Exquisitely Specific anti-KRAS Biodegraders Inform on the Cellular Prevalence of Nucleotide-Loaded States

Abstract

Mutations to RAS proteins (H-, N-, and K-RAS) are among the most common oncogenic drivers, and tumors harboring these lesions are some of the most difficult to treat. Although covalent small molecules against KRAS G12C have shown promising efficacy against lung cancers, traditional barriers remain for drugging the more prevalent KRAS G12D and KRAS G12V mutants. Targeted degradation has emerged as an attractive alternative approach, but for KRAS, identification of the required high-affinity ligands continues to be a challenge. Another significant hurdle is the discovery of a hybrid molecule that appends an E3 ligase-recruiting moiety in a manner that satisfies the precise geometries required for productive polyubiquitin transfer while maintaining favorable druglike properties. To gain insights into the advantages and feasibility of KRAS targeted degradation, we applied a protein-based degrader (biodegrader) approach. This workflow centers on the intracellular expression of a chimeric protein consisting of a high-affinity target-binding domain fused to an engineered E3 ligase adapter. A series of anti-RAS biodegraders spanning different RAS isoform/nucleotide-state specificities and leveraging different E3 ligases provided definitive evidence for RAS degradability. Further, these established that the functional consequences of KRAS degradation are context dependent. Of broader significance, using the exquisite degradation specificity that biodegraders can possess, we demonstrated how this technology can be applied to answer questions that other approaches cannot. Specifically, application of the GDP-state specific degrader uncovered the relative prevalence of the “off-state” of WT and various KRAS mutants in the cellular context. Finally, if delivery challenges can be addressed, anti-RAS biodegraders will be exciting candidates for clinical development.

Unifying Catalysis Framework to Dissect Proteasomal Degradation Paradigms

“… 81 Genetic tools such as haloTag and GFP fusions have enabled the early assessment of Ub ligase compatibility, 82 while biodegraders have enabled swift scanning of POI degradation fitness for a number of E3 ligases 83 and targeting specific conformational states of KRas. 84 Collectively, these technologies offer a diverse menu to select tractable degrader starting points in live cells.…”

RAS specific protease induces irreversible growth arrest via p27 in several KRAS mutant colorectal cancer cell lines

“…Despite this success, the strategy of selective inhibition has problems of being applicable to only a limited range of cancers integrated with personalized medicine and cannot be used to treat cancers that lack the specific mutation. To address this gap, new approaches are being developed to more broadly target RAS either with proteases that directly cleave RAS 18 , 19 or with linkers that target RAS for cellular degradation 20 – 23 .…”

Monobodies as tool biologics for accelerating target validation and druggable site discovery

“…Genetically encoded protein-based degraders have become increasingly popular as a proof-of-concept tools for demonstrating the validity of degrading a drug target. 48,[163][164][165] In contrast to other binding protein technology, the monobody degraders undergo minimum degradation by itself. 48,63 This may be due to the characteristic that the monobody scaffold is small and contains few lysine residues, which decrease its chance for being ubiquitinated.…”

“…Recently, several studies have used monobodies developed by our group for generating proteinbased degraders against several oncogenic targets by fusing a monobody directly to a subunit of E3 ligase. 48,[163][164][165] Sapkota et al first demonstrated the utility of monobodybased degraders termed AdPROMs by fusing a monobody selective to SHP2 to the VHL domain. 166 They demonstrated the feasibility of degrading endogenous RAS using NS1 monobodybased AdPROMs.…”