DOI: 10.1002/cmdc.201402284
Full Papers
Identification of Green Tea Catechins as Potent Inhibitors of the Polo-Box Domain of Polo-Like kinase 1 Hong-Mei Shan,[a] Yanxia Shi,[a] and Junmin Quan*[a, b]
Polo-like kinase 1 (PLK1) plays crucial functions in multiple stages of mitosis and is considered to be a potential drug target for cancer therapy. The functions of PLK1 are mediated by its N-terminal kinase domain and C-terminal polo-box domain (PBD). Most inhibitors targeting the kinase domain of PLK1 have a selectivity issue because of a high degree of structural conservation within kinase domains of all protein kinases. Here, we combined virtual and experimental screenings to identify green tea catechins as potent inhibitors of the PLK1 PBD. Initially, ( )-epigallocatechin, one of the main components of green tea polyphenols, was found to significantly
block the binding of fluorescein-labeled phosphopeptide to the PBD at a concentration of 10 mm. Next, additional catechins were evaluated for their dose-dependent inhibition of the PBD and preliminary structure–activity relationships were derived. Cellular analysis further showed that catechins interfere with the proper subcellular localization of PLK1, lead to cell-cycle arrest in the S and G2M phases, and induce growth inhibition of several human cancer cell types, such as breast adenocarcinoma (MCF7), lung adenocarcinoma (A549), and cervical adenocarcinoma (HeLa). Our data provides new insight into understanding the anticancer activities of green tea catechins.
Introduction Polo-like kinase 1 (PLK1) is a master regulator of mitosis, involved in multiple stages of mitotic progression including mitotic entry, centrosome maturation, spindle assembly, chromatin segregation, mitotic exit and cytokinesis.[1] Overexpression of PLK1 is observed in many cancers and is often correlated with poor prognosis.[2] Furthermore, interference with PLK1 functions by small-molecule inhibitors[3–7] or small interfering RNAs[8] causes decreased proliferation in a broad spectrum of cancer cell lines and tumor xenografts but not in normal cells,[9] suggesting that PLK1 is an attractive drug target for cancer therapy.[10] More recently, the novel PLK1 inhibitor volasertib[11] was awarded “Breakthrough Therapy” designation by the US Food and Drug Administration (FDA) for its promising performance in the treatment of acute myeloid leukemia (AML), which further supports the potential of PLK1 inhibitors in the treatment of cancers. On the other hand, PLK2 and PLK3 have been shown to play roles in checkpoint-mediated cellcycle arrest to prevent genetic instability and oncogenic transformation,[12, 13] development of PLK1-specific inhibitors is therefore essential for cancer therapy to avoid potential offtarget effects.
PLK1 is characterized by an N-terminal kinase domain (KD) and a C-terminal polo-box domain (PBD). The KD is similar to the kinase domains of other protein kinases,[14, 15] while the PBD is unique to the PLKs family. Most PLK1 inhibitors targeting the KD also significantly inhibit the other two PLK family members PLK2 and PLK3 because of a high degree of structural conservation within the KDs of the PLK family members.[16] Alternatively, the PBD that mediates the subcellular localization[17] of PLKs is relatively divergent in structure, and thus represents an attractive target for discovering PLK1-specific inhibitors by selectively blocking the protein–protein interaction between the PBD and its partners.[18, 19] Since the discovery of the PBD as a phosphopeptide-binding module,[20] various phosphopeptide ligands have been reported to selectively bind the PBD of PLK1.[21–26] All these peptides are characterized by a conserved phosphorylated SpT motif that serves as the anchor to tightly bind with the pincer residues His 538 and Lys 540 of the PBD,[27] but the negatively charged phosphate group leads to the low cellular efficacy of these phosphopeptide ligands,[22, 26] highlighting the need to discover small-molecule PBD inhibitors with higher cellular permeability. In contrast to the discovery of various phosphopeptide ligands, only a few small-molecule PBD inhibitors have been identified through high-throughput screening (HTS) assays over the past several years.[28–30] These small-molecule PBD inhibitors interfere with the subcellular localization of PLK1 and induce cancer cell mitotic arrest, but the modest selectivity and drug-likeness of these compounds highlight the unmet need for the discovery of novel PBD inhibitors with diverse chemical scaffolds.
[a] H.-M. Shan, Dr. Y. Shi, Prof. J. Quan Key Laboratory of Chemical Genomics School of Chemical Biology & Biotechnology Peking University, Shenzhen Graduate School Shenzhen 518055 (China) E-mail: [email protected] [b] Prof. J. Quan Key Laboratory of Structural Biology School of Chemical Biology & Biotechnology Peking University, Shenzhen Graduate School Shenzhen 518055 (China)
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Full Papers In the present study, we combined virtual screening and fluorescence polarization screening assays to identify green tea catechins as potent inhibitors of the PLK1 PBD. The catechins interfere with the proper subcellular localization of PLK1, lead to cell-cycle arrest in the S and G2M phases, and induce growth inhibition of several human cancer cell types such as breast adenocarcinoma (MCF7), lung adenocarcinoma (A549), and cervical adenocarcinoma (HeLa).
Structure–activity relationships of green tea catechins as PBD inhibitors To understand the interaction between EGC and the PBD, we further determined the dose-dependent inhibition for other major green tea catechins including ( )-epigallocatechin gallate (EGCG), (+)-gallocatechin (GA), and (+)-catechin (CAT) (Figure 1).[34] Compared with EGC, EGCG has higher inhibitory
Results and Discussion Combined virtual and experimental screenings The efficiency of HTS is generally limited by the scaffold diversity of the screened chemical library,[31] which could be overcome by virtual screening of large chemical databases. We performed receptor-based virtual screening to identify potential binding hits for the PLK1 PBD based on a mixed chemical database with five million molecules.[32] As shown in Scheme 1, through virtual screening and further molecular dynamics simulation optimization, we finally selected fifty candidates for experimental test by using fluorescence polarization assay. Among these candidates, we found that ( )-epigallocatechin (EGC) (Figure 1), one of the main components of green tea polyphenols,[33] significantly blocks the binding of fluoresceinlabeled phosphopeptide to the PBD at a concentration of 10 mm.
Figure 1. Structures of green tea catechins.
activity, indicating that the additional gallate group contributes to the binding with the PBD (Figure 2). The relatively lower inhibitory activity for GA suggests that the configuration of 3-hydroxy group plays a role in the binding of catechins with the PBD. More strikingly, CAT has a much lower inhibitory activity compared with EGC, which indicated that the hydroxy groups on the B ring are essential for the inhibition of PBD-dependent recognition. To characterize the molecular mechanism underlying the SARs of catechins, all catechins were docked into the substrate binding pocket of the PBD (Figure 3). The docked structures indicated that all the catechins bind with the PBD in a similar mode— the hydroxy groups on the B ring form hydrogen bonds with the important pincer residues His 538/ Lys 540 and the backbone carbonyl oxygen of Leu 491 directly or mediated by a water molecule. Compared with EGC, CAT is predicted to form one less hydrogen bond with the backbone carbonyl oxygen of Leu 491 and may have weaker binding affinity with the PBD. In addition, the unfavorable contact between the polar 3-hydroxy group and the hydrophobic side chain of Leu 490 may further weaken the binding between CAT and the PBD. Such unfavorable contact might also be the reason that GA has weaker binding affinity with the PBD compared with EGC. On the other hand, the gallate group of EGCG is Scheme 1. Flow diagram for the discovery of polo-box domain (PBD) inhibitors through predicted to form an additional hydrogen bond with combined virtual and experimental screenings. ChemMedChem 2015, 10, 158 – 163
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Full Papers pressors.[12, 13] The selectivity of the inhibitors against different PLK family members is therefore very important to the drug discovery for cancer therapy.[7, 9] As EGCG has been well studied and shown to modulate multiple signal transduction pathways,[36, 37] we thus focused on evaluating in vitro and cellular activities for EGC in the following testing. Using a fluorescence polarization assay, we examined the inhibition of EGC on the binding of phosphopeptides with the PBDs from PLK1, PLK2, and PLK3. As shown in Figure 4, EGC most efficiently blocked the binding of phosphopeptide to the PLK1 PBD, and also blocked binding to the PLK2 PBD but with about five-fold weaker potency. EGC did not significantly inhibit the binding of phosphopeptide to the PBD of PLK3 at the concentration of 100 mm, indicating that it is a selective inhibitor of the PLK1 PBD. Figure 2. Inhibition of FITC-phosphopeptide binding to the polo-box domain (PBD) by green tea catechins: EGC (&), EGCG (*), GA (~), and CAT (!). Error bars represent the standard deviation (SD). FITC, fluorescein isothiocyanate.
Subcellular localization disruption and cell-cycle arrest
The PBD plays important roles in the subcellular localization and functions of PLK1, the inhibitors of the PBD are expected to interfere with the subcellular localization of PLK1 and cell-cycle progression.[17, 20] As expected, PLK1 mainly located to centrosomes and kinetochores in metaphase of control cells, while the localization of PLK1 to centrosomes and kinetochores was significantly decreased in the metaphase of EGCtreated cells (Figure 5 a). Furthermore, we also observed chromosome congression defects due to the mislocalization of endogenous PLK1 in EGC-treated cells (Figure 5 b). Most chromosomes congressed to the metaphase plate, but some chromosomes were unaligned and dispersed to the spindle poles. In line with the functions of PLK1 in mitosis, fluorescence-activated cell sorting (FACS) showed EGC induced a dose-dependent increase of cells in the G2M phase and decrease of cells in the G0/G1 phase (Figure 6 a). Interestingly, we observed a significant increase of cells in the S phase for EGC, which is consistent with the recent reports that PLK1 also plays important roles outside of mitosis,[38] but we didn’t exclude the possibility of EGC interacting with other Sphase targets. Furthermore, cell cycle arrests in the S and G2M phases also manifested by an increase in Figure 3. Predicted binding modes of catechins on the substrate binding groove of the the concentration of PLK1 in EGC-treated cells (Figpolo-box domain (PBD) determined by docking. Only key residues involved in interactions are displayed, and hydrogen bonds are shown by dashed blue lines. ure 6 b,c). Anticancer activity
the backbone amide of Leu 490 mediated by a water molecule and also has p–p stacking with His 538, which partially explains its higher binding affinity with the PBD.
To evaluate the anticancer activity of the PBD inhibitor EGC, we employed a sulforhodamine B (SRB) colourimetric assay[39] to test the inhibitory effect of EGC on the growth of human cancer cell lines that overexpress PLK1, including HeLa, A549, and MCF-7. The results indicated that EGC induced dose-dependent inhibition of the growth of these three tested cancer cell lines (Figure 7). In particular, the human breast cancer cell line MCF-7 was found to be the most sensitive to EGC treatment (GI50 ~ 50 mm). In contrast, the growth of the normal
Selectivity of EGC against PLK family members PLK family members PLK1, PLK2, and PLK3 share similar structures, but have distinct functions in the cell-cycle progression and development of tumor cells.[15, 35] PLK2 and PLK3 act as regulators to prevent DNA damage and serve as tumor supChemMedChem 2015, 10, 158 – 163
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Full Papers indicates that the PLK1 PBD might be one of the potential targets of green tea catechins including EGCG, EGC, and GA.
Conclusions We identified green tea catechins including EGCG, EGC, and GA as potent inhibitors of the PBD of PLK1 by combined virtual and experimental screenings. Docking study rationalized the SAR of green tea catechins against the PBD of PLK1. Results of a fluorescence polarization assay indicated that EGC is more selective against the PBD of PLK1 than those of PLK2 and PLK3. Further cellular analysis showed that EGC interfered with the proper subcellular localization of PLK1, led to the cell-cycle arrest in the S and G2M phases, and induced growth inhibition of several cancer cell types such as MCF7, A549, and HeLa. This work provides new insights into understanding the anticancer activities of green tea catechins and designing new inhibitors against the PLK1 PBD based on the catechin scaffold.
Figure 4. Effect of EGC on the binding of FITC-phosphopeptide binding to the polo-box domain (PBD) from PLK1 (&), PLK2 (*), and PLK3 (~), determined by fluorescence polarization. Error bars represent the standard deviation (SD). FITC, fluorescein isothiocyanate.
Experimental Section Virtual screening
Figure 5. a) HeLa cells treated with or without EGC (50 mm; 24 h) were fixed for immunofluorescence assay. EGC disrupts the subcellular localization of PLK1 (green) to centrosomes (g-tubulin; red) and kinetochores in metaphase. b) EGC induces chromosome (DNA; blue) congression defects in HeLa cells.
The mixed database was constructed by compounds selected from various chemical libraries including: NIH, Zinc, Maybridge and Drugbank.[40, 41] AutoDock version 3.0 was used for the docking simulation and the Lamarckian genetic algorithm (LGA) for ligand conformational searching.[42] The PDB file was prepared using published coordinates (PDB: 1UMW).[20] The ligand’s translation, rotation, and internal torsions are defined as its state variables, and each gene represents a state variable. The ligand file was setup for docking using the ADT (AutoDockTools) package, including assigning Gasteiger atomic charges, defining rigid root and rotatable bonds. The grid maps were centered on the phosphopeptide binding site, with 60 60 60 grid-points of 0.375 spacing. The genetic algorithm was used with standard parameters: trials of 50 dockings, a population size of 50 individuals; a maximum number of 1.5 106 energy evaluations and a maximum number of 27 000 generations; an elitism value of 1; a mutation rate of 0.02 and a crossover rate of 0.80. In the analysis of the docked conformations, the clustering tolerance for the root-mean-square positional deviation was set to 0.5 . The top 250 compounds with the best simulated binding energies within the standard deviation of 2.1 kcal mol 1 were selected manually. The illustrated structures were generated by using PyMOL.[43]
Plasmid construction and protein expression DNA sequences coding for the PBD domain of human PLK1 326–603, human PLK2 355–685, and human PLK3 335–646 were amplified by polymerase chain reaction (PCR) and cloned into the modified GST-His6 expression vector pGEX-4T-1. Proteins were expressed from BL21DE3plys and purified as previously described.[44] BL21DE3plys cells were transformed and grown to an OD600 value of 0.6 at 37 8C. The cells were cooled to 20 8C, and expression was induced with 0.1 mm isopropyl b-d-1-thiogalactopyranoside (IPTG) for 12 h. Cells were harvested and suspended in lysis
human embryonic kidney (HEK293) cells was not significantly affected by EGC even at the concentration of 100 mm, indicating that the PBD inhibitor EGC is selectively effective against the growth of cancer cells rather than normal cells. Considerable evidences have demonstrated that green tea catechin EGCG is a potent anticancer agent that targets multiple signal transduction pathways, but the anticancer activities and targets for other green tea catechins are less studied. This work ChemMedChem 2015, 10, 158 – 163
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Full Papers Soluble protein was added to the Talon@ Metal Affinity Resins. The resin was wash four times, and the polyhistidine-tagged protein was eluted with 200 mm imidazole.
Fluorescence polarization assay Binding experiments were performed using an Envision multilabel reader (PerkinElmer). Competition assays were performed using the following fluorophore-labeled peptide: 5 nm FITCGPMQSpTPLNG for PLK1; 5 nm FITC-GPMQTSpTPKNG for PLK2; 5 nm FITC-GPLATSpTPKNG for PLK3.[45, 46] 100 nm PBD protein, and the test compounds at concentration up to 200 mm. The compound (+)-gallocatechin (GA), ( )-epigallocatechin gallate (EGCG), ( )-epigallocatechin (EGC) and (+)-catechin (CAT) were purchased from Sigma. All FITC-peptides were synthesized (GL Biochem Shanghai Ltd) and checked by mass spectrometry. All experiments were performed in 96-well black plates at 25 8C. Plates were read 1 h after mixing of all assay components.
Sulforhodamine B assays Cells were seeded into 96-well plates containing 5 % fetal bovine serum and maintained in a humidified incubator at 37 8C in 5 % CO2. The cells were treated with increasing concentrations of EGC for 72 h. The assay was terminated by addition of cold trichloroacetic acid. After three-time washout with deionized water, sulforhodamine B solution at 0.4 % (w/v) in 1 % acetic acid was added to each well, and plates were incubated for 10 min at room temperature. The unbound dye was removed by three-time washing with 1 % acetic acid, and the plates were air dried. Bound sulforhodamine B was subsequently solubilized with 10 mm of trizma base (pH 10.5), and the absorbance was read at a wavelength of 515 nm.
Figure 6. a) HeLa cells were synchronized by thymidine block and released into medium containing EGC at the indicated concentration. EGC induces cell cycle arrests at S (&) and G2M (&) phases but not in the G0/G1 (&). Cell-cycle progression was monitored by fluorescence-activated cell sorter analysis. b) Western blot analysis of cell lysates treated with different concentration of EGC for 24 h. EGC induces an increase in the concentration of Polo-like kinase 1 (PLK1) in HeLa cells. c) The relative protein level of PLK1 was quantified and standardized to GAPDH expression by the use of Gel-Pro Analyzer (version 4.0) software.
Western blotting and immunofluorescence assay HeLa cells were synchronized by G1/S boundary by a double-thymidine treatment and then released into thymidine-free medium containing the indicated concentration of EGC for 24 h. For Western blot analysis, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mm Tris pH 8.0, 150 mm NaCl, 1 % NP-40, 0.25 % sodium deoxycholate, sodium orthovanadate, sodium fluoride, EDTA, 0.1 % sodium dodecyl sulfate, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and protease inhibitor). Rabbit anti-PLK1 polyclonal antibody (1:1000, Abcam) and rabbit polyclonal antibody against GAPDH (1:1000, cell signaling) were used as primary antibody. For immunofluorescence assay, HeLa cells were grown on coverslips for 24 h, then were fixed and permeabilized in methanol for 15 min at 20 8C. Cells were washed in Tris-buffered saline (TBS) buffer (20 mm Tris 200 mm NaCl pH 7.6) and incubated for 60 min in blocking solution (TBS with 0.1 %Triton X-100, 5 % bovine serum albumin) for 60 min. Primary antibodies used were rabbit anti-PLK1 polyclonal antibody (1:1000, Abcam), mouse anti-g-tubulin (1:500, Santa Cruz Biotechnology, Inc.), rabbit anti-a-tubulin (1:500, Cell Signaling). Images were taken with an AxioImager A1 microscope and AxioCam digital camera (Zeiss, Oberkochen, Germany).
Figure 7. Dose-dependent inhibition of the growth of human cancer cells by EGC. Cells were cultured in the presence of increasing concentration of EGC for 72 h and analyzed by sulforhodamine B assays.
Cell-cycle analysis
buffer composed of 20 mm HEPES pH 7.4, 50 mm NaCl, 0.1 % NP40, 1 mg mL 1 lysozyme, 1 mm MgCl2 and DNase I. Lysates were cleared by high-speed centrifugation at 4 8C, 35 000 g for 40 min.
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HeLa cells were harvested and washed twice with cold phosphatebuffered saline. After centrifugation at 4 8C, 1000 g for 5 min,
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Full Papers washed cells were resuspended and fixed in chilled 70 % ethanol at 4 8C for 30 min. The cells were stained with 100 mg mL 1 propidium iodide and treated with 1 mg mL 1 of RNase-A (Sigma) for 30 min at 37 8C. The DNA content of 10 000 cells was determined with a FACScan flow cytometer (BD Biosciences). The data were analyzed with Modfit LT2.0.
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Acknowledgements This work was financially supported by the Chinese Ministry of Science and Technology (2012CB722602), the National Natural Science Foundation of China (NSFC21290183, NSFC81373326), and the Shenzhen Municipal Government (China) (JCYJ20130331144947526 and ZDSY20130331145112855). Keywords: cancer · catechins · inhibitors · natural products · polo-box domain · polo-like kinase 1 · screening [1] M. Petronczki, P. Lenart, J. M. Peters, Dev. Cell 2008, 14, 646 – 659. [2] K. Strebhardt, A. Ullrich, Nat. Rev. Cancer 2006, 6, 321 – 330. [3] Y. Liu, K. R. Shreder, W. Gai, S. Corral, D. K. Ferris, J. S. Rosenblum, Chem. Biol. 2005, 12, 99 – 107. [4] M. Steegmaier, M. Hoffmann, A. Baum, P. Lenart, M. Petronczki, M. Krssak, U. Gurtler, P. Garin-Chesa, S. Lieb, J. Quant, M. Grauert, G. R. Adolf, N. Kraut, J. M. Peters, W. J. Rettig, Curr. Biol. 2007, 17, 316 – 322. [5] A. Jimeno, J. Li, W. A. Messersmith, D. Laheru, M. A. Rudek, M. Maniar, M. Hidalgo, S. D. Baker, R. C. Donehower, J. Clin. Oncol. 2008, 26, 5504 – 5510. [6] S. Keppner, E. Proschak, G. Schneider, B. Spnkuch, ChemMedChem 2009, 4, 1806 – 1809. [7] a) S. Keppner, E. Proschak, M. Kaufmann, K. Strebhardt, G. Schneider, B. Spnkuch, Cell Cycle 2010, 9, 761 – 773; b) S. Keppner, E. Proschak, G. Schneider, B. Spnkuch, Cell Cycle 2011, 10, 708 – 720. [8] B. Spnkuch-Schmitt, J. Bereiter-Hahn, M. Kaufmann, K. Strebhardt, J. Natl. Cancer Inst. 2002, 94, 1863 – 1877. [9] a) X. Liu, M. Lei, R. L. Erikson, Mol. Cell. Biol. 2006, 26, 2093 – 2108; b) M. Raab et al., Nat. Commun. 2011, 2, 395. [10] K. C. Goh, H. Wang, N. Yu, Y. Zhou, Y. Zheng, Z. Lim, K. Sangthongpitag, L. Fang, M. Du, X. Wang, A. B. Jefferson, J. Rose, B. Shamoon, C. Reinhard, B. Carte, M. Entzeroth, B. Ni, M. L. Taylor, W. Stnkel, Drug Develop. Res. 2004, 62, 349 – 361. [11] D. Rudolph, M. Steegmaier, M. Hoffmann, M. Grauert, A. Baum, J. Quant, C. Haslinger, P. Garin-Chesa, G. R. Adolf, Clin. Cancer Res. 2009, 15, 3094 – 3102. [12] Y. Yang, J. Bai, R. Shen, S. A. Brown, E. Komissarova, Y. Huang, N. Jiang, G. F. Alberts, M. Costa, L. Lu, J. A. Winkles, W. Dai, Cancer Res. 2008, 68, 4077 – 4085. [13] N. Syed, P. Smith, A. Sullivan, L. C. Spender, M. Dyer, L. Karran, J. O’Nions, M. Allday, I. Hoffmann, D. Crawford, B. Griffin, P. J. Farrell, T. Crook, Blood 2006, 107, 250 – 256. [14] M. Kothe, D. Kohls, S. Low, R. Coli, A. C. Cheng, S. L. Jacques, T. L. Johnson, C. Lewis, C. Loh, J. Nonomiya, A. L. Sheils, K. A. Verdries, T. A. Wynn, C. Kuhn, Y. H. Ding, Biochemistry 2007, 46, 5960 – 5971. [15] K. Strebhardt, Nat. Rev. Drug Discov. 2010, 9, 643 – 660. [16] R. N. Murugan, J. E. Park, E. H. Kim, S. Y. Shin, C. Cheong, K. S. Lee, J. K. Bang, Mol. Cells 2011, 32, 209 – 220. [17] Y. J. Jang, C. Y. Lin, S. Ma, R. L. Erikson, Proc. Natl. Acad. Sci. USA 2002, 99, 1984 – 1989.
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Received: July 9, 2014 Published online on September 5, 2014
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Full Papers
Identification of Green Tea Catechins as Potent Inhibitors of the Polo-Box Domain of Polo-Like kinase 1 Hong-Mei Shan,[a] Yanxia Shi,[a] and Junmin Quan*[a, b]
Polo-like kinase 1 (PLK1) plays crucial functions in multiple stages of mitosis and is considered to be a potential drug target for cancer therapy. The functions of PLK1 are mediated by its N-terminal kinase domain and C-terminal polo-box domain (PBD). Most inhibitors targeting the kinase domain of PLK1 have a selectivity issue because of a high degree of structural conservation within kinase domains of all protein kinases. Here, we combined virtual and experimental screenings to identify green tea catechins as potent inhibitors of the PLK1 PBD. Initially, ( )-epigallocatechin, one of the main components of green tea polyphenols, was found to significantly
block the binding of fluorescein-labeled phosphopeptide to the PBD at a concentration of 10 mm. Next, additional catechins were evaluated for their dose-dependent inhibition of the PBD and preliminary structure–activity relationships were derived. Cellular analysis further showed that catechins interfere with the proper subcellular localization of PLK1, lead to cell-cycle arrest in the S and G2M phases, and induce growth inhibition of several human cancer cell types, such as breast adenocarcinoma (MCF7), lung adenocarcinoma (A549), and cervical adenocarcinoma (HeLa). Our data provides new insight into understanding the anticancer activities of green tea catechins.
Introduction Polo-like kinase 1 (PLK1) is a master regulator of mitosis, involved in multiple stages of mitotic progression including mitotic entry, centrosome maturation, spindle assembly, chromatin segregation, mitotic exit and cytokinesis.[1] Overexpression of PLK1 is observed in many cancers and is often correlated with poor prognosis.[2] Furthermore, interference with PLK1 functions by small-molecule inhibitors[3–7] or small interfering RNAs[8] causes decreased proliferation in a broad spectrum of cancer cell lines and tumor xenografts but not in normal cells,[9] suggesting that PLK1 is an attractive drug target for cancer therapy.[10] More recently, the novel PLK1 inhibitor volasertib[11] was awarded “Breakthrough Therapy” designation by the US Food and Drug Administration (FDA) for its promising performance in the treatment of acute myeloid leukemia (AML), which further supports the potential of PLK1 inhibitors in the treatment of cancers. On the other hand, PLK2 and PLK3 have been shown to play roles in checkpoint-mediated cellcycle arrest to prevent genetic instability and oncogenic transformation,[12, 13] development of PLK1-specific inhibitors is therefore essential for cancer therapy to avoid potential offtarget effects.
PLK1 is characterized by an N-terminal kinase domain (KD) and a C-terminal polo-box domain (PBD). The KD is similar to the kinase domains of other protein kinases,[14, 15] while the PBD is unique to the PLKs family. Most PLK1 inhibitors targeting the KD also significantly inhibit the other two PLK family members PLK2 and PLK3 because of a high degree of structural conservation within the KDs of the PLK family members.[16] Alternatively, the PBD that mediates the subcellular localization[17] of PLKs is relatively divergent in structure, and thus represents an attractive target for discovering PLK1-specific inhibitors by selectively blocking the protein–protein interaction between the PBD and its partners.[18, 19] Since the discovery of the PBD as a phosphopeptide-binding module,[20] various phosphopeptide ligands have been reported to selectively bind the PBD of PLK1.[21–26] All these peptides are characterized by a conserved phosphorylated SpT motif that serves as the anchor to tightly bind with the pincer residues His 538 and Lys 540 of the PBD,[27] but the negatively charged phosphate group leads to the low cellular efficacy of these phosphopeptide ligands,[22, 26] highlighting the need to discover small-molecule PBD inhibitors with higher cellular permeability. In contrast to the discovery of various phosphopeptide ligands, only a few small-molecule PBD inhibitors have been identified through high-throughput screening (HTS) assays over the past several years.[28–30] These small-molecule PBD inhibitors interfere with the subcellular localization of PLK1 and induce cancer cell mitotic arrest, but the modest selectivity and drug-likeness of these compounds highlight the unmet need for the discovery of novel PBD inhibitors with diverse chemical scaffolds.
[a] H.-M. Shan, Dr. Y. Shi, Prof. J. Quan Key Laboratory of Chemical Genomics School of Chemical Biology & Biotechnology Peking University, Shenzhen Graduate School Shenzhen 518055 (China) E-mail: [email protected] [b] Prof. J. Quan Key Laboratory of Structural Biology School of Chemical Biology & Biotechnology Peking University, Shenzhen Graduate School Shenzhen 518055 (China)
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Full Papers In the present study, we combined virtual screening and fluorescence polarization screening assays to identify green tea catechins as potent inhibitors of the PLK1 PBD. The catechins interfere with the proper subcellular localization of PLK1, lead to cell-cycle arrest in the S and G2M phases, and induce growth inhibition of several human cancer cell types such as breast adenocarcinoma (MCF7), lung adenocarcinoma (A549), and cervical adenocarcinoma (HeLa).
Structure–activity relationships of green tea catechins as PBD inhibitors To understand the interaction between EGC and the PBD, we further determined the dose-dependent inhibition for other major green tea catechins including ( )-epigallocatechin gallate (EGCG), (+)-gallocatechin (GA), and (+)-catechin (CAT) (Figure 1).[34] Compared with EGC, EGCG has higher inhibitory
Results and Discussion Combined virtual and experimental screenings The efficiency of HTS is generally limited by the scaffold diversity of the screened chemical library,[31] which could be overcome by virtual screening of large chemical databases. We performed receptor-based virtual screening to identify potential binding hits for the PLK1 PBD based on a mixed chemical database with five million molecules.[32] As shown in Scheme 1, through virtual screening and further molecular dynamics simulation optimization, we finally selected fifty candidates for experimental test by using fluorescence polarization assay. Among these candidates, we found that ( )-epigallocatechin (EGC) (Figure 1), one of the main components of green tea polyphenols,[33] significantly blocks the binding of fluoresceinlabeled phosphopeptide to the PBD at a concentration of 10 mm.
Figure 1. Structures of green tea catechins.
activity, indicating that the additional gallate group contributes to the binding with the PBD (Figure 2). The relatively lower inhibitory activity for GA suggests that the configuration of 3-hydroxy group plays a role in the binding of catechins with the PBD. More strikingly, CAT has a much lower inhibitory activity compared with EGC, which indicated that the hydroxy groups on the B ring are essential for the inhibition of PBD-dependent recognition. To characterize the molecular mechanism underlying the SARs of catechins, all catechins were docked into the substrate binding pocket of the PBD (Figure 3). The docked structures indicated that all the catechins bind with the PBD in a similar mode— the hydroxy groups on the B ring form hydrogen bonds with the important pincer residues His 538/ Lys 540 and the backbone carbonyl oxygen of Leu 491 directly or mediated by a water molecule. Compared with EGC, CAT is predicted to form one less hydrogen bond with the backbone carbonyl oxygen of Leu 491 and may have weaker binding affinity with the PBD. In addition, the unfavorable contact between the polar 3-hydroxy group and the hydrophobic side chain of Leu 490 may further weaken the binding between CAT and the PBD. Such unfavorable contact might also be the reason that GA has weaker binding affinity with the PBD compared with EGC. On the other hand, the gallate group of EGCG is Scheme 1. Flow diagram for the discovery of polo-box domain (PBD) inhibitors through predicted to form an additional hydrogen bond with combined virtual and experimental screenings. ChemMedChem 2015, 10, 158 – 163
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Full Papers pressors.[12, 13] The selectivity of the inhibitors against different PLK family members is therefore very important to the drug discovery for cancer therapy.[7, 9] As EGCG has been well studied and shown to modulate multiple signal transduction pathways,[36, 37] we thus focused on evaluating in vitro and cellular activities for EGC in the following testing. Using a fluorescence polarization assay, we examined the inhibition of EGC on the binding of phosphopeptides with the PBDs from PLK1, PLK2, and PLK3. As shown in Figure 4, EGC most efficiently blocked the binding of phosphopeptide to the PLK1 PBD, and also blocked binding to the PLK2 PBD but with about five-fold weaker potency. EGC did not significantly inhibit the binding of phosphopeptide to the PBD of PLK3 at the concentration of 100 mm, indicating that it is a selective inhibitor of the PLK1 PBD. Figure 2. Inhibition of FITC-phosphopeptide binding to the polo-box domain (PBD) by green tea catechins: EGC (&), EGCG (*), GA (~), and CAT (!). Error bars represent the standard deviation (SD). FITC, fluorescein isothiocyanate.
Subcellular localization disruption and cell-cycle arrest
The PBD plays important roles in the subcellular localization and functions of PLK1, the inhibitors of the PBD are expected to interfere with the subcellular localization of PLK1 and cell-cycle progression.[17, 20] As expected, PLK1 mainly located to centrosomes and kinetochores in metaphase of control cells, while the localization of PLK1 to centrosomes and kinetochores was significantly decreased in the metaphase of EGCtreated cells (Figure 5 a). Furthermore, we also observed chromosome congression defects due to the mislocalization of endogenous PLK1 in EGC-treated cells (Figure 5 b). Most chromosomes congressed to the metaphase plate, but some chromosomes were unaligned and dispersed to the spindle poles. In line with the functions of PLK1 in mitosis, fluorescence-activated cell sorting (FACS) showed EGC induced a dose-dependent increase of cells in the G2M phase and decrease of cells in the G0/G1 phase (Figure 6 a). Interestingly, we observed a significant increase of cells in the S phase for EGC, which is consistent with the recent reports that PLK1 also plays important roles outside of mitosis,[38] but we didn’t exclude the possibility of EGC interacting with other Sphase targets. Furthermore, cell cycle arrests in the S and G2M phases also manifested by an increase in Figure 3. Predicted binding modes of catechins on the substrate binding groove of the the concentration of PLK1 in EGC-treated cells (Figpolo-box domain (PBD) determined by docking. Only key residues involved in interactions are displayed, and hydrogen bonds are shown by dashed blue lines. ure 6 b,c). Anticancer activity
the backbone amide of Leu 490 mediated by a water molecule and also has p–p stacking with His 538, which partially explains its higher binding affinity with the PBD.
To evaluate the anticancer activity of the PBD inhibitor EGC, we employed a sulforhodamine B (SRB) colourimetric assay[39] to test the inhibitory effect of EGC on the growth of human cancer cell lines that overexpress PLK1, including HeLa, A549, and MCF-7. The results indicated that EGC induced dose-dependent inhibition of the growth of these three tested cancer cell lines (Figure 7). In particular, the human breast cancer cell line MCF-7 was found to be the most sensitive to EGC treatment (GI50 ~ 50 mm). In contrast, the growth of the normal
Selectivity of EGC against PLK family members PLK family members PLK1, PLK2, and PLK3 share similar structures, but have distinct functions in the cell-cycle progression and development of tumor cells.[15, 35] PLK2 and PLK3 act as regulators to prevent DNA damage and serve as tumor supChemMedChem 2015, 10, 158 – 163
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Full Papers indicates that the PLK1 PBD might be one of the potential targets of green tea catechins including EGCG, EGC, and GA.
Conclusions We identified green tea catechins including EGCG, EGC, and GA as potent inhibitors of the PBD of PLK1 by combined virtual and experimental screenings. Docking study rationalized the SAR of green tea catechins against the PBD of PLK1. Results of a fluorescence polarization assay indicated that EGC is more selective against the PBD of PLK1 than those of PLK2 and PLK3. Further cellular analysis showed that EGC interfered with the proper subcellular localization of PLK1, led to the cell-cycle arrest in the S and G2M phases, and induced growth inhibition of several cancer cell types such as MCF7, A549, and HeLa. This work provides new insights into understanding the anticancer activities of green tea catechins and designing new inhibitors against the PLK1 PBD based on the catechin scaffold.
Figure 4. Effect of EGC on the binding of FITC-phosphopeptide binding to the polo-box domain (PBD) from PLK1 (&), PLK2 (*), and PLK3 (~), determined by fluorescence polarization. Error bars represent the standard deviation (SD). FITC, fluorescein isothiocyanate.
Experimental Section Virtual screening
Figure 5. a) HeLa cells treated with or without EGC (50 mm; 24 h) were fixed for immunofluorescence assay. EGC disrupts the subcellular localization of PLK1 (green) to centrosomes (g-tubulin; red) and kinetochores in metaphase. b) EGC induces chromosome (DNA; blue) congression defects in HeLa cells.
The mixed database was constructed by compounds selected from various chemical libraries including: NIH, Zinc, Maybridge and Drugbank.[40, 41] AutoDock version 3.0 was used for the docking simulation and the Lamarckian genetic algorithm (LGA) for ligand conformational searching.[42] The PDB file was prepared using published coordinates (PDB: 1UMW).[20] The ligand’s translation, rotation, and internal torsions are defined as its state variables, and each gene represents a state variable. The ligand file was setup for docking using the ADT (AutoDockTools) package, including assigning Gasteiger atomic charges, defining rigid root and rotatable bonds. The grid maps were centered on the phosphopeptide binding site, with 60 60 60 grid-points of 0.375 spacing. The genetic algorithm was used with standard parameters: trials of 50 dockings, a population size of 50 individuals; a maximum number of 1.5 106 energy evaluations and a maximum number of 27 000 generations; an elitism value of 1; a mutation rate of 0.02 and a crossover rate of 0.80. In the analysis of the docked conformations, the clustering tolerance for the root-mean-square positional deviation was set to 0.5 . The top 250 compounds with the best simulated binding energies within the standard deviation of 2.1 kcal mol 1 were selected manually. The illustrated structures were generated by using PyMOL.[43]
Plasmid construction and protein expression DNA sequences coding for the PBD domain of human PLK1 326–603, human PLK2 355–685, and human PLK3 335–646 were amplified by polymerase chain reaction (PCR) and cloned into the modified GST-His6 expression vector pGEX-4T-1. Proteins were expressed from BL21DE3plys and purified as previously described.[44] BL21DE3plys cells were transformed and grown to an OD600 value of 0.6 at 37 8C. The cells were cooled to 20 8C, and expression was induced with 0.1 mm isopropyl b-d-1-thiogalactopyranoside (IPTG) for 12 h. Cells were harvested and suspended in lysis
human embryonic kidney (HEK293) cells was not significantly affected by EGC even at the concentration of 100 mm, indicating that the PBD inhibitor EGC is selectively effective against the growth of cancer cells rather than normal cells. Considerable evidences have demonstrated that green tea catechin EGCG is a potent anticancer agent that targets multiple signal transduction pathways, but the anticancer activities and targets for other green tea catechins are less studied. This work ChemMedChem 2015, 10, 158 – 163
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Full Papers Soluble protein was added to the Talon@ Metal Affinity Resins. The resin was wash four times, and the polyhistidine-tagged protein was eluted with 200 mm imidazole.
Fluorescence polarization assay Binding experiments were performed using an Envision multilabel reader (PerkinElmer). Competition assays were performed using the following fluorophore-labeled peptide: 5 nm FITCGPMQSpTPLNG for PLK1; 5 nm FITC-GPMQTSpTPKNG for PLK2; 5 nm FITC-GPLATSpTPKNG for PLK3.[45, 46] 100 nm PBD protein, and the test compounds at concentration up to 200 mm. The compound (+)-gallocatechin (GA), ( )-epigallocatechin gallate (EGCG), ( )-epigallocatechin (EGC) and (+)-catechin (CAT) were purchased from Sigma. All FITC-peptides were synthesized (GL Biochem Shanghai Ltd) and checked by mass spectrometry. All experiments were performed in 96-well black plates at 25 8C. Plates were read 1 h after mixing of all assay components.
Sulforhodamine B assays Cells were seeded into 96-well plates containing 5 % fetal bovine serum and maintained in a humidified incubator at 37 8C in 5 % CO2. The cells were treated with increasing concentrations of EGC for 72 h. The assay was terminated by addition of cold trichloroacetic acid. After three-time washout with deionized water, sulforhodamine B solution at 0.4 % (w/v) in 1 % acetic acid was added to each well, and plates were incubated for 10 min at room temperature. The unbound dye was removed by three-time washing with 1 % acetic acid, and the plates were air dried. Bound sulforhodamine B was subsequently solubilized with 10 mm of trizma base (pH 10.5), and the absorbance was read at a wavelength of 515 nm.
Figure 6. a) HeLa cells were synchronized by thymidine block and released into medium containing EGC at the indicated concentration. EGC induces cell cycle arrests at S (&) and G2M (&) phases but not in the G0/G1 (&). Cell-cycle progression was monitored by fluorescence-activated cell sorter analysis. b) Western blot analysis of cell lysates treated with different concentration of EGC for 24 h. EGC induces an increase in the concentration of Polo-like kinase 1 (PLK1) in HeLa cells. c) The relative protein level of PLK1 was quantified and standardized to GAPDH expression by the use of Gel-Pro Analyzer (version 4.0) software.
Western blotting and immunofluorescence assay HeLa cells were synchronized by G1/S boundary by a double-thymidine treatment and then released into thymidine-free medium containing the indicated concentration of EGC for 24 h. For Western blot analysis, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mm Tris pH 8.0, 150 mm NaCl, 1 % NP-40, 0.25 % sodium deoxycholate, sodium orthovanadate, sodium fluoride, EDTA, 0.1 % sodium dodecyl sulfate, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and protease inhibitor). Rabbit anti-PLK1 polyclonal antibody (1:1000, Abcam) and rabbit polyclonal antibody against GAPDH (1:1000, cell signaling) were used as primary antibody. For immunofluorescence assay, HeLa cells were grown on coverslips for 24 h, then were fixed and permeabilized in methanol for 15 min at 20 8C. Cells were washed in Tris-buffered saline (TBS) buffer (20 mm Tris 200 mm NaCl pH 7.6) and incubated for 60 min in blocking solution (TBS with 0.1 %Triton X-100, 5 % bovine serum albumin) for 60 min. Primary antibodies used were rabbit anti-PLK1 polyclonal antibody (1:1000, Abcam), mouse anti-g-tubulin (1:500, Santa Cruz Biotechnology, Inc.), rabbit anti-a-tubulin (1:500, Cell Signaling). Images were taken with an AxioImager A1 microscope and AxioCam digital camera (Zeiss, Oberkochen, Germany).
Figure 7. Dose-dependent inhibition of the growth of human cancer cells by EGC. Cells were cultured in the presence of increasing concentration of EGC for 72 h and analyzed by sulforhodamine B assays.
Cell-cycle analysis
buffer composed of 20 mm HEPES pH 7.4, 50 mm NaCl, 0.1 % NP40, 1 mg mL 1 lysozyme, 1 mm MgCl2 and DNase I. Lysates were cleared by high-speed centrifugation at 4 8C, 35 000 g for 40 min.
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HeLa cells were harvested and washed twice with cold phosphatebuffered saline. After centrifugation at 4 8C, 1000 g for 5 min,
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Full Papers washed cells were resuspended and fixed in chilled 70 % ethanol at 4 8C for 30 min. The cells were stained with 100 mg mL 1 propidium iodide and treated with 1 mg mL 1 of RNase-A (Sigma) for 30 min at 37 8C. The DNA content of 10 000 cells was determined with a FACScan flow cytometer (BD Biosciences). The data were analyzed with Modfit LT2.0.
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Acknowledgements This work was financially supported by the Chinese Ministry of Science and Technology (2012CB722602), the National Natural Science Foundation of China (NSFC21290183, NSFC81373326), and the Shenzhen Municipal Government (China) (JCYJ20130331144947526 and ZDSY20130331145112855). Keywords: cancer · catechins · inhibitors · natural products · polo-box domain · polo-like kinase 1 · screening [1] M. Petronczki, P. Lenart, J. M. Peters, Dev. Cell 2008, 14, 646 – 659. [2] K. Strebhardt, A. Ullrich, Nat. Rev. Cancer 2006, 6, 321 – 330. [3] Y. Liu, K. R. Shreder, W. Gai, S. Corral, D. K. Ferris, J. S. Rosenblum, Chem. Biol. 2005, 12, 99 – 107. [4] M. Steegmaier, M. Hoffmann, A. Baum, P. Lenart, M. Petronczki, M. Krssak, U. Gurtler, P. Garin-Chesa, S. Lieb, J. Quant, M. Grauert, G. R. Adolf, N. Kraut, J. M. Peters, W. J. Rettig, Curr. Biol. 2007, 17, 316 – 322. [5] A. Jimeno, J. Li, W. A. Messersmith, D. Laheru, M. A. Rudek, M. Maniar, M. Hidalgo, S. D. Baker, R. C. Donehower, J. Clin. Oncol. 2008, 26, 5504 – 5510. [6] S. Keppner, E. Proschak, G. Schneider, B. Spnkuch, ChemMedChem 2009, 4, 1806 – 1809. [7] a) S. Keppner, E. Proschak, M. Kaufmann, K. Strebhardt, G. Schneider, B. Spnkuch, Cell Cycle 2010, 9, 761 – 773; b) S. Keppner, E. Proschak, G. Schneider, B. Spnkuch, Cell Cycle 2011, 10, 708 – 720. [8] B. Spnkuch-Schmitt, J. Bereiter-Hahn, M. Kaufmann, K. Strebhardt, J. Natl. Cancer Inst. 2002, 94, 1863 – 1877. [9] a) X. Liu, M. Lei, R. L. Erikson, Mol. Cell. Biol. 2006, 26, 2093 – 2108; b) M. Raab et al., Nat. Commun. 2011, 2, 395. [10] K. C. Goh, H. Wang, N. Yu, Y. Zhou, Y. Zheng, Z. Lim, K. Sangthongpitag, L. Fang, M. Du, X. Wang, A. B. Jefferson, J. Rose, B. Shamoon, C. Reinhard, B. Carte, M. Entzeroth, B. Ni, M. L. Taylor, W. Stnkel, Drug Develop. Res. 2004, 62, 349 – 361. [11] D. Rudolph, M. Steegmaier, M. Hoffmann, M. Grauert, A. Baum, J. Quant, C. Haslinger, P. Garin-Chesa, G. R. Adolf, Clin. Cancer Res. 2009, 15, 3094 – 3102. [12] Y. Yang, J. Bai, R. Shen, S. A. Brown, E. Komissarova, Y. Huang, N. Jiang, G. F. Alberts, M. Costa, L. Lu, J. A. Winkles, W. Dai, Cancer Res. 2008, 68, 4077 – 4085. [13] N. Syed, P. Smith, A. Sullivan, L. C. Spender, M. Dyer, L. Karran, J. O’Nions, M. Allday, I. Hoffmann, D. Crawford, B. Griffin, P. J. Farrell, T. Crook, Blood 2006, 107, 250 – 256. [14] M. Kothe, D. Kohls, S. Low, R. Coli, A. C. Cheng, S. L. Jacques, T. L. Johnson, C. Lewis, C. Loh, J. Nonomiya, A. L. Sheils, K. A. Verdries, T. A. Wynn, C. Kuhn, Y. H. Ding, Biochemistry 2007, 46, 5960 – 5971. [15] K. Strebhardt, Nat. Rev. Drug Discov. 2010, 9, 643 – 660. [16] R. N. Murugan, J. E. Park, E. H. Kim, S. Y. Shin, C. Cheong, K. S. Lee, J. K. Bang, Mol. Cells 2011, 32, 209 – 220. [17] Y. J. Jang, C. Y. Lin, S. Ma, R. L. Erikson, Proc. Natl. Acad. Sci. USA 2002, 99, 1984 – 1989.
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Received: July 9, 2014 Published online on September 5, 2014
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