Construction and Biological Characterisation of a Custom Engineered Protein Degradation Tool

Development of non-small molecule drugs has expanded over recent years, with biologics such as antibodies now seen as a viable treatment for some diseases. Protein and peptide drugs benefit from higher specificities than small molecules, and there is a much wider range of targets to which they can be applied. One such target is Aurora Kinase A, which is overexpressed in many cancer types and acts as a driver of cancer by phosphorylating substrates. AURKA is overexpressed in cancers such as breast, colon and neuroblastomas and targeting AURKA by inhibition does affect cancer growth but there is not complete reversal of the cancer phenotype. Targeting AURKA for degradation using the ubiquitin-proteasome system has potential for a better clinical response. PROTACs (PROteolysis Targeting Chimeras) are a new class of drug, that induce degradation of target molecules via a bifunctional small molecule, with a flexible linker region in the middle. Degradation occurs via the Ubiquitin Proteasome System, using chains of ubiquitin as the signalling molecule.

There are a range of scaffolds used in this work, including TetratrioPeptide Repeats, Recombinase ATPase domain, and Monobodies. These scaffold proteins have AURKA binding regions inserted and degrons that bind E3 ligases elsewhere in the sequence. These bifunctional molecules should induce Aurora A degradation using the Ubiquitin Proteasome System. Work has been preformed using cancer cells treated with these constructs, and biophysical work to characterise their structure and interactions.