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

Synthetic protein binders reveal a cryptic regulatory pocket on Aurora A for selective allosteric inhibition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Synthetic protein binders reveal a cryptic regulatory pocket on Aurora A for selective allosteric inhibition Jack P. Roberts*, James Holder*, Gamaliel B. Shami-Inkindi, Isha A. Mohan, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7565154/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Cell biology/Cell division/Checkpoints Biological sciences/Molecular biology/Cell division/Mitosis Full Text Additional Declarations There is NO Competing Interest. 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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. 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