Inhibition of SEB Intoxication by a MyD88 Mimetic Figure 6. EM-163 inhibits MyD88-mediated signaling and cytokine production. EM-163 inhibition was measured by monitoring LPS-induced SEAP activity via a MyD88-mediated NF-kB driven signaling pathway. HEK 293 stable transfected cell line (TLR4-MD2-NF-kB-SEAP) was activated with LPS (TLR-4 ligand) and treated with varying concentrations of EM-163 (1 mM to 50 mM). Culture supernatants were tested for SEAP activity and compared to levels in the absence of EM- 163. (A) Inhibition of SEAP reporter activity, (B) inhibition of TNF-a and IL-1b production, (C) Inhibition of TNF-a and IL-1b production by EM- 163 after SEB stimulation of HEK 293 cells transfected with HLA-DR (a/b) and FLAG-MyD88. Results represent three experiments. doi:10.1371/journal.pone.0040773.g006 more effective than Compound 1(IC50 200 mM to 1.2 mM) [19]. The cytokine inhibitory effect of EM-163 on cultured cells was consistent among populations of MNCs isolated from different donors (Figure S2). It is also worth noting that the cytokine inhibitory concentration (IC50) of EM-163 measured in human primary cells, which was 15 mM to 400 mM for most of the cytokines were consistent with the concentration of EM-163 that inhibited SEAP reporter activity in our earlier experiments. At IC50 concentration EM-163 did not show cytotoxicity in MNCs (data not shown). Cell viability (metabolically active cells) was examined by measuring ATP content using a chemiluminescence method as described elsewhere [19]. EM-163 also inhibited NF-kB p50 activation (46% to 62% at concentration 250 mM to 10 mM) in MNCs isolated from different donors treated with SEB (Figure S3). These results suggest that the dimeric BB-loop mimetic was effective in inhibiting cytokine production in human primary cells when applied either pre- or post-exposure to SEB, without affecting cellular viability. EM-163 Abrogates Pro-inflammatory Cytokine Response in vivo and Protects Mice when Administered Either Pre- or Post-exposure to Lethal SEB Challenge The results presented above suggest that observed changes in signaling due to EM-163 treatment translate into a decrease in cytokine production in primary culture. We therefore examined if EM-163 treatment would likewise lead to a decrease in cytokines in a live animal and if this would protect the animals from a lethal SEB challenge. To address this, we used the LPS potentiation Figure 7. Pre or post exposure treatment of EM-163 inhibits TNF-a, IFN-c, IL-2 and IL-1b production. Human MNCs (56105/well) were treated with EM-163 for 30 min before (pre-exposure) or 30 min after (post-exposure) being exposed to SEB(200 ng/ml) and varying concentrations of EM-163 (1 mM, 500 mM, 100 mM and 10 mM). Culture supernatants were collected after 16 h and analyzed for cytokine release using a MSD assay. The IC50 value was calculated as the concentration required for inhibition of cytokine production by 50% relative to the control. Data represent five experiments. doi:10.1371/journal.pone.0040773.g007 PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40773