RIPK1-dependent cell death: a novel target of the Aurora kinase inhibitor Tozasertib (VX-680)
The Aurora kinase family, which includes Aurora A, B, and C, plays a vital role in regulating various mitotic processes, such as cytokinesis. Overexpression of these kinases has been linked to tumor development, and several Aurora kinase inhibitors (e.g., AT9283, AZD1152, Danusertib, MLN8054) are currently being tested in clinical trials. In this study, we demonstrate that the pan-Aurora kinase inhibitor Tozasertib (also known as VX-680 and MK-0457) not only disrupts cytokinesis by inhibiting Aurora kinases but also serves as a potent inhibitor of necroptosis, a cell death mechanism governed by the RIPK1, RIPK3, and MLKL signaling pathways. Tozasertib inhibits both RIPK1-dependent necroptosis and cytokinesis within the same concentration range, with IC50 values of 1.06 µM and 0.554 µM, respectively.
A structure-activity relationship (SAR) analysis of 67 Tozasertib analogues, modified at four different positions, revealed compounds with enhanced specificity for either cytokinesis inhibition (Aurora kinase inhibition) or necroptosis inhibition (RIPK1 inhibition). These findings suggest that Tozasertib independently targets both RIPK1 and Aurora kinases. Supporting this, more selective Aurora kinase inhibitors did not affect necroptosis, and Necrostatin-1s, a RIPK1 inhibitor, did not cause cytokinesis defects, confirming the distinct regulation of these cellular processes.
Additionally, Tozasertib was shown to inhibit recombinant human RIPK1, Aurora A, and Aurora B kinase activities but did not affect RIPK3. The specificity of the newly developed Tozasertib analogues, as observed in cellular assays, aligned with the results from ADP-Glo kinase activity assays. In conclusion, this study demonstrates that Tozasertib targets both Aurora kinases and RIPK1 independently, and we were able to design analogues with greater selectivity for either target.