Discovery of pan-KRAS molecular glue degraders via protein-first coevolution-guided E3 ligase recruitment

Background: KRAS is one of the most frequently mutated oncogenes in human cancer; however, the absence of deep hydrophobic pockets renders it challenging to drug by conventional inhibitors. Molecular glue degraders (MGDs) that induce proximity between KRAS and an E3 ubiquitin ligase represent a promising modality to achieve loss of KRAS function through proteasomal degradation. Methods: We employed proprietary MGD platform, which initiates with a protein-first, coevolution-driven selection of E3 ligases. Multiple sequence alignments of KRAS and human E3 ligases were analyzed to identify co-mutating amino acid pairs that preserve physiologically relevant protein-protein interaction (PPI) interfaces. Predicted KRAS-E3 complexes were modeled using state-of-the-art protein folding algorithms and molecular dynamics simulations. Ternary complex-compatible sites were then screened in-silic o against the commercially available chemical space using flexible, structure-based docking. Hit compounds were evaluated in HEK293T cells stably expressing HiBiT-tagged KRASWT, KRASG12D, KRASG12V, or KRASG13D. Target degradation was quantified by HiBiT luminescence, confirmed by automated capillary electrophoresis (Jess/WES) western blotting of both HiBiT-tagged and endogenous KRAS, and validated for proteasome dependence using MG132. Cytotoxicity was assessed in KRAS-dependent (AsPC-1) and KRAS-independent (RKO BRAFV600E) cell lines over 7 days. Results: The platform delivered multiple chemically tractable series of small-molecule (<500 Da) MGDs capable of potent, proteasome-dependent degradation of KRAS. Lead compounds induced >70% degradation of HiBiT-tagged KRASWT, G12D, G12V, and G13D at 10 µM after 24 h, with DC50 values in the high-nanomolar to low-micromolar range. Degradation of endogenous untagged KRAS in wild-type HEK293T cells reached 30-40% at 10 µM after 18 h treatment. Rescue experiments with proteasome inhibitors fully reversed degradation, confirming a ubiquitin-proteasome mechanism. Selected degraders exhibited favorable selectivity profiles, with CC50 values >5 µM in long-term proliferation assays and minimal cytotoxicity in KRAS-independent lines. Conclusions: Our coevolution-guided, protein-first MGD platform enables rapid discovery of small molecules that induce pan-KRAS degradation across major oncogenic mutants. These compounds achieve degradation of KRAS protein through a proteasome-dependent mechanism, distinguishing them from covalent KRASG12C inhibitors and RAS(ON) tri-complex inhibitors. The identified pan-KRAS molecular glue degraders represent a novel therapeutic modality with potential to address the majority of KRAS-driven cancers previously considered undruggable.