Phenotypic screening identifies a new oxazolone inhibitor of necroptosis and neuroinflammation

Abstract

Necroptosis is a regulated form of necrosis, which may be critical in the pathogenesis of neurodegenerative diseases. Neuroinflammation, characterized by the activation of glial cells such as microglia, is closely linked with neurodegenerative pathways and constitutes a major mechanism of neural damage and disease progression. Importantly, inhibition of necroptosis results in disease improvement, unveiling an alternative approach for therapeutic intervention. In the present study, we screened a small library of new molecules, potentially inhibitors of necroptosis, using two cellular models of necroptosis. A new oxazolone, Oxa12, reduced tumour necrosis factor α (TNF-α)-induced necroptosis in mouse L929 fibrosarcoma cells. Notably, Oxa12 strongly inhibited zVAD-fmk-induced necroptosis in murine BV2 microglial cells. Moreover, Oxa12 blocked phosphorylation of mixed-lineage kinase domain-like protein (MLKL), and interfered with necrosome complex formation, indicating that Oxa12 targets components upstream of MLKL. In fact, in silico molecular docking studies revealed that Oxa12 is occupying a region similar to the 1-aminoisoquinoline type II kinase inhibitor inside the receptor-interacting protein 1 (RIP1) kinase domain. Finally, in microglial cells, Oxa12 attenuated zVAD-fmk- and lipopolysaccharide (LPS)-induced inflammatory processes, as revealed by a marked decrease of TNF-α and/or IL-1β expression. More specifically, Oxa12 negatively targeted c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways, as well as NF-κB activation. Overall, we identified a strong lead inhibitor of necroptosis that is also effective at reducing inflammation-associated events. Oxa12 is a promising candidate molecule for further development to target disease states dependent on RIP kinase activity.

Discovery of a Necroptosis Inhibitor Improving Dopaminergic Neuronal Loss after MPTP Exposure in Mice

“…The anti-necroptotic effects, however, may not be solely dependent on this target. These results are in line with our previous work, where we showed the sequestration of key necroptosis mediators, RIP1, RIP3, and p-MLKL, in the insoluble fraction in zVAD-fmk-treated BV2 cells, while Oxa12 abolished necrosome assembly and MLKL phosphorylation [ 22 ]. Moreover, Oxa12 strongly rescued zVAD-fmk-induced cell death, as observed by microscopy analysis of cell morphology [ 22 ].…”

“…These results are in line with our previous work, where we showed the sequestration of key necroptosis mediators, RIP1, RIP3, and p-MLKL, in the insoluble fraction in zVAD-fmk-treated BV2 cells, while Oxa12 abolished necrosome assembly and MLKL phosphorylation [22]. Moreover, Oxa12 strongly rescued zVAD-fmk-induced cell death, as observed by microscopy analysis of cell morphology [22]. Furthermore, our previous in silico molecular docking calculations for Oxa12 inside the RIP1 kinase domain demonstrated that Oxa12 is occupying a region similar to the co-crystallized inhibitor, suggesting a similar interaction pattern and indicating that Oxa12 most likely targets RIP1 to some extent [22].…”

“…Previous studies have demonstrated that the pan-caspase inhibitor zVAD-fmk induces necroptosis in different cellular models, including in the L929 fibrosarcoma and in the BV2 murine microglial cell lines, by a mechanism that most likely depends on TNF autocrine secretion [ 12 , 21 , 22 ]. Here, we used BV2 cells incubated with zVAD-fmk as an in vitro model of necroptosis and screened a total of 31 small compounds potentially inhibitors of this type of cell death.…”

“…These compounds are part of a larger in-house library containing mainly the heterocyclic core of 4-methylene-2-aryloxazol-5(4H)-one and are represented in Figure 2 . The remaining compounds have been tested in previous papers [ 12 , 21 , 22 ]. Cells were incubated with 25 µM zVAD-fmk alone or in combination with 30 µM test compounds for 24 h. BV2 cells treated with DMSO were used for data normalization.…”

Necroptosis in inflammatory bowel disease and other intestinal diseases

“…Rats with cognitive impairment induced by severe acute pancreatitis show increased levels of RIPK3 and RIPK1, and berberine can partly treat this cognitive impairment through attenuation of neuronal necroptosis[ 47 ]. Oliveira et al[ 48 ] have found that Oxa12, a new oxazolone, can reduce TNF-α-induced necroptosis in mouse L929 fibrosarcoma cells and zVAD-fmk-induced necroptosis in murine BV2 microglial cells. They have confirmed that Oxa12 disturbs the phosphorylation of MLKL and formation of necrosome complexes, which provides a new insight into the treatment of other inflammatory diseases.…”