2386 Jadomycin B, an Aurora-B kinase inhibitor discovered through virtual screening Da-Hua Fu,1 Wei Jiang,1 Jian-Ting Zheng,2 Jadomycin B is a new Aurora-B kinase inhibitor worthy of Gui-Yu Zhao,1 Yan Li,1 Hong Yi,1 Zhuo-Rong Li,1 further investigation. [Mol Cancer Ther 2008;7(8):2386–93] Jian-Dong Jiang,1 Ke-Qian Yang,2 Yanchang Wang,3 and Shu-Yi Si1 Introduction In mammals, there are three Aurora kinases, Aurora-A, B, 1Institute of Medicinal Biotechnology, Peking Union Medical College & Chinese Academy of Medical Sciences, and 2Institute and C, which display >60% sequence identity (1). Although of Microbiology, Chinese Academy of Sciences, Beijing, China; the catalytic domains of the three kinases are highly and 3Department of Biomedical Sciences, College of Medicine, conserved, they show distinct subcellular localization and Florida State University, Tallahassee, Florida biological function (2, 3). Aurora-A, which is required for centrosome maturation and separation, localizes to centro- somes from early S phase to late M phase (4). Aurora-B is a Abstract component of chromosomal passenger complex, whose Aurora kinases have emerged as promising targets for function in mitosis has been extensively studied. In cancer therapy because of their critical role in mitosis. addition to Aurora-B, the chromosomal passenger complex These kinases are well-conserved in all eukaryotes, and contains Survivin, Borealin, and inner centromere protein IPL1 gene encodes the single Aurora kinase in budding (5–7). The chromosomal passenger complex associates yeast. In a virtual screening attempt, 22 compounds were with centromeric regions of chromosomes in the early identified from nearly 15,000 microbial natural products as stages of mitosis, but it translocates to microtubules after potential small-molecular inhibitors of human Aurora-B the onset of anaphase. When cells undergo cytokinesis, the kinase. One compound, Jadomycin B, inhibits the growth chromosomal passenger complex accumulates at the of ipl1-321 temperature-sensitive mutant more dramati- spindle midzone and finally concentrates at the midbody. cally than wild-type yeast cells, raising the possibility that As a serine/threonine kinase, Aurora-B phosphorylates this compound is an Aurora kinase inhibitor. Further histone H3 at Ser10, but the functional significance of this in vitro biochemical assay using purified recombinant modification remains uncertain (8). Recent data from both human Aurora-B kinase shows that Jadomycin B inhibits yeast and mammal suggest that Aurora-B kinase activity is Aurora-B activity in a dose-dependent manner. Our results necessary for correct microtubule-kinetochore attachments, also indicate that Jadomycin B competes with ATP for the chromosome alignment and segregation, as well as kinase domain, which is consistent with our docking cytokinesis (9). Aurora-C is highly expressed in testis along prediction. Like other Aurora kinase inhibitors, Jadomycin with other two human Aurora kinases and may play a role B blocks the phosphorylation of histone H3 on Ser10 in tumorigenesis, especially in the absence of p53 (10, 11). in vivo. We also present evidence suggesting that Recent data indicate that Aurora-C acts such as Aurora-B in Jadomycin B induces apoptosis in tumor cells without its localization during mitosis, and it is able to complement obvious effects on cell cycle. All the results indicate that Aurora-B kinase function (12). Accumulating evidence indicates that Aurora kinases are overexpressed in a wide range of tumor cells, including breast cancer (13, 14), colon cancer (15–17), pancreatic Received 1/14/08; revised 4/23/08; accepted 5/6/08. cancer (18), ovarian cancer (19), and gastric cancer (20). A Grant support: International Cooperation program "two bases" of National recent systematic analysis of Aurora kinase mRNA levels in Natural Science Foundation of China (grant no.30440420592 and 30670017). multiple primary tumors indicates that Aurora-A and B are The costs of publication of this article were defrayed in part by the significantly overexpressed (21). Because aberrant Aurora payment of page charges. This article must therefore be hereby marked kinases can lead to errors in chromosome alignment and advertisement in accordance with 18 U.S.C. Section 1734 solely to segregation, Aurora kinases are promising targets for indicate this fact. antitumor drugs. Moreover, Aurora kinases are only Note: D-H. Fu and W. Jiang equally contributed to this work. expressed during mitosis, thus the inhibition of Aurora Requests for reprints: Shu-Yi Si, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences/Peking Union Medical College, kinases will have no effect on quiescent cells, which makes Tiantan Xili #1, Beijing 100050, P.R. China. Phone: 8610-6318-0604; Aurora kinases more attractive targets for anticancer Fax: 8610-6318-0604. E-mail: [email protected] and Yanchang Wang, Department of Biomedical Sciences, College of Medicine, Florida therapy (22). State University, Tallahassee, FL 32306. Phone: 850-644-0402; Fax: An effective approach to inhibit kinases is the blockage of 850-644-5781. E-mail: [email protected] and Ke-Qian Yang, their interaction with substrates. Therefore, molecules that State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China. Phone: 8610- show similar structure to the kinase substrates may 6480-7457; Fax: 8610-6480-7459. E-mail: [email protected] function as competitive inhibitors. Three small-molecular Copyright C 2008 American Association for Cancer Research. inhibitors of Aurora kinases, ZM447439, Hesperadin, and doi:10.1158/1535-7163.MCT-08-0035 VX-680, have recently been described (23–25). All three Mol Cancer Ther 2008;7(8). August 2008 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Molecular Cancer Therapeutics 2387 molecules show antitumor activity in vitro and some into YPD (1% yeast extract, 2% peptone, and 2% glucose) in vivo, indicating the great potential of Aurora kinase medium containing tested compound at final concentration inhibitors as antitumor drugs (26, 27). We are interested in 10 Ag/mL. After incubation at 25jC for 24 h, the growth of identifying new Aurora kinase inhibitors from microbial the yeast cultures in the presence of tested compounds was natural products. The available crystal structure of Aurora- determined by measuring OD600. B kinase was used for virtual database screening (28). In vitro Aurora-B Kinase ActivityAssays Jadomycin B was found to be able to occupy the ATP To determine the inhibition of Aurora-B kinase activity binding pocket of Aurora-B kinase by forming hydrogen by Jadomycin B, the Aurora-B kinase activity assay kit bonds with amino acid residues around the pocket. The (Cell Signaling Technology)was used according to the subsequent biochemical and genetic analysis confirms that manufacturer’s instruction. Briefly, 100 ng purified recom- Jadomycin B is an Aurora-B inhibitor. First, a yeast mutant binant human Aurora-B kinase was added to a 100 AL allele that exhibits compromised Ipl1 (the yeast homologue reaction mixture containing 1Â kinase buffer and of Aurora-B kinase)showed more dramatic sensitivity to 25 Amol/L cold ATP in the presence of different À Jadomycin B. We also showed that Jadomycin B inhibited concentrations of Jadomycin B (ranging from 10 4 to À the kinase activity of Aurora-B in vivo and in vitro. Finally, 10 10 mol/L). After incubation at room temperature for we observed that Jadomycin B induced apoptosis at lower 15 min, biotinylated peptide substrate (Cell Signaling concentration without disturbing cell cycle progression. Technology)was added to each reaction mixture at a final Further investigations are necessary to explore the potential concentration of 1.5 Amol/L, and the mixtures were of Jadomycin B as an anticancer drug. further incubated for 30 min. A parallel control experi- ment was done under the same conditions without Jadomycin B. The reaction was stopped by addition of Materials and Methods 50 Amol/L EDTA (pH 8). Then, 25 AL reaction mixture Structure-Based Virtual Screening was transferred to a streptavidin-coated 96-well plate To identify potential inhibitors of Aurora-B kinase, the (PerkinElmer, Inc.)and incubated at room temperature for crystal structure of Aurora-B solved at 1.8-A˚ resolution was 60 min. After washing thrice with PBS/T, the phospho- retrieved from the Protein Data Bank (PDB ID code 2BFY). PLK (Ser10)antibody (Cell Signaling Technology)was The compound database used in our virtual screening is added to the plate for further incubation at 37jC for Microbial Natural Product Database developed by Institute 120 min. After washing, FITC-labeled secondary antibody of Medicinal Biotechnology, Chinese Academy of Medical (Santa Cruz)was added. After incubation at room Sciences, which contains the structural information of temperature for 30 min, the plate was finally washed five f15,000 microbial natural products. In this study, the times and the fluorescence signal was determined with ATP-binding pocket of Aurora-B was the target of interest BMG Polarstar Galaxy (Germany)at 535 nm. The and the active sites were defined as all atoms within a inhibition ratio by Jadomycin B at each concentration radius of 6.5 A˚ from Hesperadin cocrystallized in the ATP- was calculated according to the following equation: Â À binding pocket. A docking program FlexX encoded in %Inhibition = 100 (1 countstreated/countscontrol). The SYBYL7.1 (Tripos Inc)that uses an incremental construc- inhibition curve was then fitted by OriginPro7.5 program, tion algorithm was applied to optimize the interaction and the IC50 value of Jadomycin B was determined. between flexible ligands and the rigid binding sites. To To determine the mode of inhibition of Aurora-B by obtain an optimal starting conformation, all ligands were Jadomycin B, the kinase activity was also examined in the minimized using Tripos standard force field and Gasteiger- presence of different concentrations of ATP (25, 50, 75, 100, Hu¨ckel atomic partial charges with termination gradient and 200 Amol/L).