules to accentuate oxidative stress in mitochondria. PhD thesis, University of Glasgow.

Abstract

Mitochondria are the hubs of cellular metabolism and redox signalling. Reactive oxygen species (ROS), produced as by-products of normal metabolism, regulate many signalling pathways essential to cell function. However, when present at supraphysiological levels, referred to as oxidative stress, ROS damage vital biomolecules, which threatens cell survival and contributes to the onset of various diseases. Compounds that accentuate mitochondrial oxidative stress can help to provide an understanding of redox signalling and can serve as leads for drugs that aim to exert a toxic effect towards malfunctioning mitochondria in disease states. One such compound, MitoCDNB, is the mitochondriatargeted analogue of 1 chloro-2,4-dinitrobenzene (CDNB), a common substrate for glutathione S-transferase (GST) enzymes. Owing to its triphenylphosphonium (TPP) cation, MitoCDNB accumulates into the mitochondrial matrix, where it selectively depletes mitochondrial glutathione (mGSH) via catalysis by mitochondrial GSTs (mGSTs). This disrupts mitochondrial thiol redox status and increases ROS production. The primary aim of this work was to build upon the MitoCDNB platform by leveraging the GSH/GST mechanism to selectively deliver probes and drugs to mitochondria. The initial focus was to deliver biguanides to induce ROS generation through inhibition of complex I. Chapter 2 explores the expression of three mGST isoforms and the synthesis of chloronitrobenzene derivatives for kinetics studies to investigate the enzymes’ substrate specificity. hGSTA1- 1 was found to be the most active enzyme for the derivatives, while MitoCDNB was the most reactive mitochondria-targeted substrate. Chapter 3 describes the synthesis and kinetics studies of sulfonamide derivatives, largely based on the structure of MitoCDNB, which aimed to release amine and phenol payloads. Although the release of a biguanide or an arylamine was unsuccessful, alkanesulfonamides enabled GSH/GST-mediated release of alkylamines. Subsequently, sulfonamides, which were attached to a phenolic fluorophore via an ethylenediamine linker, were synthesised to investigate the release of phenols. Kinetic studies showed that these ethylenediamine sulfonamides reacted rapidly with GSH in the absence of GST to release the fluorophore. Following this, ethylenediamine sulfonamide prodrugs of two phenolic mitochondrially bioactive compounds were synthesised. Chapter 4 explores the synthesis of two photoaffinity probes of a biguanide for use in a chemoproteomics study to identify the protein targets of biguanides. These probes are expected to advance the understanding of biguanides’ complex mechanisms of action and may uncover new therapeutic targets.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from EPSRC and the Wellcome Trust.
• Subjects: Q Science > QD Chemistry; Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Funder's Name: Wellcome Trust (WELLCOTR), Engineering and Physical Sciences Research Council (EPSRC)
• Supervisor's Name: Hartley, Professor Richard C.
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85361
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 08 Aug 2025 09:04
• Last Modified: 27 May 2026 11:34
• Thesis DOI: 10.5525/gla.thesis.85361
• URI: https://theses.gla.ac.uk/id/eprint/85361

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