New NLRP3 inhibitor blocks inflammation through a different route than MCC950

Inflammation underlies many age-related conditions, including neurodegeneration, metabolic disease and cardiovascular disease. The NLRP3 inflammasome is a central driver, triggering release of IL-1β and IL-18 and a form of inflammatory cell death called pyroptosis. Current options are biologics that block IL-1 signaling. Small-molecule NLRP3 inhibitors could also block IL-18 and pyroptosis and reach the brain. Most clinical-stage candidates are sulfonylureas or MCC950 derivatives, which have off-target effects (such as carbonic anhydrase 2 inhibition) and are weak against certain NLRP3 mutations. This makes chemically distinct inhibitors valuable.

The team characterized BAL-0028, an indazole identified from a DNA-encoded library screen of more than 500 billion compounds, plus a derivative, BAL-0598. They tested the compounds in THP-1 cells, primary human monocytes and macrophages, iPSC-derived microglia and macrophages, and human whole blood. Readouts included IL-1β and LDH release, caspase-1 cleavage and ASC speck formation. They also ran cross-species assays, in vitro ATPase assays, and binding studies (nanoDSF, SPR, DARTS). Finally, they tested BAL-0598 in humanized NLRP3 mice.

BAL-0028 inhibited NLRP3-dependent IL-1β release with an IC50 of 57.5 nM in THP-1 cells (MCC950: 14.3 nM). It was active across multiple stimuli (nigericin, ATP, MSU) and cell types, and equipotent with MCC950 in iPSC-derived microglia. It blocked caspase-1 activation, IL-1β processing and ASC specks without affecting LPS priming, and was not cytotoxic. It did not inhibit AIM2, NAIP/NLRC4 or NLRP1 inflammasomes below 10 μM, though partial off-target effects appeared at 10 μM. Potency dropped more than 100-fold in mouse cells and was absent in rat, dog and rabbit cells, yet it was retained in African green and cynomolgus monkey cells and in mouse cells expressing human NLRP3. A cluster of residues in the FISNA region differs between primates and other mammals and may explain this.

Mechanistically, BAL-0028 did not inhibit NLRP3 ATPase activity, unlike MCC950. It bound the NACHT domain (SPR KD ~96–113 nM). In competition nanoDSF it further stabilized MCC950-bound NACHT, and the two compounds acted additively rather than synergistically in cells, supporting a distinct binding site. BAL-0598 was designed to reduce plasma protein binding (about 98.4% versus 99.9% bound). It kept the same species selectivity, reversibility and NACHT binding (KD ~193 nM). It inhibited NLRP3 in vivo in a peritonitis model in humanized mice, and both compounds inhibited select hyperactive NLRP3-AID mutants more potently than MCC950.

These compounds represent a new modality for NLRP3 inhibition, potentially useful where MCC950-type drugs fail. Caveats include very high plasma protein binding, the need for humanized models because of species selectivity, partial off-target activity at high concentrations, and the lack of human data. The full-text excerpt was truncated before the in vivo and mutation results were presented in detail.