An engineered protein antagonist of K-Ras/B-Raf interaction

Abstract

Ras is at the hub of signal transduction pathways controlling cell proliferation and survival. Its mutants, present in about 30% of human cancers, are major drivers of oncogenesis and render tumors unresponsive to standard therapies. Here we report the engineering of a protein scaffold for preferential binding to K-Ras G12D. This is the first reported inhibitor to achieve nanomolar affinity while exhibiting specificity for mutant over wild type (WT) K-Ras. Crystal structures of the protein R11.1.6 in complex with K-Ras WT and K-Ras G12D offer insight into the structural basis for specificity, highlighting differences in the switch I conformation as the major defining element in the higher affinity interaction. R11.1.6 directly blocks interaction with Raf and reduces signaling through the Raf/MEK/ERK pathway. Our results support greater consideration of the state of switch I and provide a novel tool to study Ras biology. Most importantly, this work makes an unprecedented contribution to Ras research in inhibitor development strategy by revealing details of a targetable binding surface. Unlike the polar interfaces found for Ras/effector interactions, the K-Ras/R11.1.6 complex reveals an extensive hydrophobic interface that can serve as a template to advance the development of high affinity, non-covalent inhibitors of K-Ras oncogenic mutants.

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

“…Although the RBDvs are a unique tool for studying Ras-dependent signaling processes, several intracellular affinity reagents targeting GTP-bound Ras have been reported (13, [15][16][17]. Compared with the previously reported Rastargeted reagents, the RBDvs were effective at lower concentrations.…”

“…An alternative strategy for inhibiting Ras signaling with engineered proteins is to prevent the recruitment of downstream effectors or block the activation of Ras by GEFs. To this end, affinity reagents have been generated that compete with Ras effectors by selectively binding to the active or inactive state of oncogenic Ras (13)(14)(15)(16)(17)(18)(19). However, several required high concentrations to be effective in cells (16,17,20), and their selectivity for members of the Ras family is unknown.…”

Competitive Inhibitors of Ras Effector Binding

“…Although the RBDvs are a unique tool for studying Ras-dependent signaling processes, several intracellular affinity reagents targeting GTP-bound Ras have recently been published 6,8,9,10 . This raises the question of how the RBDvs compare to these engineered proteins.…”

“…This raises the question of how the RBDvs compare to these engineered proteins. For example, the affinity reagent R11.1.6, based on a DNA-binding domain from a thermophilic archaeon, was engineered to bind Ras in an active conformation and was shown to compete with effector binding 8 . Although initial experiments in HEK293T cells suggested otherwise, surprisingly, R11.1.6 did not affect Ras-mediated signaling in a broad range of cancer cell lines 13 .…”

“…An alternative, more direct strategy for inhibiting Ras-signaling using engineered proteins is to prevent the recruitment of downstream effectors or block the activation of Ras by GEFs. To this end, several affinity reagents have been generated that compete with Ras effectors by selectively binding to the active or inactive state of Ras 6,7,8,9,10,11,12 . Interestingly, recent work showed that a subset of these affinity reagents had only effects in cells at high concentrations 9,10,13 .…”