Modern biomedical research has unveiled many of the complicated processes that underlie life at the most basic of level of cells. This enterprise has shown reversible protein phosphorylation, mediated by kinases, is an integral process whose mis-regulation causes many diseases, including cancer. Current therapeutic strategies targeting kinases have been focused on inhibiting enzymatic activity, and this has led to many approved therapies that extend the life and livelihood of many people.
This thesis explores the limitations of these strategies and a novel chemical biology tool to overcome them. In the first chapter, a brief history of kinases highlights how modern thinking focuses on kinase activity but ignores additional functions of protein kinases. The onco-kinase BCR/Abl is one such example: non-kinase roles of this protein are implicated in maintaining the disease and preventing cure even when the kinase activity is efficiently inhibited. A second example is the pseudokinase ROR2 and pseudokinases in general. These proteins share common structural features of kinases yet are enzymatically inactive and participate in important signaling programs within the cell. These two examples illustrate how inhibition is a limited paradigm of drug discovery.
Chapter two exemplifies recent advances in a strategy to overcome the limitations of inhibition. This strategy is called proteolysis targeting chimera, or PROTAC, and is based on heterobifunctional small molecules which bind to the protein target and recruit it to E3 ubiquitin ligases. The target protein is then ubiquitinated and degraded. While previous iterations of PROTACs have been rather unimpressive, this chapter highlights the degradation of a protein kinase as well as a nuclear hormone receptor. These PROTAC molecules are unprecedented in their potency, selectivity, and drug-likeness. The chapter concludes by discussing the many recent examples of PROTACs and their application in research and, soon, therapeutic interventions.
Chapter three then asks a basic and important question about PROTAC design. In designing potent degraders, minor structural changes in the molecule can lead to drastic effects on protein degradation. This chapter explores that phenomena, first by using a model system in which PROTAC geometry is finely tuned for degradation. It is shown that the discriminating factor between poor and potent PROTACs is the ability to form a stable ternary complex between the target, the PROTAC, and the E3 ligase. The best PROTACs induce protein:protein interactions between the target and E3 ligase, stabilizing the complex and leading to more potent degradation. In the second part of chapter three, PROTACs are explored which bind to many different kinase targets, but only degrade a subset of possible targets. Again, the discriminating factor between degraded and non-degraded proteins appear to be protein:protein interactions unique to the degraded proteins. This chapter offers biophysical explanations for commonly observed phenomena, and aids in developing design principles for PROTAC molecules.
Having shown PROTAC molecules to be a strategy for potent protein degradation in chapter two and enhancing the understanding of that platform in chapter three, chapter four returns to the two examples listed above. First, potent degradation of BCR/Abl is achieved through a PROTAC designed to target the allosteric site. Next, these compounds are used in initial assays to explore functions of BCR/Abl that are affected by either inhibition or degradation of the protein. Finally, initial studies in patient-derived stem cells are presented. While the viability of these cells is reduced by PROTACs, more nuanced work must be done to highlight differences between degradation and inhibition of BCR/Abl.
Second, initial efforts are made to develop ligands for the pseudokinase ROR2. While these compounds may not have activity on their own, they could be converted into PROTAC molecules which would deactivate all functions of ROR2. A thermal shift assay is used to identify potential ligands of ROR2 which bind with modest affinity. Future work will explore these compounds as well as developing high-throughput screens for pseudokinases in general. While previous iterations were limited in potency, this study demonstrates that PROTAC molecules can be versatile chemical tools.
While outside the scope of this thesis, PROTACs also show promise as therapeutic interventions. By degrading the entire protein rather than just inhibiting one functionality, PROTACs may expand what is currently considered druggable. Many literature examples point to this possibility. With the first PROTAC molecules soon to enter clinical trials, this study highlights the reasons for the considerable excitement surrounding this technology.
|Advisor:||Crews, Craig M|
|School Location:||United States -- Connecticut|
|Source:||DAI-B 79/12(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Biochemistry|
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