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ACS Chem Biol. 2017 Nov 17;12(11):2906-2914. doi: 10.1021/acschembio.7b00537. Epub 2017 Oct 30.

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

Author information

1
Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester , Leicester, LE1 9HN, United Kingdom.
2
Diamond Light Source, Harwell Science and Innovation Campus , Didcot, OX11 0DE, United Kingdom.
3
University of Chemistry and Technology , Technická 5, Prague 6 - Dejvice, Prague, 166 28, Czech Republic.
4
Institute of Organic Chemistry and Biochemistry , Flemingovo nám. 542/2, Prague 6, Prague, 166 10, Czech Republic.
5
LifeArc (Formerly MRC Technology), Stevenage Bioscience Catalyst , Gunnels Wood Road, Stevenage, SG1 2FX, United Kingdom.
6
Astbury Centre for Structural and Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds , Leeds LS2 9JT, United Kingdom.
7
Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford , Roosevelt Drive, Oxford, OX3 7DQ, United Kingdom.
8
Department of Biochemistry, University of Johannesburg , Auckland Park, 2006, South Africa.

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.

PMID:
29045126
DOI:
10.1021/acschembio.7b00537
[Indexed for MEDLINE]

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