Fig 1.
Necroptosis signaling pathway and selectivity of compounds in this study for one or more kinases with this pathway.
(A) Necroptosis signaling pathway includes RIPK1, RIPK3 and MLKL (B) Analysis of cpd 1% inhibition of 403 non-mutant kinases when tested at 1 μM concentration (the size of the red circle indicates a higher % inhibition). (C) Cpd 4 inhibits none of the 403 non-mutant kinases when tested at 1 μM concentration, but does inhibit pseudokinase MLKL.
Table 1.
Definition of the terms used to calculate degree of rescue or viability, based on presence or absence of compound and TNF.
Fig 2.
Chemical structure of MLKL binders described in text.
Table 2.
Compound binding affinities for MLKL, RIPK1 and RIPK3, differential thermal shift values and Necroptosis Inhibition.
Fig 3.
Necroptosis assay using FADD-deficient Jurkat cells measuring compound-dependent rescue from cell death.
The different compounds described in text were tested for rescue in dose-response curves: (A) cpd 1 (B) cpd 2 (C) cpd 3 (D) cpd 4 (E) Crizotinib (F) Necrostatin (Nec1s). The blue line represents the rescue experiment from necroptosis in the presence of TNF and the yellow line represents the viability experiment in the absence of TNF.
Fig 4.
Changes in SHG intensity over time upon compound binding.
(A) full length (FL) MLKL and (B) pseudokinase (PK) domain SHG intensity with 10 μM compound addition. (C) Quantitation of % change overall for the kinetic curves.
Fig 5.
Superposition of the crystal structures of MLKL pseudokinase bound to cpd 1 (green) or cpd 4 (magenta).
The C-terminal domain of both structures were superimposed. Phe 350 of the GFE motif in hMLKL is highlighted to demonstrate the conformational changes induced by binding the type II cpd 1. The catalytic lysine (K230) which is labeled by the SHG-sensitive dye is also highlighted.
Fig 6.
MSD assay monitoring RIPK3-dependent phosphorylation of the MLKL activation loop.
(A) GSK872 compound that specifically inhibits RIPK3 inhibits phosphorylation of MLKL as positive control. (B) Crizotinib binding to MLKL does not impact its phosphorylation by RIPK3. (C) Cpd 1 binding to MLKL does not impact its phosphorylation by Ripk3. (D) Cpd 4 binding to MLKL does not impact its phosphorylation by RipK3. Data is normalized against a reaction in the presence of DMSO alone (100% activity).