kn-62 and Inflammation

Novel 2,5-dioxoimidazolidine-based conformationally constrained analogues of KN62 (1) were developed as P2X7 receptor (P2X7R) antagonists using a rigidification strategy of the tyrosine backbone of 1. SAR analysis of the 2,5-dioxoimidazolidine scaffold indicated that piperidine substitution at the N3 position and no substitution at N1 position were preferable. Further optimization of the substituents at the piperidine nitrogen and the spacer around the skeleton resulted in several superior antagonists to 1, including 1-adamantanecarbonyl analogue 21i (IC50 = 23 nM in ethidium uptake assay; IC50 = 14 nM in IL-1β ELISA assay) and (3-CF3-4-Cl)benzoyl analogue (-)-21w (54 nM in ethidium uptake assay; 9 nM in IL-1β ELISA assay), which was more potent than the corresponding (+) isomer. Compound 21w displayed potent inhibitory activity in an ex vivo model of LTP-induced pain signaling in the spinal cord and significant anti-inflammatory activity in in vivo models of carrageenan-induced paw edema and type II collagen-induced joint arthritis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Arthritis, Experimental; Carrageenan; Cattle; Collagen Type II; Drug Discovery; Edema; Enzyme-Linked Immunosorbent Assay; HEK293 Cells; Humans; Hydantoins; Immunoblotting; Inflammation; Interleukin-1beta; Long-Term Potentiation; Macrophages; Male; Mice, Inbred DBA; Molecular Structure; Monocytes; Neuralgia; Purinergic P2X Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X7; Structure-Activity Relationship; Sulfonic Acids; Tissue Distribution

Interleukin-1 (IL-1), the 'gatekeeper' of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1β shares a β-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1β was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1β pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animal Fins; Animals; Caspase 1; Caspase Inhibitors; Conserved Sequence; Down-Regulation; Embryo, Nonmammalian; Humans; Inflammation; Interleukin-1beta; Leukocytes; NF-kappa B; Rosaniline Dyes; Signal Transduction; Up-Regulation; Zebrafish

Nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in NAD biosynthesis, has recently been identified as a novel mediator of innate immunity. In the present study, we report that treatment of LPS-primed monocytes with ATP greatly enhanced the secretion of NAMPT in a time- and concentration-dependent manner without displaying any cytotoxic effect. NAMPT release was suppressed by pretreatment with the P2X(7) receptor (P2X(7)R) inhibitors oxidized ATP (oxATP) and KN-62, indicating the engagement of P2X(7)Rs. Furthermore, P2X(7)R was found to be involved in mediating cell permeability caused by the addition of ATP. To define a role of endogenous ATP in NAMPT secretion, LPS-primed monocytes were incubated in the presence of oxATP and KN-62, as well as the ATP-hydrolyzing enzymes apyrase and hexokinase. With the exception of oxATP, neither substance led to a decrease in NAMPT release, suggesting that autocrine/paracrine ATP is unlikely to be responsible for the LPS-induced release of NAMPT. In conclusion, the enhanced release of NAMPT by extracellular ATP described here indicates the requirement of a second stimulus for the efficient secretion of NAMPT. This mode of secretion, which also applies to IL-1β, might represent a general mechanism for the release of leaderless secretory proteins at locally restricted sites.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cellular Microenvironment; Cytokines; Extracellular Space; Humans; Immunity, Innate; Inflammation; Lipopolysaccharides; Monocytes; Nicotinamide Phosphoribosyltransferase; Receptors, Purinergic P2X7; Signal Transduction

P2X(7) receptor (P2X(7)R), upon its stimulation with extracellular ATP, modulates several inflammatory responses in different cell types. No information is available on its presence in human adipocytes and its potential involvement in the chronic inflammation associated with metabolic syndrome (MS). Therefore, we evaluated P2X(7)R presence and functional activity in adipocytes from visceral (VAT) and subcutaneous (SAT) adipose tissue of patients with MS and controls (CTL).. Adipocyte gene expression of TNFα, IL-6 and PAI-1 (by realtime-PCR) and their plasma concentrations (ELISA); P2X(7)R expression (realtime-PCR, Western blot and immunofluorescence); P2X(7)R functional activity (intracellular calcium fluxes by fluorimetry); cytokine release from adipocytes (ELISA). The inflammasome components were also determined.. In VAT, TNFα, IL-6 and PAI-1 were more expressed in MS than in CTL. These differences were confirmed in SAT for IL-6 and PAI-1. Plasma IL-6, PAI-1 and TNFα levels were higher in MS. P2X(7)R mRNA and protein, identified in both VAT and SAT, were more abundant in MS than in CTL. Immunofluoresce confirmed the typical "ring-like" arrangement of P2X(7)R at the plasma membrane. Benzoyl-benzoyl-ATP raised intracellular calcium both in VAT and SAT, and induced IL-6, TNFα and PAI-1 release in both MS and CTL cells. This effect was partially inhibited by KN62, specific human P2X(7)R blocker, or by P2X(7)R gene silencing. The inflammasome was more activated in MS than in CTL adipocytes.. Human adipocytes express functionally active P2X(7)R, which modulate the release of inflammatory cytokines, at least in part via inflammasome activation. Adipocytes from MS patients show an enhanced P2X(7)R expression, which might contribute to the subclinical inflammatory status characterizing these patients and conferring them an increased CV risk.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Adipocytes; Aged; Blotting, Western; Calcium Signaling; Case-Control Studies; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Inflammasomes; Inflammation; Inflammation Mediators; Interleukin-6; Intra-Abdominal Fat; Italy; Metabolic Syndrome; Middle Aged; Plasminogen Activator Inhibitor 1; Purinergic P2X Receptor Agonists; Purinergic P2X Receptor Antagonists; Real-Time Polymerase Chain Reaction; Receptors, Purinergic P2X7; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Subcutaneous Fat; Tumor Necrosis Factor-alpha; Up-Regulation

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature