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Title: Attacking Ras-driven cancers : engineering a peptide inhibitor for Cdc42
Author: Tetley, George Jeremy Norman
ISNI:       0000 0004 7229 2698
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Prior work has indicated that preventing Cdc42 interacting with its downstream effector proteins can reverse Ras-driven oncogenesis. The experiments demonstrating this used the G protein binding region (GBD) of the Cdc42-specific effector ACK, which is 42 amino acids long and binds with 40 nM affinity. The aim of this work was to find a peptide which exhibits similar effects in reversing oncogenic characteristics in cells but with more promising therapeutic properties: for example, a smaller size, improved binding and improved protease resistance. Firstly, the Cdc42-ACK binding interface was characterised thermodynamically, producing a comprehensive dataset of the contribution of individual ACK residues to complex affinity. This information revealed regions of the ACK GBD with higher concentrations of vital interactions but also residues that were unimportant for maintaining affinity. An \emph{in vitro} display technology (CIS-display) was employed to select for shorter peptides with improved binding to Cdc42. By selecting the best binders from libraries based on the ACK GBD, peptides have been generated that are half the length of the full binding domain but bind with affinities approaching the wild-type ($\approx$100 nM). More interestingly, a serendipitous discovery in a na\"{\i}ve cyclic library revealed a 16-residue sequence that binds with 350 nM affinity, subsequently called C1. The biophysical properties of selected peptides were characterised and demonstrate that C1 is reliant on an intramolecular disulphide bond for tight binding. An N-terminal nona-arginine sequence was added to C1 to facilitate entry into cells: internalisation was confirmed by confocal microscopy. Subsequently it was found that C1 reduced signalling through the ACK and PAK effector pathways, negatively impinging on MAPK signalling. Levels of signalling returned to baseline within 24 hours, however, and the transiency of the effect was thought to be linked to peptide degradation: an effect likely in a construct dependent on a disulphide bond for cyclisation and binding affinity. Subsequent maturation of C1 using a focussed CIS-display library has selected second generation peptides. These have been characterized and display binding affinities to Cdc42 of $\approx$20 nM: a 17-fold increase in binding over C1. This has enhanced the affinity to a level even higher than wild-type ACK and should improve the cellular potency of the peptides identified in this selection. %any more?! Having successfully selected peptides both shorter in length and with higher affinity than the wild-type sequence, efforts were channelled into developing a method to cyclize the peptide by a chemical linkage that would be stable in a cellular environment. The most successful approach involved desulphurisation of the disulphide bond to a thioether, making the cyclic linkage non-labile in the reducing environment of the cytosol, while retaining original binding affinity. The peptides developed in this work bind to Cdc42 with nanomolar affinity \emph{in vitro} and can enter cells and impact upon signalling processes connected with oncogenesis. As such, they provide a potential therapeutic lead for future development.
Supervisor: Owen, Darerca Sponsor: MRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Cancer ; Cdc42 ; Ras ; Display technology ; Peptide