Dissecting Signalling Pathways with PROTAC Chemical Probes.

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Proteolysis targeting chimeras (PROTACs) are hetero-bifunctional molecules where one fragment interacts with the protein of interest (POI) and the other binds to a component of an ubiquitin ligase. This results in the poly-ubiquitination of the POI, which then undergoes irreversible proteasomal degradation, in a catalytic manner. This strategy to reduce cellular protein levels has generated huge interest and excitement in biology, and at present, there is significant research aimed at developing PROTACs to target proteins and signalling pathways previously thought undruggable using classical protein-protein interaction inhibitors. This project aims to develop/characterise new classes of cell permeable PROTACs, as novel probes to interrogate important signalling pathways and their key protein effectors. Examples include the Wnt/Beta-catenin signalling pathway and chromatin remodelling complexes, which play multiple pivotal functions in human physiology. These critical roles are underscored by the observation that their dysregulation leads to multiple cancer types. First, the candidate will focus on the development of PROTAC probes targeting key POIs at regulatory nodes. This will involve the molecular design, chemical synthesis and in vitro biophysical characterisation (NMR, ITC, MS) of the PROTACs. A second objective will focus on elucidating the cellular mode of action of the new PROTACs. Their selectivity and ability to deplete the POIs will be quantified by WB and live imaging using GFP constructs; and their downstream effects determined by monitoring mRNA levels of downstream genes known to respond to POI inhibition across a panel of cancer cell lines. This project builds on i)recent proof-of-concept results in the Baud and Linclau laboratories showing that it is possible to enhance PROTAC properties (e.g. solubility/lipophilicity) and selectivity through synthetic alteration, circumventing an important historical bottleneck in the field; and ii)expertise in cancer cell biology and signalling in the Giamas laboratory (e.g. identification/deciphering of new genes/proteins implicated in important biological processes).