Abstract:
Phosphoinositide 3-kinases (PI3Ks) play crucial roles within the cell, orchestrating multiple signalling pathways and regulating various cellular processes, misregulation of PI3K signalling is critical in human diseases including cancer. Blocking the PI3K-Gβγ protein-protein interaction (PPI) offers a way to block the activation of PI3Kβ and –γ isoforms by GPCRs to dissect out the signalling pathways related to different types of activating input, distinct from the catalytic activity. The PI3K-Gβγ PPI is studied by site-specific mutations that prevent the interaction occurring or by knockout of one of the proteins, this limits the number of cell lines studied. Chemical tools targeting the PI3K-Gβγ PPI would be better suited to investigation across multiple cells lines, possibly also avoiding the risk of cells adapting to genetic changes. Available tools include a small molecule Gβγ inhibitor, Gallein, which exhibits poor effector selectivity, and a p110β-derived peptide which shows selective inhibition of the PI3Kβ-Gβγ PPI. The aim of this thesis was to develop a cell-permeable chemical tool able to block the PI3K-Gβγ PPI and stop downstream Akt phosphorylation to further explore the different inputs to the PI3K signalling pathways. Linear peptides derived from the catalytic subunit p110γ and the regulatory subunit p101 of PI3Kγ were explored as inhibitors of the PI3Kγ-Gβγ PPI. Decapeptides derived from the Gβγ-binding region of PI3Kγ showed weak inhibition of Gβγ-stimulated PI3Kγ activity in biochemical assays, yet did not show the same potential for PPI inhibition as an analogous p110β-derived peptide targeting the PI3Kβ-Gβγ interface. A structure-activity relationship (SAR) study of the known Gβγ-blocker 15mer peptide SIGK determined the amino acids that mediate the inhibition of the PI3Kγ-Gβγ PPI. No analogues proved to be stronger inhibitors of the PI3Kγ-Gβγ PPI. This study also revealed Tyr11 as an amino acid that could influence the Gβγ-effector selectivity of the SIGK peptide. A thiol-ene stapling technique was developed that incorporates novel divinyl ester building blocks on a known Axin-derived peptide resulting in stapled Axin analogues with improved helicity compared to the unstapled linear peptide. The stapling technique was applied to the SIGK peptide to produce stapled SIGK analogues. A SAR study revealed that a backbone staple could be installed between Gly3 and Lys7 without interfering with the inhibitory activity of the peptide in biochemical assays. These analogues exhibited cell permeability without cytotoxicity and showed inhibitory activity in the PI3K signalling pathway at 40 μM. Molecular dynamics simulations were employed to predict the α-helicity conferred by different staples, however the results generated by this technique were not corroborated by experimental circular dichroism measurements. These stapled SIGK peptides represent new chemical tools that can be employed for studying the PI3K signalling pathway in numerous cell lines. Additionally, the new thiol-ene stapling technique established in this thesis is expected to be applicable to other α-helical peptides to generate cellpermeable inhibitors for other therapeutic targets.