Small-molecule approaches to interrogate the druggability of the VHL E3 Cullin RING Ubiquitin Ligase

  • Pedro Soares

Student thesis: Doctoral ThesisDoctor of Philosophy


Protein-protein interactions (PPIs) are prevalent in Nature and so are attractive targets for chemical biology and drug discovery. Modulating PPIs is challenging because of the nature of their interacting surfaces. Nevertheless, recent progress has led to the discovery of small molecule modulators of PPIs as chemical probes or lead compounds that have entered clinical trials. These findings have motivated more drug discovery programs to target PPIs. E3 ubiquitin ligases are attractive targets within the ubiquitin proteasome system, which function via PPIs. The von Hippel-Lindau protein (VHL) forms part of an E3 ubiquitin ligase for which the main biological function is to recognize and ubiquitinate the protein hypoxia inducible factor alpha subunit (HIF-α), its specific substrate, marking it for degradation by the proteasome. The VHL ligase is an important target for small drug development in two distinct approaches: in their own right, VHL inhibitors that block VHL catalytic activity could mimic a hypoxic response inside cell; moreover, VHL ligands can be conjugated into bifunctional degrader molecules (also known as PROTACs) to hijack VHL activity to induce targeted protein degradation. This work aimed to develop novel small molecules that target two different binding sites on the surface of VHL: 1) the hydroxyproline recognition site of HIF-α; and 2) a newly identified pocket on the VHL surface, and not involved in the VHL-HIF-α PPI. For the first binding site, I describe the structure-guided design and synthesis of a series inhibitors of the VHL:HIF-α PPI interaction, followed by their biophysical and cellular screening. These efforts led to the discovery of the first inhibitors of this interaction showing double-digit nanomolar affinities and good cellular activity. This work also led to the disclosure of compound VH298 as a potent and selective chemical probe of the hypoxia signalling pathway. Additionally, a series of thioamide containing analogues of VH298 were designed to probe the hydroxyproline recognition of VHL ligands. On the second VHL pocket starting from a weak-affinity fragment hit (Kd > 1 mM) I performed iterative cycles of synthesis, biophysical binding evaluation and fragment growing that yielded fragments with improved binding affinities to VHL. The most promising fragments achieved affinities around 100 µM to wild-type VHL. They also exhibited enhanced affinities for an R200W mutant, one of the most common mutations associated with the rare disease of Chuvash polycythemia. The developed efforts on this project disclose to the scientific community the first selective chemical probe of the hypoxia signalling pathway, and new ligands useful for the development of a new generation of PROTACs. The improved fragments targeting the second pocket could in future be developed into more potent and selective ligands targeting the disease-relevant mutation, and could also be used for novel PROTAC conjugation.
Date of Award2018
Original languageEnglish
SponsorsEuropean Research Council & Fundação para a Ciência e a Tecnologia
SupervisorAlessio Ciulli (Supervisor)

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