Nitroxoline inhibits pyroptosis by directly binding to the NACHT domain of NLRP3

Introduction Uncontrolled activity of the NLRP3 inflammasome contributes to diseases associated with acute inflammatory responses and chronic sterile inflammation including atherosclerosis and reperfusion injury after myocardial infarction. Purpose Currently, no selective inflammasome inhibitors are available for human use. Therefore, we aimed to identify and validate novel inhibitors. Materials and Methods Medium-throughput screens of 6,280 drugs and drug-like compounds were performed using propidium iodide fluorescence as a pyroptosis readout. Among 22 potential candidates, the antibiotic nitroxoline, one of the most promising candidates to inhibit lipopolysaccharide (LPS)- and nigericin-induced pyroptosis, was further characterised. Results Nitroxoline (80 μM) reduced IL-1β secretion by 95% (from 197.8 pg/ml to 11.0 pg/ml) in THP-1 monocytes (Fig.1 A). Celltiter-Glo assay indicated that there was no significant effect on cell viability at this concentration. 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) assessed via flow cytometry and western blot. This finding was confirmed in cultured human PBMCs, where nitroxoline abrogated IL-1β secretion. Mechanistically, nitroxoline directly interacts with the NLRP3 NACHT domain, inhibiting inflammasome assembly (drug affinity responsive target assay, 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 Nitroxoline is a novel, NLRP3-specific inhibitor of pyroptosis that interferes with inflammasome assembly, potently reducing IL-1β release. Given its established safety profile as an antibiotic approved for long-term use, nitroxoline warrants clinical investigation for NLRP3-driven cardiovascular diseases. Figure legend: 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 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.