The reactivity-driven biochemical mechanism of covalent KRASG12C inhibitors

Abstract

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.

Modeling the Interplay of Poor Inhibitor Binding and Efficient Formation of a Covalent Adduct upon the Interaction of ARS Compounds with KRASᴳ¹²ᶜ

“…Complicated kinetics is observed for the KRAS G12C •GDP interaction with the ARS compounds. 13 These inhibitors satisfy the emerging concepts in the covalent drug design showing the low binding affinity of inhibitor (K i ) and high rate constant of the covalent bond formation (k inact ). 14,15 It should be noted that ARS compounds target small and flexible binding pocket, and other proteins with similar binding pockets can be targeted as well.…”

“…Particularly, for the formation of the covalent KRAS G12C ·GDP-ARS-853 three quantities are obtained in the experimental kinetic studies. 13 The only kinetic parameter that is measured directly is the observed rate constant, k obs , of the formation of the covalent KRAS G12C ·GDP-ARS-853 complex. Then the kinetic model for the covalent inhibition is utilized.…”

“…We simulate the set of kinetic curves of the KRAS G12C •GDP covalent engagement with the ARS-853 with different initial concentrations like in the experimental study. 13 The initial KRAS G12C •GDP concentration is set to 1 μM and the initial concentration of ARS-853 varies from 10 -5 M to 10 -1 M.…”

The phylogenetic functional conservation ofDrosophilaSeven-In-Absentia (SINA) E3 ligase and its two human paralogs, SIAH1 and SIAH2, inDrosophilaeye development

“…Persistent K-RAS/EGFR/HER2 pathway activation is prevalent in chemo-resistant, relapsed and metastatic human cancer [2,[10][11][12][13]. The clinical reality is that oncogenic K-RAS-driven malignant tumors remain "undruggable," despite the recent development of small molecule inhibitors that covalently and irreversibly bind to a mutant cysteine in hyperactive K-RAS G12C [14][15][16][17][18][19][20]. Although these high-affinity cysteine-targeting covalent K-RAS G12C inhibitors have shown promising antitumor efficacy in phase I/II studies [21][22][23], their clinical efficacy remains to be demonstrated.…”

Discovery of covalent enzyme inhibitors using virtual docking of covalent fragments

“…Recently covalent K-Ras G12C inhibitor ARS-853 has been discovered that has weak binding affinity to K-Ras G12C (K i 200 ± 90 μM) but fast k inact 0.05 s −1 (k inact /K i 250 ± 20 M −1 s −1 ) due to the electrophile activation by K-Ras. 14 When profiled against 2,740 cellular cysteines this inhibitor displayed remarkable selectivity and covalently modified Cys 12 of K-Ras and only two other proteins: FAM213A and Reticulon-4. 15 Further improvement of ARS-853 has led to ARS-1620 (k inact /K i 1100 ± 300 M −1 s −1 ) active in vivo .…”