Here we report discovery of highly

Here we report discovery of highly selective pan-Aurora kinase inhibitors through phenotypic screening. The co-crystal structure of Pazopanib Hydrochloride bound in the ATP binding site of Aurora A is described, and provides insight into the possible structural basis for the selectivity of the interaction. To identify anti-leukemic compounds, a library of pyrimido-benzodiazepinones was tested for anti-proliferative activity in Jurkat cells, a T-cell acute lymphocytic leukemia (T-ALL) cell line. We previously used this phenotypic screening and profiling approach to identify PI3K-δ/γ inhibitors. Compound was identified as a cytotoxic molecule with an IC of 370nM. Kinase profiling against a panel of 468 human wild-type and mutant kinases using KINOME® at 1μM revealed that compound is a highly selective pan-Aurora inhibitor with exclusively on-target activity detected across the panel at 1μM compound concentration (). The Aurora A, B and C activity was confirmed using biochemical kinase assays (C). The Aurora kinases do not contain cysteine residues proximal to their ATP binding sites, therefore we hypothesized that the interaction of compound with Aurora kinases is reversible, and that the acrylamide functionality could be removed without loss of potency. To confirm this we incubated compound at 10-fold excess, with recombinant purified Aurora A protein, and observed no labeling by LC-MS/MS analysis. Compound was synthesized and exhibited similar potency and selectivity to compound . Both compounds have moderate off-target activity on PLK4 (IC compound =43nM, IC compound =82nM) but otherwise display excellent kinome selectivity. The ABL1 Y253F activity of compound from the kinome screen was not reproduced in a biochemical kinase assay and is likely to be a false positive (IC>10,000nM). Inhibitors based on this scaffold have been reported to bind to LRRK2 and BRD4, proteins not covered in the selectivity panel. Compounds and were tested in biochemical assays against these targets and gratifyingly both molecules were inactive (). We have previously described pan-Aurora inhibitors based upon the same chemical scaffold (XMD12-1). However, compounds and have significantly improved kinome selectivity (), making them some of the most selective pan-Aurora kinase inhibitors reported to date. The cellular activity of compounds and were assessed by their ability to inhibit the phosphorylation of biomarkers of Aurora A and Aurora B by western blot in HCT116 cells. Compounds and were cell permeable and inhibited Aurora A auto-phosphorylation and Aurora B mediated histone H3 S10 phosphorylation at concentrations below 40nM (A, C, ). These compounds showed improved cellular inhibition of Aurora A auto-phosphorylation and histone H3 S10 phosphorylation compared to the control compound SAR156497. To investigate the effects of compounds and on the cell cycle, we performed FACS cell cycle analysis in HCT116 cells after 48h of 500nM compound treatment (B, ). Treatment with compounds , , or the control pan-Aurora inhibitor SAR156497 led to failed cytokinesis and accumulation of cells that contain 4N DNA (polyploidy), consistent with cellular Aurora B inhibition. To assess the cytotoxicity of lead compound , we treated a panel of human cancer cell lines in dose response, and assessed cell viability after 72h (, ). The HeLa cell line is highly sensitive to pan-Aurora inhibition, the IC of compounds , and control compound SAR156497 are below 10nM in this cell line. In HCT116, PC3 and HEK293 cells, compound is more potent than SAR156497, consistent with it’s improved cellular inhibition of the Aurora kinases signaling seen in A, C. When compound was tested in the cell line panel, it was highly toxic in HeLa cells. In other cells lines, cell viability loss plateaued at 50%, when tested at concentrations up to 33.3μM, indicating possible cell cycle arrest (). It is known that the time from mitotic arrest to apoptotic cell death is highly variable between cancer cell lines, which may account for some of these differences. Compound is more active against Aurora A (cellular), and PLK4 (biochemical) than compound , which may account for it’s additional toxicity in this assay. Furthermore, compound and SAR156497 may have additional uncharacterized off-target activity that contributes to their toxicity.