BAL-0598 inhibits the activation of human and monkey NLRP3 and is a non-covalent inhibitor . Related to Figs. 5 and 7. (A) BAL-0598 in IL-1β release assay from PMA-differentiated THP-1 cells stimulated with LPS and MSU. (B) BAL-0598 effect on ASC speck formation assessed by fluorescence microscopy in PMA-differentiated THP-1 ASC-GFP cells stimulated with LPS and nigericin. (C and D) Comparison of BAL-0598, BAL-0028, and MCC950 in an LDH release assay in primary peritoneal macrophages isolated from (C) WT 129S6 or (D) 129S6 mouse promoter-NLRP3 mice stimulated with LPS and ATP. Graph symbols show average LDH values ± SEM from N = 2 independent experiments performed in duplicate. (E and F) BAL-0598 in IL-1β release assays from cells stimulated with LPS and nigericin. African green monkey CD14⁺ monocytes (E) and PBMCs (F). (A, B, E, and F) Graph symbols show average values relative to vehicle control ± SEM (A, N = 2) or SD (B, E, and F, N = 1) from independent experiments performed in duplicate (E) or triplicate (A, B, and F); IC₅₀ curve was fitted by nonlinear regression analysis. (G) Comparison of BAL-0028, BAL-0598, and MCC950 in an IL-1β release assay from PMA-differentiated THP-1 cells stimulated with LPS and nigericin. Cells were treated with compounds before nigericin stimulation, and compounds were left on or were washed out for 1 min before nigericin addition. The graph shows average IL-1β values ± SD from one experiment performed in triplicate. (H) Schematic illustration of U937 NLRP3 and NLRP3-AID mutant cell model.