Targeting Oncogenic Kinases and Pseudokinases with Proteolysis Targeting Chimeras

Date of Award

Spring 2021

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Crews, Craig


Targeted protein degradation (TPD) has surfaced as a novel and innovative therapeutic modality. By co-opting protein degradation pathways, TPD facilitates complete removal of protein molecules from within or outside the cell. While the pioneering Proteolysis Targeting Chimera (PROTAC) technology and molecular glues hijack the ubiquitin-proteasome system, newer modalities co-opt autophagy (AUTAC/ATTEC) or the endo-lysosomal pathway (LYTAC). Using this mechanism, TPD is posited to largely expand the druggable space beyond small molecule inhibitors. The first chapter is a primer for TPD. I introduce the field of PROTACs and discuss the early studies that set the foundation for degradation-based therapies. Next, I discuss the major components of PROTACs, namely the linker and E3 ligase recruiting elements; I explain their importance and the parameters within TPD they control. I also discuss the role of structural and computational biology in developing successful PROTACs. I examine how PROTACs can aid in overcoming resistance to traditional small molecules. Furthermore, as PROTACs enter the clinic, I highlight studies that have explored how resistance to PROTACs will arise. Lastly, I review different branches of TPD including molecular glues, LYTACS, AUTACs and ATTECs. In Chapter 2, I present BRAF targeting PROTACs. Over 300 BRAF missense mutations have been identified in patients, yet currently approve drugs target V600 mutants alone. Moreover, acquired resistance inevitably emerges, primarily due to RAF lesions that prevent inhibition of BRAF V600 with current treatments. In this chapter, I use PROTAC technology to address the limitations of BRAF inhibitor-based therapies. Using vemurafenib-based PROTACs, I show that we can achieve sub-nanomolar degradation of all classes of BRAF mutants, but spare degradation of WT RAF family members. Our lead PROTAC outperforms vemurafenib in inhibiting cancer cell growth and shows in vivo efficacy in a Class 2 BRAF xenograft model. Through mechanistic studies, I reveal that BRAFWT is spared due to weak ternary complex formation in cells owing to its quiescent inactivated conformation, and activation of BRAFWT sensitizes it to degradation. This study highlights the degree of selectivity achievable using degradation-based therapies by targeting mutant BRAF-driven cancers while sparing BRAFWT and thus expanding the therapeutic window using a new anti-tumor drug modality. In Chapter 3, I present an attempt to expand PROTAC technology to a traditionally “undruggable” oncogenic target, Protein Tyrosine Kinase 7 (PTK7). PTK7 is a receptor tyrosine pseudokinase first identified in melanocytes and found overexpressed in colon cancer. Furthermore, loss of PTK7 has been shown to induce apoptosis, further implicating PTK7 as a promising target for colorectal cancer therapy. Despite this, there is no understanding of the role of PTK7 through Wnt signaling in upregulating colon cancer. The absence of such an investigation may be in part due to PTK7’s lack of an active kinase domain which has resulted in a limited capacity for pharmacological studies of PTK7 and pseudokinases in general. Therefore, to allow for further investigation of PTK7 and its signaling in colorectal cancer there is a pressing need for small molecule tools to target the protein. To this end, I present my efforts to identify a small molecule tool to target PTK7. Through Differential Scanning Fluorimetry (DSF) , I identify bisindolylmaleimide X (Bis X) as a possible ligand for PTK7. This ligand likely binds with very weak affinity, and thus could serve as a scaffold one can incorporate into a probe for displacements assays. In Chapter 4, I introduce a second effort to target pseudokinases using PROTAC technology. Human epidermal growth factor receptor (HER3) is a receptor tyrosine pseudokinase in the EGFR family with immense potential as an oncogenic target. There have been several clinical trials that attempt to target HER3 using mABs, however clinical benefit has yet to be shown. As PROTACs remove the entire protein molecule, we hypothesize that it may have a more pronounce clinical benefit. To this end I perform a TR-FRET screen to identify HER3 ligands. The hit compound, I13, is a derivative of dabrafenib and binds HER3 with nanomolar affinity. I also find that I13 has some selectivity for HER3 over EGFR. By incorporating I13 into PROTACs, we achieve selective degradation of HER3. However, our lead PROTAC does not cause a decrease in HER3 downstream signaling, namely ERK phosphorylation. Our mechanistic studies highlight that this is due to engagement of BRAFWT which induces paradoxical activation of the MAPK pathway. Thus, despite our promising HER3 degradation, our PROTAC is limited due to off target effects. I end this work with a summary of my findings and my perspective on the future of TPD.

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