Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88 Teri L. Kissner1, Gordon Ruthel1, Shahabuddin Alam1, Enrique Mann 2 , Dariush Ajami2, Mitra Rebek2, Eileen Larkin1, Stefan Fernandez1, Robert G. Ulrich1, Sun Ping 3 , David S. Waugh3, Julius Rebek, Jr.3, 1 Kamal U. Saikh * 1 Department of Immunology, United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, United States of America, 2 Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America, 3 Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, Maryland, United States of America Abstract Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD882/2 mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- a, interferon (IFN)-c, interleukin (IL)-1b, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB- driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-a and IL-1b production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication. Citation: Kissner TL, Ruthel G, Alam S, Mann E, Ajami D, et al. (2012) Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88. PLoS ONE 7(7): e40773. doi:10.1371/journal.pone.0040773 Editor: Guillermo H. Giambartolomei, National Council of Sciences (CONICET), Argentina Received May 2, 2012; Accepted June 13, 2012; Published July 27, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the Defense Threat Reduction Agency (DTRA; WWW.jpras.us) grant CBM.THRTOX.03.10.RD.006 to Kamal U. Saikh. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kamal.saikh@amedd.army.mil Introduction The profound clinical consequences of staphylococcal entero- toxin B (SEB)-induced toxicity are known to stem from an excessive pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. Strong pro-inflammatory responses by antigen-presenting cells (APCs) and T cells are triggered by the binding of SEB to MHC class II molecules on APCs and subsequent cross-linking to T-cell receptors (TCR) [1–5]. SEB is listed by the Centers for Disease Control and Prevention (CDC) as a Category B select agent because of its potential use as an aerosolized biological weapon. There are currently no small-molecule therapeutics available to treat SEB exposure, making the development of such medical countermeasures an important goal. In a mouse model, the biological effects of SEB are potentiated by lipopolysaccharide (LPS), a bacterial factor that binds to toll- like receptor 4 (TLR4) on the surface of cells. SEB and LPS synergistically amplify pro-inflammatory cytokine production, leading to severe toxicity [6–7]. Consequently, both super- antigenic exotoxins (SEs) and bacterial LPS (endotoxin) have been implicated in the pathogenesis of TSS, supported by their identification in the bloodstream of critically ill patients with septic shock [8–9]. Results from our laboratory demonstrated that myeloid differentiation protein 88 (MyD88) gene knockout (MyD882/2) mice were resistant to lethal SEA or SEB challenge and showed a concomitant reduction in serum levels of pro- inflammatory cytokines [10–11]. We also reported that the binding of SEA or SEB to MHC class II activates MyD88- mediated pro-inflammatory cytokine signaling in human primary cells [12]. Consistent with our results, a recent report indicated that deficiency in MHC class II resulted in impaired TLR- triggered production of pro-inflammatory cytokines and pro- tected mice from an otherwise lethal challenge with TLR ligands and live Gram-negative bacteria [13]. This study also concluded that both the TLR-and MHC –mediated responses engage MyD88 [14]. MyD88 is an adaptor protein that functions to recruit signaling proteins to members of the Toll-like and interleukin-1 receptor (TLR/IL-1R) [15–16] family as well as the IFN -c receptor [17]. Activation of the MyD88-mediated pro-inflammatory signaling pathway by this class of receptors is very important for several PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40773