title: Development and implementation of a miniaturized high-throughput time-resolved fluorescence energy transfer assay to identify small molecule inhibitors of polo-like kinase 1.
abstract: Polo-like kinase (Plk) 1 is a key enzyme involved in regulating the mammalian cell cycle that is also a validated anticancer drug target. Nonetheless, there are relatively few readily available potent and selective small molecule inhibitors of Plk1. To increase the availability of pharmacologically valuable Plk1 inhibitors, we describe herein the development, variability assessment, validation, and implementation of a 384-well automated, miniaturized high-throughput time-resolved fluorescence energy transfer screening assay designed to identify Plk1 kinase inhibitors. Using a small molecule library of pharmaceutically active compounds to gauge high-throughput assay robustness and reproducibility, we found nine general kinase inhibitors, including H-89, which was selected as the minimum control. We then interrogated a 97,101 compound library from the National Institutes of Health repository for small molecule inhibitors of Plk1 kinase activity. The initial primary hit rate in a single 10 microM concentration format was 0.21%. Hit compounds were subjected to concentration-response confirmation and interference assays. Identified in the screen were seven compounds with 50% inhibitory concentration (IC50) values below 1 microM, 20 compounds with IC50 values between 1 microM and 5 microM, and eight compounds with IC50 values between 5 and 10 microM, which could be assigned to seven distinct chemotype classes. Hit compounds were also examined for their ability to inhibit other kinases such as protein kinase D, focal adhesion kinase, rho-associated coiled coil protein kinase 2, c-jun NH2-terminal kinase 3, and protein kinase A via experimentation or data-mining. These compounds should be useful as probes for the biological activity of Plk1 and as leads for the development of new selective inhibitors of Plk1.
authors.affiliation: Department of Pharmacology, Pittsburgh Molecular Libraries Screening Center, and Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Sharlow
authors.forename: Stephanie
authors.initials: S
authors.lastname: Leimgruber
authors.forename: Tong Ying
authors.initials: TY
authors.lastname: Shun
authors.forename: John S
authors.initials: JS
authors.lastname: Lazo
mesh_terms: D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D003627:Data Interpretation, Statistical; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008904:Miniaturization; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D016622:Silver Staining; D013379:Substrate Specificity; D013696:Temperature; D000097869:Polo-Like Kinase 1
chemical_list: D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1; C089212:protein kinase D