Walking over the inflammasome

The NLRP3 inflammasome contributes to pathogenic inflammation in a broad range of diseases, making it a highly relevant drug target. Two studies published in this issue found an inhibitor of NLRP3 inflammasome activation to directly bind NLRP3 within its central NACHT domain, interfering with ATP hydrolysis and structural changes critical for NLRP3 oligomerization and subsequent inflammasome formation.

Inflammasomes are cytoplasmic danger-sensing platforms in immune cells controlling the activity of leaderless IL-1 family cytokines and, thereby, inflammation. Whereas other inflammasomes react to pathogens, NLRP3 is primarily triggered by sterile, host-derived or environmental danger signals. As such, it plays a causative role in numerous acquired and hereditary disease conditions¹. Development of therapeutics that specifically inhibit NLRP3 while leaving the protective activity of other types of inflammasome intact has been obstructed by the lack of precise structural information and the missing understanding of the mode of action of the established inhibitor MCC950. The Schroder² and Pelegrin³ laboratories now independently provide compelling evidence for direct inhibition of NLRP3 by binding to its Walker B motif and promoting autoinhibition (Fig. 1).

Fig. 1: MCC950 action on NLRP3.

Together, the studies of Coll et al.² and Tapia-Abellán et al.³ suggest that the small-molecule compound MCC950 inhibits NLRP3 inflammasome activity by directly binding to the Walker B motif within the NACHT domain of NLRP3, preventing ATP hydrolysis and enforcing a closed, inactive conformation.

Like other members of the nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) family, NLRP3 harbors ATPase activity in its central nucleotide-binding and oligomerization (NACHT) domain. It contains Walker A and Walker B motifs, responsible for binding and hydrolysis of ATP, respectively. Though functional ATP binding was reported as critical for NLRP3 activation⁴, mutations in the NACHT domain of patients are associated with spontaneous NLRP3 activation and an autoinflammatory disease spectrum known as cryopyrin-associated periodic syndromes (CAPS)^1,5. Several laboratories have sought NLRP3 inhibitors⁶. MCC950 was originally described as one of several diarylsulfonylurea-containing compounds interfering with IL-1β production⁷ and was later identified as a potent NLRP3 inhibitor capable of ameliorating pathology in animal models of inflammation and autoimmunity^8,9.

Using an elegant, bioluminescence resonance energy transfer (BRET)-based proximity assay sensitive to intramolecular structural changes, Pelegrin and colleagues now provide evidence that MCC950 directly binds and inactivates NLRP3 by interfering with conformational changes³. While inactive NLRP3 is reported to have a closed conformation, the sensor protein opens up during activation and is then thought to oligomerize into a disc-like structure nucleating aggregation of the central inflammasome component ASC (Fig. 1). The ASC complexes generated act as scaffolds for the subsequent activation of caspase-1, controlling both maturation and release of inflammatory IL-1 family cytokines. These authors show that MCC950 is capable not only of preserving an inactive NLRP3 conformation but also of closing an a priori open state observed in CAPS-associated gain-of-function mutations in patients. Using homology modeling of the NACHT domain along with blind docking and molecular dynamics simulations of MCC950–NLRP3 interactions, the authors were able to pinpoint the Walker B motif as a likely target of MCC950. Amino acid substitution within this motif abolished MCC950 binding to the constitutively active mutant NLRP3 and prevented its transition to a closed conformation, supporting this notion.

Independently, and using different complementary methods, the Schroder laboratory likewise reports evidence for direct binding of MCC950 to the ATP-binding region within the NACHT domain as the mechanistic basis of its inhibition of NLRP3 (ref. ²). The authors used a drug affinity responsive target stability (DARTS) approach, which reveals small-molecule–protein interactions through protection from proteolysis. MCC950 protected NLRP3 in both its inactive and activated state, indicating that inhibitor binding does not require activation, while wash-out experiments suggested that it is reversible. As only the central NACHT domain of NLRP3 was stabilized in DARTS assays when expressed in cells, this domain turned out to be the likely target. Using a sophisticated photoaffinity labeling strategy, these authors provide evidence for direct binding of MCC950. This is confirmed by surface plasmon resonance with an immobilized recombinant version of NLRP3, which determined an affinity in the nanomolar range with a rapid off-rate. Mutations in the Walker B ATP hydrolysis motif mitigated protection from proteolysis, suggesting proximal binding of the compound. However, MCC950 did not compete with ATP for NLRP3 binding, yet suppressed ATP hydrolysis. Importantly, when the NLRP3 NACHT domain was swapped with that of the related protein NLRP12, MCC950 no longer protected NLRP3 from proteolysis, emphasizing a highly specific interaction between MCC950 and this domain.

These two studies jointly identify MCC950 as a direct NLRP3 inhibitor by reversibly binding to the Walker B ATP-hydrolysis motif of its NACHT domain and maintaining or inducing a closed, inactive conformation. Together with a recently published report on the reversible NLRP3 inhibitor CY-09, which instead binds the Walker A motif of the NACHT domain¹⁰, these studies suggest that the ATP-binding domain of NLRP3 is the primary druggable site for modulation of inflammasome activity. These insights provide a basis for the rational development of new, superior NLRP3-inhibiting compounds to target this inflammasome in conditions of acute and chronic inflammation and connected pathologies.