Towards Tumor Cell Specific Proteolysis Targeting Chimeras: Identification of Oncogenic KRASG12C, DcpS, and MAGE-A3 Degraders

Date of Award

Spring 2022

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Crews, Craig


Targeted Protein Degradation (TPD) is a nascent, rapidly evolving field in which cellular proteolysis pathways are co-opted to induce degradation of disease-causing proteins. PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional small molecules that recruit an E3 ubiquitin ligase to induce protein degradation. This pioneering technology has paved the way for other bifunctional modalities, like lysosome targeting chimeras (LYTACs) and autophagy targeting compounds (AUTAC/ATTEC), as well as monovalent “glue” degraders, and has drastically changed the drug discovery landscape. In Chapter 1 I review important PROTAC discovery milestones and provide an outlook on the future of PROTAC design. After a general introduction I highlight several “hard to drug” protein classes that have been degraded using PROTACs. Next, I discuss two emerging technologies, chemoproteomics and DNA encoded library (DEL) screening, that will enable identification of novel ligands for PROTAC design. This is followed by a discussion of the E3 ligases currently recruited for TPD and how engaging new E3 ligases could lead to PROTACs with tissue/disease specific degradation. Finally, I discuss how our knowledge of PROTAC ternary complexes, married with improved computational approaches, may enable the rational design of PROTACs. In Chapter 2 I present my work identifying a novel KRASG12C degrading PROTAC. RAS proteins are mutated in ~20% of human cancers and are highly sought-after drug targets. Recently, covalent inhibitors of KRASG12C have entered the clinic and showed efficacy in treating disease. However, resistance to these inhibitors has been observed highlighting the need for alternative means of therapeutic intervention. Therefore, we set out to develop a KRASG12C degrading PROTAC. I identified LC-2 as the first KRASG12C degrader that induces rapid, sustained degradation of KRASG12C via a bona fide PROTAC mechanism leading to inhibition of growth signaling. This discovery is a major step for the field as previous efforts to degrade KRASG12C had been unsuccessful. Moreover, LC-2 spares wild type KRAS and could therefore serve as a blueprint for the development of future tumor cell specific KRAS mutant degraders. In Chapter 3 I present my work that led to the identification of the first DcpS degrading PROTAC. DcpS is a mRNA decapping enzyme recently found to be a dependency in acute myeloid leukemia (AML) by CRISPR screening. Genetic knockdown of DcpS in untransformed immune cells does not significantly affect their viability. Therefore, a DcpS degrading PROTAC has the potential to induce AML cell death with minimal off target effects. We synthesized a library of >20 PROTACs that linked a RG3039 derivative, which binds DcpS, to small molecules that recruited either the VHL or cereblon (CRBN) E3 ligase. I identified JCS-1, which recruits VHL, as the most potent DcpS degrader. The PROTAC induces on mechanism degradation of DcpS leading to decreased expression of AML relevant RNA transcripts and cell death. JCS-1 will serve as a tool compound to further interrogate the role of DcpS in normal and diseased tissue. In Chapter 4 the focus shifts from degrading new classes of proteins to E3 ligase ligand discovery. Melanoma Antigen (MAGE) proteins interact with really interesting new gene (RING) domain containing proteins to form active MAGE RING E3 ligase (MRL) complexes. Type I MAGE proteins are cancer testis antigens; their expression is restricted to the male germline but can be re-expressed during tumorigenesis. Therefore, identification of ligands that recruit a MRL could be used to develop PROTACs that induce degradation in a tumor cell specific. In this chapter we use a DEL approach to identify a novel MAGE-A3 ligand, MJBIA9836, which I synthesized and validated. We were unsuccessful in recruiting MAGE-A3 for TPD. However, I did identify KL465, a VHL recruiting PROTAC which induces on mechanism degradation of MAGE-A3 and causes cancer cell death. Future work aims to improve the potency of MJBIA9836 for use in both MAGE-A3 recruiting and targeting PROTACs, which could serve as the foundation for the development of clinically active tumor cell specific degraders. Finally, in Chapter 5 I present my work developing a novel synthesis for 3,4-unsubstituted isoquinolin-1(2H)-ones. Isoquinolones are present in many pharmacologically active compounds, creating a need for rapid, inexpensive syntheses. We improve upon our previously published method by developing a facile two-step synthesis that uses mild reaction conditions to efficiently generate isoquinolones harboring a wide variety of functional groups. I synthesized six of the disclosed isoquinolones, including two compounds that could not be generated in high yields using other published synthetic approaches. Our novel synthesis is currently being used in several projects in the lab, highlighting the importance of this new synthetic route.

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