title: 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.
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Christian O
authors.initials: CO
authors.lastname: de Groot
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Judy E
authors.initials: JE
authors.lastname: Hsia
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: John V
authors.initials: JV
authors.lastname: Anzola
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Amir
authors.initials: A
authors.lastname: Motamedi
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Yoon
authors.affiliation: Laboratory of Chromosome Biology, Ludwig Institute for Cancer Research, La Jolla, CA, USA; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.
authors.forename: Yao Liang
authors.initials: YL
authors.lastname: Wong
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: David
authors.initials: D
authors.lastname: Jenkins
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Hyun J
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Mallory B
authors.initials: MB
authors.lastname: Martinez
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Davis
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Gahman
authors.affiliation: Laboratory of Chromosome Biology, Ludwig Institute for Cancer Research, La Jolla, CA, USA; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.
authors.forename: Arshad
authors.initials: A
authors.lastname: Desai
authors.affiliation: Small Molecule Discovery Program, Ludwig Institute for Cancer Research , La Jolla, CA , USA.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Shiau
keywords: AZD1152; Aurora kinase inhibitors; Hesperadin; MK-5108; MK-8745; MLN8054; MLN8237; ZM447439