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Published on: December 1, 2020
Characterization of Three Druggable Hot-Spots in the Aurora-A/TPX2 Interaction Using Biochemical, Biophysical, and
Patrick J McIntyre1, Patrick M Collins2, Lukáš Vrzal3,4
1Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester , Leicester, LE1 9HN, United Kingdom.
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.
Insights
Researchers identified key interactions between Aurora-A kinase and its partner TPX2 (Targeting Protein for Xenopus kinesin-like protein 2). This work advances the development of novel anticancer drugs targeting the Aurora-A/TPX2 oncogenic holoenzyme.
Area of Science:
- Molecular Biology
- Biochemistry
- Drug Discovery
Background:
- Aurora-A kinase and TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, forming an oncogenic holoenzyme.
- TPX2 activates Aurora-A during mitosis, making their interaction a potential target for anticancer therapies.
Purpose of the Study:
- To investigate the Aurora-A/TPX2 interface for novel drug development.
- To identify key residues and binding pockets involved in the Aurora-A/TPX2 interaction.
Main Methods:
- Coprecipitation assays and isothermal titration calorimetry to analyze TPX2 residue contributions.
- High-throughput X-ray crystallography screen (XChem) of 1255 fragments against Aurora-A.
Main Results:
- Identified TPX2 residues Tyr8, Tyr10, Phe16, and Trp34 as crucial for complex formation.
- Discovered that phosphorylation of Aurora-A at Thr288 is necessary for high-affinity binding.
- Identified 59 fragment hits, with over 75% binding to identified Aurora-A pockets, validating druggability.
Conclusions:
- The Aurora-A/TPX2 interaction interface is druggable, with specific pockets identified for therapeutic targeting.
- High-throughput crystallography is effective for identifying inhibitors of protein-protein interactions.
- Findings accelerate the development of chemical inhibitors for the Aurora-A/TPX2 interaction as cancer therapeutics.
