Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders

Alexander Hanzl, Ryan Casement, Hana Imrichova, Scott J. Hughes, Eleonora Barone, Andrea Testa, Sophie Bauer, Jane Wright, Matthias Brand, Alessio Ciulli (Lead / Corresponding author), Georg E. Winter (Lead / Corresponding author)

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Abstract

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined 'functional hotspots'. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

Original languageEnglish
Number of pages26
JournalNature Chemical Biology
Early online date3 Nov 2022
DOIs
Publication statusE-pub ahead of print - 3 Nov 2022

Keywords

  • Chemical genetics
  • X-ray crystallography

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