https://doi.org/10.1007/s00392-025-02625-4
1Universitätsklinikum Leipzig Klinik und Poliklinik für Kardiologie Leipzig, Deutschland; 2Universität Keipzig Rudolf-Boehm-Institut für Pharmakologie und Toxikologie Leipzig, Deutschland
Introduction: Aberrant activity of the NLRP3 inflammasome contributes to diseases associated with acute inflammatory responses and chronic sterile inflammation. Activation of the NLRP3 inflammasome mediates pyroptotic cell death and the release of pro-inflammatory cytokines. To this date, no selective inhibitor of inflammasome activity is available for the use in humans.
Materials and Methods: We conducted a medium throughput screening including 6280 drugs or drug-like compounds that identified novel inhibitors of the NLRP3 inflammasome using propidium iodide fluorescence as readout for pyroptosis and lipopolysaccharides and nigericin as activators. Effects on cell proliferation, viability, and chemosensitivity were measured by a Celltiter-Glo assay. IL-1β concentrations were quantified in cell supernatants using an ELISA. NFκB-activity was assessed by immunoblotting to determine the ratio of p-IκBα to IκBα and p65 immunostaining, revealing translocation of p65 to the nucleus. Binding to NLRP3 was confirmed using a DARTS assay and Co-Immunoprecipitation of NLRP3 and ASC. Molecular docking analysis was performed by Creative Proteomics. Ex vivo experiments were performed in isolated peripheral blood mononuclear cells (PBMCs).
Results: Among the screening hits, nitroxoline was characterized in detail because the drug is approved for antibiotic treatment in humans. Nitroxoline exhibited no toxicity over a wide range of concentrations. Treatment of THP-1 monocytes with 80 µM nitroxoline markedly reduced the secretion of the pro-inflammatory cytokine IL-1β by 95 % from 197.8 pg ml-1 to 11.0 pg ml-1 (Fig.1 A). Nitroxoline reduced downstream events of inflammasome activation including caspase-1 activity (FAM-Flica+/7AAD+ cells control 57.1±9.4% vs. nitroxoline 27.9±15.5%) and gasdermin D cleavage (ratio cleaved/uncleaved control 8.7±4.3 vs. nitroxoline 1.3±1.3, p<0.05). The data were confirmed in cultured human PBMC where nitroxoline abrogated IL-1β secretion. Mechanistically, drug affinity responsive target assays revealed that nitroxoline directly interacts with the NACHT domain of the NLRP3 protein, inhibiting inflammasome assembly (Fig.1 B/C). Nitroxoline did not affect NF-κB-dependent gene expression analyzed by nuclear p65 translocation and IκBα phosphorylation and did not inhibit the NLR-family member NLRC4 or the AIM2 inflammasomes indicating specificity for NLRP3.
Conclusion: The data suggest nitroxoline as a new NLRP3-specific inhibitor of pyroptosis that interferes with inflammasome assembly and thereby potently reduces the release of IL-1β. Together with the promising data on toxicity, tolerability and safety, the data set the stage for clinical testing of the effects of nitroxoline on the activation of the NLRP3 inflammasome in in diseases involving chronic inflammation.
Figure 1: (A) IL-1β release from THP-1 WT cells treated with LPS and nigericin with the addition of 80 µM nitroxoline or equal volumes of DMSO as solvent control (n=4). Data are presented as mean + SD. Data were analyzed using Student’s t-test. *p<0.05. Global (B) and pose view (C) of molecular docking analysis between NLRP3 (PDB: 6NPY) presented as a cartoon model and molecule as a stick model of nitroxoline. Red dashed lines indicate hydrogen bonds, the orange dashed line π-π interactions, and yellow dashed lines van-der-Waals interactions.