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Updated: Jun 20, 2025
Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein
Published on: January 16, 2020
Natural Product Graveoline Modulates Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) Membrane Association:
Gabriel Cornilescu1, Lakshman Bindu1, Louise Sternicki2
1NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States.
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.
Insights
Researchers identified graveoline, a small molecule that disrupts KRAS protein
Area of Science:
- Molecular biology
- Biochemistry
- Pharmacology
Background:
- KRAS gene mutations are key drivers in many cancers, promoting cell proliferation via the MAPK pathway.
- Targeting KRAS signaling pathways is a critical area for cancer therapy development.
- Understanding KRAS-membrane interactions is crucial for developing novel therapeutic strategies.
Purpose of the Study:
- To identify small molecules that disrupt KRAS protein's interaction with the plasma membrane.
- To investigate the potential of these molecules in inhibiting mutant RAS signaling.
- To elucidate the molecular mechanisms of KRAS-ligand interactions.
Main Methods:
- Screening using native mass spectrometry (MS) and surface plasmon resonance (SPR) to identify KRAS-interacting compounds.
- Nuclear magnetic resonance (NMR) and Förster resonance energy transfer (FRET) studies for detailed interaction analysis.
- Utilizing nanodiscs as membrane mimetics to study KRAS-membrane association in a biochemical context.
Main Results:
- Identified graveoline as a compound that preferentially binds to the hypervariable region (HVR) of KRAS.
- Graveoline's interaction with KRAS is dependent on C-terminal O-methylation and involves multiple binding sites.
- Demonstrated graveoline's ability to perturb KRAS membrane interaction using biophysical techniques.
Conclusions:
- Graveoline effectively disrupts KRAS membrane association, offering a potential therapeutic strategy.
- The findings provide insights into KRAS-ligand interactions and KRAS-associated pathophysiology.
- This research opens avenues for developing a new class of RAS therapeutics targeting KRAS membrane association.
