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.

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-1β and pro–IL-18, as well as the subsequent release of biologically active IL-1β, IL-18, and other soluble mediators of inflammation. In this study, we further define the pharmacology of the previously reported NLRP3 inflammasome–selective, IL-1β 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-1β, 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-1β 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.

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)-1β 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-1β-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.

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N-Acetylcysteine partially improved neuronal survival when administered before or after ischemia following transient cerebral ischemia (mean arterial blood pressure, 45 mm Hg) but not with a more severe ischemic insult of 10 minutes of transient cerebral ischemia with a mean arterial blood pressure of 30 mm Hg.

A temporal profile of lateral and fourth ventricle rat choroid plexus (LVCP and 4VCP, respectively) tissue injury and recovery was determined using alterations in K, Na, and H2O content and ultrastructure after 10 min of transient forebrain ischemia (TFI). At 0.5 h postischemia the LVCP displayed a maximum reduction in K content by 32% and a significant increase in Na content by 85% and H2O content by 22%. LVCP tissue K, Na, and H2O content returned to sham values by 24 h postischemia. Ultrastructural changes appeared more severe between 0.5 and 12 h postischemia, whereas by 24 h, normal ultrastructure was restored. Elevations in 4VCP tissue Na (P < 0.05) and H2O content, which were less than those in LVCP, gradually reached a maximum by 24 h compared with sham. No change in 4VCP tissue ultrastructure was observed. These results indicate that the LVCP tissue is more vulnerable than 4VCP in the bilateral carotid artery occlusion model but that it recovers in a timely manner after TFI. Furthermore, the ability of the LVCP tissue to rapidly recover suggests its functional importance in helping to restore brain homeostasis.

1. Transient forebrain ischemia in adult rats, induced by 10 min of bilateral carotid occlusion and an arterial hypotension of 40 mmHg, caused substantial damage not only to CA-1 neurons in hippocampus but also to epithelial cells in lateral ventricle choroid plexus. 2. When transient forebrain ischemia was followed by reperfusion (recovery) intervals of 0 to 12 hr, there was moderate to severe damage to many frond regions of the choroidal epithelium. In some areas, epithelial debris was sloughed into cerebrospinal fluid (CSF). Although some epithelial cells were disrupted and necrotic, their neighbors exhibited normal morphology. This patchy response to ischemia was probably due to regional differences in reperfusion or cellular metabolism. 3. Between 12 and 24 hr postischemia, there was marked restoration of the Na+, K+, water content, and ultrastructure of the choroid plexus epithelium. Since there was no microscopical evidence for mitosis, we postulate that healthy epithelial cells either were compressed together on the villus or migrated from the choroid plexus stalk to more distal regions, in order to "fill in gaps" along the basal lamina caused by necrotic epithelial cell disintegration. 4. Epithelial cells of mammalian choroid plexus synthesize and secrete many growth factors and other peptides that are of trophic benefit following injury to regions of the cerebroventricular system. For example, several growth factors are upregulated in choroid plexus after ischemic and traumatic insults to the central nervous system. 5. The presence of numerous types of growth factor receptors in choroid plexus allows growth factor mediation of recovery processes by autocrine and paracrine mechanisms. 6. The capability of choroid plexus after acute ischemia to recover its barrier and CSF formation functions is an important factor in stabilizing brain fluid balance. 7. Moreover, growth factors secreted by choroid plexus into CSF are distributed by diffusion and convection into brain tissue near the ventricular system, e.g., hippocampus. By this endocrine-like mechanism, growth factors are conveyed throughout the choroid plexus-CSF-brain nexus and can consequently promote repair of ischemia-damaged tissue in the ventricular wall and underlying brain.