The BAL-0028 derivative BAL-0598 inhibits human NLRP3 activation and binds directly to the NLRP3 NACHT domain. (A) Structure of BAL-0598. (B and C) BAL-0598 in IL-1β release assay from PMA-differentiated THP-1 cells (B) or human monocytes (C) stimulated with LPS and nigericin. Graph symbols show average IL-1β values relative to vehicle control ± SEM from independent experiments performed in triplicate; IC₅₀ curve were fitted by nonlinear regression analysis. (D and E) Comparison of BAL-0598, BAL-0028, and MCC950 in IL-1β release assay from (D) iMacs and (E) HMDM. Graph symbols show average values relative to vehicle control from independent experiments (indicated by different symbols) performed in triplicate ± SEM. (F and G) Comparison of BAL-0598, BAL-0028, and MCC950 in an IL-1β release assay in primary peritoneal macrophages isolated from WT 129S6 (F) or 129S6 mouse promoter-NLRP3 (G) mice stimulated with LPS and ATP. Graph symbols show average IL-1β values relative to vehicle control ± SEM from independent experiments performed in duplicate. (H) nanoDSF measurement of 3 μM NLRP3^NACHT incubated with 10 μM BAL-0598. Graph symbols show T_m or change in T_m relative to DMSO vehicle control ± SEM from N = 3 independent experiments, each performed in duplicate. (I and J) SPR analysis of BAL-0598 binding to NLRP3^NACHT. The sensorgram of N = 6 injections in the single cycle measurement mode yields a kinetic K_D of 189 nM (I) and a steady-state K_D of 193 nM derived from the affinity plot (J) for the binding of BAL-0598 to NLRP3. N = 3 (B, G, and H) and N = 2 (C–F).