Rab27a: ligand and interactome discovery for a secretory GTPase
Rab27a is a small GTPase belonging to the Ras superfamily. Normally involved in vesicle transport and docking, numerous studies from the past two decades have documented a positive correlation between its overexpression in tumour cells and the invasiveness of the cancer. These findings strongly suggest that Rab27a could be a novel cancer drug target, yet, the dearth of clinically successful drugs targeting other oncogenic members of the Ras superfamily reflects the difficulty of applying
... al medicinal chemistry techniques to this class of proteins, structurally flexible and have complex protein-protein interaction (PPI) networks. The development of new biophysical methods in drug discovery holds promise in delivering where past methods have struggled. In this thesis, two top small molecular hits from a fragment assay against Rab27a were developed as part of structure-activity relationship (SAR) studies using a crystallised protein construct as guidance. Peptide binders were also sought, and a dipeptide was identified as the first positive control for Rab27a with a kd of ~8 mM by SPR, and its affinity was improved by lactam bridging. The PPIs of Rab27a were also investigated as part of the development of photocleavable crosslinkers for a novel chemical biology approach to interactome proteomics. A prototype lysine-targeting covalent crosslinker was designed, synthesised and validated with recombinant protein followed by Rab27a-spiked lysate. It was subsequently incorporated into a bespoke proteomic protocol and tested against HeLa lysate transfected with Twin-Step Tag (TST)-Rab27a. The results were analysed using label-free quantification (LFQ) in MaxQuant and peptide modifications inspected with PEAKS. The analysis workflow is discussed and critically analysed, offering many fruitful avenues for the future iterations of the protocol.