Supplemental Information. Converting a Staphylococcus aureus Toxin. into Effective Cyclic Pseudopeptide Antibiotics

Transcription

1 Chemistry & Biology, Volume 22 Supplemental Information Converting a Staphylococcus aureus Toxin into Effective Cyclic Pseudopeptide Antibiotics Olivia Solecki, Amor Mosbah, Michèle Baudy Floc'h, and Brice Felden

2 Supplemental Text and Figure Converting a Staphylococcus aureus Toxin into Effective Cyclic Pseudopeptide Antibiotics Olivia Solecki 1, Amor Mosbah 2, Michèle Baudy Floc'h*,2 and Brice Felden*,1 1 U835 Inserm, Pharmaceutical Biochemistry, 2 Av. du Prof. Léon Bernard and 2 Institut des Sciences Chimiques de Rennes, UMR 6226 CNRS-Université de Rennes 1, Campus de Beaulieu, Rennes Cedex, France

3 EXPERIMENTAL PROCEDURES Abbreviations. DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMF, N,Ndimethylformamide; Fmoc, 9-fluorenylmethoxycarbonyl; TBTU, 2-(1H-benzotriazole-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; SPPS, solid phase peptide synthesis; TFA, trifluoroacetic acid; TIS, triisopropylsilane. Chemicals. Fmoc-protected amino acids were from Novabiochem and Iris Biotech (Marktredwitz, Germany). H-Rink amide ChemMatrix resin was from Sigma-Aldrich, TBTU was from Iris Biotech. Solvents for peptide synthesis and HPLC were from Carlo Erba-SdS (Sabadell, Spain). TFA was from Fluorochem Ltd (Derbyshire, UK). All other chemicals were purchased from Sigma-Aldrich at the highest quality available. Pep C-ter was from Proteogenix. Automated solid-phase synthesis. Synthesis of linear peptides and pseudopeptides including N α -Fmoc-amino acids, N β -Fmoc-aza- 3 -amino acids N β -Fmoc-aza-β 3 -homohydroxythreonine, was accomplished using our previously described synthesis (Laurencin et al 2009) and a Rink Amide resin ( mesh; 0.79 mmol/g), except for the native peptide PepA1 that we use H-Rink amide Chem Matrix resin. (0.5 mmol/g). Peptides were synthesized at 100 µmol scale. Peptides were assembled on the resin by automated Fmoc synthesis protocols run on a Liberty-12-channel synthesizer (CEM Waves). Briefly, Fmoc groups were removed with piperidine/dmf (20%) using a short (40 W, 75 ºC, 60 s) followed by a long cycle (40 W, 75 ºC, 180 s). After DMF washings, coupling was carried out with a 0.2 M solution of Fmocamino acid, in the presence of 0.5 M TBTU and 2 M DIPEA at 30 W, 68 ºC for 5 min. Fmoc- Arg (Pbf) required an additional coupling step consisting of a long cycle at 0 W, 25 ºC for 25 min followed by one short cycle at 30W, 68 ºC for 5 min. For Fmoc-His(Trt), deprotection was achieved at 50 ºC to avoid racemization, and a specific coupling cycle, again to avoid racemization, was used, consisting of an initial step at 0W, 50 ºC for 2 min followed by another at 30W, 50 ºC for 4 min. After synthesis completion, the N-deblocked peptide resins were side-chain deprotected and cleaved from the resin using TFA/H2O/TIS (95:2.5:2.5, 3 h) with mild orbital shaking. After filtration of the mixtures, the peptides were precipitated by addition of chilled diethyl ether, centrifuged at 4 C (3000 g; 10 min) and the supernatants were discarded. Peptides were analyzed by RP-HPLC on a XTerra C18 column ( mm, 3.5 m) using a Waters 2696 system equipped with a 600 PDA and Empower software. Solvents A and B were 0.1% and 0.08% (v/v) TFA in H2O and MeCN, respectively. Linear 5-60% gradients of B into A over 40 min and 60 95% B into A in 10 min were used for elution at 1 ml/min flow rate were used for elution, with UV detection at 215 nm. Preparative RP-HPLC was done on a XTerra C18 column ( mm, 10 m, Waters) using a Waters 600 system. Solvents A and B were 0.1% TFA (v/v) in H2O and 0.08% TFA (v/v) in MeCN, respectively. Linear 5-60% gradients of B into A over 40 min and 60 95% B into A in 10 min were used for elution, at 10 ml/min flow rate, with UV detection at 215 nm, Due to the hydrophobicity of PepA1 and Pep N-ter, methanol was used instead of the acetonitrile in the same conditions. The homogeneity and identity of synthetic peptides were assessed by analytical C18 reversed-phase HPLC. Peptides were satisfactorily checked for identity by mass spectrometry on MALDI- TOF (matrix-assisted laser-desorption ionization time-of-flight) Daltonics microflex LT (Bruker). H-MLIFVHIIAPVISGCAIAFFSYWLSRRNTK-NH2 (PepA1): Yield after purification 4 %; white powder; RP-HPLC Rt: 40.6 min; MALDI-TOF MS mass: m/z found

4 [M+H + ]; calc.: [M+H + ]. H-MLIFVHIIAPVISGCAIA-NH2 (Pep N-Ter): Yield after purification 10 %; white powder; RP-HPLC Rt: 30.5 min; MALDI-TOF MS mass: m/z found [M+H + ]; calc.: [M+H + ]. H-FFSYWLSRRNTK-NH2 (Pep1): Yield after purification 45%; white powder; RP-HPLC Rt: 26.8 min; MALDI-TOF MS mass: m/z found [M+H + ]; calc. : [M+H + ]. H-FFSYWLSRRTK-NH2 (Pep2): Yield after purification 32%; white powder; RP-HPLC Rt: 30.5 min; MALDI-TOF MS mass: m/z found [M+H + ]; calc.: [M+H + ]. H-FFSWLSRRTK-NH2 (Pep3): Yield after purification 41%; white powder; RP-HPLC Rt: 21.6 min; MALDI-TOF MS mass: m/z found [M+H + ]; calc. : [M+H + ]. H-FFWLSRRTK-NH2 (Pep4): Yield after purification 31%; white powder; RP-HPLC Rt: 24.8 min; MALDI-TOF MS mass: m/z found [M+H + ]; calc.: [M+H + ]. H-FFWLSRTK-NH2 (Pep5): Yield after purification 58%; white powder; RP-HPLC Rt: min; MALDI-TOF MS mass: m/z found [M+H + ]; calc.: [M+H + ]. H-FFWLRR HytK-NH2 (Pep9): Yield after purification 28%; white powder; RP-HPLC Rt: 18 min; MALDI-TOF MS mass: m/z found [M+H + ]; calc.: [M+H + ]. For cyclic peptides a 2-chlorotrityl chloride (CTC) resin was used. Typically, 2-chlorotrityl chloride resin ( mesh; 1.2 mmol/g, 1g) was swelled in dry dichloromethane (DCM) (10 ml) for 10 min. The first monomer was attached onto the resin by adding a solution of Nα-Fmoc-amino acid (1.2 equiv) in dry DCM (10 ml) and DIPEA (4 equiv) with mild orbital shaking for 4 h at room temperature under nitrogen. The loading resin was washed with dimethylformamide (DMF) (5 10 ml), dry DCM (3 10 ml), and then with a mixture of DCM/ MeOH/DIPEA (17/2/1) (2 10 ml), and finally with DMF (3 10 ml). The yield of the loading step was determined on the absorption of dibenzofulvene-piperidine adduct (λmax = 301 nm). Cyclic peptides were synthesized with the same protocol as described previously. At the end of the synthesis, the resin was washed with DCM, dried, and then treated with a solution of 3% TFA in DCM (30 ml) for 20 min at room temperature. The resin slurries were then filtered. The cleavage solution was neutralized with a solution of N-methyl morpholine and the resulting solution was diluted in DCM (170 ml). The linear peptides were slowly added to a solution of EDC, HOBt (4 equiv), and DIEA (4 equiv) in DCM (to a final concentration of 10 4 M). The resulting mixture was stirred for 2 days. The crude mixture was partially concentrated in vacuo (100mL). The resulting solution was washed with 0.5 M HCl, water, and saturated sodium chloride. The organic phases were dried under sodium sulfate then concentrated. Side chain deprotection with a solution of 10mL of TFA/H2O/TIS (95:2.5:2.5, 3 h), purification and characterisation of peptides were performed with the same as described previously. c[ffwlsrrtk] (Pep6): Yield after purification 3%; white powder; RP-HPLC Rt: 33.0 min; MALDI-TOF MS mass : m/z found [M+H + ]; calc. : [M+H + ]. c[ffwsrrtk] (Pep7): Yield after purification 12%; white powder; RP-HPLC Rt: 26.8 min; MALDI-TOF MS mass : m/z found [M+H + ]; calc. : [M+H + ]. c[ffwrrtk] (Pep8): Yield after purification 8%; white powder; RP-HPLC Rt: 26.8 min; MALDI-TOF MS mass: m/z found [M+H + ]; calc.: [M+H + ]. c[ffwlrr HytK] (Pep10): Yield after purification 6%; white powder; RP-HPLC Rt: 29.2 min; MALDI-TOF MS mass : m/z found [M+H + ]; calc. : [M+H + ]. c[ffwrr HytK] (Pep11): Yield after purification 16%; white powder; RP-HPLC Rt: 26.8 min; MALDI-TOF MS mass : m/z found [M+H + ]; calc.: [M+H + ]. Peptides stability in human sera. The stability of Nisin, Melittin and synthetic peptides were tested in vitro, as described (Laurencin et al, 2012). Typically, 1.35 ml of 25% Human serum in a 1.5 ml Eppendorf tube was preincubated at 37 ± 1 C for 15 min prior to addition of 150 μl of peptide or pseudopeptide stock solution (10 2 M in water) to make the final peptide

5 concentration 10 4 mmol/ml. The initial time was recorded, and at known time intervals, 100 µl of reaction solution was removed and quenched by adding 200 µl of 95% EtOH and adjusted at ph=2 to eliminate the effect of peptides adsorption to the protein of the human serum. The cloudy reaction sample was cooled (4 C) for 15 min and then spun at rpm (Eppendorf centrifuge) for 4 min to pellet the precipitated serum proteins. After centrifugation, the supernatants were collected, evaporated, and diluted in 100 μl of H2O and then analyzed by RP-HPLC on a C18 XTerra (4.6 mm 250 mm, 5 μm) column using water 0.08% TFA (A)/acetonitrile 1% TFA (B) linear gradient (5 95% B in 45 min, 1 ml/min, 25 C, 214 nm and 280 nm). For melittin, nisin and synthetic peptides, HPLC peak areas were used to calculate the percentage of intact compound remaining at the various time points during the incubation. Antibacterial assays. The antibacterial activity of peptide was measured using a standard microdilution assay. Two bacterial species were tested, Staphylococcus aureus strain Newman and Escherichia coli strain K12 XL-Blue. Bacterial strains were subcultured in Mueller-Hinton broth. It was checked that both strains grown with the same efficiency in the same physical conditions in this medium. Bacteria were grown to an optical density (600 nm) of 0.5 and diluted 10 3 times in MH broth. Cell density in wells was 10 5 cfu/ml. 100 µl of this suspension was mixed with 100 µl of 2-fold serial dilutions of peptides in the well of a microtitre plate. Purified peptides were first dissolved in appropriate solvent to a final concentration of 2 mg/ml, in the microtitre plate, dilutions were made in MH broth from 1 to mg/ml. MIC defined as the lowest concentration of purified peptide that inhibited growth, was determined after 20 hours incubation at 37 C in a BioTek Plate Reader. Nisin (nisin from Lactococcus lactis, Sigma-Aldrich) was also tested on bacterial strains for reference. For tests against E. coli, EDTA was added to nisin to a final concentration of 20 mm. To determine minimum bactericidal concentration, bacterial suspensions in wells around MIC were plated out on BHI agar and incubated 24 hours at 37 C. The peptide concentration at which no colonies were observed was defined as the MBC. Results obtained in massic concentrations were converted to molar concentrations for proper comparisons. Tests were performed in triplicate. Lytic activity against human erythrocytes. Human blood was collected from EFS ( Etablissement Français du Sang ) in Rennes (France). Red blood cells (RBC) were recovered to a final concentration of 3 % in PBS buffer. 100 µl of this suspension was mixed with 100 µl of 2-fold serial dilutions of purified peptide in microtiter plate with v-bottom, from 1 to mg/ml in PBS buffer. The plates were incubated 2 hours at 37 C. After centrifugation, the absorbance of the supernatant was measured at 414 nm. Three wells were positive controls, where the RBC were mixed with water 1% Triton X-100, in these wells the totality of RBC were hemolysed and the release of hemoglobin was considered to be 100%. Three wells were negative controls, where the RBC were mixed with PBS buffer, in these wells hemolysis was considered to be null. The percentage of hemolysis in each test well was calculated by comparing with controls. For each peptide, bloods from three different donors were tested. Permeation assays. E. coli outer and inner membrane permeability was determined by using E. coli strain ML-35p, which is lactose permease deficient. Nitrocefin, β-lactam is excluded by the outer cell membrane but, if able to pass this barrier thanks to the action of an AMP, it will be cleaved by the β-lactamase localized within the periplasm. ONPG, passes through lac permease and is cleaved by the cytoplasmic β-galactosidase, however with the ML-35p E. coli strain, ONPG is excluded unless permeabilisation is achieved. The permeabilization

6 assays were carried out with 100 µl of bacteria in MH broth at A600 nm (10 7 cfu/ml, density necessary to detect degradation products). Following addition of the tests molecules at concentrations of 6 µm and 12 µm against 10 5 cfu/ml (sub-mic and supra-mic respectively), nitrocefin (20 µg/ml final concentration) cleavage was monitored by light absorption measurements at 500 nm. ONPG was added to a 100 μg/ml final concentration and substrate cleavage was monitored by light absorption measurements at 420 nm. polymixin B at 8 µm against 10 7 cfu/ml was used as positive control. Bacterial suspension in MH broth plus substrates was used as negative control (untreated cells). Scanning electron microscopy (SEM). Bacterial strains were cultured in MH broth to an optical density (600 nm) of 0.5 corresponding to 10 8 cfu/ml, a bacterial density necessary for SEM analysis. Red blood cells were recovered to a final concentration of 3 % in PBS, the same concentration as for the hemolytic assays, a density sufficient for SEM analysis. Following addition of peptides, PepA1 and Pep11, samples were left 2 hours at room temperature before centrifugation for SEM preparation. Preliminary assays were performed with nisin against S. aureus, different concentrations i.e. 3, 6, 10, 30 and 100 µm against 10 5 cfu/ml were tested. Only the slight sub-mic concentration of 6 µm allowed obtaining suitable images on the effect on membrane. With the 3 µm concentration, effects could be observed but were rare and with increase concentrations, elevated bursted cells were observed. Therefore we chose to expose the two bacterial species to slight sub-mic concentrations i.e. 6 µm for PepA1 against both bacterial species and 6µM and 2 µm for Pep11 against S. aureus and E. coli respectively. Concerning RBC, we knew that no hemolysis occurred at MIC for Pep11, therefore concentrations used against bacteria were not adapted to observe membrane effect on RBC. We also wanted to compare both peptides by using the same concentration of 0.2 mg/ml at which 50 % hemolysis for PepA1 was expected and 10 % for Pep11. This corresponds to 50 µm and 160 µm for PepA1 and Pep11 respectively. Preparations for SEM examination were obtained after centrifugation of cell suspensions ( g during 5 min). Samples were fixed for 4h or 20h for RBC or bacteria respectively with 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, ph 7.2, rinsed then post fixed for 2h with 1% OsO4 in the same buffer and eventually washed. Samples were carefully embedded in agar 2% then dehydrated in a graded series of increasing concentrations of ethanol (50, 70, 90 and 100% v/v). For SEM observation, samples were obtained after preparation with Leica EM CPD 300 to reach drying in the critical point. Samples were metalized with gold paladium during 30 s (few nanometres on the surface) in a Leica EM ACE 200. The observation of samples was performed using a SEM JEOL JSM 6301F at 7 kv with a work distance of 15mm.

7 Percentage of hemoglobin released from human erythorocytes Supplemental Figure 1. PepA1 derivatives with reduced lytic activities against human erythrocytes (Figure S1, related to Table 1). The data presented are the mean values of triplicates from three different human blood samples and the standard deviation of the mean are indicated. Peptides concentrations (µm)