Evaluation of Protein Kinase Inhibitors with PLK4 Cross-Over Potential in a Pre-Clinical Model of Cancer.

Polo-like kinase 4 (PLK4) is a cell cycle-regulated protein kinase (PK) recruited at the centrosome in dividing cells. Its overexpression triggers centrosome amplification, which is associated with genetic instability and carcinogenesis. In previous work, we established that PLK4 is overexpressed in pediatric embryonal brain tumors (EBT). We also demonstrated that PLK4 inhibition exerted a cytostatic effect in EBT cells. Here, we examined an array of PK inhibitors (CFI-400945, CFI-400437, centrinone, centrinone-B, R-1530, axitinib, KW-2449, and alisertib) for their potential crossover to PLK4 by comparative structural docking and activity inhibition in multiple established embryonal tumor cell lines (MON, BT-12, BT-16, DAOY, D283). Our analyses demonstrated that: (1) CFI-400437 had the greatest impact overall, but similar to CFI-400945, it is not optimal for brain exposure. Also, their phenotypic anti-cancer impact may, in part, be a consequence of the inhibition of Aurora kinases (AURKs). (2) Centrinone and centrinone B are the most selective PLK4 inhibitors but they are the least likely to penetrate the brain. (3) KW-2449, R-1530 and axitinib are the ones predicted to have moderate-to-good brain penetration. In conclusion, a new selective PLK4 inhibitor with favorable physiochemical properties for optimal brain exposure can be beneficial for the treatment of EBT.

Multiplexing terbium- and europium-based TR-FRET readouts to increase kinase assay capacity.

Identification and characterization of kinase inhibitor potency and selectivity is often an iterative process in which a library of compounds is first screened against a single kinase, and hits from that screen are then profiled against other kinases to determine specificity. By developing kinase assays that employ either a terbium- or a europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) readout, one can take advantage of the distinct emission properties of these labels to develop assays for 2 kinases that can be performed simultaneously in the same well. This not only increases the information content provided per assay well but can immediately provide information on compound specificity. The authors have applied this strategy to the development of multiplexed assays for 2 examples systems: EGFR and IKKbeta, as well as lipid kinase family members mTOR and PIK3C3. They demonstrate the ability of these multiplexed assays to characterize selective kinase inhibitors in a dose-response mode, with no difference in results obtained from traditional single kinase assays performed separately.

A toolbox approach to high-throughput TR-FRET-based SUMOylation and DeSUMOylation assays.

The posttranslational modification of target substrates by the ubiquitin-like proteins, specifically the small ubiquitin-like modifier (SUMO), has emerged as an essential mechanism to regulate protein function and control intracellular trafficking. Traditional methods for monitoring either the attachment or removal of SUMO, such as gel electrophoresis or western blot, are effective but typically suffer from a lack of throughput. Here, we report the development and application of time-resolved Förster resonance energy transfer (TR-FRET)-based assays capable of detecting SUMOylation or deSUMOylation in a high-throughput screening (HTS) format. Using Ran GTPase-activating protein (RanGAP1) as a model target substrate, we have demonstrated that the SUMOylation of this protein can be detected using LanthaScreen (Invitrogen, Carlsbad, CA) TR-FRET technology. Additionally, we have generated reagents useful for assessing the deSUMOylation activity of a sentrin-specific protease. All assays are performed in 384-well format and display excellent statistical data (Z' > 0.7) with high signal-to-background levels. Together, this collection of tools can be utilized in a modular approach to develop HTS assays for inhibitors of SUMOylation or deSUMOylation.