A Cell Biologist’s Field Guide to Aurora Kinase Inhibitors

Abstract

Aurora kinases are essential for cell division and are frequently misregulated in human cancers. Based on their potential as cancer therapeutics, a plethora of small molecule Aurora kinase inhibitors have been developed, with a subset having been adopted as tools in cell biology. Here, we fill a gap in the characterization of Aurora kinase inhibitors by using biochemical and cell-based assays to systematically profile a panel of 10 commercially available compounds with reported selectivity for Aurora A (MLN8054, MLN8237, MK-5108, MK-8745, Genentech Aurora Inhibitor 1), Aurora B (Hesperadin, ZM447439, AZD1152-HQPA, GSK1070916), or Aurora A/B (VX-680). We quantify the in vitro effect of each inhibitor on the activity of Aurora A alone, as well as Aurora A and Aurora B bound to fragments of their activators, TPX2 and INCENP, respectively. We also report kinome profiling results for a subset of these compounds to highlight potential off-target effects. In a cellular context, we demonstrate that immunofluorescence-based detection of LATS2 and histone H3 phospho-epitopes provides a facile and reliable means to assess potency and specificity of Aurora A versus Aurora B inhibition, and that G2 duration measured in a live imaging assay is a specific readout of Aurora A activity. Our analysis also highlights variation between HeLa, U2OS, and hTERT-RPE1 cells that impacts selective Aurora A inhibition. For Aurora B, all four tested compounds exhibit excellent selectivity and do not significantly inhibit Aurora A at effective doses. For Aurora A, MK-5108 and MK-8745 are significantly more selective than the commonly used inhibitors MLN8054 and MLN8237. A crystal structure of an Aurora A/MK-5108 complex that we determined suggests the chemical basis for this higher specificity. Taken together, our quantitative biochemical and cell-based analyses indicate that AZD1152-HQPA and MK-8745 are the best current tools for selectively inhibiting Aurora B and Aurora A, respectively. However, MK-8745 is not nearly as ideal as AZD1152-HQPA in that it requires high concentrations to achieve full inhibition in a cellular context, indicating a need for more potent Aurora A-selective inhibitors. We conclude with a set of “good practice” guidelines for the use of Aurora inhibitors in cell biology experiments.

AURKA destruction is decoupled from its activity at mitotic exit but suppresses interphase activity

“…We then used the same antibody to look by immunofluorescence (IF) in fixed mitotic cells, marking centrosomes with γ-tubulin staining. All phospho-epitope signal on the centrosomes and spindle poles was abolished by treatment with AURKA inhibitor MLN8237, whereas the signal at the midbody (where the same antibody recognizes pT232 of AURKB) was insensitive to 100 nM MLN8237, indicating that the centrosomal pT288 signal is specific to AURKA as expected ( Figure 1B, Supplementary Figure S1) [34,35]. We quantified fluorescence intensity associated with pT288 at different stages of mitosis scored according to DNA morphology.…”

“…(Supplementary Figure S3). This finding was not unexpected for a diffusible biosensor, since some of the specificity in substrate phosphorylation by Aurora kinases is proposed to reside in the colocalization of kinase with substrates [3,35]. We transfected this biosensor into U2OS and FZR1 KO cells to measure Aurora kinase activity at mitosis and during mitotic exit.…”

ERK-Induced Activation of TCF Family of SRF Cofactors Initiates a Chromatin Modification Cascade Associated with Transcription

“…Hesperadin is an indolinone inhibitor of Aurora-B (half-maximal inhibitory concentration [IC 50 ] = 250 nM; de Groot et al., 2015), while LLY-507 is a potent inhibitor of KMT3C (IC 50  < 15 nM, >100-fold selectivity over other MTs; Nguyen et al., 2015). Short pretreatments with either compound inhibited TPA-induced PolII recruitment and histone modifications at Egr1 , Egr3 , and Fosl1 (Figure 7A).…”

The Aurora kinase A inhibitor TC-A2317 disrupts mitotic progression and inhibits cancer cell proliferation

“…MK-5108 (VX-689) inhibits proliferation of breast, colon, and non-small cell lung cancer, leukemia, nasopharyngeal carcinoma, uterine leiomyosarcoma, and ovarian cancer stem cells in vivo and in vitro . The duration of G2 is prolonged by MK-5108 treatment, as also observed in cells treated with siRNA targeting Aurora A [24]. MK-5108 treatment also prolongs mitosis [25, 26] and increases the proportion of H3-pS10–positive mitotic cells [25, 27–30].…”