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

Abstract

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.

Fragment-to-Lead Medicinal Chemistry Publications in 2021

“…Additionally, an NMR fragment screen against a regulator of G-protein signaling, RGS17, identified fragments that bind potentially at a site on RGS17 that was previously unknown . Moreover, an NMR fragment screen identified binders of mixed lineage kinase domain-like protein (MLKL) at a site distinct from previously reported sites …”

Recent advances in targeting protein kinases and pseudokinases in cancer biology

“…In contrast, mutations mimicking RIP3mediated phosphorylation within the PsKD promoted the ON state (Petrie et al, 2018), however, in human MLKL they were not sufficient to induce necroptosis in a cell model. This knowledge has been the basis for the development of small molecules (Rübbelke et al, 2020;Rübbelke et al, 2021).…”

“…Moreover, many pseudokinases did not retain their nucleotide-binding ability, such as the PEAK family, or even lack a defined binding pocket that could be targeted by ATPcompetitive molecules. Therefore, other binding surfaces outside the kinase-domain are searched for (Rübbelke et al, 2021), and novel therapeutic strategies, such as ProTACs or hydrophobic tagging (HyT) that induce degradation of specific proteins are also being pursued (Kung and Jura, 2019).…”

Discovery of a New Class of Uracil Derivatives as Potential Mixed Lineage Kinase Domain-like Protein (MLKL) Inhibitors

“…However, the ability of these compounds to inhibit necroptosis at the cellular level could not be demonstrated. 27 Therefore, there is still a need to develop new MLKL inhibitors with better drug profiles.…”

“…Noncovalent MLKL binders targeting the opposite end of Cys86 in the N-terminal domain have recently been reported (Figure ). However, the ability of these compounds to inhibit necroptosis at the cellular level could not be demonstrated . Therefore, there is still a need to develop new MLKL inhibitors with better drug profiles.…”