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 KRASG12D 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 KRASG12D 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 (R11ΔLys) and a "lysine-lite" variant retaining only the three lysines implicated in KRAS engagement (R11Lite)-and demonstrate that both retain nanomolar affinity for wild-type KRAS and KRASG12D. 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.

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