Identification of an H-Ras nanocluster disrupting peptide.

Hyperactive Ras signalling is found in most cancers. Ras proteins are only active in membrane nanoclusters, which are therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with the Ras binding domain (RBD) of Raf. Here, we establish that the B-Raf preference of Gal1 emerges from the divergence of the Raf RBDs at their proposed Gal1-binding interface. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B- and C-Raf-RBDs. Its 23-mer core fragment is sufficient to interfere with H-Ras nanoclustering, modulate Ras-signalling and moderately reduce cell viability. These latter two phenotypic effects may also emerge from the ability of L5UR to broadly engage with several RBD- and RA-domain containing Ras interactors. The L5UR-peptide core fragment is a starting point for the development of more specific reagents against Ras-nanoclustering and -interactors.

Eliminating oncogenic RAS: back to the future at the drawing board.

RAS drug development has made enormous strides in the past ten years, with the first direct KRAS inhibitor being approved in 2021. However, despite the clinical success of covalent KRAS-G12C inhibitors, we are immediately confronted with resistances as commonly found with targeted drugs. Previously believed to be undruggable due to its lack of obvious druggable pockets, a couple of new approaches to hit this much feared oncogene have now been carved out. We here concisely review these approaches to directly target four druggable sites of RAS from various angles. Our analysis focuses on the lessons learnt during the development of allele-specific covalent and non-covalent RAS inhibitors, the potential of macromolecular binders to facilitate the discovery and validation of targetable sites on RAS and finally an outlook on a future that may engage more small molecule binders to become drugs. We foresee that the latter could happen mainly in two ways: First, non-covalent small molecule inhibitors may be derived from the development of covalent binders. Second, reversible small molecule binders could be utilized for novel targeting modalities, such as degraders of RAS. Provided that degraders eliminate RAS by recruiting differentially expressed E3-ligases, this approach could enable unprecedented tissue- or developmental stage-specific destruction of RAS with potential advantages for on-target toxicity. We conclude that novel creative ideas continue to be important to exterminate RAS in cancer and other RAS pathway-driven diseases, such as RASopathies.