Suppression of NFAT5-mediated Inflammation and Chronic Arthritis by Novel κB-binding Inhibitors

Nuclear factor of activated T cells 5 (NFAT5) has been implicated in the pathogenesis of various human diseases, including cancer and arthritis. However, therapeutic agents inhibiting NFAT5 activity are currently unavailable. To discover NFAT5 inhibitors, a library of >40,000 chemicals was screened for the suppression of nitric oxide, a direct target regulated by NFAT5 activity, through high-throughput screening. We validated the anti-NFAT5 activity of 198 primary hit compounds using an NFAT5-dependent reporter assay and identified the novel NFAT5 suppressor KRN2, 13-(2-fluoro)-benzylberberine, and its derivative KRN5. KRN2 inhibited NFAT5 upregulation in macrophages stimulated with lipopolysaccharide and repressed the formation of NF-κB p65-DNA complexes in the NFAT5 promoter region. Interestingly, KRN2 selectively suppressed the expression of pro-inflammatory genes, including Nos2 and Il6, without hampering high-salt-induced NFAT5 and its target gene expressions. Moreover, KRN2 and KRN5, the latter of which exhibits high oral bioavailability and metabolic stability, ameliorated experimentally induced arthritis in mice without serious adverse effects, decreasing pro-inflammatory cytokine production. Particularly, orally administered KRN5 was stronger in suppressing arthritis than methotrexate, a commonly used anti-rheumatic drug, displaying better potency and safety than its original compound, berberine. Therefore, KRN2 and KRN5 can be potential therapeutic agents in the treatment of chronic arthritis. •We identify a novel NFAT5 suppressor KRN2, 13-(2-fluoro)-benzylberberine, and its derivative KRN5 to inhibit NFAT5 activity.•KRN2 inhibits the transcriptional activation of NFAT5 and the pro-inflammatory responses.•KRN2 and KRN5 suppress experimentally induced arthritis in mice. NFAT5 has been implicated in the pathogenesis of arthritis. However, therapeutic agents specifically inhibiting NFAT5 activity are currently unavailable. To discover NFAT5 inhibitors, a library of >40,000 chemicals was screened, leading to the discovery of novel berberine-based NFAT5 suppressors, KRN2 and its oral derivative KRN5. KRN2 inhibited the transcriptional activation of NFAT5 by blocking NF-κB binding to the NFAT5 promoter region, thereby reducing the expression of pro-inflammatory genes. Moreover, KRN2 and KRN5 ameliorated experimentally induced arthritis in mice without serious adverse effects. Therefore, we propose that KRN2 and KRN5 may be potential therapeutic agents