Abstract
The Aurora kinase family (Aurora A, B and C) are crucial regulators of several mitotic events, including cytokinesis. Increased expression of these kinases is associated with tumorigenesis and several compounds targeting Aurora kinase are under evaluation in clinical trials (a.o. AT9283, AZD1152, Danusertib, MLN8054). Here, we demonstrate that the pan-Aurora kinase inhibitor Tozasertib (VX-680 and MK-0457) not only causes cytokinesis defects through Aurora kinase inhibition, but is also a potent inhibitor of necroptosis, a cell death process regulated and executed by the RIPK1, RIPK3 and MLKL signalling axis. Tozasertib’s potency to inhibit RIPK1-dependent necroptosis and to block cytokinesis in cells is in the same concentration range, with an IC50 of 1.06 µM and 0.554 µM, respectively. A structure activity relationship (SAR) analysis of 67 Tozasertib analogues, modified at 4 different positions, allowed the identification of analogues that showed increased specificity for either cytokinesis inhibition or for necroptosis inhibition, reflecting more specific inhibition of Aurora kinase or RIPK1, respectively. These results also suggested that RIPK1 and Aurora kinases are functionally non-interacting targets of Tozasertib and its analogues. Indeed, more specific Aurora kinase inhibitors did not show any effect in necroptosis and Necrostatin-1s treatment did not result in cytokinesis defects, demonstrating that both cellular processes are not interrelated. Finally, Tozasertib inhibited recombinant human RIPK1, human Aurora A and human Aurora B kinase activity, but not RIPK3. The potency ranking of the newly derived Tozasertib analogues and their specificity profile, as observed in cellular assays, coincide with ADP-Glo recombinant kinase activity assays. Overall, we show that Tozasertib not only targets Aurora kinases but also RIPK1 independently, and that we could generate analogues with increased selectivity to RIPK1 or Aurora kinases, respectively.
Highlights
Mitosis is a multi-step process that is tightly regulated by several classes of kinases, like cyclin-dependent kinases (CDKs) and Aurora kinases[1]
Increased expression or gene amplification of Aurora A/B has been observed in a number of cancers[6,8,36,37] and its oncogenic potential has been illustrated by the fact that Aurora A overexpression induces transformation of mammalian fibroblasts[38]
Other kinase targets that have been described for Tozasertib are the oncogenic kinases Flt-3 and Abl (both wild-type Abl kinase and imatinib-resistant Abl mutant (T315I)) and JAK 2 kinase, with the latter playing a role in imatinib resistance in chronic myelogenous leukaemia[3,5,14,15,45,48]
Summary
Mitosis is a multi-step process that is tightly regulated by several classes of kinases, like cyclin-dependent kinases (CDKs) and Aurora kinases[1]. Aurora A and B are expressed in most cell types and play important roles in centrosome maturation, mitotic spindle formation, kinetochore assembly and cytokinesis, the final step of cell division[2,6,7]. Aurora A and B have been described as oncogenes and increased expression or polymorphisms of these kinases have been observed in several types of cancer[6], like breast cancer[8,9], prostate cancer[10,11] and non-smallcell lung carcinoma[12]. Inhibition of Aurora kinase results in failure of G2/M transition, abnormal spindle formation leading to cytokinesis defects and apoptosis[13].
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