Discovery of ubiquitin-conjugation enzyme inhibitors from marine natural products

Ubiquitination is a key biochemical event which regulates most cell processes. Proteins are modified with ubiquitin, or ubiquitinated, through a series of reactions involving the E1, E2 and E3 enzymes. Ubiquitination of a protein may change its stability, localisation or interactions with other molecules and plays a major role in signalling within a cell. Because of its importance for the cellular homeostasis, any perturbations in ubiquitination may result in diseases. For example, alterations of E2 or E3 enzyme activity are associated with cancer, immune disorders neurodegenerative and cardiovascular diseases. However, despite a clear clinical relevance, pharmaceutical interest and substantial research efforts, we are far from comprehensively understanding the mechanisms of ubiquitination. In addition, there are currently very few tool molecules, which would allow us to investigate these mechanisms.

To address this gap, we propose a PhD project to develop a distinctive, novel approach for discovery of tool molecules and potentially starting points for medicines related to ubiquitination. We will focus on the E2 enzyme family which, despite operating at the heart of ubiquitination reactions, have been underexplored so far. We will search for modulators of these enzymes within molecules produced by nature in the sea - marine natural products. Natural products are source of very diverse and chemically sophisticated compounds in contract to synthetic, relatively small and simple molecules typically used without much success towards the E2s.

Extracts will be provided by our collaborators at the University of California, San Diego, (UCSD) US, who have long lasting expertise in the field of chemical biology of natural products. We will use the innovative UPS-CONA technology, developed in the Auer lab as primary screening method, to identify extracts which inhibit or activate ubiquitination of one out of more than 10 disease related human E2 enzymes, which are already under investigation in the Auer lab. For any UPS-CONA selective extracts, which are confirmed in SDS-PAGE assays, we will collaborate with the UCSD group to follow up with further fractionation and functional chromatography to isolate the active molecule. Part of the project will be to transfer the functional chromatography method to the Auer lab.

For any identified E2-active new natural product, a series of biophysical methods will be applied to determine E2 binding affinity and stoichiometry, including quantitative microdialysis, ITC, NMR and fluorescence spectroscopy. Structure-based design, particularly docking methods, mass spectrometry methods like HDX and MS cross-linking, 2D-NMR and X-ray crystallography will be applied to determine binding site and complex structure of natural product hits with nanomolar affinity to an E2 enzyme. Ki values will be determined using a variety of enzyme kinetic methods.

Active molecules, identified against established disease target E2s such as the oncogenic Ube2C or Cdc34 will be further tested in a variety of cancer cell lines available in the Carragher lab (second supervisor), at the Edinburgh Cancer Centre. Mode of action of hit natural products will be further explored by the reversed phase protein array (RPPA) technology applied routinely in the Carragher lab.

At the end of the proposed project, it is expected that several high affinity, potent and selective inhibitors of the E2 enzymes from the highly diverse natural compound extracts will be identified. Identification of selective tool compounds will be of great value for the research community currently struggling with availability of selective modulators of ubiquitination pathways.

The PhD student will have gained experience in multiple techniques which are currently applied in basic and translational research and should have excellent possibilities for further career progression academia, biotech or pharma industry.