EASTBIO: Targeting fungal E3 ligases with small-molecule ligands for degrader development

The increase in global trade of agricultural product coupled with climate change have driven the emergence of new pathogen species and genotypes. The resilient and adaptive behaviour of these pathogens due to coevolution between species demand a global response to meet food security and sustainability. It is therefore important to study fungal pathology and to discover new modalities to treat fungal infection in plants.

Targeted Protein Degradation (TPD) is a new therapeutic modality validated in humans. Molecular glue and Proteolysis-targeting chimera (PROTAC) degraders are small molecules that hijack the cell’s ubiquitin-proteasome system via recruiting a ubiquitin E3 ligase to a target protein to mediate target ubiquitination and degradation. The emergence of TPD promises advancement in fungal research and development as the ubiquitin system is important for fungal cellular pathways, adaptation, and virulence.  

In this project, we will deploy a multidisciplinary approach to advance and de-risk targeting of fungal E3 ligases with small molecule binding ligands that will ultimately underpin the development of novel glue and PROTAC agents. Three consecutive workstreams are envisaged: 

1) Identification of putative E3 targets. Bioinformatic analyses in FungiDB (https://fungidb.org/fungidb/app/) will identify homologues to known E3 ligases in Botrytis cinerea and Zymoseptoria tritici (and oomycetes such as Phytophthora infestans and/or Phytophthora capsica). Alphafold and related tools (e.g. DALI) will be used to predict structural or functional features related to Cullin RING E3 ligases substrate recognition subunits. Candidates will be prioritized based on life-stage expression profile and functional evidence of ubiquitination activity, obtained from literature or focussed target validation studies. 

2) Structural and biophysical screens enablement. For the selected targets, constructs will be generated to enable protein expression and purification suitable for biophysics, crystallography and fragment-based screening. Knowledge of native substrates and degrons will be leveraged to design and identify epitope substrate peptides, that may include post-translational modifications at specific sites. Peptides will be qualified as reagents and positive controls for binding. 

3) E3 Ligand development. We will perform biophysical and crystallographic fragment screens using surface plasmon resonance (SPR), thermal shift and/or ligand/protein observed NMR spectroscopy of a ~1,000 fragment library, including both covalent and non-covalent fragments. Fragment hits will be validated, structurally characterised for their binding site and binding mode, and further optimized as starting points for molecular glue and PROTAC degrader development. 

Successful outcome from this project will identify and de-risk new fungal E3 ligases and unlock their potential to support development of degrader molecules as potential novel antifungal agents. 

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