Targeting the cell’s protein folding machinery: the development of heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) inhibitors

Heat shock proteins 90 (Hsp90) and 70 (Hsp70) are over-expressed in various cancer cells and these two chaperone systems collaborate to fold several oncogenic proteins involved in tumorigenesis. Heat shock organising protein (HOP) is a mediator protein that links the Hsp70 and Hsp90 chaperone systems together by simultaneously binding to the C-terminus of both heat shock proteins. This thesis describes the development of the C-terminal Hsp90 modulators, and the development of C-terminal Hsp70 inhibitors that target the Hsp70-HOP interaction. Modulating the Hsp70-HOP interaction is an effective strategy for simultaneously inhibiting the chaperone function of Hsp70 and the downstream Hsp90 system. Chapter 2 describes the synthesis, formulation and use of SM253 as a biological probe to understand the function of Hsp90. SM253 is a novel C-terminal modulator of Hsp90 previously developed by the McAlpine group. SM253 was synthesised and formulated for testing in tumour mice models, but was too hydrophobic to be formulated in an appropriate aqueous formulation to be delivered to mice. The most successful strategy for improving the aqueous solubility of SM253 involved encapsulating the peptide into polymeric micelles. This allowed SM253 to be delivered into cells as its active form. SM253 was also sent to collaborators to study the role of Hsp90 in membrane deformation and exosome release as well as Hsp90’s role in the hypoxic response. Chapter 3 describes the design, synthesis and biological evaluation of a peptide library derived using sequences from HOP’s TPR1 domain that interact with Hsp70. C1 was identified as the lead molecule from this library and inhibited the folding function of Hsp70 by directly stabilising the Hsp70-HOP interaction, thus preventing the dynamic nature of the protein folding complex. Chapter 4 describes the biological activity of analogues of C1 and their ability to impact the Hsp70-HOP interaction. Longer peptide analogues C7 and C8 behaved as traditional inhibitors. To investigate the binding site of C1, C7 and C8, these compounds were tested in pulldown assays with Hsp70 and its domains. The results revealed that C1 and C7 bind to the C-terminus of Hsp70 and therefore directly target the Hsp70-HOP interaction. Preliminary results also suggest that C1 displays preferential binding to the stress inducible isoform of Hsp70