KRAS was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRAS between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRAS. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRAS-specific inhibitors with promising therapeutic potential.

KRAS gain-of-function mutations occur in approximately 30% of all human cancers. Despite more than 30 years of KRAS-focused research and development efforts, no targeted therapy has been discovered for cancers with KRAS mutations. Here, we describe ARS-853, a selective, covalent inhibitor of KRAS G12C that inhibits mutant KRAS-driven signaling by binding to the GDP-bound oncoprotein and preventing activation. Based on the rates of engagement and inhibition observed for ARS-853, along with a mutant-specifi c mass spectrometry-based assay for assessing KRAS activation status, we show that the nucleotide state of KRAS G12C is in a state of dynamic fl ux that can be modulated by upstream signaling factors. These studies provide convincing evidence that the KRAS G12C mutation generates a "hyperexcitable" rather than a "statically active" state and that targeting the inactive, GDP-bound form is a promising approach for generating novel anti-RAS therapeutics. SIGNIFICANCE:A cell-active, mutant-specifi c, covalent inhibitor of KRAS G12C is described that targets the GDP-bound, inactive state and prevents subsequent activation. Using this novel compound, we demonstrate that KRAS G12C oncoprotein rapidly cycles bound nucleotide and responds to upstream signaling inputs to maintain a highly active state. Cancer Discov; 6(3); 316-29.

Activating mutations in KRAS are among the most common tumor driver mutations. Until recently, KRAS had been considered undruggable with small molecules; the discovery of the covalent KRAS inhibitors ARS-853 and ARS-1620 has demonstrated that it is feasible to inhibit KRAS with high potency in cells and animals. Although the biological activity of these inhibitors has been described, the biochemical mechanism of how the compounds achieve potent inhibition remained incompletely understood. We now show that the activity of ARS-853 and ARS-1620 is primarily driven by KRAS-mediated catalysis of the chemical reaction with Cys12 in human KRAS, while the reversible binding affinity is weak, in the hundreds of micromolar or higher range. The mechanism resolves how an induced, shallow and dynamic pocket not expected to support high-affinity binding of small molecules can nevertheless be targeted with potent inhibitors and may be applicable to other targets conventionally considered undruggable.

We describe the biochemical mechanism of the covalent KRASG12C inhibitors ARS-853 and ARS-1620. Activating mutations in KRAS are among the most common mutations found in cancer. The KRASG12C mutation in particular is observed in approximately 15 % of non-small cell lung adenocarcinoma, 3 % of colorectal adenocarcinoma and 1 % of pancreatic adenocarcinoma. Until recently, KRAS had been considered undruggable due to the lack of clearly defined pockets that might support binding of small molecules, and the difficulty of targeting the nucleotide binding site due to the high affinity of GDP and GTP. However several years ago small molecules were discovered that bind an inducible pocket near the switch II region and covalently target the mutated cysteine in KRASG12C, trapping KRASG12C in a nonproductive GDP-bound state. Subsequent optimization of these compounds yielded the recently described inhibitors ARS-853 and ARS-1620, the first compounds that directly inhibit KRAS with high potency in cells and animals. While the biological activity of the inhibitors has been described, the biochemical mechanism of how the compounds achieve potent inhibition remained incompletely understood. We now show through biochemical kinetics studies that the activity of ARS-853 and ARS-1620 is primarily driven by KRAS-mediated catalysis of the chemical step of covalent bond formation with cysteine 12 in KRASG12C, rather than by high reversible binding affinity. The reversible inhibition constant (Ki) for both ARS-853 and ARS-1620 is well above the highest compound concentration tested (64 µM, to avoid solubility limitations), likely in the hundreds of micromolar range, while the rate of the chemical step (kinact) is fast. We confirm by several independent means that there is no detectable reversible binding affinity of the inhibitors for KRAS up to at least 32 µM, and show that the rapid chemical reaction is not due to high inherent reactivity of cysteine 12 in KRAS, nor to high intrinsic reactivity of the inhibitors. The results imply that the inhibitors do bind reversibly to KRAS to enable bond formation, but that binding is weak and primarily serves to orient the electrophile. The KRAS-dependent activation of covalent bond formation of ARS-853 and ARS-1620 with the mutated cysteine 12 is reminiscent of mechanism-based or suicide covalent enzyme inhibition, and explains both the high selectivity of the inhibitors for this cysteine relative to other cellular cysteines, and their potent overall activity despite exhibiting poor reversible affinity. The mechanism described here therefore resolves how an induced, shallow and dynamic pocket that is not expected to support high affinity binding of small molecules can nevertheless be targeted with potent inhibitors, and may be applicable to other targets conventionally considered undruggable. Citation Format: Rasmus Hansen, Ulf Peters, Anjali Babbar, Yuching Chen, Jun Feng, Matthew R. Janes, Liansheng Li, Pingda Ren, Yi Liu, Patrick P. Zarrinkar. Drugging an undruggable pocket: The biochemical mechanism of covalent KRASG12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 686.

Activating mutations in KRAS have a high prevalence in human cancer. The codon 12 glycine to cysteine missense mutation (KRASG12C) is among the most common KRAS mutations, present in non-small cell lung adenocarcinoma (~15 %), colorectal adenocarcinoma (~3 %), and pancreatic adenocarcinoma (~1 %). KRASG12C was previously identified as potentially druggable by allele-specific covalent targeting of cysteine 12 near the allosteric switch II pocket (S-IIP). Building on this early work, we recently described the ARS-853 series of S-IIP KRASG12C inhibitors that covalently react with the GDP-bound state of KRASG12C, trapping KRASG12C in this inactive state. In cells, ARS-853 series compounds profoundly deplete the signaling competent GTP-bound state of KRASG12C, thereby inhibiting downstream RAS signaling. However, this series of covalent KRASG12C inhibitors exhibited modest cellular potency and/or poor pharmacokinetic properties, making them unsuitable for further evaluation of covalent KRASG12C inhibition in animal models. We now describe in further detail the discovery and characterization of a new series of structurally distinct quinazoline based S-IIP KRASG12C inhibitors with substantially improved potency and pharmacologic properties that overcome limitations of the ARS-853 series. Through structure-guided medicinal chemistry optimization we identified compound ARS-1620, a potent, orally bioavailable covalent inhibitor of KRASG12C. The co-crystal structure of ARS-1620 covalently bound to KRASG12C reveals a distinct binding mode and additional interactions, compared to ARS-853. ARS-1620 rapidly engages KRASG12C, depletes KRASG12C-GTP in tumor cell lines, and inhibits downstream RAS signaling in a dose-dependent manner. The compound potently inhibits the growth of cell lines harboring the KRASG12C mutation with little or no effect on control cell lines. ARS-1620 demonstrates robust dose-dependent efficacy with once daily oral administration across a panel of KRASG12C-positive mouse cell line (CDX) and patient-derived (PDX) tumor xenograft models, with no response observed at all doses tested in KRASG12C-negative tumor models. The anti-tumor activity of ARS-1620 correlates with target engagement in the tumors as well as with inhibition of downstream RAS signaling. The in vivo efficacy and mutant selectivity observed with ARS-1620 across a wide range of KRASG12C mouse tumor models provides the first in vivo evidence that the S-IIP targeted approach may be a promising therapeutic strategy for patients with KRASG12C mutant cancers. Citation Format: Liansheng Li, Matthew R. Janes, Jingchuan Zhang, Rasmus Hansen, Ulf Peters, Xin Guo, Yuching Chen, Anjali Babbar, Sarah J. Firdaus, Jun Feng, Jeffrey H. Chen, Shuangwei Li, Shisheng Li, Carol Thach, Yuan Liu, Ata Zarieh, Jeff M. Kucharski, Tao Wu, Ke Yu, Yi Wang, Yvonne Yao, Xiaohu Deng, Patrick P. Zarrinkar, Dashyant Dhanak, Matthew V. Lorenzi, Dana Hu-Lowe, Pingda Ren, Yi Liu. Discovery of novel covalent KRASG12C inhibitors that display high potency and selectivity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 929.