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

Target identification is a high-priority, albeit challenging, aspect of drug discovery. Diazirine-based photoaffinity probes (PAPs) can facilitate the process by covalently capturing transient molecular interactions. This can help identify target proteins and map the ligand's interactome. Diazirine probes have even been incorporated by cellular machinery into proteins. Embarking on the synthesis of customized PAPs, containing either an aliphatic or trifluoromethyl phenyl diazirine, can be a considerable endeavor, particularly for medicinal chemists and chemical biologists new to the field. This review takes a synthetic focus, aiming to summarize available routes, propose new avenues, and illuminate recent advances in diazirine synthesis. Select examples of diazirine photoaffinity labeling applications have been included throughout to provide instructive definition of the advantages and limitations of the technology while simultaneously highlighting how these reagents can be applied in a practical sense.

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The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.

Estrogen and progesterone concentrations were determined in plasma for the periods of proestrus (4 days prior to estrus) and estrus. In trial I, blood was obtained at 0630 and 1800 hr every day for 4 days preceding estrus in 10 heifers. In trial II, blood was obtained on the day prior to estrus and every 2-4 hr on the day of estrus in four cows. Estrogen concentrations were determined by radioimmunoassay. Progesterone concentration was determined by the competitive protein binding assay. Plasma estrogen concentrations were less than 10 pg/ml until the day prior to estrus when the concentrations were 15-25 pg/ml. There were transient increases between 3 and 10 pg/ml during the first 3 days of proestrus. Progesterone levels were highest (5-12 ng/ml) on the fourth day prior to estrus, then progressively decreased to a nearly nondetectable level on the day prior to estrus. An estrogen concentration greater than 10 pg/ml was not detected until the progesterone concentration was below 2 ng/ml. Upon the onset of estrus, the estrogen level was equal to that present on the day prior to estrus (15-25 pg/ml), however, it began to decrease within 2-5 hr after the onset of estrus. {Endocrinology 89: 1350, 1971)