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Inhibition of prenylated KRAS in a lipid environment
Johanna M Jansen1, Charles Wartchow1, Wolfgang Jahnke2
1Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
Abstract:
RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.
Insights
Researchers developed a new assay to study KRASG12V protein activation. This led to the discovery of compounds that inhibit KRASG12V
Area of Science:
- Chemical Biology
- Molecular Biology
- Biochemistry
Background:
- RAS mutations result in constitutively active oncogenic proteins.
- These proteins signal through multiple effector pathways, driving cancer progression.
- Understanding KRAS signaling is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To develop a novel coupled biochemical assay for measuring BRAF activation by prenylated KRASG12V.
- To discover and characterize novel inhibitors of KRASG12V function.
- To align in vitro assays with physiological conditions regulating RAS biology.
Main Methods:
- Development of a coupled biochemical assay incorporating lipid-dependent activation and prenylation.
- High-throughput screening to identify inhibitory compounds.
- Nuclear Magnetic Resonance (NMR) and biolayer interferometry for structural and biophysical characterization.
Main Results:
- Discovery of compounds inhibiting KRASG12V biochemical and cellular functions with low single-digit micromolar potency.
- Structural characterization revealed compounds stabilize the inactive conformation of KRASG12V.
- In vitro assays aligned with physiological conditions (lipid environment, prenylation) demonstrated matching potency and binding affinity.
Conclusions:
- The developed assay provides a physiologically relevant platform for studying KRAS biology.
- Novel KRASG12V inhibitors were identified, offering potential for drug discovery.
- These tools advance the study of KRAS inhibition and the development of targeted cancer treatments.