Peptido-mimetic Approach in the Design of Syndiotactic Antimicrobial Peptides
4.2 Materials and methods .1. Reagents and chemicals
Rink Amide resin, F-moc amino acids, N, N, N, N-Tetramethyl-O-(1H-benzotriazol- 1-yl) uronium hexafluorophosphate (HBTU), N, N-Dimethylformamide (DMF), m- Cresol, Glutaraldehyde, Dimethylsulphoxide (DMSO), Chloroform and Ethanol were purchased from Merck. 1-Hydroxybenzotriazole (HOBt), Diethyl ether, 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Sodium Phosphate monobasic anhydrous, Acetonitrile (ACN), Ethylene diamine tetra acetic acid (EDTA) and Piperidine were obtained from SRL laboratories. POPC (1- pamlitoyl - 2 - oleoyl - sn - glycerol- 3- phosphocholine, POPG (1- pamlitoyl- 2- oleoyl - sn- glycerol- 3- glycerol), N, N-Diisopropylethylamine (DIPEA), Thioanisole, 1, 2 Ethane dithiol (EDT), Trifluoroacetic acid (TFA), 3 - (4, 5-dimethylthiazol - 2 - yl) -2, 5- diphenyltetrazolium bromide (MTT), were purchased from Sigma-Aldrich.
Nutrient Broth and Agar were purchased from Himedia laboratories. All other reagents used were of the highest purity (≥ 99%).
4.2.2. Methodology
4.2.2.1. Modelling and simulation
The peptide molecules used in this study were designed by an in-house software PDB make and a modified version of Ribosome software from G.D. Rose laboratory.
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37 Apart from these, softwares like PyMol, Swiss PDB viewer were used for visualization of (Protein Data Base)PDB files 7, 167, 226. The backbone integrity of test peptide candidates was examined by Molecular Dynamics (MD) simulation analysis, using GROMACS program suite 228. GROMACS was used with GROMOS 96 43a1 force field 229 for carrying out MD simulations. The structural stability of the designed peptides was studied at 300 K under standard NVT conditions. The structures were energy minimized by steepest descent algorithm in vacuum in 2000 steps in a simulation box with the size relatively 1.5 nm distant from the peptide in three dimensions. Solvent molecules (SPC, water) were then added and another energy minimization procedure was carried out in water prior to the simulation run.
The 10 ns production runs for each peptide were done with an integration step of 2 fs. LINCS algorithm with a geometric accuracy of 10−4 was used as the bond length constraint. Maxwell distribution was used for initial velocity calculations with 0.1 ps of coupling relaxation step at 300 K. The non-bonded interaction cut off was set at 0.8 nm to 1.1 nm.
4.2.2.2. Electrostatic profiling
Electrostatic potential was calculated at every position by solving Finite Difference Poisson Boltzmann (FDPB) equation, which was summed up for every residue side- chain, represented collectively at the chromophoric center of the side-chain. The hetero atoms of cysteine (S), lysine (K) and methionine (M), C-alpha of glycine and C-zeta of arginine are taken as respective chromophoric centers for electrostatic profiling. On the other hand, C-beta of alanine, valine, isoleucine, serine, and threonine along with C-gamma of asparagine, aspartic acid, leucine, proline, histidine, tyrosine, phenylalanine, and tryptophan are their respective chromophoric centers. Electrostatic profiling was next accomplished by frame-wise comparison of electrostatic potential and structure similarity 7. The profiles were then plotted in a three dimensional graph using MATLAB
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4.2.2.3. Peptide synthesis and characterization
In our study, the peptides were synthesized using standard solid phase peptides synthesis procedure (Fmoc chemistry) 7. Overnight soaked resin in DMF was treated with 20 % piperidine for a period of 20 minutes to remove Fmoc group.
Following that, the resin was washed with DMF to attain neutral pH. An activated amino acid mixture comprised of amino acid, three-fold excess of HBTU, HOBT and six-fold excess of DIPEA was added to resin for amino acid coupling. This process was performed for two cycles of one hour and half hour separately. The cycle of Fmoc removal and amino acid attachment was carried for the entire sequence followed by final Fmoc removal. Post synthesis, the peptides were cleaved from the resin using a cleavage cocktail (m-Cresol: Thioanisole: EDT: TFA :: 2:2:1:20) for 12 hours in dark followed by precipitation in Diethyl ether (ice cold).
The peptides were purified by means of semi-preparative reverse-phase liquid chromatography. A chromatographic run of 10% ACN to 100% ACN with 0.1% TFA, was used for the gradient elution of peptides at a flow rate of 0.5 - 1 mL/min. The eluents were monitored at 210 nm and verified by electrospray ionization mass spectrometry (ESI-MS) and Matrix assisted laser desorption ionization (MALDI) mass spectroscopy 7, 230.
4.2.2.4. Anti-bacterial assay
In our study, mid logarithmic phase bacterial cells were washed and re-suspended in sodium phosphate buffer (10 mM, pH 7.4). The resulting suspension was diluted to a net absorbance value of 0.2 at 600 nm and 50μL of the inoculum was treated with required concentrations of peptide (Net incubation broth is 100 µL), followed by 2 hours of incubation. Post incubation, 100 µL of MTT solution (0.5 mg/mL) was added to the broth and incubated for four hours at 37ºC. Subsequently, the reaction broth was centrifuged and the bacterial pellets were mixed with DMSO. The difference in the absorbance at 570 nm and 660 nm was calculated and percent bacterial cell lysis was reported, relative to untreated bacterial cells 227, 231, 232. A relative inhibition of 80 % growth with reference to growth control was considered
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39 to be as Minimal Inhibitory Concentration, as per relative reported standard procedure 233.
4.2.2.5. Field Emission-Scanning Electron Microscopy (FE-SEM)
The FE-SEM analysis of bactericidal activity of peptide is a qualitative estimation providing a topographic interpretation of higher magnification, after peptide activity upon bacterial species. For this experiment, the microscopic analysis protocol was similar to antibacterial assay until peptide-bacteria incubation (section 4.2.5). Post incubation the bacterial suspension was mixed with glutaraldehyde up to a net concentration of 4%, followed by incubation at 4°C for 30 minutes. After that, the mixture was washed with buffer and mounted upon a glass slide. The bacterial cells were allowed to attach to the glass surface over a period of 30 minutes. Later, samples were subjected to gradient wash with 30% to 100% alcohol. The samples were dried at room temperature and coated with gold prior microscopy. The membrane active property of peptides was concluded after the comparative study of images obtained from peptide treated and untreated bacterial samples 7, 227, 234.
4.2.6. Hemolytic activity
The hemolytic potential of peptides was examined by studying, peptide interaction with human Red Blood Cells (RBC). To conduct this experiment, blood from healthy individual was collected and mixed with 2mg / mL of Ethylene diamine tetra acetic acid (EDTA) to avoid coagulation. Later the blood was washed twice with 5 mM [(4- (2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl) piperazine- N′-(2-ethanesulfonic acid)] HEPES buffer saline and 50 % cell suspension was prepared in the same buffer. 50 µL of the cellular suspensions were mixed with required concentration of peptides and incubated for 2 hours at 37±2 °C. RBC in water was considered as 100 % lysis whereas RBC in buffer as 0% lysis, which was
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