cells were then incubated with primary MyD88 antibody for 1 h, washed in PBS containing 0.5% BSA, then incubated with Alexa 568-conjugated goat anti-rabbit secondary antibody. The cells were counterstained with DAPI to detect cell nuclei. Labeled cells were mounted on glass slides using fluoromount and covered with glass cover slips. Labeled cells were imaged using a Leica (Mannheim, Germany) TCS SP5 laser scanning confocal system. Green and red fluorescence were visualized using 488-nm and 568 nm wavelength laser excitation, respectively, and DAPI images were collected using two-photon excitation from a Ti/ sapphire laser tuned to 780 nm. Synthesis of Compound EM-163 Initially, Compound 1 was used to synthesize EM-110 [20], as follows. Briefly, to a stirred solution of 2,5-pyridine dicarboxylic acid (52 mg; 0.3 mmol) in CH2Cl2 (0.5 ml) and dimethylforma- mide (10 ml), thionyl chloride (0.4 ml) was added drop-wise at 0uC. The mixture was then heated at 40uC for 1 h, and the solvent was removed under reduced pressure. The diacid chloride obtained was added to a solution of amine 2 (200 mg; 0.69 mmol) and Et3N (97 ml; 0.69 mmol) in dry CH2Cl2 (10 ml). The reaction mixture was stirred at ambient temperature overnight, the solvent was evaporated, and the residue was purified by silica gel chromatography (hexane/AcOEt mixtures) yielding compound 4 (36 mg; 17% yield) as a white solid. 1H NMR: (600 MHz, CDCl3) d 8.39 (s, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.20 (m, 4H), 7.11 (m, 2H), 6.94 (m, 2H), 6.86 (m, 2H), 5.21 (m, 2H), 3.70 (m, 4H), 3.59 (m, 2H), 3.43 (m, 2H), 3.31 (m, 4H), 2.51 (m, 2H), 2.40 (m, 2H), 2.29 (m, 2H), 1.98 (m, 4H), 1.90 (m, 4H), 1.68 (m, 2H), 1.54 (m, 2H), 1.04 (m, 12H); 13C NMR: (150 MHz, CDCl3) d169.6, 169.0, 168.5, 168.4, 155.6, 145.8, 141.4, 140.7, 135.1, 133.8, 128.9 (2C), 128.8 (2C), 128.5 (2C), 128.3 (2C), 126.4, 126.2, 123.6, 60.9, 60.4, 47.2, 47.0, 46.5 (2C), 45.2, 44.8, 33.6, 33.4, 31.8, 31.7, 28.3 (2C), 26.6 (2C), 24.6 (2C), 20.5, 20.3, 18.9, 18.8; MS: (ESI-TOF) MH+ calculated:708.4488, found: 708.4461. EM-163 was synthesized from compound EM-110 [20] as shown in Figure 1. Compound EM-110 (600 mg, 0.85 mmol) and methyl iodide (10 ml) were stirred at 70uC for 72 h. The excess methyl iodide was then eliminated under a stream of nitrogen and the residue was purified by column chromatography (AcOEt/ MeOH, 95:5) to afford compound EM163 (380 mg, 53%) as a orange solid. 1H NMR (600 MHz, CD3CN, 50uC, mixture of conformers) d 9.70 (bs, 1H), 8.58 (bs, 1H), 8.16 (bs, 1H). 7.25–6.90 (m, 10 H), 5.07 (m, 2H), 4.48 (s, 3H), 3.62 (m, 4H), 3.53 (m, 2H), 3.41 (m, 2H), 3.32 (m, 4H), 2.55 (m, 2H), 2.45 (m, 2H), 2.37 (m, 2H), 1.95 (m, 4H), 1.86–1.70(m, 8H), 1.00–0.85 (m, 12H). MS: (ESI-TOF), M+ calculated: 722.4845, found: 722.4837. Expression of MyD88 Protein The open reading frame encoding the TIR domain of human MyD88 (residues 157–296) was amplified from pCDNA3-MyD88- GFP (Addgene plasmid 13026, Addgene, Cambridge, MA, USA) and inserted into pDONR201 (Invitrogen, Carlsbad, CA,). The gene was sequence verified and then inserted into the destination vector pDEST-HisMBP [21] by Gateway recombinatorial cloning to generate expression vector pPS2218. The recombinant His-MBP-MyD88(157–296) fusion protein was expressed in E. coli BL21(DE3) Codon Plus-RIL cells (Stratagene, La Jolla, CA), which were grown in Luria broth and induced at mid-log phase with 1 mM IPTG for 4 h at 30uC. The cells were harvested by centrifugation at 4uC and frozen at 280uC until use. The cell pellet was resuspended in 50 mM sodium phosphate pH 7.1, 150 mM NaCl, 5% glycerol, 25 mM imidazole, and then the cells were disrupted using an APV Model Inhibition of SEB Intoxication by a MyD88 Mimetic G1000 homogenizer (Invensys, Roholmsvej, Denmark). The lysate was centrifuged at 15,000 rpm at 4uC, filtered, and the fusion protein was then purified by immobilized metal affinity chroma- tography (IMAC) as described [22]. Fractions containing the fusion protein were pooled, cleaved overnight with hexahistidine- tagged TEV protease, and then subjected to another round of IMAC as described [22]. The flow-through fractions were pooled, concentrated to 5 ml and applied to a 320 ml XK26/60 Sephacryl S-100 gel filtration column (GE Healthcare, Piscat- away, NJ) equilibrated in 25 mM 2-(N-morpholino) ethanesulfonic acid (MES) pH 6.3, 150 mM NaCl, and 2 mM Tris(2-carbox- yethyl) phosphine hydrochloride (TCEP). The peak fractions corresponding to MyD88 (157–296) were pooled and concentrat- ed to approximately 1 mg/ml. Microarray-based Binding Assay A microarray-based binding assay was developed and optimized to measure the binding of compound EM-163 to MyD88. Nitrocellulose-coated slides (PATH slides, Gentel Biosciences, Madison, WI) were spotted in quadruplicate with anti-MyD88 antibody or MyD88 monomer using the ArrayJet Marathon printer (Roslin, Scotland, UK). Slides were blocked overnight (,16 h) at 4 oC with blocking buffer (50 mM Hepes, 0.08% triton X-100, 50% glycerol, 140 mM NaCl and 3% BSA). Recombinant MyD88 was bio-tinylated using the EZ Link Biotin BMCC kit from Pierce (Cat# PI-21900, Pittsburg PA). Biotinlyated MyD88 (25 mg/ml) was pre-incubated with and without compound (100 mg/ml and 50 mg/ml) for 1 h on ice and then diluted with probing buffer (50 mM Hepes, 0.1%Tween, 3% human antibody serum). MyD88 samples and a probing buffer only control were added to designated wells on the microarray slide for 2 h at room temperature. Slides were then washed 6 times for 5 min with washing buffer (50 mM Hepes, 0.2%Tween, 3% human antibody serum). Streptavidin labeled with alexa fluor 647 (1:1000, Invitrogen, Carlsbad CA) was then added for 1 h at room temperature to detect MyD88 binding interaction. Slides were again washed as before and then rinsed with distilled water for 10 sec, dried and analyzed using the Genepix 4000B (Molecular Devices, Sunnyvale, CA). Cell Culture and Transfections Human embryonic kidney (HEK) 293T cells were cultured in EMEM, supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), and grown in a 37uC humidified atmosphere of 5% CO2. For co-immunoprecipitation of MyD88- Flag/HA-MyD88, HEK cells were cultured in 6-well plates and transfected by lipofectamine 2000 (Invitrogen) method with 4– 5 mg of the appropriate plasmids according to the manufacturer’s instructions. The MyD88 mimetic EM-163 was added to the medium 6 h after transfection. For SEB-induced cytokine in- hibition by EM-163, HEK 293 cells were transfected with plasmids expressing HLA-DR (a/b) and Flag-MyD88. Six h after transfection, cells were cultured with SEB in the presence or absence of EM-163. Culture supernatants were collected after 24 h for measuring cytokine production. Co-immunoprecipitation Assay HEK293T cells (transfected or Mock) were collected 48 h after transfection, washed with 2 ml of ice-cold PBS, and lysed in 80 ml of buffer [50 mM HEPES, pH7.4]. Cells were pelleted by centrifugation at 10, 000 x g for 10 min at 4uC, and cytosolic fractions were collected for immunoprecipitation. Cell extracts (1 mg total proteins) were incubated with 2 mg of mouse anti –Flag M2 conjugated with agarose attached to magnetic beads (Sigma- PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40773