Identification of cyclic peptide inhibitors of RAS/SOS1 complex that disrupts RAS signaling in cancer cells

Abstract

The RAS protein plays a central role in regulating cell signaling pathways involved in cell growth and survival. Oncogenic mutations in RAS render the protein constitutively active, leading to uncontrolled cellular proliferation. Among the three isoforms of RAS, KRAS is the most frequently mutated in cancer, with KRAS(G12D) mutation being particularly prevalent and highly deleterious across various cancers. Recent advances in the development of KRAS inhibitors have resulted in the FDA approval of a drug targeting the KRAS(G12C) mutation. However, effective treatments for other, more widespread KRAS mutations are still unavailable. Efforts to directly target KRAS and its key interactions, such as with SOS1, continue to present a significant challenge in drug discovery.

This thesis describes the identification of cyclic hexapeptides capable of disrupting the KRAS(G12D)-SOS1 interaction using the high-throughput Split Intein Circularization of Peptides and Proteins (SICLOPPS) platform. Reverse two-hybrid systems were constructed to mimic the KRAS-SOS1 interaction, enabling the screening process. Multiple rounds of screening, combined with next-generation sequencing, were conducted to enrich for peptides with inhibitory activity against the KRAS-SOS1 association. The enriched cyclic hexapeptide inhibitors were subsequently synthesized via solid-phase synthesis and subjected to a series of assays to characterize their activity.

Several cyclic peptides were identified as promising inhibitors, with some exhibiting binding affinities in the low- to mid-micromolar range. Most of these peptides disrupted nucleotide exchange and reduced the phosphorylation levels of downstream effectors, such as ERK and AKT, in KRAS-driven cancer cells, as observed in preliminary Western blot assays. Two promising candidates were selected for alanine mutagenesis, which identified key amino acids essential for the interaction. Additional structural insights were gained by modeling their interaction with the target proteins. In both complexes, the binding mode was predicted to occur at the KRAS-SOS1 interface, suggesting a competitive mechanism of inhibition. Notably, c-CWITGK was predicted to exhibit specificity for the KRAS(G12D) mutant. 3

These findings establish a foundation for the identification and design of cyclic peptide inhibitors targeting the KRAS-SOS1 interaction. By characterizing their binding affinities, activities, and mechanisms of action, this work provides a solid framework for developing peptide inhibitors of the KRAS-SOS1 interaction. Future directions include further in vivo validation of these peptides, optimization of their pharmacodynamic properties, and exploration of their potential as selective inhibitors for the KRAS(G12D) mutation.

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Identifiers

ORCID for Juan Antonio Sarmiento Muro: orcid.org/0009-0001-8598-5077

ORCID for Ali Tavassoli: orcid.org/0000-0002-7420-5063

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