unstimulated cells after treatment with EM-163 only at 4 hr. Similar effects of EM-163 on T cells (CD3+MyD88+) were observed when examined by dual color flow cytometric analysis (data not shown). A possible explanation for these results is that EM-163, having two BB-loop mimetic groups, capable of binding to the basal MyD88 molecules in the cells and stabilized to allow accumulation within the cell. At low concentrations, EM-163 would be expected to be more likely to bind two MyD88 monomers than at saturating concentrations, where there would be enough EM-163 molecules to bind MyD88 monomer at a 1:1 ratio. We therefore examined if this prediction would be borne out experimentally. In addition, because SEB stimulation induces up regulation of MyD88, we wished to test if newly synthesized MyD88 would be targeted by EM-163. To this end, HEK 293T cells were co-transfected with plasmids pCMV-HA-MyD88 and MyD88-Flag and 6 h later were treated with varying concentrations of EM-163. In a co-immuno- precipitation assay using anti-Flag antibody, followed by SDS- PAGE and immunoblot analysis with anti-HA antibody, EM-163 treatment inhibited the presence of 62 kDa protein hence, increased the accumulation of 31 kDa proteins in a dose-de- pendent manner (Figure 5A). While controls, i.e., no compound treated pCMV-HA-MyD88 and MyD88-Flag transfected cells (Figure 5A) or only DMSO treated cells had no inhibitory effect of 62 kDa (data not shown), because more accumulation of 31 kDa band was observed compared to EM-163 treatment. Using the same experimental set up as for the results shown in Figure 5A, the presence of 31 kDa MyD88 was confirmed by reprobing with anti- MyD88 antibody (data not shown). These results suggest that EM- 163 prevented the association of Flag-MyD88 -HA-MyD88 complex (Figure 5B) in a dose-dependent manner by targeting to newly synthesized MyD88. To further confirm that newly expressed MyD88 are targeted by EM-163, we performed an Inhibition of SEB Intoxication by a MyD88 Mimetic additional experiment using MyD88 KO HEK293 cell line (HEK293-I3A). Similar to results shown in Figure 5, co- immunoprecipitation assay using anti-Flag antibody, followed by SDS-PAGE and immunoblot analysis demonstrated that EM-163 treatment inhibited in a dose dependent mannar MyD88 dimer, and thereby, allowed accumulation of 31 kDa MyD88 (Figure S1). It may likely be that the monomeric form of the MyD88 TIR domain, with its exposed BB loop, is the molecular target of EM- 163. Thus, EM-163 appeared able to target newly expressed MyD88 and to prevent MyD88 complexes. However, it was important to ascertain if the interaction of EM-163 with MyD88 could inhibit downstream signaling. EM-163 Blocks MyD88-mediated Signaling To determine if the inhibition of TIR-TIR interaction demonstrated above had an effect on signaling, we utilized a cell- based reporter assay. A stable co-transfected (TLR4-MD2-NF-kB/ SEAPorterTM) HEK 293 cell line was used to detect a MyD88- mediated NF-kB driven SEAP reporter response induced by LPS. As shown in Figure 6A, EM-163 inhibited the LPS-induced TLR4- MD2-MyD88-NF-kB-SEAP response in a dose-dependent manner. In contrast, TLR3 ligand poly IC stimulation, which does not utilize MyD88, had no effect on SEAP response (data not shown). In addition to inhibition of the SEAP response to LPS stimulation, EM-163 also blocked LPS-induced TNF-a and IL-1b production by these cells in a dose-dependent manner (Figure 6B). We also confirmed that EM-163 was capable of inhibiting cytokine production stimulated by SEB in a similar HEK 293 cell model. HEK 293 cells were co-transfected with HLA-DR (a/b) and Flag- tagged MyD88 plasmids and stimulated with SEB in the presence or absence of EM-163. Culture supernatants were then tested for the presence of TNF-a and IL-1b. Results shown in Figure 6C demonstrate SEB-stimulated production of TNF-a and IL-1b by Figure 4. SEB stimulation of human monocytes in the presence of EM-163 leads to intracellular accumulation of MyD88. Increased accumulation of MyD88 in CD14+ monocytes treated with SEB in the presence of EM-163 at 1 h and 4 h. Confocal images show expression of nuclei (blue) and intracellular MyD88 (red) proteins in CD14+ monocytes. Monocytes were untreated or treated with SEB, EM-163 or SEB plus EM-163 (250 mM). Cell nuclei were labeled with DAPI (blue). Scale bar = 10 mm. Data reperesent three similar experiments with different donors. doi:10.1371/journal.pone.0040773.g004 PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40773