Discovery and Structure-Based Optimization of Fragments Binding the Mixed Lineage Kinase Domain-like Protein...

Necroptosis is a form of programmed cell death that in case of misregulation can lead to inflammatory diseases. Mixed lineage kinase domain-like protein (MLKL), the effector protein in the canonical necroptosis signaling pathway, becomes activated by phosphorylation. Here, we report the identification of novel reversible binders of the MLKL executioner domain by a protein NMR-detected fragment-based screen. Determination of protein fragment costructures using NMR spectroscopy revealed a small molecule binding site that is distinct from the previously identified binding site of covalent MLKL inhibitors. Affinity optimization of the initially prioritized hit with millimolar affinity was achieved by NMR-guided structure-based design and yielded fragment-like molecules with a K_D of 50 μM. Furthermore, we demonstrate that the improved fragment competes for the same binding site as nonyl-maltoside, a detergent that in conjunction with phytic acid activates the MLKL executioner domain.