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.

Fig. 1. zVAD-fmk induces necroptosis in BV2 cells at 24 h of incubation.

a BV2 cells were pre-exposed to 100 ng/mL LPS for 24 h and then incubated with the pan-caspase inhibitor zVAD-fmk (25 µM) for additional 24 h. Nec-1 (30 µM) was added 1 h before zVAD-fmk. Cell metabolic activity was determined by the MTS metabolism assay and cell membrane integrity by the LDH activity assay. Results are presented as the mean value ± SEM of three independent experiments performed in duplicates and normalized to control cells. *p < 0.001 vs. control; ^§p < 0.001 vs. LPS; ^Ɨp < 0.001 vs. zVAD-fmk; ^ǂp < 0.001 vs. LPS/zVAD-fmk. b BV2 cells were co-incubated with zVAD-fmk (25 µM) plus Nec-1 (30 µM) for 24 h. Detergent soluble and insoluble fractions were prepared for Western blot analysis of RIP1 and RIP3. β-actin was used as loading control. Representative immunoblots are presented

Fig. 2. Drug screening identifies Oxa12 as necroptosis inhibitor in BV2 microglia cells.

a Schematic overview of the drug screening workflow. b Normalized necroptosis inhibition values depicted as percentage of control (DMSO) for compounds (30 µM) tested on BV2 cells exposed to 25 µM zVAD-fmk for 24 h. Results are presented as the mean value ± SEM of at least three independent experiments performed in duplicates. c Oxa12 chemical structure. d BV2 cells were incubated with Oxa12 (0.1–50 µM) plus zVAD-fmk (25 µM) for 24 h. e BV2 cells were incubated with Oxa12 (1–150 µM) for 24 h. Cell viability was determined by the MTS metabolism assay. The results are presented as the mean value ± SEM of three independent experiments performed in duplicates and normalized to vehicle control (DMSO)

Fig. 3. Drug screening identifies Oxa12 as necroptosis inhibitor in L929 cells.

a Schematic overview of the drug screening workflow. b Normalized necroptosis inhibition values depicted as percentage of control (DMSO) for compounds (30 µM) tested on L929 cells exposed to 30 µM TNF-α for 8 h. Results are presented as the mean value ± SEM of at least three independent experiments performed in duplicates. c L929 cells were incubated with Oxa12 (0.1 to 50 µM) plus TNF-α (30 µM) for 24 h. d L929 cells were incubated with Oxa12 (1–150 µM) for 24 h. Cell viability was determined by the MTS metabolism. The results are presented as the mean value ± SEM of three independent experiments performed in duplicates and normalized to vehicle control (DMSO)

Fig. 4. In silico molecular docking calculations for Oxa12.

a Optimal poses obtained inside RIP1 active site (grey) for compound Oxa12 (represented in stick model and coloured blue) compared with crystallographic ligand 1-aminoisoquinoline inhibitor (PDBID: 4NEU) (yellow). b Compound Oxa12 and 4NEU co-crystallized inhibitor interacting with Asp156, Leu157, Met67, and Met95. Docking calculations were performed using the X-ray structure obtained for RIP1 complexed with 1-aminoisoquinoline inhibitor at resolution of 2.57 Å, PDBID: 4NEU, by the GOLD 5.2 software

Fig. 5. Oxa12 inhibits necroptosis in a murine microglial cell line.

a BV2 cells were incubated with zVAD-fmk (25 µM), zVAD-fmk (25 µM) plus Nec-1 (30 µM), or zVAD-fmk (25 µM) plus Oxa12 (30 µM) for 24 h. Detergent soluble and insoluble protein fractions were prepared for Western blot analysis of RIP1, RIP3, MLKL, and p-MLKL. Representative immunoblots are presented. β-actin was used as loading control. b Densitometric analysis. Values are expressed as mean ± SEM of three independent experiments. ^ǂp < 0.05 vs. control; *p < 0.05 vs. zVAD-fmk. c Bright-field microscopic images of BV2 cells incubated with zVAD-fmk (25 µM) in the presence of absence of Nec-1 (30 µM) or Oxa12 (30 µM) for 24 h. Microscopy photographs were taken at ×100 with a Primo Vert microscope. Scale bar=100 µm

Fig. 6. Oxa12 decreases TNF-α gene expression and protein secretion levels.

a BV2 cells were incubated with zVAD-fmk (25 µM) in the presence or absence of Nec-1 (30 µM) or Oxa12 (30 µM) for 24 h. TNF-α mRNA levels were measured by qRT-PCR and secreted TNF-α by ELISA. Results are expressed as mean ± SEM from three independent experiments. *p < 0.001 vs. control; ^ǂp < 0.001 vs. zVAD-fmk. b BV2 cells were incubated with 100 ng/mL LPS, LPS plus zVAD-fmk (25 µM) in the presence or absence of Nec-1 (30 µM) or Oxa12 (30 µM) for 24 h. TNF-α and IL-1β mRNA levels were measured and results are expressed as mean ± SEM from three independent experiments. *p < 0.001 vs. control; ^Ɨp < 0.001 vs. LPS; ^¶p < 0.001 vs. LPS/zVAD-fmk

Fig. 7. Oxa12 decreases zVAD-fmk-induced JNK and p38 MAPK activation in BV2 cells.

BV2 cells were treated with zVAD-fmk (25 µM) in the presence or absence of Nec-1 (30 µM) or Oxa12 (30 µM) for 24 h. Representative immunoblots are presented, together with the respective densitometric analysis of the p-JNK/JNK, p-p38/p38 and p-Akt/Akt ratios. a p-JNK (Thr183/Tyr185) and total JNK. b p-p38 (Thr180/Tyr182) and total p38. c p-Akt (Ser473) and total Akt. Results are expressed as mean ± SEM of three independent experiments. *p < 0.001 vs. control; ^ǂp < 0.05 vs. zVAD-fmk; ^Ɨp < 0.01 vs. control; ^§p < 0.001 vs. zVAD-fmk

Fig. 8. Oxa12 reduces NF-κB/IκB ratio and NF-κB p65 nuclear translocation when compared to zVAD-fmk-treated cells.

a BV2 cells were incubated with zVAD-fmk (25 μM) in the presence or absence of Nec-1 (30 µM) or Oxa12 (30 µM) for 24 h. Representative immunoblots of total NF-κB are presented together with the respective densitometric analysis of NF- κB/IκB ratio. β-actin was used as loading control. Results are expressed as mean ± SEM from three independent experiments. *p < 0.05 vs. control; ^ǂp < 0.05 vs. zVAD-fmk. b Representative images of immunofluorescence staining showing NF-κB p65 (red) nuclear translocation in BV2 cells treated with zVAD-fmk (25 µM) in the presence or absence of Nec-1 (30 µM) or Oxa12 (30 µM) for 5 h, and quantification of mean fluorescence intensity. Cell nuclei were detected by Hoechst (blue). Scale bar=1 µm