Optimized protocols for studying the NLRP3 inflammasome and assessment of potential targets of CP-453,773 in undifferentiated THP1 cells.

The NLRP3 inflammasome is a complex multimeric signaling apparatus that regulates production of the pro-inflammatory cytokine IL-1ß. To overcome both the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. Undifferentiated THP1 cells constitutively express NLRP3, and NLRP3 inflammasome activation occurred in response to canonical NLRP3 activation stimuli including nigericin, ATP, and urea crystals, culminating in pro-IL-1ß cleavage, extracellular release of mature IL-1ß, and pyroptosis. We used this THP1 cell system to investigate potential targets of the potent, NLRP3 inflammasome selective inhibitor CP-456,773. We optimized a viral shRNA transduction method for gene expression knockdown (KD), and the KD of NLRP3 itself eliminated inflammasome activation and IL-1ß production. NLRP3 inflammasome activation and CP-453,773 pharmacology were not altered in ABCb7- or ABCb10-deficient THP1 cells, eliminating these gene products as candidate pharmacological targets of CP-453,773. For ABCb10, we confirmed our results using CRISPR/CAS9-mediated ABCb10 knockout (KO) THP1 sub-lines. In summary, undifferentiated THP1 cells are fully competent for activation of the NLRP3 inflammasome and production of IL-1ß, without differentiation into macrophages, and we describe optimized KD and KO methodologies to manipulate gene expression in these cells. As an example of the utility of undifferentiated THP1 cells for investigations into the biology of the NLRP3 inflammasome, we have used this cell system to rule out ABCb7 and ABCb10 as potential targets of the NLRP3 inflammasome inhibitor CP-453,773.

Efficacy and Pharmacology of the NLRP3 Inflammasome Inhibitor CP-456,773 (CRID3) in Murine Models of Dermal and Pulmonary Inflammation.

A critical component of innate immune response to infection and tissue damage is the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome, and this pathway and its activation products have been implicated in the pathophysiology of a variety of diseases. NLRP3 inflammasome activation leads to the cleavage of pro-IL-1ß and pro-IL-18, as well as the subsequent release of biologically active IL-1ß, IL-18, and other soluble mediators of inflammation. In this study, we further define the pharmacology of the previously reported NLRP3 inflammasome-selective, IL-1ß processing inhibitor CP-456,773 (also known as MCC950), and we demonstrate its efficacy in two in vivo models of inflammation. Specifically, we show that in human and mouse innate immune cells CP-456,773 is an inhibitor of the cellular release of IL-1ß, IL-1α, and IL-18, that CP-456,773 prevents inflammasome activation induced by disease-relevant soluble and crystalline NLRP3 stimuli, and that CP-456,773 inhibits R848- and imiquimod-induced IL-1ß release. In mice, CP-456,773 demonstrates potent inhibition of the release of proinflammatory cytokines following acute i.p. challenge with LPS plus ATP in a manner that is proportional to the free/unbound concentrations of the drug, thereby establishing an in vivo pharmacokinetic/pharmacodynamic model for CP-456,773. Furthermore, CP-456,773 reduces ear swelling in an imiquimod cream-induced mouse model of skin inflammation, and it reduces airway inflammation in mice following acute challenge with house dust mite extract. These data implicate the NLRP3 inflammasome in the pathogenesis of dermal and airway inflammation, and they highlight the utility of CP-456,773 for interrogating the contribution of the NLRP3 inflammasome and its outputs in preclinical models of inflammation and disease.

An NLRP3-specific inflammasome inhibitor attenuates crystal-induced kidney fibrosis in mice.

Intrarenal crystal formation activates the Nlrp3 inflammasome in myeloid cells and triggers a profound inflammatory response. Here, we studied whether a specific inhibitor of the Nlrp3 inflammasome, CP-456,773, can prevent kidney fibrosis in a murine model of crystal nephropathy induced by diets rich in oxalate or adenine. Inflammasome activation in renal dendritic cells and the resulting interleukin (IL)-1ß and IL-18 production were markedly reduced by CP-456,773 treatment both ex vivo and in vivo. We directly visualized intrarenal inflammasome activation and its inhibition by CP-456,773 in vivo by adoptive transfer of bone marrow cells transduced with interleukin-1ß-Gaussia luciferase, a proteolytic luciferase-based reporter for inflammasome activation, into irradiated mice. CP-456,773 treatment strongly attenuated kidney fibrosis when given early in the genesis of crystal nephropathy, but was unable to reverse established crystal-induced fibrosis. The urinary IL-18 concentration appeared to be a useful noninvasive biomarker for renal inflammasome activation. Finally, NLRP3 inhibition did not compromise adaptive immune responses as previously reported for the global inhibition of IL-1 signaling. Thus, early NLRP3 inhibition by CP-456,773 may be an effective treatment for crystal nephropathy. Use of iGLuc transfected cells introduces a novel imaging technique for inflammasome activation in mice.

Identification of a chemical probe for bromo and extra C-terminal bromodomain inhibition through optimization of a fragment-derived hit.

The posttranslational modification of chromatin through acetylation at selected histone lysine residues is governed by histone acetyltransferases (HATs) and histone deacetylases (HDACs). The significance of this subset of the epigenetic code is interrogated and interpreted by an acetyllysine-specific protein-protein interaction with bromodomain reader modules. Selective inhibition of the bromo and extra C-terminal domain (BET) family of bromodomains with a small molecule is feasible, and this may represent an opportunity for disease intervention through the recently disclosed antiproliferative and anti-inflammatory properties of such inhibitors. Herein, we describe the discovery and structure-activity relationship (SAR) of a novel, small-molecule chemical probe for BET family inhibition that was identified through the application of structure-based fragment assessment and optimization techniques. This has yielded a potent, selective compound with cell-based activity (PFI-1) that may further add to the understanding of BET family function within the bromodomains.