Exploiting ELIOT for Scaffold-Repurposing Opportunities: TRIM33 a Possible Novel E3 Ligase to Expand the Toolbox for PROTAC Design.

The field of targeted protein degradation, through the control of the ubiquitin-proteasome system (UPS), is progressing considerably; to exploit this new therapeutic modality, the proteolysis targeting chimera (PROTAC) technology was born. The opportunity to use PROTACs engaging of new E3 ligases that can hijack and control the UPS system could greatly extend the applicability of degrading molecules. To this end, here we show a potential application of the ELIOT (E3 LIgase pocketOme navigaTor) platform, previously published by this group, for a scaffold-repurposing strategy to identify new ligands for a novel E3 ligase, such as TRIM33. Starting from ELIOT, a case study of the cross-relationship using GRID Molecular Interaction Field (MIF) similarities between TRIM24 and TRIM33 binding sites was selected. Based on the assumption that similar pockets could bind similar ligands and considering that TRIM24 has 12 known co-crystalised ligands, we applied a scaffold-repurposing strategy for the identification of TRIM33 ligands exploiting the scaffold of TRIM24 ligands. We performed a deeper computational analysis to identify pocket similarities and differences, followed by docking and water analysis; selected ligands were synthesised and subsequently tested against TRIM33 via HTRF binding assay, and we obtained the first-ever X-ray crystallographic complexes of TRIM33α with three of the selected compounds.

ELIOT: A platform to navigate the E3 pocketome and aid the design of new PROTACs.

Proteolysis-targeting chimeras (PROTACs) are novel therapeutics for the treatment of human disease. They exploit the enormous potential of the E3 ligases, a class of proteins that mark a target protein for degradation via the ubiquitin-proteasome system. Despite the existence of several E3 ligase-related databases, the choice of the functioning ligase is limited to only 1.6% of those available, probably due to the fragmentary understanding of their structures and their known ligands; in fact, none of the existing databases report detailed studies covering their 3D structure or their pockets. Here, we report ELIOT (E3 LIgase pocketOme navigaTor), an accurate and complete platform containing the E3 ligase pocketome to enable navigation and selection of new E3 ligases and new ligands for the design of new PROTACs. All E3 ligase pockets were characterized with innovative 3D descriptors including their PROTAC-ability score, and similarity analyses between E3 pockets are presented. Tissue specificity and their degree of involvement in patients with specific cancer types are also annotated for each E3 ligase, enabling appropriate selection for the design of a PROTAC with improved specificity. All data are available at https://eliot.moldiscovery.com.