The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

Abstract

Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.

Keywords: kinase inhibitor; necroptosis; protein-serine-threonine kinases; signalling.

Figure 1.. ABT-869 is a previously unreported inhibitor of necroptosis.

(A) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and RIPK3, and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. (B) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in (A,B) was used under a Creative Commons Attribution-Share Alike 4.0 license. (C) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl^−/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. (D) Chemical structure of ABT-869 and its analogue WEHI-615. (E) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. (F) Mlkl^−/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. (G) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.

Figure 2.. ABT-869 inhibits necroptosis in mouse and human cells.

(A,B) Wild-type mouse dermal fibroblast (MDF) cells were treated with increasing concentrations of ABT-869 or control compounds, RIPK3 inhibitors GSK′872 and GSK′843, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) (A) or TSZ (TNF, Smac-mimetic, z-VAD-fmk) (B) for 24 h to induce necroptosis. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 3 (A) or n = 4 (B) independent experiments and error bars represent SEM. (C–F) Human U937 cells were treated with increasing concentrations of ABT-869 or control compounds, MLKL inhibitor NSA and RIPK1 inhibitor GSK′481, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) for 48 h (C) or TSI (TNF, Smac-mimetic, IDN-6556) for 24 h (E) to induce necroptosis. Parallel experiments were performed to assess protection of TSQ (D) or TSI (F) induced death in the presence of the ABT-869 analogue, WEHI-615. Cell death was monitored by SPY505 (live cells) and propidium iodide (PI; dead cells) uptake using IncuCyte live cell imaging. One representative result shown from n = 4 (C,D) or n = 3 (E,F) independent experiments. See also Supplementary Figure S2A–H.

Figure 3.. ABT-869 binds to RIPK1 in mouse and human cells.

(A) Binding affinities (K_D) of ABT-869 and WEHI-615 for human full-length MLKL, RIPK1 kinase domain and RIPK3 kinase domain measured by competition binding assays from the DiscoverX KINOMEscan platform using the KdELECT service. Each value is the mean of two replicates. (B–D) Cellular Thermal Shift Assays (CETSA) in mouse and human cells. Mlkl^−/− mouse dermal fibroblast (MDF) cells expressing MLKL Q343A (B), wild-type MDF cells (C) and human U937 cells (D) were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s, RIPK3 inhibitor GSK′872 or human RIPK1 inhibitor GSK′481 (all 20 µM). Cells were subjected to an increasing temperature gradient focused around the melting temperature of the protein of interest. Following the separation of soluble and insoluble proteins, the remaining soluble proteins were detected by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 (B,C) or n = 2–3 (D) independent experiments. See also Supplementary Figure S3A–C.

Figure 4.. ABT-869 binds to mouse and human RIPK1 in vitro.

Thermal Shift Assays (TSA) with mouse and human RIPK1 and RIPK3 kinase domains. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to alter the melting temperature (T_M) of mouse RIPK1 (9.5 µg) (A,B), human RIPK1 (12 µg) (C,D), mouse RIPK3 (10 µg) (E,F) and human RIPK3 (6.5 µg) (G,H) compared with the positive controls Compound 2 [48] for mouse RIPK1, GSK′481 for human RIPK1 and GSK′872 for mouse and human RIPK3 (all 30 µM). Data represent the mean of n = 3 independent experiments and error bars represent SEM. See also Supplementary Figure S4A–H.

Figure 5.. ABT-869 inhibits RIPK1 kinase activity in vitro and in cells.

(A–H) In vitro phosphorylation assays with mouse and human RIPK1 and RIPK3 kinase domains measured by ADP-Glo Kinase Assays. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to inhibit the autophosphorylation (IC₅₀) of mouse RIPK1 (200 nM) (A,B), human RIPK1 (200 nM) (C,D), mouse RIPK3 (10 nM) (E,F) and human RIPK3 (10 nM) (G,H). Data represent the mean of n = 3 (A,B,E,F) or n = 2 (C,D,G,H) independent experiments and error bars represent SEM. (I) Cellular phosphorylation assays. Wild-type mouse dermal fibroblast (MDF) cells were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK′872 for 2 h then stimulated with TSI (TNF, Smac-mimetic, IDN-6556) for 2 h to induce autophosphorylation of RIPK1 and RIPK3. Ripk1^−/−Mlkl^−/− MDF cells and Ripk3^−/− MDF cells were included as controls. Phospho-RIPK1 and phospho-RIPK3 protein levels were detected from whole cell lysates by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 independent experiments. See also Supplementary Figure S5A–C.

Figure 6.. ABT-869 targets RIPK1 to block necroptosis.

Schematic of the ABT-869 mechanism of action in the context of necroptosis inhibition. Necroptosis mediated by the constitutively activated MLKL Q343A mutant was inhibited by ABT-869, which binds to and inhibits RIPK1, implicating RIPK1 as having a role downstream of MLKL activation in the signalling pathway. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) was used under a Creative Commons Attribution-Share Alike 4.0 license.