Centrinone-B (LCR-323)

Kinase experiment:

All kinase assays are performed in white 384-well plates. Plk4 assays use equal volumes of: (1) purified 6xHis-tagged human Plk4 kinase domain (aa 2-275) (expressed in E. coli and purified via Ni-NTA affinity chromatography) in 20 mM Tris pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM DTT; (2) 2X reaction buffer consisting of 50 mM HEPES pH 8.5, 20 mM MgCl2, 1 mM DTT, 0.2 mg/mL BSA, 16 μM ATP, and 200 μM A-A11 substrate (amino acid sequence: TPSDSLIYDDGLS). The Plk4 concentration in the final reaction is 2.5-10 nM with a final pH of 8.0. Inhibitors arrayed in dose response are added from DMSO stocks. Reactions are allowed to proceed for 4-16 hours at 25°C. Detection is performed using ADP-Glo reagent. Luminescence is measured on an Infinite M1000 plate reader. Data are fit using Prism and Kis are calculated from IC50 data[1].

Cell experiment:

The effect of centrinone B on melanoma cell line and normal melanocyte viability is determined using the CytoTox-Glo assay. Briefly, cells are counted and plated in a 96-well plate and next day, treated with centrinone B for 48 hours, followed by incubation for 15 min with AAF-Glo substrate (alanyl-alanylphenylalanyl-aminoluciferin), which determines a distinct intracellular protease activity related with cytotoxicity (dead-cell protease) via a luminescent signal. Cell viability is determined by subtracting the luminescent signals of dead cells (due to centrinone B) from total dead cells (after addition of digitonin to lyse remaining viable cells). Data are represented as relative light units (RLU) for viable cells[2].

References:

[1]. Wong YL, et al. Cell biology. Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science. 2015 Jun 5;348(6239):1155-60.
[2]. Denu RA, et al. Centriole Overduplication is the Predominant Mechanism Leading to Centrosome Amplification in Melanoma. Mol Cancer Res. 2018 Jan 12.