Birgit Spänkuch

Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo.

Cyclin B1 is the regulatory subunit of cyclin-dependent kinase 1 (Cdk1) and is critical for the initiation of mitosis. Accumulating data indicate that the deregulation of cyclin B1 is tightly linked to neoplastic transformation. To study the phenotype and the potential preclinical relevance, we generated HeLa cell lines stably transfected with the plasmids encompassing short hairpin RNA (shRNA) targeting cyclin B1. We demonstrate that the reduction of cyclin B1 caused inhibition of proliferation by arresting cells in G2 phase and by inducing apoptosis. Cells, entering mitosis, were impaired in chromosome condensation and alignment. Importantly, HeLa cells with reduced cyclin B1 were more susceptible to the treatment of small interfering RNA targeting Polo-like kinase 1 (Plk1) and to the administration of the chemotherapeutic agent taxol. Finally, HeLa cells with reduced cyclin B1 showed inhibited tumor growth in nude mice compared to that of control cells. In summary, our data indicate that cyclin B1 is an essential molecule for tumor cell survival and aggressive proliferation, suggesting that the downregulation of cyclin B1, especially in combination with other molecular targets, might become an interesting strategy for antitumor intervention.

Rational combinations of siRNAs targeting Plk1 with breast cancer drugs

Commonly used drugs for the treatment of breast cancer patients like paclitaxel and Herceptin often show severe side effects or induce resistance in clinical settings. Thus, we analysed a combination of Plk1 (polo-like kinase 1)-specific small interfering RNAs (siRNAs), a powerful tool to induce ‘mitotic catastrophe’ in cancer cells, together with these drugs to identify conditions for enhanced drug sensitivity. After transfection, the antineoplastic agents were added and cell proliferation, apoptosis and cell cycle distribution in breast cancer cells (MCF-7, SK-BR-3, MDA-MB-435 and BT-474) and in primary human mammary epithelial cells were determined. Downregulation of cellular Plk1 levels led to an elevated percentage of cells in G2/M phase. The percentage of apoptotic nuclei in MCF-7, MDA-MB-435, SK-BR-3 and BT-474 cells was clearly increased after incubation with Plk1-specific siRNAs and paclitaxel. Interestingly, the caspase pathway was activated after treatment with Plk1-specific siRNAs and paclitaxel or Herceptin. Treatment of breast cancer cells with siRNAs targeting Plk1 improved the sensitivity toward paclitaxel and Herceptin in a synergistic manner. In all experiments, very low concentrations across a wide range of clinically relevant concentrations were sufficient to induce an antiproliferative effect. The combination of Plk1-specific siRNAs with modern breast cancer drugs seems to represent rational combinations to be tested in preclinical trials.

Down-regulation of Polo-like Kinase 1 Elevates Drug Sensitivity of Breast Cancer Cells In vitro and In vivo

Human polo-like kinase 1 (Plk1) is a key player in different stages of mitosis and modulates the spindle checkpoint at the metaphase-anaphase transition. Overexpression of Plk1 is observed in various human tumors and it is a negative prognostic factor in patients suffering from diverse cancers. We used phosphorothioate antisense oligonucleotides (ASO) targeted against Plk1, together with paclitaxel, carboplatin, and Herceptin, for the treatment of breast cancer cells to identify conditions for enhanced drug sensitivity. After transfection of the breast cancer cell lines BT-474, MCF-7, and MDA-MB-435 with Plk1-specific ASOs, paclitaxel, carboplatin, or Herceptin was added and cell proliferation, cell cycle distribution, and apoptosis were measured. Whereas the dual treatment of breast cancer cells with Plk1-specific ASOs with carboplatin or Herceptin caused only a limited antiproliferative effect in breast cancer cells, we observed synergistic effects after combination of low doses of Plk1-specific ASOs with paclitaxel, which is used in a variety of clinical anticancer regimens. Plk1-specific ASOs also acted synergistically with paclitaxel in the arrest of the cell cycle at the G(2)-M phase and in the induction of apoptosis. Interestingly, in a human xenograft experiment using MDA-MB-435 cells, the combination of Plk1 ASOs with paclitaxel led to synergistic reduction of tumor growth after 3 weeks of treatment compared with either agent alone. This study suggests that antisense inhibitors against Plk1 at well-tolerated doses may be considered as highly efficient promoters for the antineoplastic potential of taxanes, such as paclitaxel, causing synergistic effects in breast cancer cells.

Effect of trastuzumab-modified antisense oligonucleotide-loaded human serum albumin nanoparticles prepared by heat denaturation.

Nanoparticles represent a promising tool for targeted drug delivery to tumour cells and are able to protect drugs against degradation. In our present study we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against Plk1 (polo-like kinase 1) prepared by heat denaturation instead of using glutaraldehyde. Glutaraldehyde can lead to an inactivation of ASOs through chemical crosslinking and is a toxic entity. We examined the ideal preparation conditions and characterised the resulting particles in terms of physico-chemical properties, ASO recovery after enzymatic degradation and stability. Stable monodisperse nanoparticles with an ASO recovery of more than 80% could be prepared at a temperature of 105 degrees C for 10 min. Furthermore we performed quantitative real-time PCR and Western blot to detect an ASO-mediated effect on Plk1 in BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression. Thus, this is the first report of ASO-loaded HSA nanoparticles prepared by heat denaturation, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells.

Conditional inhibition of cancer cell proliferation by tetracycline-responsive, H1 promoter-driven silencing of PLK1.

RNA interference (RNAi) is a powerful tool for studying gene function. We developed an inducible genetic element for short interfering RNA-mediated gene silencing. This system uses a tetracycline (Tet)-responsive derivative of the H1 promoter and the Tet repressor (TetR) for conditional expression of short hairpin RNA (shRNA) in HeLa cells. Promoter constructs were generated, which contain the Tet operator (TetO) derived from a prokaryotic Tet resistance transposon upstream and/or downstream of the TATA box. To quantify the response of controllable transcription units for shRNA expression, we examined the functional activity of polo-like kinase 1 (PLK1), a key component of mitotic progression, that is overexpressed in many human tumors. Cotransfection of plasmids for the expression of TetR and shRNA/PLK1 under the control of an H1 promoter-variant carrying TetO upstream of the TATA box did not alter PLK1 expression and proliferation properties of HeLa cells in the absence of doxycycline. Addition of the antibiotic led to marked downregulation of endogenous PLK1 accompanied by strong inhibition of cellular proliferation. Our data indicate that an inducible transcription system for shRNAs based on the human H1 promoter could be a versatile tool for controlled gene silencing in vitro.

Polo-Like Kinase 1 as Predictive Marker and Therapeutic Target for Radiotherapy in Rectal Cancer

The ability to predict tumor sensitivity toward radiotherapy may significantly impact the selection of patients for preoperative combined-modality therapy. The aim of the present study was to test the predictive value of Polo-like kinase 1 (PLK1) in rectal cancer patients and to investigate whether PLK1 plays a direct role in mediating radiation sensitivity. PLK1 expression was evaluated by immunohistochemistry (n = 76) or Affymetrix HG133 microarray (n = 20) on pretreatment biopsies of patients with advanced rectal cancer. Expression was correlated with both tumor regression in the resected specimen and long-term clinical outcome. Furthermore, we used small interfering RNAs (siRNAs) to down-regulate PLK1 expression in colorectal cancer cells and analyzed the effects of PLK1-specific siRNAs by Western blot and quantitative real-time PCR analysis, FACScan analysis, caspase 3/7 assays, and colony-forming assays. We observed that increased PLK1 protein expression was significantly related to a poorer tumor regression and a higher risk of local recurrence in uni- and multivariate analysis. A significant decrease of PLK1 expression by siRNAs in combination with ionizing radiation induced an increased percentage of apoptotic cells and increased caspase 3/7 activity. Furthermore, enhanced G(2)-M levels, decreased cellular viability, and reduced clonogenic survival were demonstrated, indicating a radiosensitizing effect of PLK1 depletion. Therefore, PLK1 may be a novel predictive marker for radiation response as well as a promising therapeutic target in rectal cancer patients.

Identification and Validation of a Potent Type II Inhibitor of Inactive Polo-like Kinase 1

The search for new therapeutic strategies is one of the main research fields in translational cancer research. The serine/ threonine kinase polo-like kinase 1 (Plk1) attracts great attention in the field of cancer therapy because it exhibits generally elevated activity in cancer cells 3] and is a negative prognostic factor for cancer patients. The importance of Plk1 activity as a measure for the aggressiveness of a tumor results from its important role in mitotic checkpoints. Plk1 inhibition by antisense oligonucleotides, small interfering RNAs, antibodies, or dominant-negative mutants has resulted in reduced Plk1 expression and activity in vitro and in vivo. A first generation of Plk1 inhibitors targeting the active conformation has entered clinical trials. Here, we present the structure-based identification and biochemical validation of a novel potent (IC50 = 200 pm) inhibitor of inactive Plk1 as a potential starting point for lead structure optimization. The high degree of conservation of kinase structure due to the same catalytic mechanism, the same cosubstrate (ATP) and similar protein folding poses the problem of inhibitor selectivity. Kinases undergo conformational changes between the active and the inactive conformation by switching crucial structural elements: the aC helix, the activation loop with the conserved DFG motif as anchor, and the glycine-rich P loop (Figure 1, figure S1 in the Supporting Information). An additional hydrophobic pocket (allosteric site), in which amino acid residues are less conserved, is accessible in the inactive conformation. As a consequence, inhibitors of kinases in the inactive conformation (type II inhibitors) are more selective over other kinases than inhibitors of the active conformation (type I). We performed structure-based virtual screening for potential type II Plk1 inhibitors using a comparative protein model (homology model) in the absence of known reference ligands—a strategy that has been successful in other hit and lead finding projects already. To cope with the structural ambiguities of the homology model, we combined pharmacophore screening and automated ligand docking methods, 24] and transferred this concept to a model of the inactive conformation of Plk1. Our homology model of Plk1 reveals some distinct structural differences in the binding site between the inactive and active conformation (Figure 1 a, cf. Supporting Information): movement of the activation loop (DFG-out), shift of the aC helix to open additional space in the hydrophobic pocket, movement of the P loop between beta-sheets b1 and b2. The resulting potential ligand-binding site of the inactive conformation is long and narrow, and might be able to accommodate stretched-out ligands like most known type II inhibitors that exhibit a common interaction profile with inactive kinase. Ligand interactions with the hinge region and the DFG motif are crucial for stabilizing an inactive kinase conformation. Therefore, we decided to develop three pharmacophore models with different interaction points in all three regions (Table 1). In total, we considered five potential pharmacophoric points within the binding pocket of Plk1 in the inactive conformation (Figure 1 b). Figure 1. Plk1 structure and homology model. a) Plk1 crystal structure (PDB 2OU7, gray) with activation loop, aC-helix, and P loop (green), superimposed with the homology model (red; inactive conformation). b) Enlarged binding site of Plk1 homology model with the five potential pharmacophore points (1: Glu 131, 2: Cys 133, 3: Cys 133, 4: Asp 194, 5: Arg 95).

Reaction-driven de novo design, synthesis and testing of potential type II kinase inhibitors

BACKGROUND De novo design of drug-like compounds with a desired pharmacological activity profile has become feasible through innovative computer algorithms. Fragment-based design and simulated chemical reactions allow for the rapid generation of candidate compounds as blueprints for organic synthesis. METHODS We used a combination of complementary virtual-screening tools for the analysis of de novo designed compounds that were generated with the aim to inhibit inactive polo-like kinase 1 (Plk1), a target for the development of cancer therapeutics. A homology model of the inactive state of Plk1 was constructed and the nucleotide binding pocket conformations in the DFG-in and DFG-out state were compared. The de novo-designed compounds were analyzed using pharmacophore matching, structure-activity landscape analysis, and automated ligand docking. One compound was synthesized and tested in vitro. RESULTS The majority of the designed compounds possess a generic architecture present in known kinase inhibitors. Predictions favor kinases as targets of these compounds but also suggest potential off-target effects. Several bioisosteric replacements were suggested, and de novo designed compounds were assessed by automated docking for potential binding preference toward the inactive (type II inhibitors) over the active conformation (type I inhibitors) of the kinase ATP binding site. One selected compound was successfully synthesized as suggested by the software. The de novo-designed compound exhibited inhibitory activity against inactive Plk1 in vitro, but did not show significant inhibition of active Plk1 and 38 other kinases tested. CONCLUSIONS Computer-based de novo design of screening candidates in combination with ligand- and receptor-based virtual screening generates motivated suggestions for focused library design in hit and lead discovery. Attractive, synthetically accessible compounds can be obtained together with predicted on- and off-target profiles and desired activities.

Drugs by Numbers: Reaction‐Driven De Novo Design of Potent and Selective Anticancer Leads

A potent and selective inhibitor of the anticancer target Polo-like kinase 1 was found by computer-based molecular design. This type II kinase inhibitor was synthesized as suggested by the design software DOGS and exhibited significant antiproliferative effects against HeLa cells without affecting nontransformed cells. The study provides a proof-of-concept for reaction-based de novo design as a leading tool for drug discovery.

Biological impact of freezing Plk1 in its inactive conformation in cancer cells

Human polo-like kinase 1 (Plk1), a key regulator of mitosis, is over-expressed in various human tumors. It is a negative prognostic factor for cancer patients and a measure for the aggressiveness of a tumor. Thus, targeting Plk1 might be a promising approach for cancer therapy. Kinase inhibitors are divided in type I inhibitors, targeting the highly conserved active conformation, and the more selective type II inhibitors, targeting the inactive conformation of kinases. We analyzed our previously identified type II Plk1 inhibitor SBE13 which is able to inhibit Plk1 activity. To determine its ability to induce cell death in cancer cells, we applied kinase assays, Western blot analyses, FACScan analyses, Caspase assays and immunofluorescence studies. We detected decreased cell proliferation, delayed progression through the cell cycle in lower SBE13 concentrations, a G2/M arrest using higher SBE13 concentrations followed by apoptosis, and abnormal mitotic figures. Notably, SBE13 did not influence activity of other kinases (Plk2, Plk3, Aurora A), indicating the selectivity of this type II Plk1 inhibitor. This study suggests that Plk1 kinase inhibitors targeting the inactive conformation of Plk1 may be considered for the development of cancer therapeutics.