Fig. 5: Nitroxoline inhibits NLRP3 by targeting the NACHT domain.

IL-1β release from THP-1 WT cells treated with 1 µg ml⁻¹ LPS and 3 µg ml⁻¹ polydA:dT (n = 3, A) or 1 µg ml⁻¹ LPS and NeedleTox (n = 5, B). Nitroxoline and the solvent control were added as indicated. C Co-immunoprecipitation from THP-1 ASC-GFP cells treated with 1 µg ml⁻¹ LPS for 3 h and 5 µg ml⁻¹ nigericin using α-GFP antibody. 120 µM nitroxoline and solvent control were added as indicated. D Schematic representation of the domains present in full-length NLRP3 and the deletion constructs NLRP3-PYD and NLRP3ΔLRR. E DARTs assay of pEGFP-C2-NLRP3-transfected HEK293 cell lysate incubated with the indicated concentration of nitroxoline or solvent control digested with pronase (28 ng µg⁻¹) for 30 min and analysed by immunoblot. DARTs assay of HEK293 cell lysates transfected with NLRP3-PYD (F) or NLRP3ΔLRR (G). Next, 160 µM nitroxoline or an equivalent volume of solvent control was added as indicated. Pronase (28 ng µg⁻¹) was added for 30 min, and digestion levels were analyzed by immunoblotting. GAPDH was used as a loading control. Global (H) and pose view (I) of molecular docking analysis between NLRP3 (PDB: 6NPY) presented as a cartoon model and the nitroxoline molecule as a stick model. Red dashed lines indicate hydrogen bonds, the orange dashed line π-π interactions, and yellow dashed lines van-der-Waals interactions. J Quantification of ASC Specks per nucleus of HEK293 cells co-transfected with ASC-Myc and either NLRP3-GFP (WT), NLRP3-GFP-D272N, NLRP3-GFP-F297A, or NLRP3-GFP-R335A. 120 µM nitroxoline or an equivalent volume of solvent control was added as indicated. Data are presented as mean + SD. Data were analyzed using Student’s t-test. *p < 0.05.