Abstract 686: Drugging an undruggable pocket: The biochemical mechanism of covalent KRASG12C inhibitors
Authors: Hansen Rasmus , Peters Ulf , Babbar Anjali , Chen Yuching , Feng Jun , Janes Matthew R. , Li Liansheng , Ren Pingda , Liu Yi , Zarrinkar Patrick P. 
Published: August 2018

Abstract
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.