Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation. Main Text: The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) is a cytosolic sensor of diverse pathogen-and host-derived molecules. Upon activation, NLRP3 oligomerises and recruits apoptosis-associated speck-like protein containing a CARD (ASC), forming a platform for the binding, dimerisation and activation of the caspase-1 protease 1. Caspase-1 then cleaves the pro-inflammatory cytokines prointerleukin-1 (IL-1) and pro-IL-18, mediating the secretion of their active cytokines. Caspase-1 also cleaves Gasdermin-D, triggering pyroptosis 2,3. NLRP3-driven inflammation is pathological in the development of many diseases including cryopyrin-associated periodic syndromes, Alzheimer's Disease, Parkinson's

Macrophage pre-treatment with bacterial lipopolysaccharide (LPS) boosts subsequent activation of the NLRP3 inflammasome, which controls caspase-1-dependent pro-inflammatory cytokine maturation. Previous work has attributed this phenomenon (known as LPS 'priming') to LPS-dependent induction of NLRP3 expression. Whilst this plays a role, here we demonstrate that rapid LPS priming of NLRP3 inflammasome activation can occur independently of NLRP3 induction, since the priming effect of LPS is still apparent at short pre-treatment times in which NLRP3 protein expression remains unchanged. Furthermore, rapid LPS priming is still evident in Nlrp3(-/-) primary macrophages with NLRP3 expression reconstituted using a constitutive promoter. Similarly, we found that LPS potentiates AIM2 inflammasome activation to submaximal doses of cytosolic DNA without concomitant upregulation of AIM2 protein expression. Our data suggest that, in addition to augmenting NLRP3 inflammasome activity via NLRP3 induction, LPS boosts caspase-1 activation by the NLRP3 and AIM2 inflammasomes by an acute mechanism that is independent of inflammasome sensor induction.

Inflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) that links the inflammasome initiator protein to the recruited caspases. ASC is normally diffuse within the cell but within minutes of inflammasome activation relocates to a dense speck in the cytosol. The dramatic redistribution of ASC can be monitored by flow cytometry using parameters of fluorescence peak height and width when immunostained or tagged with a fluorescent protein. This can be used to define cells with active inflammasomes within populations of primary macrophages and monocytes, allowing quantification of responses and flow-sorting of responding cells. Protein structural requirements for ASC speck formation and recruitment of caspases to ASC specks can be assessed by expressing components in HEK293 cells. This provides rapid quantification of responding cell number and correlation with the expression level of inflammasome components within single cells. © 2016 by John Wiley & Sons, Inc.

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