Identification of Compounds With Glucocorticoid Sparing Effects on Suppression of Chemokine and Cytokine Production by Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Abstract

In recent years target based drug discovery has expanded our therapeutic armamentarium in the treatment of inflammatory and autoimmune diseases. Despite these advances and adverse effects, glucocorticoids remain reliable agents that are used in many of these diseases. The anti-inflammatory mechanisms of glucocorticoids include the suppression of transcription factor activity like nuclear factor kappa B (NF-κB). By reanalyzing data from two prior high throughput screens (HTS) that utilized a NF-κB reporter construct in THP-1 cells, we identified 1824 small molecule synthetic compounds that demonstrated NF-κB suppressive activities similar to the glucocorticoids included in the original >134,000 compound libraries. These 1824 compounds were then rescreened for attenuating NF-κB activity at 5 and 16 h after LPS stimuli in the NF-κB THP-1 reporter cells. After a “Top X” selection approach 122 hit compounds were further tested for toxicity and suppression of LPS induced CXCL8 release in THP-1 cells. Excluding cytotoxic compounds, the remaining active compounds were grouped into chemotype families using Tanimoto based clustering. Promising representatives from clustered chemotype groups were commercially purchased for further testing. Amongst these index compounds a lead chemotype: 1H-pyrazolo [3,4 d] pyrimidin-4-amine, effectively suppressed CXCL8, and TNF production by THP-1 cells when stimulated with LPS, TNF or IL-1ß. Extending these studies to primary cells, these lead compounds also reduced IL-6 and CXCL8 production by TNF stimulated fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. Importantly a lead 1H-pyrazolo [3,4 d] pyrimidin-4-amine compound demonstrated synergistic effects with dexamethasone when co-administered to TNF stimulated THP-1 cells and RA FLS in suppressing chemokine production. In summary, a cell based HTS approach identified lead compounds that reduced NF-κB activity and chemokine secretion induced by potent immunologic stimuli, and one lead compound that acted synergistically with dexamethasone as an anti-inflammatory agent showing a dose-sparing effect.

Anti-Inflammatory Effects of a Novel Nuclear Factor–κ B Inhibitory Derivative Derived from Pyrazolo[3,4-d]Pyrimidine in Three Inflammation Models

“…In the previous study, we identified that 5 μM of INH #1 suppressed 90% of NF-κB transcriptional activity 16 hours after the stimulation and the anti-inflammatory effects were saturated above 5 μM (Hosoya, T. et al, 2020). To explore the kinase inhibitory profile, we conducted 30 major serine/threonine-specific protein kinase assays, which were critical for cellular bioactivity (Table 1).…”

“…These problems inspired us to develop agents that exhibit anti-inflammatory activity while retaining the physiologically essential bioactivities of NF-κB, which would enable us to address unmet needs for the treatment of inflammatory and autoimmune diseases. Toward this goal, we found a novel compound that inhibited inflammation-related NF-κB activity while retaining its physiological activity and with little cell toxicity using NF-κB reporter cells (Hosoya, T. et al, 2020).…”

Plasma Exosomes Transfer miR-885-3p Targeting the AKT/NFκB Signaling Pathway to Improve the Sensitivity of Intravenous Glucocorticoid Therapy Against Graves Ophthalmopathy

“…In order to further explore the functions of these exosome miRNAs and screen target miRNAs related to glucocorticoids, we performed KEGG and Go enrichment analyses of different miRNA target genes. The chemokine signaling pathway enriched by KEGG is closely related to the pharmacological action of glucocorticoids ( 26 , 27 ). The downstream protein of the chemokine signaling pathway is NFκB, and the anti-inflammatory effect of glucocorticoids is mainly achieved through the transcriptional inhibition of NFκB by GR ( 14 , 28 ).…”