Characterization of Three Druggable Hot-Spots in the Aurora-A/TPX2 Interaction Using Biochemical, Biophysical, and Fragment-Based Approaches

Abstract

The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein-protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.

Covalent Aurora A regulation by the metabolic integrator coenzyme A

“…Structural analysis of Aurora A confirms the importance of activation loop dynamics for activity and binding-partner interactions (Burgess et al, 2018). Phosphorylation, as well as TPX2 binding, stabilise the activation segement in an appropriate conformation for catalysis, and distinct Aurora A conformations can also be induced and/or stabilised by a huge variety of chemical small molecules (Dodson et al, 2010, McIntyre et al, 2017, Pitsawong et al, 2018, Tyler et al, 2007, Scutt et al, 2009, Damodaran et al, 2017. Such compounds have been extremely useful to validate dozens of cellular Aurora A substrates (Tyler et al, 2005;Sardon, 2010), which include other cell cycleregulated kinases such as PLK1 (Macurek et al, 2008, Scutt et al, 2009).…”

“…Interestingly, the TPX2 complex also protects Aurora A from Thr 288 dephosphorylation by PP1 and PP2A phosphatases in vitro , Bayliss et al, 2004, although accumulating evidence suggests that the PP6 Ser/Thr phosphatase is rate-limiting for mitotic dephosphorylation at this site in cells (Zeng et al, 2010). In addition to TPX2, Aurora A is targeted to core protein cofactors, including the centrosomal protein Cep192, the mitotic entry factor BORA, the ciliary protein Pifo and the transcription factor NMYC (Joukov and De Nicolo, 2018), whose interaction with a specific inactive Aurora A conformation is important for controlling NMYC stability in cells, and the basis for new approaches to target Aurora A output with drugs (Richards et al, 2017).…”

Construction of a Shape‐Diverse Fragment Set: Design, Synthesis and Screen against Aurora‐A Kinase

“…[25] The pocket also dominated the hits obtained from ac rystallography-based screen of ac ommercial fragment library. [26] The synthesised fragment hit 39 made three polar interactions:d irect interactions with K166 and R179 and au nique water-mediated interaction with Y199 (panelA). In contrast, the three purchased fragments each made one direct interaction with the protein:w ith R179 (hit 55)o rY 199 (hits 56 and 57).…”

An evolutionary-conserved redox regulatory mechanism in human Ser/Thr protein kinases

“…The activation loop of Aurora A itself undergoes dynamic conformational changes in response to phosphorylation and interactions with allosteric binding-partners, enabling Aurora A to transition between alternate active states (Levinson, 2018). This structural plasticity is perhaps most apparent when considering the number of distinct Aurora A conformations that have been captured in complex with small molecule inhibitors by crystallographic and NMR based techniques (Dodson et al, 2010, McIntyre et al, 2017, Pitsawong et al, 2018, Lake et al, 2018. Even in an active, phosphorylated form, the activation loop of Aurora A possesses a dynamic conformational ensemble of 'DFG-in' sub-states (Gilburt et al, 2017, Ruff et al, 2018.…”