Thesis
Next-generation transcriptional activators for the treatment of Friedreich’s Ataxia
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2023
- Thesis identifier
- T16811
- Person Identifier (Local)
- 201978623
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Pyrrole-imidazole polyamides are programmable minor groove binders which recognize specific duplex DNA sequences. Polyamides have been extensively utilized as exogenous regulators of transcription. However, their application in biology has mainly been focused on down-regulating gene expression. Friedreich’s Ataxia is a neurodegenerative disease in which transcription of the FXN gene is compromised, due to an abnormal expansion of GAA triplet repeats. A previous study has shown that the use of a GAA-targeting polyamide (FA1) linked to bromodomain inhibitor scaffolds produces a “synthetic transcription factor” effect, restoring the levels of frataxin in affected cells (1.34, Figure a). Nonetheless, the use of existing synthetic transcription factors (syn-TFs) for in vivo applications is limited by poor pharmacokinetic properties. Moreover, the complex interaction between syn-TF and its binding partners remains uncodified. Therefore, a synthetic campaign was applied to increase the affinity and druggability of the polyamide core (Figure b). Structural analogues of FA1 were produced with a particular focus on heterobicyclic incorporation at the N terminus of the polyamide backbone. These modifications increased the stabilisation of the target DNA while improving FA1 druggability. Additionally, biophysical analysis using DNA nanolevers showed that the positioning of the flexible β-alanine unit in the modified FA1 analogues greatly affected the selectivity profile of the polyamide towards the target duplex DNA. Furthermore, NMR analysis (2D-NMR, isotopic labelling and diffusion techniques) of the polyamide-DNA complex was used to investigate the specific binding mode of the polyamide structure. The results of this study will directly inform the rational design of an improved generation of syn-TFs. Chapter 1 provides an overview of recognition of DNA by small molecules and exogenous regulation of gene expression. Furthermore, it describes Friedreich’s Ataxia and its treatment. Also, limitations related to the use of existing syn-TFs to upregulate frataxin transcription are presented.Chapter 2 describes the rational design and the production of a series of FA1 analogues bearing different types of modifications. It illustrates the synthesis of novel aromatic head groups which are incorporated into linear polyamides. Also, solid-phase methodology which is used to prepare the polyamides, is described in this chapter.Chapter 3 reports on the evaluation of the binding properties of linear polyamides. Formation of polyamide-DNA complexes is assessed by UV melt analysis. A biophysical technique based on the switchSENSE® technology, is also used to get insights into the kinetic parameters and binding affinity of a selected set of polyamides.Chapter 4 describes the development of an NMR-based approach to analyze polyamides targeting repeated GAA-sequences. Limitations of current methodology are illustrated as well as alternative strategies that could facilitate the study of challenging DNA sequences.Chapter 5 provides a summary of the work carried out in this project, with a particular focus on potential future directions.
- Advisor / supervisor
- Burley, Glenn
- Resource Type
- DOI
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- Embargo Note
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