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.
Highlights
Aurora kinases were discovered in the mid-nineties in Drosophila and yeast [1, 2]
We began by analyzing the inhibitory properties of the 10 compounds in Figure 1 on the in vitro activities of full-length human Aurora A, alone or bound to an activating N-terminal peptide fragment of TPX2, and full-length human Aurora B bound to a C-terminal fragment of INCENP
We focused on additional characterization of the four inhibitors designed to target Aurora A that were not cytotoxic (MLN8054, MLN8237, MK-5108, MK-8745; Figure 4D; Figure S2B in Supplementary Material), and AZD1152-HQPA and GSK1070916, because they are chemically distinct (Figure 1) and the two most potent Aurora B inhibitors in the HeLa substrate phosphorylation assays (Figure 4A)
Summary
Aurora kinases were discovered in the mid-nineties in Drosophila and yeast [1, 2]. Whereas yeasts only have one Aurora kinase, metazoans generally have two, named Aurora A and B. Aurora A localizes to centrosomes and spindle microtubules and plays important roles in centrosome maturation, controlling spindle length and bipolarity, asymmetric cell division, and promoting mitotic entry both in unperturbed cells and following DNA damage [3, 4]. Aurora C is expressed in testis [8], where it exhibits tissue-specific functions [9, 10], and in oocytes, where it contributes to early embryonic divisions by providing functions associated with Aurora B in somatic cells [11,12,13,14]. Aurora C is aberrantly expressed in cancer cells [15]
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