Inhibition of SEB Intoxication by a MyD88 Mimetic Figure 1. Synthesize of compound EM-163. EM-163 was synthesized from compound EM-110 as described in Materials and Methods. doi:10.1371/journal.pone.0040773.g001 Aldrich) for 16 h under constant shaking at 4uC. Agarose bead- bound immunocomplexes were separated by a magnetic separa- tor, washed three times, and eluted in SDS-PAGE sample buffer for western blot analysis. Secreted Alkaline Phosphatase (SEAP) Assay TLR4/MD-2/NF-kB/SEAPorter HEK 293 cells (56105 cells/ ml/well) were cultured with LPS (1 mg/ml) or poly IC (1 mg/ml), with varying concentrations of EM-163 in a 24-well plate and incubated at 37uC for 16 h. The culture supernatant was collected and centrifuged to remove any cell debris. The Great EscAPe SEAP assay from Clonetech was used to determine the amount of alkaline phosphatase secreted into the supernatant. A 16 dilution buffer was prepared from a 56 stock solution and 75 ml of the 16 dilution buffer was mixed with 25 ml of the supernatant, incubated for 30 min at 65 oC to inactivate endogenous alkaline phospha- tase. The samples were placed on ice for 3 min and then equilibrated at room temperature. SEAP substrate solution (100 ml) was added to each sample and read at 10 min intervals using a chemiluminescence reader. Statistical Analysis The SAS program v9.2 (Cary, NC, USA) was used for statistical analysis. The planned analyses using T-tests with step-down Bonferroni adjustment to compare geometric mean NF-kB activation and cytokine levels between groups were undertaken for determining statistical significance. Kaplan- Meier survival analysis (dependent variables: survival status, time to death) and log-rank test to compare survival curves (with step-down Bonferroni adjustment for pair wise comparisons) among groups were performed. Results Design and Synthesis of a Dimeric BB-loop Mimetic of MyD88 (EM-163) Several structural and mutational studies have pointed to the BB-, DD- and EE-loop regions as mediators of the homo- or hetero-dimerization function of TIR domains in bacteria and mammals [23–26]. However, neither the homotypic nor hetero- typic interactions between TIR domains of receptors and adaptors are well understood [27]. Our earlier results indicated that a synthetic mimetic, Compound 1, modeled on a tripeptide sequence of the BB-loop [(F/Y)-(V/L/I)-(P/G)] of the TIR domain, showed promise in attenuating SEB as well as TSST1- induced pro-inflammatory cytokine production in human primary cell culture [19]. Because of the importance of homotypic and heterotypic interactions in MyD88 signaling, we hypothesized that a BB-loop mimetic with a dimeric structure might be more effective in blocking MyD88-signaling. Therefore, in an attempt to improve specificity and therapeutic efficacy, a dimeric compound EM-110 was synthesized based on the original structure of Compound 1 [20]. Further chemical modification of EM-110 by treatment with methyl iodide led to the synthesis of a quaternary ammonium salt: the stable crystalline solid compound EM-163 (Figure 2). EM-163 Binds to TIR Domain and Inhibits TIR-TIR Homotypic Interaction TIR domain interaction of MyD88 is required for its recruitment to the receptor complex, a critical step for MyD88-mediated pro-inflammatory signaling via the ensuing activation of the downstream kinases IRAK1 and IRAK4. To determine if EM-163 interferes with TIR-TIR domain in- teraction, we examined the effect of EM-163 binding to recombinant TIR domain MyD88 protein in a microarray- based binding assay. We expressed and purified the recombi- nant MyD88 TIR domain (amino acids 157–296) as a histidine- tag GST fusion protein. In SDS-PAGE a molecular mass of 16.4 kDa of the MyD88 TIR domain protein was observed, as expected. A MyD88 binding array was developed and optimized to detect inhibition of the TIR–TIR domain in- teraction. The results shown in Figure 3 indicate interaction of TIR domain protein (monomer) coated on chips with biotinylated TIR protein. Pre-incubation of immobilized TIR domain protein with EM-163 resulted in a dose-dependent reduction of the signal, suggesting that EM-163 binding to TIR domain protein inhibited the TIR–TIR domain interaction (Figure 3). PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40773