Thesis
Expanding the heterocyclic repertoire of DNA minor groove binding polyamides
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2019
- Thesis identifier
- T15177
- Person Identifier (Local)
- 2010760278
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Exogenous control of gene expression is an essential goal to better understand and ultimately treat diseases which involve dysregulation of transcription. In this effort, Pyrrole-Imidazole (Py-Im) hairpin polyamides (PAs) are minor groove binding (MGB) organic small molecules that demonstrate a versatile capability to recognize, bind, and alter transcription in vitro and, more recently, in vivo.[1] Despite their utility for distinguishing G-C/C-G base pairs, there are several limitations which impede the utilization of polyamides on scale, a major hurdle being that current monomeric pairings do not distinguish a T-A base pair from an A-T. To break this degeneracy, novel building blocks have been sought after to both improve selectivity as well as tune the pharmacokinetic properties of polyamides. Using an 8-ring hairpin scaffold that has demonstrated activity against prostate cancer, the impact of an N-isopropylimidazole monomer (iPr-Im) in the terminal position is reported and a comparison made with the N-methylimidazole (Im) analogue, along with thiazole analogues studied by Padroni et al. (isopropyl thiazole, iPr-Nt, and methyl thiazole, Me-Nt)[²]. UV absorption / fluorescence melt analyses and detailed interrogation of the binding kinetics suggest a strong affinity of hairpin polyamides incorporating the terminal iPr-Im unit, comparable with Im. The data gathered further suggest that the iPr-Im unit itself maintains specificity for G over C, A, or T. The switchSENSE® data also provided insight into the kinetic basis of the selectivity differences, showing a difference between the imidazole analogues and the thiazole analogues. A three-dimensional NMR structure of the iPr-Im-containing polyamide in complex with a 12-mer sequence of double-stranded DNA containing the targeted androgen response element 5'-WWGWWCW-3' (W = A/T) revealed an enhanced major groove compression over the previously reported Im analogue, but also a deeper minor groove penetration than previously reported iPr-Nt polyamide.[²];Chapter 1 introduces the regulation of gene expression using MGB small molecules.Chapter 2 describes the synthesis of hairpin polyamides, starting with the building blocks and then the solid phase synthetic protocol for constructing 8-ring hairpin polyamides.Chapter 3 showcases the thermal UV melt data, and examines the polyamide binding kinetics with DNA duplexes. Further insight into the binding was obtained with switchSENSE® technology, which allowed the generation of kinetic rate maps. The data outlined in this chapter enabled the profiling of the kinetic parameters that govern PA-dsDNA binding.Chapter 4 provides a structural view of a novel iPr-Im-containing polyamide-DNA complex, highlighting the closer proximity of the N-terminal iPr-Im N2 to the exocyclic amine of G5 compared with the N-terminal iPr-Nt analogue. Furthermore, the 3D NMR-restrained molecular dynamics (MD) structure indicated the major groove compression was greater for polyamide with the terminal iPr-Im monomer compared with the Me-Im analogue, highlighting the importance of a seemingly subtle change in steric bulk facing away from the minor groove for augmenting structural distortion of the DNA duplex.Chapter 5 takes the key findings of this thesis and uses them as a lens for looking to the future direction that this research may take. Namely, the potential of this novel iPr-Im monomer unit to replace imidazole as a G-selective residue that can induce greater structural distortion of the DNA duplex by polyamides.
- Advisor / supervisor
- Burley, G.A.
- Resource Type
- DOI
- Date Created
- 2019
- Former identifier
- 9912707492302996
- Embargo Note
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