Abstract
dc:description.abstractThe ras genes are the most commonly mutated oncogenes in human cancers, with mutations occurring in approximately 20% of human tumours. However, more than 30 years of attempts to target Ras proteins therapeutically have yielded no effective therapies in the clinic, leading the proteins to be widely deemed ‘undruggable’. In recent years, there has been substantial evidence implicating the Ral GTPases, RalA and RalB, which are activated downstream of Ras, as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. Therefore, targeting this pathway may provide an effective method for inhibition of oncogenic Ras signalling. Prior work identified stapled peptides based on the Ral effector, RLIP76, that can bind to the Ral GTPases and disrupt downstream signalling. To improve the affinity of these peptide sequences, an affinity maturation was performed on the Ral-binding domain of RLIP76 from which potential sequence changes were identified. The work described in this thesis aimed to identify sequences from this selection with improved affinity for Ral proteins to guide the design of second-generation stapled peptides targeting the Ral GTPases. In vitro validation of the selection sequences enabled the identification of several sequence substitutions that together improved binding to Ral proteins by more than 20-fold. The effects of individual residue substitutions on the affinity for Ral proteins were determined using biophysical assays and two 1.5 Å co-crystal structures of the tightest-binding mutants in complex with RalB revealed the key interactions formed. The sequences were successfully translated into stapled peptides based on RLIP76, resulting in peptides with improved affinity compared to the wild-type parent sequence. The peptides have been shown to be selective for the active form of Ral, with undetectable binding to a panel of related small GTPases in in vitro assays. The binding site of the lead peptide on RalB has been determined by NMR and was found to overlap with multiple Ral-effector interactions. The peptides were able compete with multiple Ral-effector interactions in vitro and in cellular lysates. This work demonstrates how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein-protein interaction.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2020
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Hurd, Catherine
- Advisors dc:contributor.advisor
-
- Owen, Darerca
- Mott, Helen
Subjects
dc:subject × 6Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
-
0000-0003-0978-5425
0000-0002-7890-7097 - OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/316829