Necroptosis is a process of programmed cell death and has been described as part of disease progression in neurodegeneration. The final step in the molecular pathway of necroptosis is RipK3’s phosphorylation of MLKL within its activation loop (Ser358), which triggers its translocation to the cell membrane, oligomerization, and pore formation resulting in the holes that cause cell death. MLKL is a pseudokinase, and contains an ATP-binding domain and a pore-forming domain. In this study, we investigate the conformational changes involved in binding of an ATP-pocket MLKL inhibitor (cpd 1), the only MLKL ATP-pocket inhibitor that prevents necroptosis, and 4 MLKL-selective compounds. We utilize a new technology, SHG, to measure real-time conformational changes of a labelled protein upon ligand binding. An MLKL phosphorylation assay, structure-based visualization of inhibitor binding, cell-based necroptosis assay and kinase selectivity panels were also used to understand the effect of the compounds on MLKL phosphorylation and subsequent necroptosis rescue upon inhibitor binding to MLKL. The data indicate that SHG can distinguish type I and II ATP-pocket inhibitors of MLKL with distinct SHG signal changes (positive or negative). But the necroptosis assay reveals that none of these inhibitors can rescue necroptosis, possibly because inhibitor binding-induced conformational changes of MLKL are not sufficient to prevent MLKL phosphorylation at Ser358. The reported necroptosis rescue effect of cpd 1 is likely due to its inhibition of upstream RIPK1. Our study indicate SHG is a sensitive technology to triage HTS hits and perform targeted screens; it provides conformational change information rather than KDs obtained from conventional biochemical and biophysical technologies.

MLKL: A study of conformational changes

2019 Spring National Meeting

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