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.

Discovery of Orally Bioavailable SOS1 Inhibitors for Suppressing KRAS-Driven Carcinoma

“…HPLC purity is >95%; 1 H NMR (600 MHz, methanol-d 4 ): δ 7.75 (s, 1H), 7.56 (t, J = 7.4 Hz, 1H), 7.43 (t, J = 7.1 Hz, 1H), 7.19 (t, J = 7.7 Hz, 1H), 7.10−6.88 (m, 2H), 5.81 (q, J = 7.1 Hz, 1H), 4.67 (td, J = 11.1, 1.9 Hz, 1H), 4.24−4.15 (m, 1H), 3.47−3.39 (m, 1H), 3.18 (d, J = 11.8 Hz, 1H), 3.14 (q, J = 9.9 Hz, 2H), 3.09 (td, J = 6.2, 3.0 Hz, 1H), 3.03 (d, J = 10.7 Hz, 1H), 2.85− 2.78 (m, 1H), 2.73 (t, J = 10.5 Hz, 1H), 2.62 (t, J = 10.1 Hz, 1H), 2.33 (s, 3H), 2.22−2.14 (m, 1H), 1.90−1.82 (m, 1H), 1.65 (d, J = 7.1 Hz, 3H). 13 [1,4]oxazepino [3,2-g]…”

“…3 Due to the relatively smooth surface, direct KRAS blockade by smallmolecule inhibitors has been proved challenging. 4 Inhibition of downstream effector of the RAF-MEK-ERK cascade has also shown limited efficacy in the clinic because of the feedback networks that keep the pathway in check. 5 Recently, inhibiting the interaction of son of sevenless 1 (SOS1) and KRAS has been recognized as an attractive approach against multiple KRAS mutations simultaneously.…”

Chemoselective Covalent Modification of K-Ras(G12R) with a Small Molecule Electrophile

“…However, our work presents an important first step in targeting this oncogene, and future medicinal chemistry optimization could yield more potent ligands that target the GTP-bound form. This has recently been shown with other mutants for K-Ras. , Our discovery expands our ability to selectively target a recurrent oncogenic mutant, K-Ras­(G12R), for which no direct inhibitors have been reported. The chemistry reported here may also serve as the basis for the therapeutic targeting of other acquired arginine residues in human diseases.…”

“…This has recently been shown with other mutants for K-Ras. 38,39 Our discovery expands our ability to selectively target a recurrent oncogenic mutant, K-Ras(G12R), for which no direct inhibitors have been reported. The chemistry reported here may also serve as the basis for the therapeutic targeting of other acquired arginine residues in human diseases.…”

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC–MS/MS Approach

“…One approach utilized the nanoBRET (bioluminescence resonance energy transfer) technology with a cell-permeable fluorescent probe from the SW-I/II pocket inhibitor BI-2852, which enables real-time monitoring of RAS target engagement in cells. 7 Another approach is through quantification by mass spectrometry of free KRAS G12C not covalently bound to the inhibitor; in this way, the percent of the unbound target can be calculated by normalizing to the untreated or pre-treatment control samples. 8 However, the requirement of a fluorescent probe or control samples in these assays represent a major limitation for their utility in the clinic.…”