Figure 1: Development of a no-wash CETSA for human TS.
(a) Overview of the assay principle with live K562 cells seeded into a 384-well PCR plate. The plate contains controls or library compounds that are taken up by the cells. Following a pre-incubation period the plate is transiently heated for 3 min followed by cooling and cell lysis. Part of the cell lysate is transferred to a detection plate, to which antibodies and AlphaScreen beads are added to allow measurements of remaining soluble TS. (b) CETSA derived Tagg curves for TS in K562 cells in the presence of DMSO (0.5%) (green circle), 15 μM floxuridine (blue triangle) or 1 μM raltitrexed (magenta square). All data were normalized to the response observed for each treatment condition at the lowest test temperature. The solid line represents the best fit to the Boltzmann sigmoid equation resulting in an apparent Tagg of 46.7±0.2 °C for the DMSO control, whereas both floxuridine and raltitrexed stabilized TS above 65 °C (we do not consider higher Tagg values reliable as these temperatures influence cell membrane integrity10). The vertical dotted line is at 50 °C, the temperature selected for the isothermal screen. Data are provided as the average and standard error of mean (s.e.m.) from two independent experiments performed in duplicate for raltitrexed and as individual data points from one experiment in duplicate for floxuridine. (c) ITDRFCETSA of floxuridine (blue triangle) at 50 °C based on raw data from the AlphaScreen readings. The solid line represents the best fit to a saturation binding curve resulting in an EC50 of 47±16 pM. Data are provided as two individual data points from one test occasion. (d) The corresponding ITDRFCETSA for raltitrexed (magenta square) at 50 °C resulting in an EC50 of 0.75±0.2 nM. Data are provided as two individual data points from one test occasion.
