In the present study we identified novel NFAT suppressors fo

In the present study, we identified novel NFAT5 suppressors for use in RA therapies. We screened >40,000 small molecules which suppress NO induction and then validated anti-NFAT5 activity of the candidate molecules using an NFAT5-specific reporter. As a result, we discovered the novel NFAT5 inhibitor KRN2 and its derivative KRN5. KRN2 exhibited 40 times more anti-NFAT5 activity than BBR deprived of its 13-fluorobenzyl group. KRN2 and KRN5 inhibited the expressions of TLR4-stimulated (‘inflammatory’) NFAT5 and its target genes, including Il6, Nos2, and Csf2, in RAW 264.7 macrophages. Interestingly, high salt-stimulated (‘osmotic’) NFAT5 and tonicity-associated genes, including Ar, Bgt and Smit, were unaffected by KRN2, suggesting that KRN2 selectively works under LPS-stimulated conditions, but will not disturb cellular homeostasis and cytoprotection under hypertonic conditions. To understand the mode of action of KRN2, we investigated the transcriptional regulatory mechanism of KRN2 on NFAT5. We found that KRN2 nearly completely ameliorated the upregulation of NFAT5 mRNA and protein induced by LPS, demonstrating that KRN2-induced decreases in NFAT5 protein troglitazone and nuclear translocation of NFAT5 are caused by its down-regulatory effect on NFAT5 mRNA transcription. To further understand the inhibitory mechanism of KRN2, we checked the activation of representative signaling molecules affecting NFAT5 mRNA transcription, p38 MAPK and NF-κB. p38 MAPK is phosphorylated in response to LPS stimulation (Han et al., 1993), and it then activates various transcription factors (Zarubin and Han, 2005). Especially, p38α has been identified as an important upstream molecule of NFAT5 (Ko et al., 2002; Kim et al., 2014). NF-κB has long been implicated in a pro-inflammatory signaling pathway (Ghosh and Hayden, 2008) and is recently identified as a critical regulator of NFAT5 in macrophages subjected to LPS stimulation (Buxadé et al., 2012; Kim et al., 2014). In this study, we found that p38 MAPK and its isoforms were unaffected by treatment with KRN2. The translocation and phosphorylation of NF-κB p65 upon LPS stimulation were not inhibited by KRN2 either. Furthermore, KRN2 had no effects on ROS expression and Elk-1- and CREB-dependent reporter activity, all of which are associated with NF-κB and p38 MAPK (Wen et al., 2010; Tian and Karin, 1999). Thus, we exclude the possibility that KRN2\'s actions on NFAT5 are indirect. BBR is a traditional Chinese medicine used for the treatment of gastroenteritis without serious adverse effects and is known to have potent anti-inflammatory activity (Grycova et al., 2007; Tillhon et al., 2012; Yan et al., 2012; Sarna et al., 2010; Cheng et al., 2015). In this study, we discovered novel BBR-based NFAT5 suppressors to inhibit NFAT5-dependent reporter activity, NFAT5 mRNA and protein expressions, and NFAT5 translocation to nucleus. There are many physicochemical studies on BBR binding to double-stranded DNA (Xu et al., 2012; Mazzini et al., 2003). For example, evidence has shown that BBR binds the minor groove of the AT-rich region [d(AAGAATTCTT)]2 and that this binding is related to its effect on Topoisomerase-I and -II (Mazzini et al., 2003; Kim et al., 1998). Thus, KRN2, a BBR derivative, might interact with NFAT5-binding consensus sequence (TGGAAAATTACCG) through its BBR moiety, decreasing expression of NFAT5 target genes. It has also been demonstrated that BBR directly binds to specific DNA sequences and forms a complex with DNA triplexes or G-quadruplexes [d(TGGGGT)]4 (Xu et al., 2012). In this study, we postulated that KRN2 could target two NF-κB consensus binding sites, GGGGATTTCC and GGGACTTCCC, located in the Nfat5 promoter site. We discovered BBR-based novel κB inhibitors to suppress NFAT5 expression, which have completely different mode of action from conventional NF-κB inhibitors, such as MG132 (Ortiz-Lazareno et al., 2008), that suppress I-κB expression or activity. Using a GFP reporter vector encompassing the upstream site (base pairs −3000 to +1) of Nfat5 exon 1, we demonstrated that KRN2 selectively blocked Nfat5 promoter activity induced by LPS, but not by high salt. Moreover, a ChIP assay using an anti-NF-κB antibody demonstrated a marked reduction of p65 binding to NF-κB consensus binding sites in the Nfat5 promoter. EMSA also revealed that KRN2 decreased the formation of NF-κB p65-DNA complexes in a dose-dependent manner. Given that NFAT5 expression is nearly completely dependent on NF-κB binding to its promoter (Buxadé et al., 2012), our data suggest that KRN2 inhibits transcriptional activation of NFAT5 at least partially through blocking NF-κB binding to the Nfat5 promoter. This notion is bolstered by our transcriptome data showing that NF-κB target genes significantly overlapped with the genes expressed differentially in NFAT5-deficient RAW 264.7 macrophages, particularly in the presence of LPS (Supplementary Fig. 4).