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 uniand 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. Recent data from preoperative radiochemotherapy (RCT) in rectal cancer indicate a broad variety in tumor responses, ranging from pathological complete response with no viable tumor cells left to virtually no tumor regression at all.1 Despite uniform treatment protocols, this observed heterogeneity in treatment response is most probably caused by the individual genetic accouterment of the tumor. Current research has focused on molecular markers involved in the resistance toward anticancer therapy including key elements of the apoptotic and mitotic pathways.2,3 Among these factors, the serine/threonine kinase Polo-like kinase 1 (PLK1) has emerged as prognostic factor and as promising target for cancer therapy, 4,5 because it is a key regulator for the mitotic progression of mammalian cells, 6 -8 and it is overexpressed in all human cancers analyzed up to date. -12Several preclinical approaches including the use of PLK1 antibodies, dominant-negative forms of PLK1, antisense oligonucleotides, small interfering RNA (siRNA) and small molecule inhibitors have consistently revealed cell cycle arrest at G 2 -M, and inhibition of tumor proliferation both in vitro and in vivo associated with the induction of apoptosis. [13][14][15][16][17][18][19] A growing number of small molecule inhibitors is currently under clinical investigation. 20 -24 PLK1 kinase activity is inhibited by DNA damaging agents, in an ataxia telangiectasia mutated or Rad3-related kin...

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) [1] attracts great attention in the field of cancer therapy because it exhibits generally elevated activity in cancer cells [2,3] and is a negative prognostic factor for cancer patients.[4] The importance of Plk1 activity as a measure for the aggressiveness of a tumor results from its important role in mitotic checkpoints. [5][6][7][8] 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. [8][9][10][11][12][13][14][15] A first generation of Plk1 inhibitors targeting the active conformation has entered clinical trials. [16][17][18] Here, we present the structure-based identification and biochemical validation of a novel potent (IC 50 = 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.[19] 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.[19] 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). [19] 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. [20][21][22] To cope with the structural ambiguities of the homology model, we combined pharmacophore screening and automated ligand docking methods, [23,24] 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 [19] that exhibit a common interaction profile with inactive kinase.[19] Ligand interactions with the hinge region and the DFG motif are crucial for stabilizing an inactive kinase conform...

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.

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