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 (SII-P), 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 SII-P is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a 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 SII-Ps of many KRAS hotspot (G12, G13, Q61) mutants are accessible using noncovalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the SII-P as a privileged drug-binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

Fig. 1. In vitro noncovalent binding to the KRAS SII-P determined by NMR spectroscopy.

a, Chemical structures of AMG510 (1), MRTX849 (4), MRTX1257 (5) and MRTX-EX185 (6). b, Summary of the effects of SII-P binders on ¹H−¹⁵N HSQC NMR spectra of RAS proteins. c, Examples of CSPs of GDP-loaded KRAS in the presence of MRTX849 and comparison of irreversible binding to KRAS(G12C) and reversible binding to KRAS(G12D). Spectra recorded at pH 7.4 and 298 K with 100 µM U-¹⁵N protein and 200 µM ligand.

Fig. 2. Target engagement assay for RAS.

a, Chemical structure of SI/II-P RAS BRET probe (7). b, Illustration of the RAS cellular target engagement assay. A luminescent complex is formed between RAS multimers in live cells; competitive displacement of the BRET probe reduces BRET signal while leaving the NanoBiT complex intact. Target engagement leads to competitive displacement of the BRET probe at RAS dimers. c, Removal of the hyper variable region or mutation at C185S results in a loss of luminescence in HEK293 cells compared with full-length KRAS WT, supporting RAS lipidation signals as critical to assay signal (left). Results are the mean ± s.e.m. of three independent experiments (n = 3). d, RAS BRET probe dose response for KRAS WT. BRET fold change is calculated by normalizing the BRET ratio of each point by the BRET ratio in the absence of BRET probe (instrument background). The BRET signal was competed by 20 µM BI-2852 (2), demonstrating specificity. Data are the mean ± s.e.m. of three independent experiments (n = 3). e, Live cell target engagement is observed at WT KRAS with BI-2852, but not with AMG510 (1). BRET is normalized relative to a saturating (20 µM) dose of BI-2852, as marked. RLU stands for relative luminescence units. Source data

Fig. 3. Measuring SII-P engagement of KRAS(G12C).

a, The SI/II BRET probe is used to quantify binding of unmodified SII-P ligands to RAS in live cells. Image created with BioRender.com from PDB 6OIM (AMG510-KRAS(G12C)). b, Protracted KRAS(G12C) engagement is observed for AMG510 (1). Data are representative of two independent experiments (n = 2), and individual data points are the mean ± s.d. of four technical replicates. c, Comparison of BRET target engagement data after 2 h at KRAS(G12C) versus p-ERK from MIA PaCa-2 cells as reported. Data are means of four independent experiments ± s.e.m. (n = 4). Source data

Fig. 4. SII-P binders engage KRAS WT and G12 hotspot mutants.

a−c, BRET target engagement profiles of MRTX849 (4) (a), MRTX1257 (b) and noncovalent methylsulfonamide derivative 14 (c). BRET is normalized relative to a saturating (20 µM) dose of BI-2852, as marked. a,b, Data are representative of three independent experiments, and each data point is the mean of three or four technical replicates ± s.d. (n = 3). c, Each data point is the mean of three independent experiments ± s.e.m. (n = 3). Source data

Fig. 5. Profiling in-cell target engagement of SII-P binding molecules.

a, Chemical structures of MRTX849 (4) and noncovalent derivatives 9–14. b, Summary of BRET target engagement across KRAS hotspot mutants and HRAS. pIC₅₀ values were calculated as –log₁₀(IC₅₀ [M]). Combinations that exhibited incomplete engagement at the highest concentration tested (10⁻⁴ M) were grouped as pIC₅₀ < 5.0 (white cells). Source data

Fig. 6. Characterization of engagement of KRAS with MRTX-EX185.

a, BRET target engagement profiles for MRTX-EX185 (6) at KRAS(G12D) and KRAS(G12V). BRET is normalized relative to a saturating (20 µM) dose of BI-2852 (2), as marked. Data are means of three independent experiments ± s.e.m. (n = 3). b, EX185-driven antiproliferation (CellTiter-Glo) is observed in SW-1990 (KRAS(G12D)) but not in HEK293 (KRAS-independent) cells. Data are the means ± s.e.m. of three independent experiments, each performed with at least three technical replicates (n = 3). c, EX185 inhibits p-ERK. Data are means ± s.e.m. of three independent experiments (n = 3). Source data