Construction and Biological Characterisation of Custom Engineered Protein Degradation Tools.

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Stockton, Sian 

Event-driven pharmacology is a new paradigm in disease treatment for a wide range of diseases, including cancer, that have failed by traditional occupancy-driven pharmacology. One target that has been resistant to conventional inhibition approaches is Aurora A (AurA). Degradation rather than inhibition of AurA should be able to block non-catalytic functions that cannot be inhibited. Event-driven pharmacology can be effected using bifunctional constructs to recruit the cell’s degradation machinery to target proteins such as AurA via targeted protein degradation. This work explores the design and characterisation of bifunctional protein constructs (Polyproxins) with two grafted binding regions to recruit E3 ubiquitin ligases to the target AurA. By simultaneously binding these two proteins there should be transfer of ubiquitin to AurA and proteasome-mediated degradation in the cell. In this study, three different scaffold proteins are used to display a number of different AurA- and E3-binding sequences: tetratricopeptide repeats (TPRs), RAD protein, and monobodies. The structures, stabilities and binding of the grafted scaffolds were investigated. Characterisation included circular dichroism spectroscopy to ensure correct folding and thermal and chemical denaturation assays. Binding assays were also performed, both qualitative with pull downs and quantitative with florescence polarisation and isothermal titration calorimetry. Purification of the binding partners, especially AurA, needed some optimisation. Binding was seen with monobodies and RAD proteins to AurA and and the E3 ligase Keap1; however another E3 ligase UBR5 was not observed to bind. Cell-based experiments were performed, and microscopy and luminescence assays were designed to test for AurA degradation by the polyproxins. Low expression of the polyproxins in mammalian cells and their weak binding to AurA and E3 ligases prevented successful knock-down of AurA. For the TPR proteins, in silico optimisation of scaffold stability around a grafted binding peptide was performed, and the predictions were compared with the experimentally determined data. Two of these designs showed remarkable increases in stability compared with the original consensus-designed TPR protein. The work described provides a starting point for the development of polyproxins by peptide grafting onto stable scaffolds for targeted protein degradation

Itzhaki, Laura
Aurora A, PROTACs
Awarding Institution
University of Cambridge
BBSRC (1943410)
Biotechnology and Biological Sciences Research Council (1943410)