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STIP1 homology and U-Box containing protein 1 (STUB1) plays a key role in maintaining cell health during stress and aging. Recent evidence suggested STUB1 also helps regulate immunity with the potential of clearing malignant cells. Indeed, we and others have shown that STUB1 is a pivotal negative regulator of interferon gamma sensing – a process critical to the immunosurveillance of tumors and pathogens. Thus far, investigation of STUB1’s role relies mostly on genetic approaches as pharmacological inhibitors of this protein are lacking. Identification of a STUB1 tool compound is important as it would allow therapeutically relevant target validation in a broader sense. Accordingly, we leveraged phage display and computational modeling to identify and refine STUB1 binders. Screening of >10E9 macrocyclic peptides resulted in several conserved motifs as well as structurally diverse leads. Co-crystal structure of the peptide hit and STUB1 has enabled us to employ structure-based in silico design for further optimization. Of the modifications employed, replacing the hydrophilic solvent-exposed region of the macrocyclic peptides with a hydrophobic scaffold improved cellular permeability, while the binding conformation was maintained. Further substitution of the permeability-limiting terminal aspartic acid with a tetrazole bioisostere retained the binding to certain extent while improving permeability, suggesting a path forward. The current lead, although not optimal for cellular study, provides a valuable template for further development into selective tool compounds for STUB1 to enable target validation.

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 KRASG12C 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 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., Biodegraders, engineered proteins that fuse a high-affinity binder (e.g., RBD, K27, R11.1.6, and NS1) to an E3 ligase (e.g., SPOP), prompt degradation of RAS and reveal novel biological insights.

Recent COVID-19 vaccines unleashed the potential of mRNA-based therapeutics. mRNA optimization is indispensable for reducing immunogenicity, ensuring stability, and maximizing protein output. We present mRNAid, an experimentally validated software for mRNA optimization and visualization that generates mRNA sequences with comparable if not superior characteristics to commercially optimized sequences. To encompass all aspects of mRNA design, we also interrogated the impact of uridine content, nucleoside analogs and UTRs on expression and immunogenicity.

Mutations 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.