Y its activity in vivo against P. aeruginosa [13]. For M33-D 
we propose the following 15900046 mechanism of action. M33-D binds LTA and persists on the bacterial surface for some time by virtue of its resistance to bacterial proteases, causing membrane perturbation that kills the bacteria. Concluding, we identified a new form of the peptide M33, which is strongly active against S. aureus and retains its antimicrobial activity irrespective of strain-resistance phenotypes and mechanisms. MRSA and S. aureus strains with altered susceptibility to glycopeptides pose a CI 1011 custom synthesis serious clinical threat and major therapeutic challenge. In this context, development of a new broad-spectrum therapeutic agent with no cross-resistance to available drugs would be a major achievement.(AG1-X8, 100?00 mesh, 1.2 meq/ml capacity, Bio-Rad). The resin-to-peptide ratio was 2000:1, resin and peptide were stirred for 1 h, the resin was filtered off, washed extensively and the peptide recovered and freeze-dried. Final peptide purity and identity were confirmed by reversed phase chromatography on ?a Phenomenex Jupiter C18 analytical column (300 A, 5 mm, 25064.6 mm) and by mass spectrometry with a Bruker Daltonics ultraflex MALDI TOF/TOF.MIC TestingMICs were determined using a standard microdilution assay as recommended by the Clinical and Laboratory Standards Institute. Assays were performed in triplicate using cation-supplemented Mueller-Hinton (MH) broth (Becton Dickinson, Franklin Lakes, NJ, USA) and a bacterial inoculum of 5×104 CFU/well, in a final volume of 100 ml. The tested concentrations ranged from 0.1 mM to 24 mM for both peptides. Results were recorded after 18?0 h of incubation at 37uC.Materials and Methods Peptide SynthesisSolid-phase synthesis was carried out by standard Fmoc chemistry on Fmoc4-Lys2-Lys-b-Ala Wang resin with a Syro multiple peptide synthesizer (MultiSynTech, Witten, Germany). Side chain protecting groups were 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for R, t-butoxycarbonyl for K and t-butyl for S. M33-L was synthesized using Fmoc-L-aminoacids, and M33-D with Fmoc-D-aminoacids with the exception of the three lysins of the branched core which were Fmoc-L-Lys(Fmoc)-OH (M33-D is consequently a diastereomer). The final products were cleaved from the solid support, deprotected by treatment with TFA containing triisopropylsilane and water (95/2.5/2.5), and precipitated with diethyl ether. Crude peptides were purified by reversed-phase chromatography on a Phenomenex Jupiter C18 ?column (300 A, 10 mm, I-BRD9 250610 mm) in linear gradient form for 30 min, using 0.1 TFA/water as eluent A and methanol as eluent B. Purified peptides were obtained as trifluoroacetate salts (TFacetate). The exchange from TFacetate to acetate form was carried out using a quaternary ammonium resin in acetate formSurface Plasmon ResonanceBiotinylated peptides were immobilized on SA coated flow cells. M33-L and M33-D peptides, diluted to 10 mg/ml in HBS-EP+ buffer (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.05 polysorbate 20 pH 7.4), were injected for 90 sec at a flow rate of 10 ml/min, obtaining 550 RU and 580 RU for M33-L and M33D respectively. LTA and LPS molecules from different species (LPS from E. coli, K. pneumonia, P. aeruginosa and LTA from S. aureus and S. faecalis, were obtained from Sigma-Aldrich: L-3012, L-4268, L9143, L2515 and L4015, respectively) were diluted in HBSEP+ buffer at the concentration of 10 mg/ml and injected for 180 sec with a flow rate of 30 ml/min ove.Y its activity in vivo against P. aeruginosa [13]. For M33-D we propose the following 15900046 mechanism of action. M33-D binds LTA and persists on the bacterial surface for some time by virtue of its 
resistance to bacterial proteases, causing membrane perturbation that kills the bacteria. Concluding, we identified a new form of the peptide M33, which is strongly active against S. aureus and retains its antimicrobial activity irrespective of strain-resistance phenotypes and mechanisms. MRSA and S. aureus strains with altered susceptibility to glycopeptides pose a serious clinical threat and major therapeutic challenge. In this context, development of a new broad-spectrum therapeutic agent with no cross-resistance to available drugs would be a major achievement.(AG1-X8, 100?00 mesh, 1.2 meq/ml capacity, Bio-Rad). The resin-to-peptide ratio was 2000:1, resin and peptide were stirred for 1 h, the resin was filtered off, washed extensively and the peptide recovered and freeze-dried. Final peptide purity and identity were confirmed by reversed phase chromatography on ?a Phenomenex Jupiter C18 analytical column (300 A, 5 mm, 25064.6 mm) and by mass spectrometry with a Bruker Daltonics ultraflex MALDI TOF/TOF.MIC TestingMICs were determined using a standard microdilution assay as recommended by the Clinical and Laboratory Standards Institute. Assays were performed in triplicate using cation-supplemented Mueller-Hinton (MH) broth (Becton Dickinson, Franklin Lakes, NJ, USA) and a bacterial inoculum of 5×104 CFU/well, in a final volume of 100 ml. The tested concentrations ranged from 0.1 mM to 24 mM for both peptides. Results were recorded after 18?0 h of incubation at 37uC.Materials and Methods Peptide SynthesisSolid-phase synthesis was carried out by standard Fmoc chemistry on Fmoc4-Lys2-Lys-b-Ala Wang resin with a Syro multiple peptide synthesizer (MultiSynTech, Witten, Germany). Side chain protecting groups were 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for R, t-butoxycarbonyl for K and t-butyl for S. M33-L was synthesized using Fmoc-L-aminoacids, and M33-D with Fmoc-D-aminoacids with the exception of the three lysins of the branched core which were Fmoc-L-Lys(Fmoc)-OH (M33-D is consequently a diastereomer). The final products were cleaved from the solid support, deprotected by treatment with TFA containing triisopropylsilane and water (95/2.5/2.5), and precipitated with diethyl ether. Crude peptides were purified by reversed-phase chromatography on a Phenomenex Jupiter C18 ?column (300 A, 10 mm, 250610 mm) in linear gradient form for 30 min, using 0.1 TFA/water as eluent A and methanol as eluent B. Purified peptides were obtained as trifluoroacetate salts (TFacetate). The exchange from TFacetate to acetate form was carried out using a quaternary ammonium resin in acetate formSurface Plasmon ResonanceBiotinylated peptides were immobilized on SA coated flow cells. M33-L and M33-D peptides, diluted to 10 mg/ml in HBS-EP+ buffer (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.05 polysorbate 20 pH 7.4), were injected for 90 sec at a flow rate of 10 ml/min, obtaining 550 RU and 580 RU for M33-L and M33D respectively. LTA and LPS molecules from different species (LPS from E. coli, K. pneumonia, P. aeruginosa and LTA from S. aureus and S. faecalis, were obtained from Sigma-Aldrich: L-3012, L-4268, L9143, L2515 and L4015, respectively) were diluted in HBSEP+ buffer at the concentration of 10 mg/ml and injected for 180 sec with a flow rate of 30 ml/min ove.
