A high-throughput MS platform was used to accurately detect and quantitate different covalent modifications of proteins, including KRAS G12C, which contain one or more reactive cysteines, lysines, or other nucleophilic residues. We employed the Agilent RapidFire system to rapidly quantitate the extent of covalent protein-inhibitor adduct formation by MS for several proteins, including KRAS G12C. We used this approach to screen large numbers of potential covalent inhibitors in an automated fashion and to test medicinal chemistry compounds as part of a regular lead optimization cycle for KRAS G12C leading to the development of AMG-510.

We examined the utility of MRTX849, a covalent KRAS G12C inhibitor, in treating KRASG12C mutated cancers and comprehensively profiled sensitive and partially resistant preclinical models. We identified mechanisms likely responsible for limiting the single agent activity of MRTX849 in partially resistant KRASG12C-dependent models and identified clinically actionable combination strategies to augment MRTX849 in NSCLC and CRC patient populations.

The NCI RAS Initiative, led by scientific director, Frank McCormick of UCSF, combines novel drug discovery approaches (covalent tethering, computational modeling, and structure-guided fragment-based screening) to identify new compounds that directly target KRAS and its oncogenic mutants. These techniques provide a potential set of new targets in RAS drug discovery which have not yet been fully explored and will be discussed in this presentation.

An iterative medicinal chemistry/screening campaign identified a novel group of indenes with unique chemical and biological selectivity to inhibit the growth of cancer cells harboring mutant RAS. Lead optimization resulted in ANC 007 with IC50 values in the low nanomolar range and selectivity indices >100 fold. ANC 007 inhibited the growth of a large panel of human cancer cell lines having the most prevalent RAS mutational codons of all three RAS isozymes. Cancer cells with activated RAS from mutations in receptor tyrosine kinases or NF1 were also sensitive, while cancer cells having low activated RAS levels or cells from normal tissues were essentially insensitive. The mechanism of action involves suppression of activated RAS levels and MAPK/AKT signaling followed by mitotic arrest and apoptosis. ANC 007 also decreased levels of activated RAS and MAPK signaling in vivo at dosages that suppressed tumor growth and were well-tolerated. Treatment of immunocompetent mice with ANC 007 also suppressed PD-L1 levels on cancer cells and activated antitumor immunity. Experiments with recombinant RAS show direct binding of ANC 007 to the protein by NMR spectroscopy, inhibition of GTP binding, and blockage of RAS-effector interactions.

We share the discovery of scaffolded miniproteins (“Griptides”) that were engineered to bind the Ras proteins with picomolar affinity, and the subsequent characterization of their binding mode by solving high-resolution crystal structures. These structures reveal Ras trapped in a conformation that exposes a large binding pocket in the effector domain that connects two smaller pockets, which suggests that Ras may possess a surface pocket sufficiently large for effective small-molecule targeting.

The development of K-Ras G12C covalent inhibitors has led to clinical candidates. The overwhelming majority of Ras mutants and all Ral proteins do not have an accessible cysteine. We report one compound class that forms a covalent bond with non-catalytic residue Tyr-82. High-resolution crystal structures at 1.2-Å resolution revealed a deep, hydrophobic and druggable pocket, suggesting that it could be used to develop therapeutics targeting oncogenic Ras lacking cysteine.

The most frequently mutated oncogene in cancer is KRAS, which utilizes alternative fourth exons to generate two gene products, KRAS4A and KRAS4B, that differ only in their C-terminal membrane-targeting region. Because oncogenic mutations occur in exons 2 or 3, when KRAS is activated by mutation two constitutively active KRAS proteins are encoded, each capable of transforming cells. No functional distinctions among the splice variants have been established. Oncogenic KRAS alters tumor metabolism. Among these alterations is increased glucose uptake and glycolysis, even in the presence of abundant oxygen (the Warburg Effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes, direct regulation of metabolic enzymes has not been examined. We report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, establishing HK1 as an effector of KRAS4A. The interaction is unique to KRAS4A because the palmitoylation/depalmitoylation cycle of this RAS isoform permits co-localization with HK1 on the outer mitochondrial membrane (OMM). In recent, unpublished work we have explored the effect of KRAS4A on multimerization of HK1 on the OMM as a mechanism for controlling allosteric regulation of the enzyme. KRAS4A expression in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.