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KRAS is one of the most frequently mutated genes in cancer and was long considered undruggable until the recent discovery of inhibitors that bind the inactive (GDP-bound) form of KRASG12C. The most clinically advanced of these first-generation molecules have demonstrated clinical response rates of 30-45% and approximately 6-month progression-free survival in lung cancer patients. While significant, a majority of patients fail to achieve a clinical response and acquired resistance is common. Resistance to first-generation inhibitors can be driven by upregulation of the activated (GTP-bound) form of KRASG12C, which remains an undrugged form of the oncoprotein. Here we report the discovery of FMC-376, a novel inhibitor of the activated, GTP-bound, form of KRASG12C, which also potently inhibits the inactive, (GDP-bound), form of KRASG12C. FMC-376 was discovered through the FrontierTM platform, which integrates chemoproteomics, machine-learning, and covalent fragment-based drug discovery. FMC-376 binds KRAS in the switch II pocket, rapidly forming a covalent bond with cysteine 12 in the presence of either GDP or GTP. X-ray crystallography demonstrated that Cys12 adopts a novel confirmation in forming a covalent bond with FMC-376. This results in potent inhibition of RAF1 and PI3Kα effector interactions (IC50 = 0.007 μM for both respectively at 2 h) in contrast to sotorasib or adagrasib (IC50 > 50 and ~ 5 μM respectively). FMC-376 treatment results in potent anti-tumor activity across a panel of KRASG12C mutant tumor cell lines, sparing non- KRASG12C cell lines. To model resistance mediated by activated KRASG12C, a mutation that abrogates GTPase activity (A59G) was introduced into KRASG12C. This upregulation of GTP-bound KRASG12C drives significant (>10-fold) resistance to both adagrasib and sotorasib in tumor cell viability assays whereas FMC-376 remains equipotent in settings where GTP-bound KRASG12C is upregulated. Evaluation of FMC-376 in models where EGFR signaling (a suspected mechanism of clinical resistance) is induced demonstrated rapid and durable target engagement in contrast to both sotorasib and adagrasib which show decreased effectiveness after EGF stimulation. Further evaluation of FMC-376 in vivo has demonstrated rapid and durable KRASG12C target occupancy (>90%) and pathway inhibition in tumors, resulting in regression of CDX/PDX tumor models. FMC-376, an inhibitor of both active and inactive forms of KRASG12C, provides a differentiated mechanism of action with the potential for broader and more durable response in the clinic. Citation Format: Snahel Patel, Barun Bhhatarai, Philamer Calses, Daniel Erlanson, Robert Everley, Susan Fong, Phil Gerken, Johannes C. Hermann, Tiep Le, Li-kai Liu, Evan McMahon, Richard M. Neve, Tony Phan, Allison Roberts, Mikayla Shanafelt, Sophie Siemsgluess, Jocelyn Staunton, Yan Wang, Weiru Wang, Monika Williams, Kevin R. Webster. Discovery of FMC-376 a novel orally bioavailable inhibitor of activated KRASG12C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1142.

a) Many cancer drivers are considered “undruggable” and without targeted treatments because they lack binding sites for conventional small molecules. Here, we introduce The FRONTIER™ Platform applying machine learning (ML), chemoproteomics and covalent chemistry to identify binding sites and cell-active covalent fragments across the human proteome, including against most cancer drivers and previously “undruggable” targets. Molecules tested in functional assays are active and can serve as starting points for new drug discovery initiatives. b) We are using mass spectrometry and data analysis workflows to perform high-throughput chemoproteomic profiling experiments. These experiments identify hits across the proteome using different cancer-relevant cell backgrounds and characterize binding sites for drug discovery. Customized ML algorithms using chemoproteomic, genomic and structural data to characterize and prioritize identified binding sites for covalent drug discovery. The performance of the platform allows the profiling of thousands of compounds from a custom-built library that has been optimized by ML for covalent fragment-based drug discovery. The nature of the fragment hits and the ability to map them to and focus on preferred binding sites for covalent drugs enables accelerated lead generation. c) We show details of the platform highlighting library design concepts and the hotspot map with binding site prioritization algorithms. We show coverage and applicability across important cancer target classes and signaling pathways. Covalent fragment hits have been identified for multiple difficult cancer targets, including KRAS, p53, STAT3, KEAP1, PTPN11 and others. The platform also identifies ligands for novel allosteric binding sites in established oncology targets such as CDK4, PI3KCA and BTK. We will highlight how discovered ligands show functional activity in orthogonal assays, demonstrating their fitness for drug discovery campaigns and target validation experiments. d) Undruggable targets across a variety of cancer target classes have become druggable. Citation Format: Johannes C. Hermann, Robert Everley, Laura Marholz, Matthew Berberich, Tzu-Yi Yang, Yu-Hsin Chao, Abduselam K. Awol, Michael Shaghafi, Han Yoon, Rohan Varma, Reed Stein, Karsten Krug, Emily Lachtara, Daniel Erlanson, Chris Varma, Kevin R. Webster. Combining chemoproteomics with machine learning identifies functionally active covalent fragments for hard-to-drug cancer drivers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5333.

KRAS is one of the most frequently mutated genes in cancer with alterations occurring in > 14% of all tumors. Recent advances have led to the discovery and development of inhibitors that bind the inactive (GDP-bound) form of KRASG12C. The most advanced of these first-generation molecules demonstrated clinical response rates of 30-45% and approximately 6-month progression-free survival in lung cancer patients. While significant, a majority of patients failed to achieve a clinical response and acquired resistance can be rapid. One hypothesis to explain tumor resistance is the failure of existing inhibitors to recognize the activated (GTP-bound) form of KRASG12C that can be upregulated in response to these first-generation inhibitors. Here we report the discovery of a series of novel inhibitors that effectively inhibit both the GTP- and GDP-bound forms of KRASG12C. These “dual-acting” inhibitors bind in the switch II pocket of both GTP-bound and GDP-bound KRASG12C and rapidly form a covalent bond with cysteine 12. This results in significantly increased inhibition of RAF1 and PI3Kα effector interactions (IC50 < 5 nM at 2 hrs.) in comparison to inactive state inhibitors. Dual targeting of both GTP- and GDP-bound KRASG12C results in potent cellular activity in models that are both sensitive (NCI-H358 and MIA PaCa-2) and resistant (NCI-H2122) to adagrasib and sotorasib. In contrast to adagrasib and sotorasib which are less effective in the NCI-H2122 cell line model, dual-acting inhibitors of GTP- and GDP-bound KRASG12C elicit rapid inhibition of pERK in < 1 hour with sustained inhibition of MAPK signaling through 48 hours. To model resistance to first generation inhibitors, an A59G mutation was introduced into KRASG12C, abrogating GTPase activity. This decreases the activity of both adagrasib and sotorasib in tumor cell viability assays by more than an order of magnitude whereas dual-acting inhibitors of GTP- and GDP-bound KRASG12C are equally effective in the G12C/A59G and parental G12C cell lines. Evaluation of dual-acting inhibitors of KRASG12C in vivo demonstrated rapid and > 90% KRASG12C target occupancy, resulting in regression of MIA PaCa-2 tumors. Dual-acting inhibitors of both the active and inactive states of KRASG12C may provide the potential for broader and more durable responses in the clinic. Citation Format: Philamer Calses, Sam Clark, Jacob Corpuz, Susan Fong, Phil Gerkin, Mohammad Hekmatnejad, Evan McMahon, Megan Murray, Truc Nguyen, Tony Phan, Allison Roberts, Phillip Schwartz, Mikayla Shanafelt, Hiroko Tanaka, Jennifer Tomczyk, John Widen, Monika Williams, John Eksterowicz, Daniel Erlanson, Marie Evangelista, Johannes Hermann, Richard M. Neve, Snahel Patel, Kevin R. Webster. Discovery of novel dual-acting KRASG12C inhibitors that target both the active and inactive forms of the protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3601.