Jadomycin B, an Aurora-B Kinase Inhibitor Discovered Through Virtual Screening

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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

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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 experiment 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). The inhibition ratio of Aurora-B kinase assigned 0.42 J/(molÁnm)before docking. As for Aurora-B activity by Jadomycin B at various concentrations was protein, all crystal water molecules were removed from the calculated as described above, and the inhibition curve was original structure, hydrogen was added using Biopolymer fitted. The IC50 value of Jadomycin B at each ATP module in SYBYL, and standard AMBER atomic partial concentration was then determined and a linear fit was K charges were assigned. During soft docking simulations, made with all five IC50 values. The i was determined the free energy of binding was calculated mainly by the from the intercept of the plot of [ATP] versus IC50 values K K K sum of hydrogen bonds and hydrophobic interactions. The (IC50 = i/ m[ATP]+ i). sum of the lowest estimated free energy from various Cell Growth Assay binding conformations of each ligand was calculated and A549, HeLa, and MCF-7 cell lines were cultured in ranked by CScore function in SYBYL with default variables. 96-well tissue culture plates at a cell density of 5,000 cells One hundred fifty compounds with top docking score were per well in RPMI 1640 or MEM containing 10% fetus selected as candidates. Among them, 22 compounds were bovine serum and 2 mmol/L L-glutamine. After attach- obtained. ment overnight, the medium was replaced and cells were Determination of the Activity of Selected Com- incubated with increasing concentrations of Jadomycin B pounds Using BuddingYeast or its two derivatives, Jadomycin S and T (ranging from À À Wild-type and ipl1-321 mutant yeast strains were used 10 4 to 10 8 mol/L). After treatment for 48 h, 3-(4, for this purpose. Saturated yeast cells were 1/100 diluted 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

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Figure 1. Schematic diagrams of modeling of structure-based virtual screening. A, the structure of a subunit of Aurora-B cocrystallized with Hesperadin that is rendered by a ball-and-stick model. The structure is based on Protein Database Bank entry 2bfy. B, the predicted docking model of Jadomycin B to the ATP-binding pocket of Aurora-B. Jadomycin B is rendered by a stick model (red, oxygen atoms; white, carbon atoms; blue, nitrogen atoms; aqua, hydrogen atoms). The molecular surface of kinase domain is colored with electrostatic potentials: blue, negatively charged regions. C, specific hydrogen bonds (yellow broken lines) formed between Jadomycin B (stick model) and residues (violet ball-and-stick model) around the ATP-binding site. B and C was generated by SYBYL7.1. D, the structure of Jadomycin B.

assays were carried out in triplication. The concentration- the indicated times were fixed and stained with Propidium viability curves were fitted and IC50 values were Iodide. Fluorescence-activated cell sorting (FACS)analysis determined. Cells were also plated at a cell density of was done to determine the percentage of apoptotic cells 10,000 cells per well in 6-well tissue culture plates. After and cell cycle distribution by using the EPICS XL flow attachment overnight, cells were treated with 0, 5, or cytometer and System II software. 10 Ag/mL Jadomycin B for 96 h. Cells were detached Detection of Chromatin Condensation and Apoptotic every 24 h by trypsinization to count the cell number. All Body experiments were repeated thrice. Hochest 33342 is a fluorescence dye that can be Fluorescence-Activated Cell Sorting embedded in DNA double helix and emit blue fluorescence A549 cells in exponential growth phase were treated with when excited with UV light. Treated and untreated A549 0or5Ag/mL Jadomycin B for up to 48 h. Cells collected at cell samples with 5 Ag/mL Jadomycin B for 48 h were

Figure 2. The growth of ipl1-321 mutant cells is more sensitive to Jadomycin B than that of wild-type yeast cells. A, the ATP-binding pocket of Aurora kinase is well-conserved. Shadows, residues surrounding the ATP-binding site; panes, different residues between Ipl1 and Aurora-B. B, yeast cells were treated with 10 Ag/mL Jadomycin B and other compounds at 25jC for 24 h, and the OD600 of the cell cultures was then determined. Here, we show the percentage of the OD600 of treated yeast cells versus that of untreated cells. The results are the means and SDs from three separate experiments. *, P < 0.05. C, wild-type and ipl1-321 cells show different sensitivity to Jadomycin B. The cells were treated with Jadomycin B at final concentrations of 0.4, 2, 5, 20, and 100 Ag/mL, respectively. The OD600 was determined after incubation at 25jC for 24 h. The percentage of growth inhibition was determined as described in (B).

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Results Identification of Jadomycin B through Structure- Based Virtual Screening The crystal structure of Aurora-B shows that Aurora-B is composed of two homotypical subunits. Each subunit has two protein kinase domains, the NH2-terminal lobe (residues 86–174)rich in h-strands and COOH-terminal lobe (residues 175–347)that mainly consists of a a-helix. The NH2-terminal lobe offers the binding sites of nucleo- tides and kinase regulators, whereas the COOH-terminal lobe contains the active center where substrates are docked and catalytic reactions occur. The ATP-binding pocket that is composed of several spatially close residues (Lys122, Lys103, Glu171, Ala173, Leu99, Val107, and Glu177)lies at the interface between the two lobes. Figure 1A shows the crystal structure of Aurora-B with Hesperadin, a known Aurora-B–competitive inhibitor docked in the ATP-binding pocket. The indolinone moiety of Hesperadin occupies the catalytic cleft with the oxygen and nitrogen atoms of this moiety hydrogen bonded to Glu171 and Ala173 (28). Also, the sulfur and oxygen atoms of the sulfonamide moiety are hydrogen bonded to Lys122 and Lys103, occupying the position where the a-phosphate of ATP should be. The central phenyl ring of Hesperidin occupies the entry site to the catalytic cleft by van der Waals contact with other residues. Figure 3. The inhibition of human recombinant Aurora-B kinase by The protein structure was extracted from the 2bfy.pdb Jadomycin B. A, the inhibition of Aurora-B kinase by Jadomycin B at various file, and structure-based virtual screening was done. One concentrations in the presence of 25 Amol/L ATP. The experiment was carried out as described in Materials and Methods, and here, we show the hundred fifty compounds with top docking score were fitted inhibition curve and IC50 value. B, ATP-dependent IC50 values of selected as candidates. Among them, 22 were obtained, Jadomycin B. The IC50 of the inhibition of Aurora-B kinase by Jadomycin B in including Jadomycin B, a natural product (MW 563) the presence of various concentrations of ATP was determined as described Streptomyces venezuelae in Materials and Methods. A linear fit was made and the Ki was calculated. isolated from (Fig. 1D). The docking data showed that Jadomycin B could fit into the catalytic cleft of Aurora-B kinase and bind strongly to the residues stained with 100 ng/mL Hochest 33342 for 10 min in dark. surrounding the cleft (Fig. 1B). Elaborate docking indicates Stained cells were visualized with a Nikon ECLIPSS that Jadomycin B occupies the ATP-binding pocket of the TE2000-U fluorescence microscope (Japan)at Â100 and active center of Aurora-B with multiple interactions with Â400 magnification. Apoptotic cells show brightly stained the residues around the pocket: the two hydroxyls of nuclei because of the chromosome condensation and the L-digitoxose are hydrogen bonded to Glu171 and Ala173, appeared apoptotic bodies. whereas the two oxygen atoms of the oxazolone ring are Western Blotting hydrogen bonded to Lys122 and Lys103, respectively. At A549 cells in the exponential growth phase were the same time, the hydroxyl of the solvent-exposed angular treated with 0, 1.25, 2.5, 5, 7.5, or 10 Ag/mL Jadomycin phenyl ring in Jadomycin B binds to Glu177 with hydrogen B for 24 h, whereas HeLa and HepG2 cells were treated bond, occupying the entry site to the catalytic cleft in with 0 or 10 Ag/mL Jadomycin B. Cells were washed Aurora-B (Fig. 1C). with cold PBS and whole-cell extracts were prepared with The Growth of Yeast Cells Is inhibited by Jadomycin B radioimmunoprecipitation assay lysis buffer containing Aurora-B is a conserved protein kinase, and its homo- 60 Ag/mL phenylmethylsulfonyl fluoride. Equal amounts logue in budding yeast is Ipl1 (29). We reason that the of protein (30 Ag)were resolved with 15% SDS-PAGE. ATP-binding pocket should be conserved well between After transfer, the membranes were incubated over- these two kinases. As the crystal structure of Ipl1 is not night with primary antibodies (1:1,000)against phosphor- available, we compared the amino acid residues around ylated histone H3 (Ser10; Cell Signaling Technology)and the ATP binding site of Ipl1 kinase with that of human h-tubulin (Santa Cruz Biotechnology)at 4 jC. The mem- Aurora-B and found that most of the residues are identical branes were then incubated with horseradish peroxidase– except two residues that play minor roles in ATP binding conjugated goat anti-rabbit secondary antibody (Santa (Fig. 2A). On the basis of this similarity, we speculate that Cruz)at 1:200 followed by Chemiluminescence staining yeast Ipl1 kinase should also be sensitive to the identified (Millipore). Aurora-B inhibitors from the virtual screen.

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Figure 4. Jadomycin B inhibits the growth of several human cancer cell lines in a dose-dependent manner. A, A549, HeLa, and MCF-7 cells were exposed to increasing concentrations of Jadomycin B, Jadomycin S, and Jadomycin T for 48 h. The IC50 values were determined as described in Materials and Methods; columns, mean from three independent experiments; bars, SD. B, A549, HeLa, and MCF-7 cells were treated with different concentrations of Jadomycin B for 96 h. Cells were detached by trypsinization and counted every 24 h as described in Materials and Methods. JB, Jadomycin B.

BecauseIpl1kinaseisessentialforchromosome Jadomycin B Inhibits the Kinase Activity of Purified segregation, its inhibition will stop cell growth. ipl1-321 Recombinant Aurora-B is a temperature-sensitive mutant that grows well at As the budding yeast Ipl1 kinase is the homologue of 25jC but fails to grow at the restrictive temperature mammalian Aurora-B kinase, the result from yeast cells (37jC; ref. 30). Published data indicate that the Ipl1 suggests that Jadomycin B might inhibit the kinase activity kinase activity is compromised even when incubated at of mammalian Aurora-B. To test this possibility, we the permissive temperature 25jC (31). We reason that examined the inhibition of purified human Aurora-B ipl1-321 mutant cells should exhibit more dramatic kinase by Jadomycin B using ELISA (Enzyme-linked sensitivity to Aurora-B inhibitors. Therefore, we exam- ImmunoSorbent Assay). The kinase activity of Aurora-B, ined the growth of wild-type and ipl1-321 cells in the as indicated by fluorescence counts, was inhibited by presence of 10 Ag/mL of the 22 compounds, including micromolar concentration of Jadomycin B in a dose- A Jadomycin B. After 24 h of incubation, the presence of dependent fashion, with an IC50 value of 10.5 mol/L 10 Ag/mL Jadomycin B inhibited the growth of ipl1-321 (Fig. 3A). As controls, we also tested the inhibitory effect of cells almost completely compared with the untreated Jadomycin S and Jadomycin T on Aurora-B under the same control, whereas the growth of wild-type cells was condition, no inhibitory activity was detected (data not uninhibited. Other compounds did not show any toxicity shown). to yeast cells or exhibited similar toxicity to wild-type We next sought to determine the mode of Jadomycin and ipl1-321 mutant cells (Fig. 2B). We also tested the B-dependent inhibition of Aurora-B kinase. We speculate effect of two Jadomycin derivatives, Jadomycin S and that Jadomycin B inhibits Aurora-B by preventing the Jadomycin T, on the growth of ipl1-321 mutant cells, but access of ATP to the kinase domain based on the neither showed inhibitory effect at 10 Ag/mL (data not structure analysis. Therefore, the inhibition of the kinase shown). activity by Jadomycin B was analyzed in the presence of To further determine whether the toxicity of Jadomycin B ATP at different concentrations (25, 50, 75, 100, or 200 A is due to its inhibition of Ipl1 kinase, we compared the mol/L). The IC50 values with a given concentration of growth inhibition on wild-type and ipl1-321 mutant cells by ATP was determined by fitting the inhibition curve and Jadomycin B at different concentrations. At 5 Ag/mL, they were 12.46 Amol/L (25 Amol/L ATP), 16.60 Amol/L Jadomycin B inhibited 50% of the growth of ipl1-321 (50 Amol/L ATP), 24.50 Amol/L (75 Amol/L ATP), 32.16 mutant cells, but there is little growth inhibition for wild- Amol/L (100 Amol/L ATP), and 53.09 Amol/L (200 Amol/ type cells. In the presence of 100 Ag/mL of Jadomycin B, the L ATP; Fig. 3B). The titration experiments using 5 growth of wild-type cells was inhibited by 60% (Fig. 2C). different concentrations of ATP revealed a competitive The different sensitivity of wild-type and ipl1-321 cells inhibition of Aurora-B kinase by Jadomycin B with K indicates that Jadomycin B could be an unidentified respect to ATP, and the i of Aurora-B by Jadomycin B Aurora-B inhibitor. is 6.8 Amol/L.

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Jadomycin B Inhibits the Proliferation of Cancer Cells in a specific cell cycle stage was observed, indicating that It has been reported that Jadomycin B inhibits the Jadomycin B does not block cell cycle at this concentration proliferation of IM-9, IM-9/Bcl-2, HepG2, and H460 cells (Fig. 5B). Instead, we found increased cell population with (32). We examined the effects of Jadomycin B on the growth sub-G1 content of DNA (Fig. 5A), suggesting that apoptosis of other three cancer cell lines, A549, HeLa, and MCF-7. occurs. After 3 hours of incubation, there was almost no The growth curves indicated that all these cell lines were difference in the percentage of apoptotic cells between the sensitive to Jadomycin B and the growth inhibition is in a treated and untreated cells. After longer exposure to dose-dependent manner (Fig. 4B). The IC50 values Jadomycin B, however, the portion of apoptotic cells in of Jadomycin B and its two derivatives determined by treated samples increased to 17.8%, 44%, and 71.7% at 12, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro- 24, and 48 hours, respectively, whereas those of untreated mide assays against these cancer cell lines are shown in Fig. sample remained at <5% (Fig. 5A). Hochest 33342 staining 4A. Although A549 cells are most sensitive to Jadomycin B, confirmed that more and more cells underwent apoptosis the inhibitory potency did not differ significantly among after long-time exposure to Jadomycin B, as indicated by these cell lines. the appearance of brightly stained compressed chromo- Jadomycin B Induces Apoptosis without Blocking somes (Fig. 5C). Meanwhile, the apoptotic body could be Cell Cycle detected at a Â400 magnification under a fluorescence FACS analysis was used to examine the mechanism of microscope (Fig. 5C). cell growth inhibition by Jadomycin B. Jadomycin B was Jadomycin B Inhibits Histone H3 Phosphorylation added to A549 cell cultures in the exponential growth It has been shown that Aurora-B kinase phosphorylates phase to a final concentration of 5 Ag/mL. Treated and histone H3 on serine 10. We reason that this phosphory- untreated cell samples were taken at 3, 12, 24, and 48 hours lation should be impaired in the presence of Jadomycin B if and fixed for FACS analysis. Although treatment with it inhibits the activity of Aurora-B kinase. To test this idea, 5 Ag/mL Jadomycin B induced an increase of cells in S we examined the phosphorylation of histone H3 in A549 phase by 2%, the difference between treated and untreated cells after 24 hours of treatment with Jadomycin B cells was so negligible that no obvious accumulation of cells at different concentrations. In untreated cells, a clear

Figure 5. Jadomycin B induces apoptosis in A549 cells without cell cycle disturbance. A, A549 cells treated with 0 or 5 Ag/mL Jadomycin B were collected at indicated times and fixed for FACS analysis as described in Materials and Methods. Jadomycin B (5 Ag/mL) induces apoptosis in A549 cells. Dotted line, the portion of apoptotic cells. B, A549 cells treated with 0 or 5 Ag/mL Jadomycin B were harvested at indicated times. FACS analysis was done. The percentage of viable population in each phase of the cell cycle was calculated; points, mean from three independent experiments; bars, SD. C, nuclear staining with Hochest 33342. The cells were stained with Hochest 33342 and examined by fluorescence microscopy. The nuclei of apoptotic cells shrank, turned round, and were stained brightly. Apoptotic bodies can be detected with a Â400 magnification.

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Discussion Recent studies have showed that Aurora kinases are implicated in tumorigenesis, therefore they are thought to be promising targets for anticancer drug development (33, 34). Although Aurora-A and B are always coexpressed, Aurora-B is believed to be the primary target on the basis of the observation that inhibition of this kinase results in a catastrophic mitosis (35, 36). Because the Aurora kinases are highly conserved, and the function of Aurora-B in mammals is similar with that of Ipl1 in budding yeast (36), it is reasonable to use yeast cells to evaluate the bioactivity of the compounds obtained. In this study, the compound that showed inhibitory effect on ipl1-321 mutant was subsequently proved to be able to inhibit the purified Aurora-B kinase. This testified the functional parallelism and credibility of yeast cells in screening Aurora-B inhibitors. Recently, budding yeast has been used as a model system in our laboratory to investigate the antitumor mechanism of DH334, a h-carboline derivative. The data from yeast indicate that DH334 inhibits the activity of cyclin-dependent kinase (37). Because of the availability of the powerful genetic and biochemical tools and the short doubling time, yeast cells can be used for extensive screen and mechanistic studies for anticancer drugs. In this study, we used computational screening to identify small molecules that inhibit Aurora-B kinase from 15,000 microbial natural products. Among the 22 candidate compounds tested, Jadomycin B exhibited specific inhibition of Aurora-B kinase on the basis of the following observations. First, Jadomycin B is more toxic to ipl1-321 Figure 6. Jadomycin B inhibits Aurora-B – dependent H3 phosphorylation in a dose-dependent manner. A, Jadomycin B inhibits H3 phosphor- mutant cells, in which the kinase activity of Ipl1 (Aurora-B ylation in A549 cells. Jadomycin B was added into the cell cultures to homologue in budding yeast)is compromised. Second, the different concentrations. After incubation for 24 h, cells were harvested kinase activity of recombinant human Aurora-B kinase is and 30 Ag protein from each sample was subjected to SDS-PAGE and in vitro Western blot analysis with the anti – phospho-H3 (Ser10) antibody. B, H3 inhibited by Jadomycin B . Finally, we showed that phosphorylation in HeLa and HepG2 cells treated with Jadomycin B. Cells Aurora-B–dependent phosphorylation of histone H3 at were cultured in the absence and presence of 10 Ag/mL Jadomycin B for serine 10 decreased in the presence of Jadomycin B. 24 h. The cells were then harvested for protein preparation. Thirty micrograms of protein were subjected to SDS-PAGE and Western blot Therefore, we conclude that Jadomycin B is a new analysis with anti – phospho-H3 antibody. C, quantitative analysis of the Aurora-B inhibitor. inhibition of H3 phosphorylation in A549 cells by Jadomycin B. H3 Several Aurora kinase inhibitors have been identified, phosphorylation in the presence of different concentrations of Jadomycin including Hesperadin, ZM447439, and VX-680 (23–25). All B was measured by grayscale scanning after Western blotting. The percentage of phospho-H3 (P-H3) phosphorylation was determined by these compounds inhibit Aurora-B kinase–dependent H3 using untreated cells as control; columns, mean (n = 3); bars, SD. phosphorylation and prevent cell division. Moreover, VX- 680 induces apoptosis in several tumor cell lines. Here, we showed that Jadomycin B also induced apoptosis at A phospho-H3 band was observed after Western blot analysis 5 g/mL, but no obvious cell cycle disturbance was with anti–phospho-H3 antibody. In contrast, A549 cells did observed. For instance, FACS analysis did not show not express any detectable phosphorylated histone H3 after polyploidy cells after treatment with 5 Ag/mL Jadomycin treatment with 10 Ag/mLJadomycinB(Fig.6A).A B. One possibility is that Jadomycin B inhibits other kinases significant decrease in the levels of phosphorylated histone more efficiently and results in apoptosis. Another possible H3 upon treatment with Jadomycin B was also observed in explanation is that Jadomycin B does not compromise the HeLa and HepG2 cells (Fig. 6B). Further quantitative spindle checkpoint function as other inhibitors do. There- analysis showed that Jadomycin B inhibits histone H3 fore, more work needs to be done to determine the phosphorylation in a dose-dependent manner. Treatment specificity of Jadomycin B against other kinases, including with 2 or 10 Ag/mL Jadomycin B reduced H3 phosphor- Aurora-A, Aurora-C, PLK et al. In addition to Jadomycin ylation by 28% and 73%, respectively (Fig. 6C). Collectively, B, Jadomycin S and T also showed inhibitory effects these data show that Jadomycin B inhibits the kinase on proliferation of IM-9, IM-9/Bcl-2, HepG2, and H460 activity of Aurora-B. cells (32). In this study, we found that Jadomycin S and

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T inhibited the growth of A549, HeLa, and MCF-7 cells, 16. Katayama H, Ota T, Jisaki F, et al. Mitotic kinase expression and colorectal cancer progression. J Natl Cancer Inst 1999;91:1160 – 2. although the inhibition was not as dramatic as Jadomycin 17. Takahashi T, Futamura M, Yoshimi N, et al. Centrosomal kinases, B. Unlike Jadomycin B, neither the kinase activity of HsAIRK1 and HsAIRK3, are overexpressed in primary colorectal cancers. Aurora-B, nor the growth of ipl1-321 cells, was inhibited Jpn J Cancer Res 2000;91:1007 – 14. by Jadomycin S and T. The antitumor effect of Jadomycin S 18. Li D, Zhu J, Firozi PF, et al. Overexpression of oncogenic STK15/ BTAK/Aurora A kinase in human pancreatic cancer. Clin Cancer Res 2003; and T is probably caused by the inhibition of other targets 9:991 – 7. other than Aurora-B. In summary, we identified a new 19. Gritsko TM, Coppola D, Paciga JE, et al. 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Mol Cancer Ther 2008;7(8). August 2008

Da-Hua Fu, Wei Jiang, Jian-Ting Zheng, et al.

Mol Cancer Ther 2008;7:2386-2393.

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