Fragment-based approaches to probe the surface of the VHL E3 ligase

Abstract

Among the proteins involved in the control of protein homeostasis, E3 ubiquitin ligases are emerging as interesting targets for the development of chemical probes and drugs. Small-molecule modulation of E3 ligases can provide several advantages over other members of the ubiquitin-proteasome system (UPS), as E3 ligases are responsible for specifically recognising substrates for ubiquitylation. Another strategy of increasing interest consists of developing bivalent molecules capable of bringing an E3 ligase in close proximity to a desired protein, inducing the ubiquitylation and subsequent degradation of an unnatural substrate of the E3 ligase.

In this work, interactions of components of the von Hippel-Lindau (VHL) E3 ligase were investigated using small-molecules. By applying an integrated set of biophysical and structural biology techniques, the main goals of this project were to provide chemical tools and novel assays to probe interactions at known sites of the VHL E3 ligase, as well as discovering novel binders and pockets by fragment screening. A competition assay for probing interactions in the substrate recognition domain of the VHL E3 ligase was developed by fluorine nuclear magnetic resonance. Thorough characterisation of the fluorinated reporter molecules revealed features that are important for enhancing assay sensitivity. This assay has a wide detection range and was applied to both fragment screening and studying high-affinity interactions, and the main advantages and limitations were explored. Additionally, after extensive characterisation, several fragment hits were identified in a binding site in the elongin C protein (eloC), and adaptor subunit part of the VHL E3 ligase. Preliminary fragment merging approaches revealed promising vectors for the design of eloC ligands. Altogether, this study highlights multiple possibilities for investigating interactions on the surface of the VHL E3 ligase, with applications to the identification of novel biological functions of members of this complex and in the design of inhibitors and chemical degraders.

Date of Award	2019
Original language	English
Sponsors	Coordenação de Aperfeiçoamento de Pessoal de Nível Superior & European Research Council
Supervisor	Alessio Ciulli (Supervisor)