Mutations in small GTPases RAS is responsible for 15% of all human cancers; the majority of these are due to defect in the KRAS gene. KRAS is a very dynamic allosteric enzyme, and previous studies revealed that it harbors four allosteric ligand-binding pockets and suggested that targeting KRAS directly is a viable strategy to abrogate its abnormal functions. Existing KRAS focused structure-based drug design studies suffer from limitations in dealing with its dynamic nature. To address this, we conducted extensive multi-conformer, multi- target hierarchical virtual screening (VS) against all four allosteric pockets of KRAS using purchasable chemical libraries and prioritized lead compounds through post docking analysis, biophysical and cellular assays. We also characterized the druggability of these pockets and the favored chemical scaffolds to each pocket. Our VS approach yielded a list of 785 potential hits. 217 of these were procured for experimental testing and about 100 were tested for their ability to bind to GDP-bound KRAS using N15-labeled heteronuclear single quantum coherence (HSQC) NMR. Of the 100 tested, 10 compounds showed significant amide chemical shift perturbation at several residues, suggesting potential binding. Among these 10 hits we, herein report a small-molecule V14 found to have low µM to nM affinities for wild type and different mutants of KRAS. V14 inhibits KRAS signaling in cells primarily by inhibiting RAS-Raf complex formation but also has inhibitory effect on nucleotide exchange reaction. This compound also showed dose dependent decrease in cell proliferation for pancreatic cancer cells upon treatment. Our work not only represent a successful route to target difficult proteins like KRAS but also report a novel KRAS inhibitor V14 as an excellent candidate for lead optimization and drug development for the treatment of cancer.

Successful application of ensemble-based, multi-target hierarchical virtual screening approach to discover novel allosteric KRAS inhibitor validated through experiments

256th National Meeting (2018)

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