Background
VER-246608 is a pan-inhibitor of pyruvate dehydrogenase kinase (PDHK; IC50s = 35, 84, 40, and 91 nM for PDHK1, -2, -3, and -4, respectively).1 It is selective for PDHKs over heat shock protein 90 (Hsp90; IC50 = >100 ?M), as well as a panel of 97 kinases at 10 ?M. VER-246608 (10 and 20 ?M) decreases the production of L-lactate, a marker of glycolytic activity, in PC3 cells cultured in D-glucose- and L-glutamine-depleted media. It reduces the growth of, and induces cell cycle arrest at the G1 phase in, serum-starved PC3 cells when used at a concentration of 20 ?M.
1.Moore, J.D., Staniszewska, A., Shaw, T., et al.VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and induces context-dependent cytostasis in cancer cellsOncotarget5(24)12862-12876(2014)
Protocol
Kinase experiment: | DELFIA assay reagents (assay buffer, wash buffer, enhancement solution and anti-rabbit IgG-Eu-N1 secondary antibody) and plates are used. Test compounds are subjected to a 10 point tripling dilution in DMSO, diluted in MOPS buffer (60 mM MOPS pH7.2, 15 mM Magnesium acetate, 60 mM KCl) and added to the enzyme mix (10 nM PDK-1, 2 and 3 or 20 nM PDK-4, 300 nM E1, 0.1 mg/mL BSA, 1 mM DTT) in 96-well V-bottom plates. The reaction is initiated by the addition of ATP to a final concentration of 5 μM followed by a 1 h incubation at 30°C. The reaction is then stopped by the addition of STOP solution (50 mM Carbonate-Bicarbonate Buffer, pH 9.6), and then transferred to 96 well DEFLIA yellow plates. The plates are then sealed and incubated o/n at 4°C. Detection and quantification of p(Ser293)E1α levels is then achieved through incubation with anti-p(Ser293)E1α primary antibody followed by anti-rabbit secondary IgG-Eu-N1 antibody and addition of enhancement solution. The time-resolved fluorescent signal is then measured using a Victor2 plate reader. The data is fitted by non-linear regression using XLFIT4 within a custom ABASE (IDBS) protocol in order to determine IC50 values[1]. |
Cell experiment: | Compound cytotoxicity is determined using the Sulforhodamine B assay for cells cultured as a monolayer. For spheroid growth experiments, PC-3 cells are seeded (500 cells/well) into 96 well round bottom plates in RPMI-1640 media containing 2.5% (w/v) Matrigel. The resultant spheroids are treated with VER-246608 (2.5, 5, 10, 20, and 40 μM) 48 h post-seeding. Spheroid volumes are determined by obtaining diameter measurements from images taken on a Zeiss Axiovert 200 M inverted microscope using the axiovision software[1]. |
参考文献: [1]. Moore JD, et al. VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate dehydrogenase kinase, disrupts Warburg metabolism and induces context-dependent cytostasis in cancer cells. Oncotarget. 2014 Dec 30;5(24):12862-76. |
没有评价数据