Adhiron reagents as novel genetic and cell biology tools to modulate the protein-protein interactions of Aurora kinase A

Abstract
Aurora Kinase A (AurA) is a mitotic kinase and established therapeutic target that plays essential roles in cell division and is frequently overexpressed in cancer. Most clinical inhibitors of AurA target the conserved ATP-binding pocket, but this strategy often leads to limited selectivity and off-target toxicities due to high structural conservation across the kinome. Allosteric inhibition, by contrast, offers a promising but underexploited approach to selectively modulate kinase function via less conserved, conformationally dependent sites. In this project, Adhirons, small synthetic binding proteins, were identified and characterised as allosteric inhibitors of AurA. A ‘phage display screen against phosphorylated AurA yielded a panel of Adhirons that inhibit kinase activity through engagement of a previously uncharacterised cryptic site on the αG-helix of the C-lobe. This site, which we term the ‘T-pocket’, was revealed by X-ray crystallography to induce conformational changes in the T-loop (activation loop), a key structural element required for kinase activation. Adhiron binding stabilises a DFG-in conformation of AurA, distinct from classical ATP-competitive inhibitors, and is compatible with regulatory protein interactions such as TPX2. In vitro kinase assays confirmed direct inhibition of AurA catalytic activity, while in cellulo studies demonstrated that Adhiron expression results in impaired AurA function and prolonged mitotic progression, consistent with targeted inhibition. These findings establish the T-pocket as a novel allosteric regulatory site and demonstrate the utility of Adhirons as high-affinity, conformation-selective reagents for kinase modulation. More broadly, this work highlights the potential of using engineered binders to uncover cryptic regulatory pockets in challenging targets and guide the development of structure-informed, allosteric therapeutic strategies.