title: Targeted protein degradation for disruption of key proteins that overcome genomic instability in cancers
title: Wee1 and KIFC1
titleType: Subtitle
Creators: name: Aublette, Marine
Publisher: name: Lancaster University
Year: 2023

description: Targeted protein degradation, especially using proteolysis targeting chimeras (PROTACs), is rapidly emerging as an interesting tool to target and selectively degrade cancer driving proteins. PROTACs are bifunctional molecules composed of a target-protein ligand and a ubiquitin E3-ligase binder linked together. These form a ternary complex to degrade the target via the proteasome instead of inhibiting it. Because of the added complexity of PROTACs, they are typically found to exhibit enhanced selectivity for certain targets over others. Current inhibitors for cancer therapy are typically not very selective towards their intended targets, especially protein kinases due to their conserved ATP-binding sites. Wee1 kinase has an important role in the cell cycle as it regulates the G2/M checkpoint and is overexpressed in many cancers. It is an interesting target for which therapeutic intervention would be useful. The first chapter of results, Chapter 3, focuses on how I designed and synthesised Wee1 PROTACs using a potent but not very selective inhibitor (AZD1775) and two E3 ligase recruiters for von Hippel-Lindau (VHL) and Cereblon (CRBN), part of the Cullin-RING ubiquitin ligases. A structure activity relationship of Wee1 PROTACs around the linker was generated and their potential cytotoxic activity assessed in cancer cells, as detailed in the next chapter, Chapter 4. I established the Wee1 degradation profiles of the two series of PROTACs recruiting CRBN or VHL toward different target kinases. A range of activities were observed depending on the recruited E3 ligase and the linker (length and nature). Wee1 was degraded selectively, bringing a promising tool to target kinases selectively for cancer treatments. A corresponding decrease in downstream phosphorylation of CDK1 was observed in multiple cell lines. The target engagement and the ternary complex formation were studied to validate the PROTAC mechanism of action. The linker had a determining influence in the degradation which led to optimisation of the PROTACs, as discussed in Chapter 5. I show the synthesis of new PROTACs by removing an amide bond in the molecule and using different linkers. One PROTAC of each new series displayed significantly improved potency against Wee1. In Chapter 6, I propose the generation of a new type of 3D linkers for bifunctional molecules using photochemistry: cubanes. With the hypothesis in mind that short linkers can block the conformation of the PROTAC to possibly favour ternary complex formation for further degradation, using 1,2- and 1,3-cubanes could become a key technology to use in the PROTAC field and medicinal chemistry in general. The 1,3-disubstituted cubane was successfully obtained at the required scale, although enabling selective functionalisation of the substituents required additional optimisation. The synthesis of a 1,2-disubstituted cubane was more challenging however the “almost cubane”, before closing the last bond, was successfully obtained. Some additional synthetic effort to close the final bond of the cubane is needed to complete this work. Finally, in Chapter 7, I show the design and synthesis of KIFC1 PROTACs. KIFC1 overexpression in cancers lead to multipolar mitosis and successful cancer cell proliferation. KIFC1 inhibitor AZ82 was selected and studied to find an attachment point, despite the lack of an available crystal structure. I synthesised the PROTACs using both CRBN and VHL recruiters and different alkyl linkers. After assessment in cancer cells, one PROTAC (MA185) recruiting CRBN successfully degraded KIFC1. MA185 is the first KIFC1 PROTAC to our knowledge, which opens up exciting prospects for future development as a cancer-selective treatment. MA185 exhibits enhanced cytotoxicity as compared with AZ82 inhibitor. The influence of this first KIFC1 PROTAC on the cell cycle is currently being investigated to better understand its mechanism of action, as well as the extent of this discovery.