BAL-0028 does not inhibit NLRP3 ATPase activity and binds to the NLRP3 NACHT at a site distinct from MCC950. (A and B) Comparison of BAL-0028 and MCC950 in an ATPase activity assay with (A) recombinant MBP-ΔNLRP3-HIS protein (NLRP3ΔPYD) and (B) recombinant, full-length NLRP3 protein peak 1. Graph symbols show average values relative to vehicle control ± SEM from (A) N = 3 or (B) N = 2 independent experiments. (C) Schematic illustration of the NLRP3^NACHT recombinant protein used for nanoDSF studies. (D and E) nanoDSF analysis of 3 μM NLRP3^NACHT incubated with (D) 10 μM BAL-0028 or MCC950 or preincubated with (E) 10 μM MCC950 before addition of 10 μM BAL-0028 or 10–30 μM MCC950. Graph symbols show T_m or change in T_m relative to DMSO vehicle control or relative to MCC950-bound NLRP3^NACHT ± SEM from N = 3 independent experiments. (F and G) SPR analysis of BAL-0028 binding to NLRP3^NACHT. The sensorgram of N = 6 injections in the single cycle measurement mode yields a kinetic K_D of 113 nM (F) and a steady-state K_D of 96 nM derived from the affinity plot (G) for the binding of BAL-0028 to NLRP3.