Design and Synthesis of Multivalent Dendritic Assemblies for the Treatment of Cancer
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The importance of protein-protein interactions in regulating key biological processes, particularly in relation to apoptotic regulation and cancer, has provided many challenges for traditional small molecule approaches to drug development. Historically, the majority of small molecules targeted at protein-protein interfaces have been unable to provide the tight binding affinities and steric bulk required to disrupt such interactions, as they often occur along flat and extended protein surfaces. In this work we have investigated the combination of peptide-based therapeutics, with modularly constructed dendritic scaffolds, targeted at disrupting crucial protein-protein interactions involved in apoptosis. In particular, we have selected the interaction between the anti-apoptotic protein Mcl-1 and its pro-apoptotic, BH3 only, binding partner NOXA. The Bcl-2 family of antiapoptotic proteins, of which Mcl-1 is a member, has been linked to a wide variety of human cancers. Specifically, over-expression of Bcl-2 related proteins has been correlated to resistance of many tumours to the normal chemotherapeutic approaches in use to date. Over the past decade there has been significant research focus on the antiapoptotic proteins Bcl-2 and Bcl-XL resulting in the development of small molecule Bcl-2 inhibitors such as ABT-737. However, recent investigations have shown that knockdown of a single Bcl-2 family protein within the apoptotic cascade may be insufficient for the effective treatment of some cancers. Resistance to ABT-737 in some malignancies has been linked to the over expression of the less well-known antiapoptotic protein Mcl-1, highlighting the need for greater investigation into its potential as a therapeutic target. The potential development of a therapeutic targeted at inhibiting the anti-apoptotic effect of Mcl-1 within this work was focused on the design and synthesis of a series of NOXA derived peptides bearing a C-terminal poly arginine transport sequence (R5). The peptides were synthesised using solid phase peptide synthesis methods and investigated for their cellular transport and cytotoxicity. It was determined that the 31 residue NOXA peptide PAELEVECATQLRFRQRRRRR has an IC50 of ~3μM and that the C-terminal poly-arginine tag provided stabilisation of α-helical secondary structural motifs in addition to facilitating transport across the cellular membrane. Truncation of the NOXA derived peptide to 15 residues incorporating only the conserved BH3 region of the protein resulted in a complete loss of pro-apoptotic activity. Concurrently, studies were conducted into the assembly of dendritic scaffolds based on the principles of click chemistry and convergent dendrimer synthesis. This work was successful in producing a series of modular ‘building blocks’ including bi- and trifunctional core moieties, tri-functionalised peripheral dendrons bearing a variety of functionalities (NO2, OMe, and Maleimide) and an internal branching unit designed to facilitate the ‘generational like’ growth of the macromolecules. The array of modular components was successfully combined in the synthesis of a series of corresponding dendritic assemblies bearing 6, 9 or 12 functional groups. The dendritic assemblies bearing 6 or 9 peripheral functionalities were achieved in very good yield and high purity without the need for chromatographic purification. Application of the dendritic scaffolds for the construction of peptide dendrimer conjugates was focused on the use of the maleimide functionalised 6-mer dendritic scaffold and cysteine terminated peptides. A total of five peptide-dendrimer conjugates were assembled ranging from the attachment of small di-peptide units to a series of conjugates bearing synthetic NOXA derivatives with a molecular mass of >17kDa. The systems were unable to be investigated for cytotoxicity at this time however due to complications in purification. The results outlined within this work indicate that the neutralisation of Mcl-1 via synthetic NOXA derivatives provides a novel solution to the challenges presented by the protein-protein interactions crucial to drug resistant cancers. In particular the application of highly defined dendritic macromolecules such as those presented within this work may lead to effective co-therapies when used in conjunction with Bcl-2 inhibitors such as ABT-737. Furthermore, it highlights the flexibility of a modular synthetic design that can be adapted to incorporate any cysteine-terminated peptide of biological interest or thiol containing therapeutic.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
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The following images have been removed at the autor's request due to copyright. Chapter 1 : Figure 1-2 Chapter 1 : Figure 1-6 Chapter 1 : Figure 1-7 Chapter 1 : Figure 1-11 Chapter 1 : Figure 1-12 and 1-13 Chapter 2 : Figure 2-1 Chapter 2 : Figure 2-3