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.

Rational approach to highly potent and selective apoptosis signal-regulating kinase 1 (ASK1) inhibitors.

Many diseases are believed to be driven by pathological levels of reactive oxygen species (ROS) and oxidative stress has long been recognized as a driver for inflammatory disorders. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to be activated by intracellular ROS and its inhibition leads to a down regulation of p38-and JNK-dependent signaling. Consequently, ASK1 inhibitors may have the potential to treat clinically important inflammatory pathologies including renal, pulmonary and liver diseases. Analysis of the ASK1 ATP-binding site suggested that Gln756, an amino acid that rarely occurs at the GK+2 position, offered opportunities for achieving kinase selectivity for ASK1 which was applied to the design of a parallel medicinal chemistry library that afforded inhibitors of ASK1 with nanomolar potency and excellent kinome selectivity. A focused optimization strategy utilizing structure-based design resulted in the identification of ASK1 inhibitors with low nanomolar potency in a cellular assay, high selectivity when tested against kinase and broad pharmacology screening panels, and attractive physicochemical properties. The compounds we describe are attractive tool compounds to inform the therapeutic potential of ASK1 inhibition.

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.

Enhancement of COPD biological networks using a web-based collaboration interface.

The construction and application of biological network models is an approach that offers a holistic way to understand biological processes involved in disease. Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory disease of the airways for which therapeutic options currently are limited after diagnosis, even in its earliest stage. COPD network models are important tools to better understand the biological components and processes underlying initial disease development. With the increasing amounts of literature that are now available, crowdsourcing approaches offer new forms of collaboration for researchers to review biological findings, which can be applied to the construction and verification of complex biological networks. We report the construction of 50 biological network models relevant to lung biology and early COPD using an integrative systems biology and collaborative crowd-verification approach. By combining traditional literature curation with a data-driven approach that predicts molecular activities from transcriptomics data, we constructed an initial COPD network model set based on a previously published non-diseased lung-relevant model set. The crowd was given the opportunity to enhance and refine the networks on a website ( https://bionet.sbvimprover.com/) and to add mechanistic detail, as well as critically review existing evidence and evidence added by other users, so as to enhance the accuracy of the biological representation of the processes captured in the networks. Finally, scientists and experts in the field discussed and refined the networks during an in-person jamboree meeting. Here, we describe examples of the changes made to three of these networks: Neutrophil Signaling, Macrophage Signaling, and Th1-Th2 Signaling. We describe an innovative approach to biological network construction that combines literature and data mining and a crowdsourcing approach to generate a comprehensive set of COPD-relevant models that can be used to help understand the mechanisms related to lung pathobiology. Registered users of the website can freely browse and download the networks.

JAK inhibition with tofacitinib suppresses arthritic joint structural damage through decreased RANKL production.

OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.

Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-kappaB essential modulator binding domain peptides.

The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.

Aminopyridinecarboxamide-based inhibitors: Structure-activity relationship.

Series of aminopyridinecarboxamide-based inhibitors were synthesized and tested against human recombinant IKK-2 and in IL-1beta stimulated synovial fibroblasts. The 2-amino-5-chloropyridine-4-carboxamides were identified as the most potent inhibitors with improved cellular activity.

Novel tight-binding inhibitory factor-kappaB kinase (IKK-2) inhibitors demonstrate target-specific anti-inflammatory activities in cellular assays and following oral and local delivery in an in vivo model of airway inflammation.

Nuclear factor-kappaB (NF-kappaB) is one of the major families of transcription factors activated during the inflammatory response in asthma and chronic obstructive pulmonary disease. Inhibitory factor-kappaB kinase 2 (IKK-2) has been shown to play a pivotal role in cytokine-induced NF-kappaB activation in airway epithelium and in disease-relevant cells. Nevertheless, the potential toxicity of specific IKK-2 inhibitors may be unacceptable for oral delivery in chronic obstructive pulmonary disease. Therefore, local delivery to the lungs is an attractive alternative that warrants further exploration. Here, we describe potent and selective small-molecule IKK-2 inhibitors [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (PHA-408) and 8-(2-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-5-chloroisonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (PF-184)] that are competitive for ATP have slow off-rates from IKK-2 and display broad in vitro anti-inflammatory activities resulting from NF-kappaB pathway inhibition. Notably, PF-184 has been designed to have high systemic clearance, which limits systemic exposure and maximizes the effects locally in the airways. We used an inhaled lipopolysaccharide-induced rat model of neutrophilia to address whether inhibiting NF-kappaB activation locally within the airways would show anti-inflammatory effects in the absence of systemic exposure. PHA-408, a low-clearance compound previously shown to be efficacious orally in a rodent model of arthritis, dose-dependently attenuated inhaled lipopolysaccharide-induced cell infiltration and cytokine production. Interestingly, PF-184 produced comparable dose-dependent anti-inflammatory activity by intratracheal administration and was as efficacious as intratracheally administered fluticasone propionate (fluticasone). Together, these results support the potential therapeutic utility of IKK-2 inhibition in inflammatory pulmonary diseases and demonstrate anti-inflammatory efficacy of an inhaled IKK-2 inhibitor in a rat airway model of neutrophilia.

Pharmacokinetic and pharmacodynamic evaluation of the suitability of using fluticasone and an acute rat lung inflammation model to differentiate lung versus systemic efficacy.

Inhaled corticosteroids (ICSs) are often prescribed as the first line therapy for pulmonary diseases such as asthma. The biggest concern of using steroid therapy is the systemic side effects at high dose. To reduce the side effects, the pharmaceutical industry has been putting effort to generate new drugs with maximized topical efficacy. One of the key challenges is to differentiate efficacy from local versus systemic contribution in preclinical animal models. Fluticasone with various formulations was used as a model compound to explore the possibilities to demonstrate lung targeted efficacy by intratracheally instillation in the lipopolysaccharide induced inflammation rat model. Fluticasone formulations contained various surfactant concentrations and particle sizes to achieve lung retention and lower systemic exposure. Neutrophil infiltration in broncoalveolar lavage fluid and cytokine production in whole blood were measured to assess pulmonary efficacy versus systemic efficacy. PK/PD characterization of fluticasone with various formulations in the rat inflammation model provided an integrated approach in preclinical to evaluate lung targeted efficacy for ICS. Our study concluded that the combination of the rat LPS model and fluticasone is not suitable to use for establishing potency and dose requirement for new drug candidate designed for topical only efficacy.

A novel, highly selective, tight binding IkappaB kinase-2 (IKK-2) inhibitor: a tool to correlate IKK-2 activity to the fate and functions of the components of the nuclear factor-kappaB pathway in arthritis-relevant cells and animal models.

Nuclear factor (NF)-kappaB activation has been clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The central role that IkappaB kinase-2 (IKK-2) plays in regulating NF-kappaB signaling in response to inflammatory stimuli has made this enzyme an attractive target for therapeutic intervention. Although diverse chemical classes of IKK-2 inhibitors have been identified, the binding kinetics of these inhibitors has limited the scope of their applications. In addition, safety assessments of IKK-2 inhibitors based on a comprehensive understanding of the pharmacokinetic/pharmacodynamic relationships have yet to be reported. Here, we describe a novel, potent, and highly selective IKK-2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide]. PHA-408 is an ATP-competitive inhibitor, which binds IKK-2 tightly with a relatively slow off rate. In arthritis-relevant cells and animal models, PHA-408 suppresses inflammation-induced cellular events, including IkappaBalpha phosphorylation and degradation, p65 phosphorylation and DNA binding activity, the expression of inflammatory mediators, and joint pathology. PHA-408 was efficacious in a chronic model of arthritis with no adverse effects at maximally efficacious doses. Stemming from its ability to bind tightly to IKK-2, as a novelty, we demonstrated that PHA-408-mediated inhibition of IKK-2 activity correlated very well with its ability to modulate the fate of IKK-2 substrates and downstream transcriptional events. We ultimately directly linked IKK-2 activity ex vivo and in vivo to markers of inflammation with the inhibitor plasma concentrations. Thus, PHA-408 represents a powerful tool to further gain insight into the mechanisms by which IKK-2 regulates NF-kappaB signaling and validates IKK-2 as a therapeutic target.

Inhibition of IKK-2 by 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides.

A series of 21 novel 2-[(aminocarbonyl)amino]-5-acetylenyl-3-thiophenecarboxamides were synthesized and evaluated for the inhibition of IKK-2. In spite of their often modest activity on the enzyme, six selected analogs showed significant inhibition of the production of inflammatory cytokine IL-8 in IL-1beta stimulated rheumatoid arthritis-derived synovial fibroblasts, demonstrating their potential usefulness as NF-kappaB regulators.

A selective IKK-2 inhibitor blocks NF-kappa B-dependent gene expression in interleukin-1 beta-stimulated synovial fibroblasts.

NF-kappa B-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. I kappa B kinase (IKK) is the converging point for the activation of NF-kappa B by a broad spectrum of inflammatory agonists and is thus a novel target for therapeutic intervention. We describe a small molecule, selective inhibitor of IKK-2, SC-514, which does not inhibit other IKK isoforms or other serine-threonine and tyrosine kinases. SC-514 inhibits the native IKK complex or recombinant human IKK-1/IKK-2 heterodimer and IKK-2 homodimer similarly. IKK-2 inhibition by SC-514 is selective, reversible, and competitive with ATP. SC-514 inhibits transcription of NF-kappa B-dependent genes in IL-1 beta-induced rheumatoid arthritis-derived synovial fibroblasts in a dose-dependent manner. When the mechanism of NF-kappa B activation was evaluated in the presence of this inhibitor, several interesting observations were found. First, SC-514 did not inhibit the phosphorylation and activation of the IKK complex. Second, there was a delay but not a complete blockade in I kappa B alpha phosphorylation and degradation; likewise there was a slightly slowed, decreased import of p65 into the nucleus and a faster export of p65 from the nucleus. Finally, both I kappa B alpha and p65 were comparable substrates for IKK-2, with similar Km and Kcat values, and SC-514 inhibited the phosphorylation of either substrate similarly. Thus, the effect of SC-514 on cytokine gene expression may be a combination of inhibiting I kappa B alpha phosphorylation/degradation, affecting NF-kappa B nuclear import/export as well as the phosphorylation and transactivation of p65.