Discovery of a series of ester-substituted NLRP3 inflammasome inhibitors.

The NLRP3 inflammasome is a component of the innate immune system involved in the production of proinflammatory cytokines. Aberrant activation by a wide range of exogenous and endogenous signals can lead to chronic, low-grade inflammation. It has attracted a great deal of interest as a drug target due to the association with diseases of large unmet medical need such as Alzheimer's disease, Parkinson's disease, arthritis, and cancer. To date, no drugs specifically targeting inhibition of the NLRP3 inflammasome have been approved. In this work, we used the known NLRP3 inflammasome inhibitor CP-456,773 (aka CRID3 or MCC 950) as our starting point and undertook a Structure-Activity Relationship (SAR) analysis and subsequent scaffold-hopping exercise. This resulted in the rational design of a series of novel ester-substituted urea compounds that are highly potent and selective NLRP3 inflammasome inhibitors, as exemplified by compounds 44 and 45. It is hypothesized that the ester moiety acts as a highly permeable delivery vehicle and is subsequently hydrolyzed to the carboxylic acid active species by carboxylesterase enzymes. These molecules are greatly differentiated from the state-of-the-art and offer potential in the treatment of NLRP3-driven diseases, particularly where tissue penetration is required.

Substrate specificity and inducibility of TACE (tumour necrosis factor alpha-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity.

Tumour necrosis factor alpha (TNF alpha)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor alpha, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.

TACE/ADAM-17 enzymatic activity is increased in response to cellular stimulation.

Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease disintegrin that cleaves a variety of membrane proteins, releasing ("shedding") their extracellular domains from cells. Most TACE-mediated shedding events occur at low basal rates that are enhanced by treatment of cells with a variety of stimuli. To study the mechanism of induced shedding, we developed a peptide-cleavage assay that measures the cellular TACE activity. In unstimulated cells, cleavage of a TNFalpha processing-site peptide was mediated mainly by enzymes other than TACE. However, stimulation of cells with phorbol-12-myristate-13-acetate (PMA) increased peptide cleavage in a TACE-dependent manner. PMA treatment did not increase the amount of TACE on the cell surface. Moreover, the cytoplasmic domain of TACE was not required for the induced activity. Based on these observations, induction of TACE-mediated shedding events occurs at least in part via an increase in the enzymatic activity of cellular TACE, independent of its cytoplasmic domain.