Inhibition of SEB Intoxication by a MyD88 Mimetic Meier survival analysis and log-rank tests were performed to compare survival curves with stepdown Bonferroni adjustment for pair wise comparisons between groups. (A), Pre-treatment of EM-163, 0.42 mg/mouse, or 0.86 mg/mouse protected mice against lethal SEB (0.5 mg/mouse,1 LD50) challenge, p = 0.0015; Pre-treatment of EM-163 protected mice challenged with SEB (5 mg/mouse equivalent to10 LD50), p#0.0261; (B). Administration of EM-163 in mice inhibited pro-inflammatory cytokine response. Similar to experimental settings as in Figure 7A, mice (n = 6) were bled at 4 h, serum were pooled from each group [LPS, SEB+LPS, EM-163 (1.7 mg/mouse)] and measured serum cytokines. (C). Post-exposure to SEB (0.5 mg I LD50), EM- 163 treated mice were protected, p = 0.0102; Post-exposure to SEB challenge, EM-163 delayed death (5 mg = 10 LD50), p#0.0129. doi:10.1371/journal.pone.0040773.g008 mediated signaling. In both cases, targeting of MyD88 by EM-163 inhibited pro-inflammatory cytokine signaling and protected mice from SEB intoxication. EM-163 treatment caused intracellular accumulation of MyD88 in primary monocytes when stimulated with SEB. It is likely that EM-163 binds to MyD88, including MyD88 that is newly synthesized in response to SEB, rendering it incapable of downstream signal transduction. EM-163 may or may not be capable of disrupting dimers of the MyD88 TIR domain, but it is clear that stable, monomeric forms of MyD88 must exist since they engage interchangeably in both homo and heterotypic interactions. It seems likely that the monomeric form of the MyD88 TIR domain, with its exposed BB loop, is the molecular target of EM-163. In summary, our results provide evidence that a synthetic BB- loop mimetic, EM-163, that targets the TIR-domain of MyD88 can limit hyper-inflammation and prevent toxic shock. The effectiveness of this mimetic when administered either pre- or post-SEB exposure further supports its therapeutic potential. An ongoing effort is underway to further refine EM-163 by chemical modification to increase potency, bioavailability and drug-like properties for potential clinical use against toxic shock. Supporting Information Figure S1 EM-163 targets newly expressed MyD88. MyD88 KO HEK293 (HEK293-I3A) cell line were co-transfected with plasmids MyD88 flag and pCMV-HA-MyD88. Seven hours after transfection, cells were incubated for 13 h with or without EM-163 (100 mM to 1 mM). At the end of incubation, cells were lysed, and cytoplasmic fractions were separated. (A), MyD88 expression was detected by Western blot analysis using anti- MyD88 antibody. (B), Cell extracts were immunoprecipitated (IP) with anti-Flag antibody, and immune-precipitated proteins were analyzed in Western blot with anti-MyD88 antibody. (C) Immunoblot (B) was stripped and reprobed with anti-HA antibody. The results are representative of two independent experiments. (TIF) Figure S2 EM-163 inhibits SEB-induced cytokine re- sponse in different donors. MNCs (16106) from two normal donors (Donor 1 and Donor 2) were cultured with SEB (200 ng/ ml) with or without EM-163 (500 mM to 10 mM) for 20 h. The culture supernatants were collected and measured for cytokine by MSD assay. The IC50 value was calculated as the concentration required for inhibition of cytokine production by 50% relative to the control. Data are representative of three experiments. (TIF) References 1. Fraser JD (1989) High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR. Nature 339: 221–223. 2. Dinges MM, Orwin PM, Schlievert PM (2000) Exotoxins of Staphylococcus aureus. Clin Microbiol Rev 13: 16–34. 3. Scholl P, Diez A, Mourad W, Parsonnet J, Geha RS, et al. (1989) Toxic shock syndrome toxin 1 binds to major histocompatibility complex class II molecules. Proc Natl Acad Sci USA 86: 4210–4214. 4. Ulrich RG, Bavari S, Olson MA (1995) Bacterial superantigens in human disease: structure, function and diversity. Trends Microbiol 3: 463–468. Figure S3 Inhibition of NF-kB p50 activation in the presence of EM-163 in MNCs stimulated with SEB. Activation of NF-kB in primary mononuclear cells treated with SEB in the absence or presence of different concentration EM-163 was determined as described elsewhere [12]. Data are presented in the figure as percentage increase over the control and represent one of three experiments using separate donors. Significant differences (p#0.005) are indicated for MNCs treated with SEB vs MNCs treated SEB in the presence of EM-163 (*). (TIF) Figure S4 EM-163 attenuates pro-inflammatory cyto- kines and SEA induced lethality in mice. (A), Administra- tion of EM-163 protected mice from toxic shock induced death challenged with lethal dose of SEA. C57BL/6 mice mice (n = 4) were injected with EM-163 (0.85 mg, or 1.7 mg, 100 ml volume/ mouse), 30 min later injected with SEB (5 mg/mouse) followed by LPS 2 h later. Mice were observed for survival. Control mice injected with 150 mg of LPS or 1 mg of SEB survived. Data are representative of two separate experiments; (B), Administration of EM-163 in mice inhibited pro-inflammatory cytokine response, mice were bled at 24 h, serum were pooled from each group and measured serum cytokines. (TIF) Acknowledgments The authors thank Thomas Plummer for his help with animal experiments, Lorraine Farinick for figure preparation and Diana Fisher for statistical analysis. We are also grateful to the investigators at Addgene for providing plasmids originally published in Proc. Natl. Acad. Sci 2006 Jul.18.103(29): 10961–6. Author Contributions Conceived and designed the experiments: Performed cytokine measure- ment and micro-array experiments: TLK. Partly involved in writing the manuscript: TLK. Performed the confocal image data collection and edited the manuscript: GR. Performed the transfection, co-immunoprecipitation and animal experiments: SA. Contributed in the synthesis of compounds: EM DA. Contributed optimization and data analysis of micro-array based binding experiments: EL SF RU. Contributed design and synthesis of BB- loop mimetic: MR. Contributed expression and purification of TIR domain recombinant protein: SP DSW. Designed and supervised overall synthetic chemistry: JR. Designed and supervised the research and wrote the manuscript: KUS. Performed the experiments: TLK GR SA EM DA EL SF SP KUS. Analyzed the data: TLK GR SA EM DA MR EL SF SP DSW JR KUS. Contributed reagents/materials/analysis tools: RGU. Wrote the paper: TLK SP MR KUS. 5. Stiles BG, Bavari S, Krakauer T, Ulrich RG (1993) Toxicity of staphylococcal enterotoxins potentiated by lipopolysaccharide: major histocompatibility com- plex class II molecule dependency and cytokine release. Infect Immun 61: 5333– 5338. 6. Blank C, Luz A, Bendigs S, Erdmann A, Wagner H, et al. (1997) Superantigen and endotoxin synergize in the induction of lethal shock. Eur J Immunol 27: 825–833. 7. Schlievert P (1982) Enhancement of host susceptibility to lethal endotoxin shock by staphylococcal pyrogenic exotoxin type C. Infect Immun 36: 123–128. PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e40773