Hit identification of IKKβ natural product inhibitor
© Leung et al.; licensee BioMed Central Ltd. 2013
Received: 15 November 2011
Accepted: 21 December 2012
Published: 7 January 2013
The nuclear factor-κB (NF-κB) proteins are a small group of heterodimeric transcription factors that play an important role in regulating the inflammatory, immune, and apoptotic responses. NF-κB activity is suppressed by association with the inhibitor IκB. Aberrant NF-κB signaling activity has been associated with the development of cancer, chronic inflammatory diseases and auto-immune diseases. The IKK protein complex is comprised of IKKα, IKKβ and NEMO subunits, with IKKβ thought to play the dominant role in modulating NF-κB activity. Therefore, the discovery of new IKKβ inhibitors may offer new therapeutic options for the treatment of cancer and inflammatory diseases.
A structure-based molecular docking approach has been employed to discover novel IKKβ inhibitors from a natural product library of over 90,000 compounds. Preliminary screening of the 12 highest-scoring compounds using a luciferase reporter assay identified 4 promising candidates for further biological study. Among these, the benzoic acid derivative (1) showed the most promising activity at inhibiting IKKβ phosphorylation and TNF-α-induced NF-κB signaling in vitro.
In this study, we have successfully identified a benzoic acid derivative (1) as a novel IKKβ inhibitor via high-throughput molecular docking. Compound 1 was able to inhibit IKKβ phosphorylation activity in vitro, and block IκBα protein degradation and subsequent NF-κB activation in human cells. Further in silico optimization of the compound is currently being conducted in order to generate more potent analogues for biological tests.
The nuclear factor-κB (NF-κB) proteins are a small group of heterodimeric transcription factors that play an important role in regulating inflammatory, immune, and apoptotic responses [1–3]. NF-κB is ubiquitously present in the cytoplasm and its activity is normally suppressed by association with inhibitor IκB . The intracellular NF-κB signaling cascade is initiated by a variety of inducers including proinflammatory cytokines TNF-α, IL-1 or endotoxins [5, 6]. The aberrant activity to the NF-κB signaling pathway has been implicated in the development of a number of human diseases including cancer, auto-immune and chronic inflammatory conditions [3, 7, 8]. Therefore, inhibitors of the NF-κB signaling pathway could offer potential therapeutic value for the treatment of such diseases [9, 10].
The IκB kinase is a multi-component complex composed of two catalytic subunits, IKKα and IKKβ and a regulatory unit NF-κB essential modulator (NEMO) [11–13]. Although both catalytic units are able to phosphorylate IκB, IKKβ has been shown to play the dominant role in activating NF-κB signaling in response to inflammatory stimuli [14, 15]. Phosphorylated IκB is subsequently tagged by the E1 ubiquitin enzyme and degraded by the proteasome to liberate active NF-κB. Free NF-κB then translocates into the nucleus, where it binds to its cognate DNA site and enhances the expression of a number of genes related to the immune response, cell proliferation and survival [16, 17]. Consequently, IKKβ represents an attractive target in the NF-κB pathway for the development of anti-inflammatory or anti-cancer therapeutics.
Virtual screening (VS) has emerged as a powerful tool in drug discovery complementing the vast array of popular but relatively costly high-throughput screening technologies [18, 19]. Using virtual screening, the number of compounds to be evaluated in vitro could be dramatically decreased, which could greatly reduce the time and resource costs of drug discovery efforts. Meanwhile, natural products (NPs) have long provided a valuable source of inspiration to medicinal chemists due to the diversity of their molecular scaffolds, favourable biocompatibility and evolutionarily validated bioactive substructures [20, 21]. Combining these two ideas, our group has previously identified natural product or small molecule inhibitors antagonizing cancer or inflammation-related targets using virtual screening [22–28]. For example, we have successfully identified natural product or natural product-like compounds targeting the c-myc oncogene G-quadruplex, tumor necrosis factor-alpha (TNF-α) and NEDD8-activating enzyme (NAE) [29–34].
Results and Discussion
High-throughput virtual screening
Molecular modeling analysis
Our molecular docking analysis revealed that the top-scoring binding mode of the natural product derivative 1 to the IKKβ complex is similar to that of the reference compound. The bound inhibitor in the co-crystal structure of IKKβ interacts with the ATP binding pocket in shape-driven manner . While the structure of the reference compound contains the anilinopyrimidine motif that is found in other kinase inhibitors such as imatinib , no detectable hydrogen bonds between the hinge region of IKKβ and the anilinopyrimidine moiety of the reference compound were recorded. The aromatic rings of the reference compound span the hinge loop while its terminal chlorine atom points towards the gatekeeper residue Met96 (Figure 2b).
By comparison, the benzoic acid moiety of 1 is situated at the end of the hinge loop with predicted hydrogen bonding interactions between the carboxyl oxygen and amide oxygen atoms of 1 with the phenolic hydrogen atom of Tyr98 and the backbone amino group of Gly102, respectively (Figure 2c). The pendant side chain of 1 is predicted to be situated in a hydrophobic binding pocket also occupied by the reference compound. We envisage that 1 could act as a reversible inhibitor of IKKβ by blocking the nucleotide recognition domain that binds ATP . The binding score for 1 with the IKKβ complex was calculated to be -35.28 kcal/mol, reflecting a strong interaction between the compound and the IKKβ binding site.
The other eleven compounds were also predicted to situate in the hinge region of the binding pockets in the docking analysis. Most of the compounds could form hydrogen bonds with the hinge residues including Glu97, Cys99 and Glu100. Furthermore, several of the compounds formed additional hydrogen bonds with the residues in the solvent accessible region (Arg31 and Lys106). The lowest energy binding pose of the other compounds are summarized in Additional file 1: Table S2.
We also investigated the selectivity of compound 1 for IKKβ over four other kinases (PKCα, PAK4, CaMK2α and JAK2) using molecular modeling. While compound 1 was predicted to bind at the ATP binding sites of the four other kinases, the ICM docking energies of the 1-kinase complexes were significantly less negative than that for IKKβ (Additional file 1: Table S3). Molecules exhibiting such weak binding energies would be expected to be inactive in vitro.
1 inhibits IκBα phosphorylation in vitro
1 inhibits TNF-α induced NF-κB signaling in a HepG2 cell line
Based on the results of the IKKβ assay and the molecular modeling analysis, we envisage that the inhibition of TNF-α-induced NF-κB signaling by 1 could be attributed, at least in part, to the inhibition of IKKβ activity in vitro, thus preventing the degradation of the NF-κB repressor IκBα. The slightly higher potency of 1 in the cell-based luciferase assay compared to the enzyme assay is possibly due to a multi-target effect of 1, suggesting that this compound could potentially influence other steps involved in NF-κB activation.
In conclusion, we have discovered a new small molecule IKKβ inhibitor from a large natural product library of 90,000 compounds using high-throughput structure-based molecular docking. The benzoic acid derivative 1 is able to inhibit IKKβ activity in both cell-free and system with micromolar potency. Furthermore, compound 1 could inhibit IKKβ-mediated NF-κB signaling pathway in human cancer cells. We envisage that compound 1 attenuates the in cellulo transcriptional activity of NF-κB, at least in part, by abrogating the activity of IKKβ. The discovery of this natural product-like derivative provides medicinal chemists with a structurally interesting scaffold, facilitating further chemical modifications in order to sample greater regions of the chemical space of potential IKKβ inhibitors. We are currently investigating the effects of 1 on the proteins involved in NF-κB signaling and conducting in silico lead optimization to generate more potent analogues of 1 for in vitro biological testing.
Materials and cell lines
The NP/NP-like compound collection, which includes compound 1 and the other tested compounds, was obtained from InterBioScreen (Moscow, RUS). The K-LISA™ IKKβ Inhibitor Screening Kit was obtained from Calbiochem (Darmstadt, Germany). Passive lysis buffer and luciferase assay reagent were obtained from Promega Corporation (Madison, WI, USA). HepG2 and HepG2-NF-κB-Luc cells were provided by Prof. Y.C. Cheng (Department of Pharmacology, Yale University School of Medicine, USA). Cells cultured in Minimum Essential Media containing 10% fetal bovine serum were incubated at 37°C/5% CO2 and passaged three times a week.
IKKβ enzymatic activity
IKKβ activity was determined using the ELISA-based (K-LISA™) IKKβ Inhibitor Screening Kit according to the manufacturer’s instructions. The GST-IκBα 50-amino acid peptide that includes the Ser32 and Ser36 IKKβ phosphorylation sites was used as a substrate and was incubated for 30 min at 30°C with human recombinant IKKβ in the presence of DMSO vehicle or different concentrations of 1 in a glutathione-coated 96-well plate. The phosphorylated GST-IκBα substrate was subsequently detected using anti-phospho-IκBα (Ser32/Ser36) antibody and a horseradish peroxidase-conjugated secondary antibody. The samples were finally incubated with TMB solution, and the color development was monitored at 450 nm on a plate reader (Bio-Rad).
NF-κB transactivation activity
Exponentially growing HepG2-NF-κB-Luc cells were seeded overnight at 1 × 104 cells/well in a 48-well plate. On the next day, the cells were pre-incubated with the indicated concentrations of 1 for 1 h before stimulation by 5 ng/mL of TNF-α for an additional 3 h. Passive lysis buffer (50 μL) was added to each well and the plate was incubated for 15 min with shaking. A 20 μL aliquot from each well was mixed with 70 μL luciferase assay reagent in a 96-well white plate. The transcriptional activity was determined by measuring the activity of firefly luciferase in a multi-well plate luminometer (Fusion α-FP, Perkin-Elmer).
where E vw, E el, Ehb, E hp, and E sf are Van der Waals, electrostatic, hydrogen bonding, and nonpolar and polar atom solvation energy differences between bound and unbound states, respectively. E int is the ligand internal strain, ΔS Tor is its conformational entropy loss upon binding, and T = 300 K, and αi are ligand- and receptor independent constants. The initial model of IKKβ was built from the X-ray crystal structure of the Inhibitor of kappaB kinase beta (PDB: 3RZF) according to a previously reported procedure. Hydrogen and missing heavy atoms were added to the receptor structure followed by local minimization by using the conjugate gradient algorithm and analytical derivatives in the internal coordinates. In the docking analysis, the binding site was assigned across the entire structure of the protein complex. Each compound was assigned the MMFF force field atom types and charges and was then subjected to Cartesian minimization. The ICM docking was performed to find the most favorable orientation. The resulting trajectories of the complex between the small molecules and protein complex were energy minimized, and the interaction energies were computed. Each compound was docked three times and the minimum of the three scores was used. The 12 highest scoring compounds were utilized for biological testing without further selection. The crystal structures of PAK4 (4APP), PKCα (3IW4), CAMK2α (2VZ6) and JAK2 (3IOK) were also prepared and compound 1 was docked to these molecular models individually using the aforementioned procedures.
This work is supported by Hong Kong Baptist University (FRG2/11-12/009), Centre for Cancer and Inflammation Research, School of Chinese Medicine (CCIR-SCM, HKBU), the Health and Medical Research Fund (HMRF/11101212), the Research Grants Council (HKBU/201811 and HKBU/204612), the Science and Technology Development Fund, Macao SAR (001/2012/A) and the University of Macau MYRG091(Y1-L2)-ICMS12-LCH and MYRG121(Y1-L2)-ICMS12-LCH).
- Hayden MS, Ghosh S: Shared Principles in NF-κB Signaling. Cell. 2008, 132: 344-362. 10.1016/j.cell.2008.01.020.View ArticlePubMedGoogle Scholar
- Vallabhapurapu S, Karin M: Regulation and Function of NF-κB Transcription Factors in the Immune System. Annu Rev Immunol. 2009, 27: 693-733. 10.1146/annurev.immunol.021908.132641.View ArticlePubMedGoogle Scholar
- Karin M: Nuclear factor-κB in cancer development and progression. Nature. 2006, 441: 431-436. 10.1038/nature04870.View ArticlePubMedGoogle Scholar
- Baldwin AS: The NF-κB AND IκB Proteins: New Discoveries and Insights. Annu Rev Immunol. 1996, 14: 649-681. 10.1146/annurev.immunol.14.1.649.View ArticlePubMedGoogle Scholar
- Hideshima T, Chauhan D, Richardson P, Mitsiades C, Mitsiades N, Hayashi T, Munshi N, Dang L, Castro A, Palombella V: NF-κB as a Therapeutic Target in Multiple Myeloma. J Biol Chem. 2002, 277: 16639-16647. 10.1074/jbc.M200360200.View ArticlePubMedGoogle Scholar
- Schmid JA, Birbach A: IκB kinase β (IKKβ/IKK2/IKBKB)—A key molecule in signaling to the transcription factor NF-κB. Cytokine Growth Factor Rev. 2008, 19: 157-165. 10.1016/j.cytogfr.2008.01.006.View ArticlePubMedGoogle Scholar
- Lee CH, Jeon Y-T, Kim S-H, Song Y-S: NF-κB as a potential molecular target for cancer therapy. Biofactors. 2007, 29: 19-35. 10.1002/biof.5520290103.View ArticlePubMedGoogle Scholar
- Karin M, Cao Y, Greten FR, Li Z-W: NF-κB in cancer: from innocent bystander to major culprit. Nat Rev Cancer. 2002, 2: 301-310. 10.1038/nrc780.View ArticlePubMedGoogle Scholar
- Kim HJ, Hawke N, Baldwin AS: NF-κB and IKK as therapeutic targets in cancer. Cell Death Differ. 2006, 13: 738-747. 10.1038/sj.cdd.4401877.View ArticlePubMedGoogle Scholar
- Senftleben U: Anti-inflammatory interventions of NF-κB signaling: Potential applications and risks. Biochem Pharmacol. 2008, 75: 1567-1579. 10.1016/j.bcp.2007.10.027.View ArticleGoogle Scholar
- Gilmore TD: Introduction to NF-κB: players, pathways, perspectives. Oncogene. 2005, 25: 6680-6684.View ArticleGoogle Scholar
- Perkins ND: Integrating cell-signaling pathways with NF-[kappa]B and IKK function. Nat Rev Mol Cell Biol. 2007, 8: 49-62. 10.1038/nrm2083.View ArticlePubMedGoogle Scholar
- Karin M, Ben-Neriah Y: Phosphorylation Meets Ubiquitination: The Control of NF-κB Activity. Annu Rev Immunol. 2000, 18: 621-663. 10.1146/annurev.immunol.18.1.621.View ArticlePubMedGoogle Scholar
- Karin M: How NF-κB is activated: the role of the IκB kinase (IKK) complex. Oncogene. 1999, 18: 6867-6874. 10.1038/sj.onc.1203219.View ArticlePubMedGoogle Scholar
- Strnad J, Burke JR: IκB kinase inhibitors for treating autoimmune and inflammatory disorders: potential and challenges. Trends Pharmacol Sci. 2007, 28: 142-148. 10.1016/j.tips.2007.01.005.View ArticlePubMedGoogle Scholar
- Karin M, Yamamoto Y, Wang QM: The IKK NF-κB system: a treasure trove for drug development. Nat Rev Drug Discov. 2004, 3: 17-26. 10.1038/nrd1279.View ArticlePubMedGoogle Scholar
- Gilmore TD: The Rel/NF-B signal transduction pathway: introduction. Oncogene. 1999, 18: 6842-6844. 10.1038/sj.onc.1203237.View ArticlePubMedGoogle Scholar
- Bajorath J: Integration of virtual and high-throughput screening. Nat Rev Drug Discov. 2002, 1: 882-894. 10.1038/nrd941.View ArticlePubMedGoogle Scholar
- Shoichet BK: Virtual screening of chemical libraries. Nature. 2004, 432: 862-865. 10.1038/nature03197.View ArticlePubMedPubMed CentralGoogle Scholar
- Breinbauer R, Vetter IR, Waldmann H: From Protein Domains to Drug Candidates—Natural Products as Guiding Principles in the Design and Synthesis of Compound Libraries. Angew Chem Int Ed. 2002, 41: 2878-2890. 10.1002/1521-3773(20020816)41:16<2878::AID-ANIE2878>3.0.CO;2-B.View ArticleGoogle Scholar
- Ertl P, Roggo S, Schuffenhauer A: Natural Product-likeness Score and Its Application for Prioritization of Compound Libraries. J Chem Inf Model. 2007, 48: 68-74.View ArticlePubMedGoogle Scholar
- Leung C-H: Chan DS-H, Kwan MH-T, Cheng Z, Wong C-Y, Zhu G-Y, Fong W-F, Ma D-L: Structure-Based Repurposing of FDA-Approved Drugs as TNF-α Inhibitors. ChemMedChem. 2011, 6: 765-768. 10.1002/cmdc.201100016.View ArticlePubMedGoogle Scholar
- Chan DS-H, Yang H, Kwan MH-T, Cheng Z, Lee P, Bai L-P, Jiang Z-H, Wong C-Y, Fong W-F, Leung C-H, Ma D-L: Structure-based optimization of FDA-approved drug methylene blue as a c-myc G-quadruplex DNA stabilizer. Biochimie. 2011, 93: 1055-1064. 10.1016/j.biochi.2011.02.013.View ArticlePubMedGoogle Scholar
- Yang H, Zhong H-J, Leung K-H, Chan DS-H, Ma VP-Y, Fu W-C, Nanjunda R, Wilson WD, Ma D-L, Leung C-H: Structure-based design of flavone derivatives as c-myc oncogene down-regulators. Eur J Pharm Sci. 2013, 48: 130-141. 10.1016/j.ejps.2012.10.010.View ArticlePubMedGoogle Scholar
- Ma D-L: Chan DS-H, Leung C-H: Molecular docking for virtual screening of natural product databases. Chem. Sci. 2011, 2: 1656-1665. 10.1039/c1sc00152c.View ArticleGoogle Scholar
- Ma D-L: Chan DS-H, Lee P, Kwan MH-T, Leung C-H: Molecular modeling of drug–DNA interactions: Virtual screening to structure-based design. Biochimie. 2011, 93: 1252-1266. 10.1016/j.biochi.2011.04.002.View ArticlePubMedGoogle Scholar
- Ma D-L: Ma VP-Y, Chan DS-H, Leung K-H, Zhong H-J, Leung C-H: In silico screening of quadruplex-binding ligands. Methods. 2012, 57: 106-114. 10.1016/j.ymeth.2012.02.001.View ArticlePubMedGoogle Scholar
- Ma D-L, Chan DS-H, Leung C-H: Drug repositioning by structure-based virtual screening. Chem Soc Rev. 10.1039/C2CS35357A.Google Scholar
- Leung C-H, Chan DS-H, Yang H, Abagyan R, Lee SM-Y, Zhu G-Y, Fong W-F, Ma D-L: A natural product-like inhibitor of NEDD8-activating enzyme. Chem Commun. 2011, 47: 2511-2513. 10.1039/c0cc04927a.View ArticleGoogle Scholar
- Ma D-L, Lai T-S, Chan F-Y, Chung W-H, Abagyan R, Leung Y-C, Wong K-Y: Discovery of a Drug-Like G-Quadruplex Binding Ligand by High-Throughput Docking. ChemMedChem. 2008, 3: 881-884. 10.1002/cmdc.200700342.View ArticlePubMedGoogle Scholar
- Chan DS-H, Lee H-M, Yang F, Che C-M, Wong CCL, Abagyan R, Leung C-H, Ma D-L: Structure-Based Discovery of Natural-Product-like TNF-α Inhibitors. Angew Chem Int Ed. 2010, 49: 2860-2864. 10.1002/anie.200907360.View ArticleGoogle Scholar
- Lee H-M: Chan DS-H, Yang F, Lam H-Y, Yan S-C, Che C-M, Ma D-L, Leung C-H: Identification of natural product Fonsecin B as a stabilizing ligand of c-myc G-quadruplex DNA by high-throughput virtual screening. Chem Commun. 2010, 46: 4680-4682. 10.1039/b926359d.View ArticleGoogle Scholar
- Zhong H-J: Ma VP-Y, Cheng Z, Chan DS-H, He H-Z, Leung K-H, Ma D-L, Leung C-H: Discovery of a natural product inhibitor targeting protein neddylation by structure-based virtual screening. Biochimie. 2012, 94: 2457-2460. 10.1016/j.biochi.2012.06.004.View ArticlePubMedGoogle Scholar
- Ma D-L, Chan DS-H, Fu W-C, He H-Z, Yang H, Yan S-C, Leung C-H: Discovery of a Natural Product-Like c-myc G-Quadruplex DNA Groove-Binder by Molecular Docking. PLoS One. 2012, 7: e43278-10.1371/journal.pone.0043278.View ArticlePubMedPubMed CentralGoogle Scholar
- Miller DD, Bamborough P, Christopher JA, Baldwin IR, Champigny AC, Cutler GJ, Kerns JK, Longstaff T, Mellor GW, Morey JV: 3,5-Disubstituted-indole-7-carboxamides: The discovery of a novel series of potent, selective inhibitors of IKK-β. Bioorg Med Chem Lett. 2011, 21: 2255-2258. 10.1016/j.bmcl.2011.02.107.View ArticlePubMedGoogle Scholar
- Crombie AL, Sum F-W, Powell DW, Hopper DW, Torres N, Berger DM, Zhang Y, Gavriil M, Sadler TM, Arndt K: Synthesis and biological evaluation of tricyclic anilinopyrimidines as IKKβ inhibitors. Bioorg Med Chem Lett. 2010, 20: 3821-3825. 10.1016/j.bmcl.2010.04.022.View ArticlePubMedGoogle Scholar
- Kempson J, Spergel SH, Guo J, Quesnelle C, Gill P, Belanger D, Dyckman AJ, Li T, Watterson SH, Langevine CM: Novel Tricyclic Inhibitors of IκB Kinase. J Med Chem. 2009, 52: 1994-2005. 10.1021/jm8015816.View ArticlePubMedGoogle Scholar
- Kempson J, Guo J, Das J, Moquin RV, Spergel SH, Watterson SH, Langevine CM, Dyckman AJ, Pattoli M, Burke JR: Synthesis, initial SAR and biological evaluation of 1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-4-amine derived inhibitors of IκB kinase. Bioorg Med Chem Lett. 2009, 19: 2646-2649. 10.1016/j.bmcl.2009.03.159.View ArticlePubMedGoogle Scholar
- Christopher JA, Bamborough P, Alder C, Campbell A, Cutler GJ, Down K, Hamadi AM, Jolly AM, Kerns JK, Lucas FS: Discovery of 6-Aryl-7-alkoxyisoquinoline Inhibitors of IκB Kinase-β (IKK-β). J Med Chem. 2009, 52: 3098-3102. 10.1021/jm9000117.View ArticlePubMedGoogle Scholar
- Xu G, Lo Y-C, Li Q, Napolitano G, Wu X, Jiang X, Dreano M, Karin M, Wu H: Crystal structure of inhibitor of κB kinase β. Nature. 2011, 472: 325-330. 10.1038/nature09853.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen J-J, Cho J-Y, Hwang T-L, Chen I-S: Benzoic Acid Derivatives, Acetophenones, and Anti-inflammatory Constituents from Melicope semecarpifolia. J Nat Prod. 2007, 71: 71-75.View ArticlePubMedGoogle Scholar
- Hsieh Y-H, Chu F-H, Wang Y-S, Chien S-C, Chang S-T, Shaw J-F, Chen C-Y, Hsiao W-W, Kuo Y-H, Wang S-Y: Antrocamphin A, an Anti-inflammatory Principal from the Fruiting Body of Taiwanofungus camphoratus, and Its Mechanisms. J Agric Food Chem. 2010, 58: 3153-3158. 10.1021/jf903638p.View ArticlePubMedGoogle Scholar
- Nakajima H, Fujiwara H, Furuichi Y, Tanaka K, Shimbara N: A novel small-molecule inhibitor of NF-κB signaling. Biochem Biophys Res Commun. 2008, 368: 1007-1013. 10.1016/j.bbrc.2008.01.166.View ArticlePubMedGoogle Scholar
- Ghose AK, Herbertz T, Pippin DA, Salvino JM, Mallamo JP: Knowledge Based Prediction of Ligand Binding Modes and Rational Inhibitor Design for Kinase Drug Discovery. J Med Chem. 2008, 51: 5149-5171. 10.1021/jm800475y.View ArticlePubMedGoogle Scholar
- Cowan-Jacob SW, Fendrich G, Manley PW, Jahnke W, Fabbro D, Liebetanz J, Meyer T: The Crystal Structure of a c-Src Complex in an Active Conformation Suggests Possible Steps in c-Src Activation. Structure. 2005, 13: 861-871. 10.1016/j.str.2005.03.012.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/14/3/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.