Synthetic protein binders reveal a cryptic regulatory pocket on Aurora A for selective allosteric inhibition

Aurora Kinase A (AurA) is an essential mitotic kinase and therapeutic target in cancer. Most protein kinase inhibitors target the conserved ATP-binding pocket, often resulting in poor selectivity and off-target effects. Here, we identify and characterise small synthetic protein binders, Adhirons, as allosteric inhibitors of AurA. Using ‘phage display, we isolated Adhiron reagents that bind a previously uncharacterised site on the αG-helix of the kinase C-lobe. Structural and biochemical analyses revealed that the Adhiron inhibited AurA by modulating the activation loop via this cryptic site, which we designate the T-pocket. In cells, Adhiron expression mimics the effects of small molecule inhibitors of AurA on substrate and auto-phosphorylation, while sparing Aurora kinase B and without impairing TPX2-mediated localisation of AurA to the mitotic spindle. The AurA-inhibitory Adhirons demonstrate remarkable selectivity, potency and affinity, a highly sought-after combination of properties for kinase inhibition facilitating their use as tractable research tools for probing AurA function and as pharmacophore templates for structure-based drug design. Finally, these reagents illustrate a generalisable strategy for targeting allosteric sites across the Kinome. *Jack P Roberts & James Holder contributed equally to this work.