qHTS for Inhibitors of PLK1-PDB (polo-like kinase 1 - polo-box domain): Primary Screen
The polo-like kinase 1 (Plk1) is a member of a conserved subfamily of serine / threonine protein kinases that plays a pivotal roles in cell proliferation. It has a C-terminal non-catalytic region called the polo-box domain (PBD) that plays a crucial role in the subcellular localization of the Plk1. Recent publications also revealed that Plk1 is the major kinase required for the viability of more ..
BioActive Compounds: 10182
The polo-like kinase 1 (Plk1) is a member of a conserved subfamily of serine / threonine protein kinases that plays a pivotal roles in cell proliferation. It has a C-terminal non-catalytic region called the polo-box domain (PBD) that plays a crucial role in the subcellular localization of the Plk1. Recent publications also revealed that Plk1 is the major kinase required for the viability of activated Ras or inactivated p53 mutation-bearing cancer cells, but not the respective normal cells. Furthermore, depletion of the overexpressed Plk1 by RNAi efficiently induces apoptotic cell death in cancer and reduces tumor growth in mouse xenograft models. Thus, targeting Plk1 is an appealing strategy for anti-cancer therapy.
Therefore, this project aimed to screen the Molecular Libraries Small Molecule Repository (MLSMR) using a novel approach to find inhibitors that target the Plk1 PBD instead of the conventional N-terminal catalytic domain. This project employed a fluorescence polarization (FP) assay using a FITC-conjugated phospho-9-mer (p-9-mer) peptide and purified human Plk1 PBD protein. The identified small molecule inhibitors of Plk1 PBD can then further be developed into useful tools for biomedical and clinical research that complements or replaces current relevant disease therapy.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH100728
Assay Submitter (PI): Kyung Lee, NCI
Four microliter of 25 nM FITC-p-9-mer + 60 nM Plk1 PBD in buffer solution (final concentrations 25 nM and 60 nM respectively) or 4 uL of 25 nM FITC-p-9-mer (final concentration 25 nM) in buffer will be dispensed into a 1,536 Greiner medium-binding black solid plate. Small molecules at final concentrations of 18 nM to 57 uM and positive control phospho-9-mer (sequence: Ac-PPLHSpTAI-NH2) at final concentrations 26 nM to 57 uM and phospho-13-mer (sequence: Ac-CETFDPPLHSpTAI-NH2) at final concentrations of 1 nM to 2.3 uM will be pin-transferred (23 nL) to the plate via a Kalypsys pin-tool robotics equipped with a 1,536-pin array. The reaction plate will be centrifuged for 1 min at 1000 rpm and incubated at room temperature for 10 min. The FITC fluorescence signals will be read using the PerkinElmer ViewLux plate reader at 480 / 20 nm excitation and 540 / 20 nm S and P polarization emission wavelengths. Reagent bottles will be kept submerged into 4 oC recirculating chiller bath and all liquid lines will be covered with aluminum foil to minimize degradation. Library plates will be screened starting from the lowest to the highest concentrations to minimize compound carryover. Vehicle only plates, with DMSO being pin-transferred instead of compound solutions, will be included regularly throughout the screen to record any systematic shifts in assay signal.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)