The identity of the primary peptidomimetic sequences 4a, 4b and 4c

were confirmed by high-resolution MS (Bruker MicroTOF-Q LC mass spectrometer equipped with an electrospray ionization source): compound 4a, (m/z) [M+4H]4+ obsd. = 339.9727 (calcd. = 339.9719, ΔM 2.3 ppm); compound 4b, (m/z) [M+5H]5+ obsd. = 402.0614 (calcd. = 402.0608, VX-680 molecular weight ΔM 1.4 ppm); compound 4c, (m/z) [M+6H]6+ obsd. = 443.2880 (calcd. = 443.2879, ΔM 0.2 ppm). Peptides were solubilized to a stock of 10 mg/mL in sterile MilliQ water and stored at -20°C. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The Minimum Inhibitory Concentration (MIC) of the chimeras was determined against the spectrum of bacteria using the microdilution method according to guidelines of the Clinical PRI-724 mw and Laboratory Standards Institute (CLSI) [30]. Chimera 1:2 serial dilutions were prepared from 1,024 μg/mL stock solutions to give a final range

of 512-0.5 μg/mL in the wells. This corresponds to a final range of 144 to 0.14 μM for the heaviest chimera (i.e. chimera 4c) and of 282 to 0.27 μM for the lightest chimera (i.e. chimera 4a). Colonies grown overnight (i.e. approximately 18 hours) on BHI agar were suspended in 0.9% saline to give a turbidity of 0.13 at OD546 (approximately 1 × 108 CFU/mL), and then diluted in MHB pH 7.4 to a final concentration of 5 × 105 CFU/mL in each well. Following CLSI guidelines the media for testing of Listeria monocytogenes strains were supplemented with 2.5% lysed horse blood. Polypropylene plates (Nunc 442587) were used to minimize peptide binding and incubation time was 18-20

hours at 37°C. MIC was determined PJ34 HCl in a minimum of two technical replicates as the lowest concentration of the peptide analogue where no visible growth was found. The Minimum Bactericidal Concentration (MBC) was determined by plating 10 μL of the suspension from the first three wells without growth on BHI agar and incubating these for 24 hours at 37°C. MBC was the lowest concentration at which a 99.9% reduction in CFU/mL was observed. Activity is expressed in μmol/L to enable a direct comparison of analogues with different length (= size). Killing kinetics of Staphylococcus aureus and Serratia marcescens In vitro time-kill curves for chimera 1, 2 and 3 were determined against S. aureus 8325 (MIC μM: chimera 1 5.9; chimera 2 2.8; chimera 3 18.7) and Serratia marcescens ATCC 8100 (MIC μM: chimera 1 46.8; chimera 2 45.5; chimera 3 150.0). These two bacterial strains represent organisms susceptible and tolerant to the chimeras, respectively. The bactericidal effect of the three chimeras was tested at MIC in two independent experiments; additionally the effect of chimera 2 was tested at ¼ and 1/2 times MIC.