Targeting the Aurora-A/TPX2 Interaction

The mitotic serine/threonine kinase Aurora-A and its partner protein TPX2 are both overexpressed in many different cancers. It has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for the activation and localisation of Aurora-A during mitosis, thereby ensuring proper cell division. Disruption of the Aurora-A/TPX2 interface is therefore a potential target for novel anti-cancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. This study focuses on further characterisation of the Aurora-A/TPX2 interaction and the application of multiple approaches to developing inhibitors of Aurora-A and its complex with TPX2. We identify three key hot-spot sites within the interaction. We also reveal a previously unknown functional role within the complex for the first six residues of TPX2 and the tolerance of its flexible linker region to mutation and shortening. We show that TPX2 binds to phosphorylated Aurora-A with an order of magnitude greater affinity and provide evidence for our model of how the phosphorylation state of Aurora-A is ‘signaled’ to TPX2. To aid ongoing drug discovery efforts to inhibit Aurora-A, we show the crystal structures of several ATP-competitive inhibitors bound to Aurora-A and explain the structural mechanisms behind their high selectivity for Aurora-A. We show an alternative approach to allosterically inhibiting Aurora-A; through the use of a hydrocarbon-stapled peptidomimetic of TPX2. This stapled peptide binds to Aurora-A with a greater affinity than its wild- type, recombinantly expressed analogue and activates Aurora-A autophosphorylation to the same extent. Finally, we describe our work to develop a small molecule inhibitor of the Aurora-A/TPX2 interaction using fragment-based drug design. Following a high-throughput X-ray crystallography- based fragment screen we identified many fragment hits that bound within the Aurora-A/TPX2 binding interface. Our top fragments show inhibition of Aurora- A kinase activity, inhibition and activation of Aurora-A autophosphorylation and weaken the affinity between Aurora-A and TPX2 in competition assays.