Fig. 6: AMG-47a is a dual RIPK1-RIPK3 inhibitor.
From: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL
A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 (D) or MDF (E) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 (F) and RIPK3 (G) was measured in competitive binding assays using the KINOMEscan® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the Kd and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. Kd determinations for individual replicates can be found in supplementary data (Supplementary Fig. 4A–F). H, I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.
