Abstract

The KRAS gene plays a pivotal role in numerous cancers by encoding a GTPase that upon association with the plasma membrane activates the MAPK pathway, promoting cellular proliferation. In our study, we investigated small molecules that disrupt KRAS's membrane interaction, hypothesizing that such disruption could in turn inhibit mutant RAS signaling. Native mass spectrometry screening of KRAS-FMe identified compounds with a preference for interacting with the hypervariable region (HVR), and surface plasmon resonance (SPR) further refined our selection to graveoline as a compound exhibiting preferential HVR binding. Subsequent nuclear magnetic resonance (NMR) analysis showed that graveoline's interaction with KRAS depends on C-terminal O-methylation. Moreover, our findings revealed multiple interaction sites, suggesting weak engagement with the KRAS G domain. Using nanodiscs as a membrane mimetic, further characterization through NMR and Förster resonance energy transfer (FRET) studies demonstrated graveoline's ability to perturb KRAS membrane interaction in a biochemical setting. Our biophysical approach sheds light on the intricate molecular mechanisms underlying KRAS-ligand interactions, providing valuable insights into understanding the KRAS-associated pathophysiology. These findings contribute to the translational aspect of our study, offering potential avenues for further research targeting KRAS membrane association with the potential to lead to a new class of RAS therapeutics.