Fragment Optimization of Reversible Binding to the Switch II Pocket on KRAS Leads to a Potent, In Vivo Active KRAS^G12C Inhibitor

Abstract

Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRAS G12C inhibitors. To date, KRAS G12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRAS G12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRAS G12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.

In Silico Evaluation of the Thr58-Associated Conserved Water with KRAS Switch-II Pocket Binders

“…The only exceptions are where the conserved water appears with adjacent hydration sites, such as the lower-affinity fragment GNE-2897 reported by Genentech (PDB ID: 7MDP), cocrystallized together with an antibody, and Boehringer–Ingelheim’s fragment compound 12 and BI-0474 (PDB IDs: 8AFC, 8AFB) (Table ; Figure S6). In addition to this, the BI-0474 structure (PDB ID: 8AFB) also displays two conformations of Lys16 (normal and shifted), with a probability of 0.5 for both, which in turn has an impact on the WaterMap-predicted hydration site pattern (Table ; Figure S6). WaterMap suggests that all of these several distinct compounds with the isolated hydration site exhibit truly a high-energy hydration site in this location (Δ G : >8.50 kcal/mol).…”

“…19 Electron density (blue transparent surface) displayed at 2Fo − Fc σ = 1.5. 7MDP), 47 cocrystallized together with an antibody, and Boehringer−Ingelheim's fragment compound 12 and BI-0474 (PDB IDs: 8AFC, 8AFB) 52 (Table 1; Figure S6). In addition to this, the BI-0474 structure (PDB ID: 8AFB) 52 also displays two conformations of Lys16 (normal and shifted), ARS-853, the first cell active G12C inhibitor, appears with an isolated Thr58-associated hydration site (ΔG = +5.09 kcal/mol).…”

“…7MDP), 47 cocrystallized together with an antibody, and Boehringer−Ingelheim's fragment compound 12 and BI-0474 (PDB IDs: 8AFC, 8AFB) 52 (Table 1; Figure S6). In addition to this, the BI-0474 structure (PDB ID: 8AFB) 52 also displays two conformations of Lys16 (normal and shifted), ARS-853, the first cell active G12C inhibitor, appears with an isolated Thr58-associated hydration site (ΔG = +5.09 kcal/mol). (C) ARS-1620, the first in vivo active G12C inhibitor, displays a high-energy conserved hydration site (ΔG = +10.93 kcal/mol).…”

“…Indeed, if the Thr58-associated conserved water is present in the structure, the more recent KRAS­(G12C) SII-P binders reported after ARS-853 tend to contain only isolated conserved high-energy water (a total of 32 published cocrystal structures). The only exceptions are where the conserved water appears with adjacent hydration sites, such as the lower-affinity fragment GNE-2897 reported by Genentech (PDB ID: 7MDP), cocrystallized together with an antibody, and Boehringer–Ingelheim’s fragment compound 12 and BI-0474 (PDB IDs: 8AFC, 8AFB) (Table ; Figure S6). In addition to this, the BI-0474 structure (PDB ID: 8AFB) also displays two conformations of Lys16 (normal and shifted), with a probability of 0.5 for both, which in turn has an impact on the WaterMap-predicted hydration site pattern (Table ; Figure S6).…”

“…We excluded the two structures from the SII-P binder analysis (PDB IDs: 7U8H and 8AFD) that contain SII-P-binding fragments that are cocrystallized with a secondary site covalent small molecule and with a different position 12 mutant (KRAS(G12V, S39C-BIT)+GDP). 52 WaterMap 30,31 simulations were conducted with default settings, including waters to the analysis within 10.0 Å range of the SII-P-bound ligand. For the structures without the SII-P binder, residues Thr58, Met72, and Tyr96 were selected to define the site.…”