Synthesis and selection of novel phosphoester oligomers to inhibit the interaction between KRASG12D and RAF1

Protein-protein interactions (PPIs) have been a challenging area for drug discovery, due in part to the lack of small molecule binding sites and large flat surface areas. Whilst there have been some small molecule PPI inhibitors that have successfully passed through clinical trials in recent years, other alternative approaches are being examined that could overcome these issues, including the use of peptide-based inhibition and modified oligonucleotides that can bind across the larger surface area.

Oncogenic RAS proteins are responsible for almost one-third of human cancers, with KRAS being the most prevalent isoform. Oncogenic RAS is trapped in the GTP-bound active state and PPIs with proteins such as RAF and PI3K allows for continuous signaling, leading to tumour formation. Currently, only KRASG12C has clinically approved small molecule inhibitors, which covalently modify the cysteine at position 12, but there is no way to directly inhibit other more prevalent mutations (for example KRASG12D), and a new approach is needed to target these PPIs.

The main objective of this research is to develop and synthesise oligomers incorporating non-nucleosidic monomers that can disrupt the interactions between KRASG12D and RAF1. Seven monomers that offer different chemical reactvities were synthesised and combined in an oligomer library of over 800,000 unique oligomers using the split-and-mix method. Flow cytometry was then used to select oligomers that only bind to KRAS-GMPPnP (a non-hydrolysable analogue of GTP), do not bind to the inactive KRASG12D-GDP, and disrupt interactions between KRASG12D and the RAS binding domain of RAF1, resulting in 200 top binding oligomers. Tandem mass spectrometry was used to determine the sequences of these top binding oligomers, of which six oligomers were resynthesised. Validation assays using an ELISA based RAS-RAF interaction assay showed five of the six top binding oligomers disrupted the interaction between KRASG12D GMPPnP and fluorescently tagged RAF1 and three of these five did not inhibit the interactions of wild type KRAS (KRASWT).

This work demonstrates the first example of novel oligomers synthesised and selected that act as inhibitors of the KRASG12D/RAF1 PPI and has the potential to impact both the field of RAS drug discovery as well as PPI inhibitor drug discovery overall.