Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D

Abstract

K-Ras is the most commonly mutated oncogene in human cancer. The recently approved non-small cell lung cancer drugs sotorasib and adagrasib covalently capture an acquired cysteine in K-Ras-G12C mutation and lock it in a signaling-incompetent state. However, covalent inhibition of G12D, the most frequent K-Ras mutation particularly prevalent in pancreatic ductal adenocarcinoma, has remained elusive due to the lack of aspartate-targeting chemistry. Here we present a set of malolactone-based electrophiles that exploit ring strain to crosslink K-Ras-G12D at the mutant aspartate to form stable covalent complexes. Structural insights from X-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone that resisted attack by aqueous buffer but rapidly crosslinked with the aspartate-12 of K-Ras in both GDP and GTP state. The GTP-state targeting allowed effective suppression of downstream signaling, and selective inhibition of K-Ras-G12D-driven cancer cell proliferation in vitro and xenograft growth in mice.

Targeting KRAS Diversity: Covalent Modulation of G12X and Beyond in Cancer Therapy

“…Because they used the racemate for crystallization, the authors proposed an S N 2 attack for the ring-opening mechanism (Scheme ). This pioneering work shows G12D-specific inhibitors that target a non-catalytic carboxylic side chain with malolactone warheads, which has not been described before …”

“…The authors stated that a covalent bond with Asp12 is observable as an ester bond and the free carboxyl group, showing that the lactone ring opens upon ligand binding. For the α-carbon atom, an S -configuration can be observed (Zheng et al., Figure G) . Because they used the racemate for crystallization, the authors proposed an S N 2 attack for the ring-opening mechanism (Scheme ).…”

“…Another approach presented by Zheng et al demonstrated, for the first time, a selective inhibitor that forms a stable covalent complex with KRAS G12D using a malolactone-based electrophile . As β-lactone moieties, known from KRAS G12S inhibitors, do not react with G12D, the authors designed β-lactone electrophiles bridged via a carbonyl group to the core scaffold of MRTX1133 .…”

“…Given that (RS)- 1 appears to interact with the active GTP-bound state of KRAS, the authors investigated its effect on downstream effector binding. They observed a reduced binding to Raf-Ras binding domain (Raf-RBD), which is a significant finding, particularly considering that the G12D mutant exhibits severely impaired GTP hydrolysis, meaning that it is predominantly present in the active state. , Compound (RS)- 1 was further optimized in several rounds, leading to ( R )- 7 . This compound showed mutant selective labeling and inhibition toward KRAS G12D in cellular studies but none for non-G12D mutated cancer cell lines.…”

“…(A) Known SWIIP binders can be utilized for diverse KRAS mutations in codon 12 by facilitating unique interactions with the mutated amino acid. − (B) Distribution of KRAS G12X mutations in non-small cell lung cancer (NSCLC), pancreatic, and colorectal cancer …”