KRAS, the most common oncogene in human lung, colorectal and pancreatic cancers, has been a challenging drug discovery target for decades. Recent breakthroughs with covalent small molecule inhibitors like sotorasib have rejuvenated efforts to drug mutant KRAS proteins. Macrocyclic peptides represent an emerging alternate modality that block intracellular protein-protein interactions involving high value therapeutic targets. Here, we will describe our efforts in designing cell active KRAS inhibitors starting from a known arginine-rich macrocyclic peptide, KRpep-2d. Structure-activity-relationship (SAR) studies involving macrocyclic linker replacement, stereochemical inversion, and backbone alpha-methylation, afforded peptide leads with on-target cellular activity confirmed by the strategic use of chemical and biological controls. However, the arginine-dependent cell entry mechanism limited its therapeutic potential due to the correlation between net positive charge and histamine release in an <i>ex vivo</i> mast cell degranulation assay. To mitigate this liability, we initiated studies directed towards engineering cellular permeability without the use of cation-mediated cell entry starting with identifying key residues on the arginine-less macrocycle that improved potency. Concurrently, leveraging the NanoClick permeability assay, we identified targeted optimization of lipophilicity as a primary driver of cellular activity, enabling a transition to non-cationic permeable peptides with potent on-target activity in a variety of mutant-KRAS cancer cell lines but without any mast cell degranulation liability. These cell active peptides validate a unique inhibitory epitope on KRAS and provide valuable molecular templates for the development of novel therapeutics to address KRAS-driven cancers.

Non-cationic macrocyclic peptides with on-target cellular inhibition of KRAS signaling

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ACS Fall 2022

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