Background/Purpose: We reported that nuclear factor of activated T-cells 5 (NFAT5), originally identified as an osmo-protective transcription factor, has a critical role in the pathogenesis of rheumatoid arthritis (RA). In the present study, we investigated to discover a small molecule to specifically inhibit pro-inflammatory activity of NFAT5.

Methods: We screened small molecules (>150,000) to effectively suppress biosynthesis of nitric oxide (NO), a sensitive end-product of NFAT5 in RAW 264.7 macrophages using high-throughput drug screening. Fluorescence-activated cell sorting (FACS) analysis was performed to confirm a direct NFAT5 inhibitory activity of the candidate chemicals using RAW 264.7 macrophages transfected stably with a novel NFAT5-specific GFP reporter under lipopolysaccharide (LPS) stimulation. Expression of NFAT5 and its target genes was confirmed by quantitative RT-PCR, western blot, ELSIA, and NO assay. In vivoeffect was tested in adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) mice models.

Results: NFAT5 regulated the production of NO and IL-6 by RAW 264.7 macrophages under LPS stimulation. Among the screened small molecules, filtered were 198 candidate chemicals that strongly inhibited the NO production (IC50 < 1 μM). After testing direct NFAT5 inhibitory activity and in vitro cytotoxicity, KRN2 and its oral derivative with high serum stability (KRN5) were finally selected. These compounds did not affect NFAT1-4, NF-κB, p38 MAP kinase and CREB activity, excluding the potential off-target effect of the candidate molecules. We further found out the inhibitory mechanism of KRN2 in regulating NF-κB binding capacity on the NF-κB binding site in the promoter region of NFAT5. Interestingly, these compounds inhibited LPS-induced IL-6, TNF-α, GM-CSF, iNOS, nitric oxide production in RAW 264.7 macrophages, while they did not affect AR and SMITmRNA expression involved in osmo-protection and cellular homeostasis. Effectively, KRN2 and KRN5 alleviated disease severity of arthritis and oral administration of KRN5 successfully prevented the development of arthritis in experimental mice models.

Conclusion: We identified selective inhibitors of NFAT5 (KRN2 and KRN5) that potently inhibited the production of pro-inflammatory mediators in macrophages and effectively suppressed the development of experimental arthritis. These compounds could be a good candidate for treating the chronic inflammatory arthritis.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/suppression-of-chronic-arthritis-by-a-novel-nuclear-factor-of-activated-t-cell-5-nfat5-inhibitor/