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Nanobody PROTACs for the degradation of intracellular protein targets
Secondary Supervisor(s): Dr James Hodgkinson
University of Registration: University of Leicester
BBSRC Research Themes:
Project Outline
G-protein-coupled receptors and enzymes account for approximately 70% of human drug targets, however, there are many more protein classes dysregulated in human disease. About 4000 proteins are associated with human disease, yet only 900 are drugged, leaving over 3000 undrugged proteins, which contain protein classes such as chaperones, phosphatases and transcription factors.
Targeted protein degraders, including PROTACs (PROteolysis TArgeting Chimeras) provide an alternative to enzyme inhibition and can induce targeted degradation of aberrant proteins to study cellular mechanisms and treat unmet clinical need.
We have developed a platform to produce PROTACs to exploit the relatively small size, stability and specificity of nanobodies (single-domain antibodies) to degrade intracellular targets. The mechanism by which PROTACs induce degradation means that the targeting group does not need to inhibit an active site, so can bind specific epitopes on the target protein. Nanobodies are single-domain antibodies, usually derived from camelid antibodies, which only 2 contain heavy chains, as opposed to human antibodies that comprise of 2 heavy chains and 2 light chains. Our nanobody-PROTACs are prepared via click chemistry and comprise a targeting nanobody, chemical linker and small-molecule E3 ligase ligand, such as thalidomide. Intracellular delivery is achieved using established protein transfection approaches, such as cell penetrating peptides and liposomes and cellular localisation and degradation is observed using fluorescence microscopy and western blot. Structural biology will be used to elucidate the PROTAC ternary structure required for degradation.
The aims of the project are to establish a robust platform to produce libraries of nanobody-PROTACs to undrugged protein targets to demonstrate the utility of this approach in understanding the roll of proteins in disease states and potentially develop a novel therapeutic modality for unmet clinical need.
