Background/Purpose: Target-based drug discovery has expanded our therapeutic armamentarium in the treatment of inflammatory and autoimmune diseases. Despite these advances, glucocorticoids (GC) remain reliable agents that are used in many of these conditions. In this study we utilized a discovery based approach to identify small molecular weight synthetic compounds that reduced NF-κB signaling activity in cell-based assays. We assessed the potency of specific chemotypes in reducing pro-inflammatory cytokine production by stimulated RA fibroblast-like synoviocytes (RASF) and synergy with GC.

Methods: We re-analyzed existing data from two prior high throughput screens (HTS) which utilized a library from the Small Molecule Discovery Center (UCSF) and a Forster resonance energy transfer based NF-κB reporter assay in THP-1 cells. NF-κB activation resulted in beta-lactamase production, which shifted the fluorescence emission of the substrate from 530 nm to 460 nm. Hit compounds were rescreened with LPS (10ng/ml) stimulated NF-κB reporter cells at 5 and 16 hours with an IκB kinase inhibitor (IKKI) and dexamethasone (DEX) as controls for variability. Compounds (5µM) were tested for toxicity by MTT. Selected lead compounds were commercially repurchased. CXCL8 production by THP-1 cells when stimulated with LPS (10ng/ml), TNFα (2ng/ml) or IL-1ß (2ng/ml) was measured by ELISA. Human synovial tissue derived RASF were treated with TNFα (1ng/ml) and graded amounts of lead compounds and DEX, and the levels of IL-6 and CXCL8 produced were measured by ELISA.

Results: Reanalyzing prior data we identified 1843 compounds that attenuated NF-κB activities in both HTS similar to the GCs included in the original 166,000 compound library. We performed confirmation screens with these hit compounds (5µM) for their effects on the kinetics of NF-κB activity in LPS stimulated reporter cells at peak (5 hours) and decay (16 hours) timepoints. There were 270 compounds that met the following criteria: NF-κB activity < 50 % max at 5 hours or < 25% max at 16 hours.  Excluding compounds with < 90% viability by MTT, the remaining compounds were clustered into 15 chemotype families. Promising representatives from the largest chemotype families were commercially purchased for further testing. Amongst these index compounds two chemotypes: 1H-pyrazolo[3,4-d] pyrimidin-4-amine and bis-aryl urea, effectively suppressed CXCL8 production by THP-1 cells when stimulated with LPS, TNFα or IL-1ß (p< 0.05, by  one-way ANOVA with Dunnett’s post hoc tests). These lead compounds also reduced IL-6 and CXCL8 production by TNFα stimulated RASF (p< 0.05).  Importantly a lead 1H-pyrazolo[3,4-d] pyrimidin-4-amine compound demonstrated a dose sparing effect for DEX when co-administered with TNFα stimulated THP-1 cells and had a synergistic effect with DEX in suppressing RASF chemokine production(p< 0.05, and Bliss independence-based analysis).

Conclusion: In summary, using a broad cell-based HTS approach, lead compounds were identified that reduced NF-κB activity and chemokine/cytokine secretion induced by potent immunologic stimuli, and acted synergistically with glucocorticoids as anti-inflammatory agents.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-small-molecules-with-efficacy-as-steroid-sparing-suppression-of-chemokine-and-cytokine-production-by-rheumatoid-arthritis-fibroblast-like-synoviocytes/