Abstract
Aurora kinases are a family of cell division regulators that govern the correct assembly of a bipolar mitotic spindle and the fidelity of chromosome segregation. Their overexpression is associated with genomic instability and aneuploidy, and is frequently observed in cancer. Accordingly, competitive inhibitors targeting Aurora kinase activity at the ATP-binding site are being investigated for therapeutic purposes. Despite promising pre-clinical data, these molecules display moderate effects in clinical trials and incomplete selectivity, either against distinct family members, or other kinases. As an alternative approach, protein-protein interaction inhibitors targeting mitotic kinases and their activators can be exploited to achieve increased specificity of action. In this study, a virtual screening of small molecules led to the identification of 25 potential inhibitors of the interaction between Aurora-A and its activator TPX2. In vitro experiments confirmed that 4 hits bind Aurora-A in the low micromolar range and compete for TPX2 binding. Immunofluorescence assays showed that 2 compounds also yield lowered Aurora-A activity and spindle pole defects in cultured osteosarcoma cells. The identified protein-protein interaction inhibitors of the Aurora-A/TPX2 complex might represent lead compounds for further development towards pioneering anti-cancer drugs and provide the proof-of-concept for a new exploitable strategy to target mitotic kinases.
Highlights
Targeting mitosis is a well known strategy to kill cancer cells: besides anti-microtubule (MT) drugs commonly used in chemotherapy, inhibitors of specific mitotic regulators are being developed to overcome the issues of toxicity and/or acquired resistance displayed by anti-MT molecules [1]
We describe the identification of two drug-like small molecules, showing the ability to interfere with the formation of the complex in vitro and to perturb Aurora-A activity and spindle structure in cultured osteosarcoma cells
In order to develop the rational design of small molecule inhibitors of the Aurora-A/ TPX2 interaction, we first in-depth investigated the key structural determinants of affinity and specificity at protein-protein interface
Summary
Single cell microscopy analyses recently showed that the most specific Aurora-A inhibitor in clinical trials, MLN8237 (Alisertib), has still incomplete specificity towards the related Aurora-B [7, 8] This can yield undesired effects, including lack of sustained mitotic arrest and exit from mitosis with unbalanced chromosome segregation, a condition that may contribute to protumorigenic effects [7]. Kinases often bind specific protein partners at allosteric sites to gain full activity: novel classes of protein-protein interaction (PPI) inhibitors targeting pockets remote from the ATP-binding site are rapidly moving to the forefront of kinase inhibitors research [9]. TPX2 binds Aurora-A and not Aurora-B due to a single amino acid difference within the catalytic domain [15, 16], suggesting that such PPI inhibitors would at least partially overcome the specificity issues of ATP-competitors. In the search for a new generation of more specific and effective inhibitors of Aurora-A activity, these compounds represent promising scaffolds for future hitto-lead optimization studies
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