Mechanisms and design of small molecule degraders recruiting the VHL Cullin 2 RING E3 ubiquitin ligase

Targeted protein degradation is now a proven therapeutic strategy for degrading disease-causing proteins, with almost fifty small molecule degraders in clinical trials for various disease conditions. The vast majority of small molecule degraders redirect the activity of the VHL Cullin 2 RING E3 ubiquitin ligase. In this thesis, biochemical, biophysical, and structural methods are applied to deepen our understanding of the mechanisms through which PROTACs recruit Cullin RING E3 ligases and mediate degradation of the target protein. We develop and establish in vitro assays for quantitatively assessing the key events involved in the degrader’s mechanism of action, namely degrader binary binding, degrader-mediated ternary complex formation, and degrader-targeted ubiquitination, ultimately leading to protein degradation. These in vitro assays, complimented with cellular biophysical measurements, provide a robust suite of methods to evaluate each step of the degrader mechanism. We apply this methodology to a library of PROTACs, to understand how different chemical modifications impact on each step of the PROTAC pathway. We also develop and apply an integrative structural biology approach, focusing on cryo-electron microscopy, to explore how these small molecules recruit VHL in the context of the full-length Cullin 2 RING and E2 ubiquitin-conjugating enzyme, and target substrates for selective and effective ubiquitination. Here, we reveal how small molecules mediate exquisite specificity in degrader-driven substrate recruitment, orienting key spatially optimised lysine residues for ubiquitination and degradation. Finally, we apply these methodologies to a VHL dimerising homoPROTAC system, obtain structural information and use this to guide the rational design and synthesis of a new panel of E3 degraders. The findings in this study will unveil novel insights at an unprecedented atomic and mechanistic level of detail into the degraders' mechanism of action. The understanding revealed from this work will ultimately aim to support and accelerate the development of targeted protein degrader molecules.