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.
Highlights
RAS proteins function as molecular switches to regulate cell growth, differentiation, and apoptosis through interactions with several effectors leading to multiple pathways emanating from this critical node in the cell [1, 2]
We identified KRAS-specific inhibitors following the workflow in Fig 2, which includes the KRASG12V-dependent coupled assay with the BRAFV600E counter-screen, hit-validation by protein-observed NMR, determination of cellular potency, and biophysical assessments
To further evaluate if the biochemical and cellular potencies of the compounds are consistent with binding to KRAS, and to test the hypothesis that the affinity of binding to KRAS is dependent on the presence of the C-terminal prenyl group and the presence of lipids, we explored the binding of compounds directly to prenylated KRAS using ForteBio’s biolayer interferometry (BLI) platform [38]
Summary
RAS proteins function as molecular switches to regulate cell growth, differentiation, and apoptosis through interactions with several effectors leading to multiple pathways emanating from this critical node in the cell [1, 2]. We focused on mutant KRAS4B (referred to as KRAS in the remainder of this paper), which is the KRAS splice variant that is prevalent in cancer and has been shown to require the C-terminal farnesyl group, the C-terminal poly-Lysine block and a membrane/ lipid environment with negatively charged head-groups for biological function [3, 4, 32]. Evidence of the direct binding of the inhibitors to KRAS in these biosensor-based studies, as well as the alignment between the Kds and the potencies in the biochemical and cellular assays, support the hypothesis that these assays are modulated through the direct interaction of the inhibitors with KRAS
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