Figure 2
Development of a CETSA for human RIPK1 with HT-29 cells. (a) Chemical structure of representative tool compounds. (b) Tagg curves for RIPK1 in HT-29 cells in the presence of DMSO (0.1%) (black closed circle), 10 μM compound 25 (red square), GSK-compound 27 (purple diamond), or compound 15 (orange open circle). Evaluation of 3 min and 8 min denature conditions was performed to verify the optimized conditions. All data were normalised to the response observed at DMSO-treated conditions at room temperature. The Tagg shift was analysed using the Boltzmann sigmoid equation. The vertical dotted line is at 47 °C for 8 min denature, the experimental condition selected for the ITDRF assay. Data are provided as the average and SEM performed in duplicate. (c,d) ITDRF of representative RIPK1 inhibitors at 47 °C for 8 min denature based on raw data from the Western blotting chemiluminescence readings. The chemiluminescence data are shown above the graphs. ITDRF lines are fitted with a four-parameter logistic curve. The corresponding ITDRF for compound 25 (c, black circle), Nec-1 (c, blue triangle), compound 22 (d, black circle), and GSK-compound 27 (d, blue triangle) result in EC50 of 5.0 nM (95% CI 2.8–9.1), 1,100 nM (95% CI 500–2,400), 6.5 nM (95% CI 4.3–9.8), and 1,200 nM (95% CI 810–1,700), respectively. Data are provided as the average and SEM performed in duplicate. All full-length Western blotting images are presented in Supplementary Fig. 8. CETSA, cellular thermal shift assay; DMSO, dimethyl sulfoxide; ITDRF, isothermal dose-response fingerprint; RIPK1, receptor interacting protein 1 kinase; SEM, standard error of the mean; Tagg, aggregation temperature.
