Khoo, R. | bioRxiv Science

bioRxiv2020

bioPROTACs establish RAS as a degradable target and provide novel RAS biology insights

ABSTRACTMutations to RAS proteins (H-, N-, and K-RAS) are amongst the most common oncogenic drivers and tumors harboring these lesions are some of the most difficult to treat. Although the recently discovered covalent small molecules against the KRASG12C mutant have shown promising efficacy against lung cancers, traditional barriers remain for drugging the more prevalent KRASG12D and KRASG12V 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 drug-like properties. As a tool to gain insights into the advantages and feasibility of KRAS targeted-degradation, we applied the bioPROTAC 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. We generated a series of anti-RAS bioPROTACs that span different RAS isoform/nucleotide-state specificities and leverage different E3 ligases. Overall, our results provide definitive evidence for the degradability of RAS proteins. We further elucidate the functional consequences of RAS degradation, the susceptibility and degradation kinetics of various mutant KRAS, and the prevalence of different nucleotide-states in WT and mutant KRAS. Finally, if delivery challenges can be addressed, anti-RAS bioPROTACs will be exciting candidates for clinical development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2019

bioPROTACs as versatile modulators of intracellular therapeutic targets: Application to proliferating cell nuclear antigen (PCNA)

Targeted degradation approaches have recently generated much excitement as a paradigm shift to address human disease in unprecedented ways. Amongst these, small molecule based approaches such as Proteolysis targeting chimeras (PROTACs) have attracted the lions share of attention due to their potential to tackle historically intractable targets and achieve greater potency, efficacy, and specificity over traditional small molecule inhibitors. Despite their promise, the identification of high-affinity ligands that can serve as starting points for PROTAC strategies remains challenging. As a complementary approach, we describe herein a class of intracellular biologics termed bioPROTACs. The substrate binding component of these fusion proteins consists of a peptide or an antibody-mimetic which allows for an unprecedented diversity of protein targets that can be addressed. The high-affinity binder is linked directly to an E3 ubiquitin ligase to harness the power of targeted degradation. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. Indeed, 9 out of 16 binder-E3 combinations tested resulted in greater than 70% target clearance. Through a systematic approach, we then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA), a sliding DNA clamp with critical roles in DNA replication and repair. The bioPROTAC, termed Con1-SPOP, elicited rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Compared to RNAi-based approaches which typically take days to manifest, PCNA knockdown using Con1-SPOP was evident within 4 h. The advantage of degradation versus stoichiometric inhibition was also clearly demonstrated with bioPROTAC strategies. Combining superior pharmacological inhibition and relative ease of development, bioPROTACs are powerful tools for interrogating the degradability of a substrate, for guiding the identification of the fittest E3 ligase, for studying the functional consequences associated with target protein down-regulation, and potentially for making therapeutic impacts.

cell biology↗