A designer protein binder of KRAS enables exogenous protein degradation

Abstract Targeted protein degradation has emerged as a powerful strategy for modulating intracellular proteins, particularly those lacking traditional small‐molecule binding pockets. While protein‐based degraders expand this approach to undruggable targets, they typically require intracellular expression in target cells, limiting their utility as exogenous research tools. Here, we engineer the Sso7d‐derived KRAS G12D binder R11.1.6 for use as a chemically conjugatable, exogenously deliverable degrader scaffold. We show that a C‐terminal arginine‐to‐cysteine variant can be produced in good yield and purity and conjugated to a von Hippel–Lindau (VHL) E3 ligase ligand, albeit with an approximately 10‐fold reduction in KRAS G12D affinity relative to parent R11. To reduce the number of lysines available for potential ubiquitination, we generate two lysine‐reduced variants—a fully lysine‐free variant (R11 ΔLys ) and a “lysine‐lite” variant retaining only the three lysines implicated in KRAS engagement (R11 Lite )—and demonstrate that both retain nanomolar affinity for wild‐type KRAS and KRAS G12D . Esterification of R11–VHL with an α‐aryl‐α‐diazoamide reagent enables cellular treatment with the chimeric conjugate and leads to VHL‐ligand‐dependent reduction in pan‐RAS levels in HPAF‐II cells, as shown by competition with excess free VHL ligand. Our results establish R11 variants as tractable protein‐engineering scaffolds for developing chemically conjugated biodegraders and provide design principles for engineering lysine‐reduced binders based on the Sso7d scaffold.