KRAS is vulnerable to reversible switch-II pocket engagement in cells

Abstract

Current small molecule inhibitors of KRAS (G12C) bind irreversibly in the switch-II pocket, exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the switch-II pocket is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a novel cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the switch-II pockets of many GTP hydrolysis-deficient KRAS hotspot (G12, G13, Q61) mutants are accessible using non-covalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the switch-II pocket as a privileged drug binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

Profiling oncogenic KRAS mutant drugs with a cell-based Lumit p-ERK immunoassay

“…It should be noted that while the MRTX-1133 compound was 430 developed as a KRAS G12D inhibitor, our study also shows inhibition of 431 p-ERK accumulation in KRAS G12C and G12V mutant cell lines when 432 this compound is titrated to a relatively high concentration, albeit with 433 40 and 400-fold less potency, respectively. The ability of higher concen-434 trations of MRTX-1133 to inhibit p-ERK in other KRAS mutant cell lines 435 corresponds with data obtained using BRET target engagement assays, 436 where a compound with similar structure to MRTX-1133 (EX185) was 437 found to engage both G12C and G12V mutated KRAS, but at lower po-438 tency than G12D mutated KRAS [38] . Interestingly, while most mutant 439 KRAS inhibitors target inactive GDP-bound KRAS [ 39 , 40 ], EX185 was 440 also found to engage GNP-bound (GTP RAS homolog) KRAS [38] .…”

“…The ability of higher concen-434 trations of MRTX-1133 to inhibit p-ERK in other KRAS mutant cell lines 435 corresponds with data obtained using BRET target engagement assays, 436 where a compound with similar structure to MRTX-1133 (EX185) was 437 found to engage both G12C and G12V mutated KRAS, but at lower po-438 tency than G12D mutated KRAS [38] . Interestingly, while most mutant 439 KRAS inhibitors target inactive GDP-bound KRAS [ 39 , 40 ], EX185 was 440 also found to engage GNP-bound (GTP RAS homolog) KRAS [38] . Since 441 G12V mutant KRAS has a more preferred GTP state over other KRAS 442 mutants, it has traditionally been more difficult to target.…”

“…Since 441 G12V mutant KRAS has a more preferred GTP state over other KRAS 442 mutants, it has traditionally been more difficult to target. However, our 443 data showing inhibition of G12V KRAS with MRTX-1133 combined with 444 the target engagement data for EX-185 [38] could indicate that inhibi-445 tion of G12V mutant KRAS is possible.…”

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy

“…We tested whether this combination of probes could discriminate between the inactive GDP-state and the active and inactive conformations of the GTP state. The transverse relaxation rate of probe 9 exhibited a strong dependence of the nucleotide state and conformation of KRas G12D ; R2,9 varied over 6 Hz between samples containing 6 µM of either KRas G12D GDP, KRas G12D GNP, KRas 21 In contrast to the small molecule SIIP-binders, the cyclic peptide KD2 preferentially occupied the active GNP-state. 27…”

“…[15][16][17] Furthermore, the two nucleotide-states of KRas can be resolved by proteinobserved NMR spectroscopy, enabling nucleotidecycling reactions and nucleotide-state-specific binding to be directly observed in mixed samples containing both GDP and GTP. [18][19][20][21] While information-rich, proteinobserved experiments are burdened by the requirements of high protein concentrations, long acquisition times, and isotopic labels. Ligand-observed NMR-spectroscopy alleviates these burdens; well-validated "probe" ligands can be applied to assay properties of the protein at lower concentrations, with faster acquisition times, and without the need for isotopic labelling.…”