Computational protocol: Controlling resistant bacteria with a novel class of β-lactamase inhibitor peptides: from rational design to in vivo analyses

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Protocol publication

[…] Rational design for synthetic peptide construction was mainly based on pocket volume, which included distances between the amino acid residues that compose the β-lactamases conserved motif (KSG), Ser130 (adjacent nucleophilic amino acid) and several adjacent amino acid residues around the catalytic triad (Lys73, Ser130 and Glu166). Firstly, an isosceles trapezoid was designed inside the catalytic enzyme pocket. The distances encountered inside this geometric form showed dimensions of approximately 12 (major base) × 9 (minor base) × 13 (sides) Å in a total of 13 Å2 (). Peptides were designed with ideal lengths to fit and interact with the catalytic triad and neighbouring amino acid residues localized around the catalytic site inside the trapezoid. The peptides were designed with less than five amino acid residues, flexible (presence of glycines) and soluble (presence of hydrophilic amino acid residues such as lysine and glutamic acid) (). Moreover, the output energy that could be correlated to affinity was also detected for each in silico docking simulation with β-lactamases from Gram-negative and –positive bacteria. The parameters used to rank the best peptides to be synthesized were the energy results, with values below -4.0 Kcal.mol−1 being discarded. Five of seven constructed and analysed peptides were discarded, with four of them presenting moderate or no activity toward β-lactamases (data not shown). dBLIP-1 and -2 presented values of -5.8 and -5.4 Kcal.mol−1 toward β-lactamase from E. coli, respectively, and values of -4.8 and -4.8 Kcal.mol−1 toward β-lactamase from S. aureus, respectively. The three-dimensional models for dBLIP-1 and dBLIP-2 were constructed based on the structures of 2je8 and 3pxi (pdb code), which presented 100% of identity for both peptides. Fifty theoretical tridimensional peptide structures were constructed by using Modeller v.9.8 for each peptide. Final models were evaluated using PROCHECK for analysis of stereochemical quality. The peptide structures were visualized and investigated on Delano Scientific's PyMOL To calculate the grand average of hydropathicity, named GRAVY, ProtParam was used to evaluate physical-chemical parameters for a given amino acid sequence. [...] All docking calculations were performed using the AUTODOCK 4.2 program. Docking simulations of both peptides (dBLIP-1 and dBLIP-2) were performed for two β-lactamases (EC, pdb code 1zg4 from Escherichia coli and pdb code 3blm from Staphylococcus aureus. All hydrogen atoms were added by using the AutoDockTool. Grid maps were calculated with 20 × 15 × 15 for both dBLIP-1 and -2 tested against E. coli enzyme; for the enzyme from S. aureus the grid was calculated with 35 × 35 × 15 for both dBLIP-1 and -2, and in all tests the spacing centre was 1.0 Å on the catalytic pocket of both enzymes. In order to understand the competitive in vitro inhibition observed, the simulation was optimized in a reduced region around the catalytic pocket. A Lamarckian genetic algorithm was used as the search method to find the best peptide–protein complex. Fifty docking runs were performed for each peptide, where the maximum freedom to side chains was unlocked due to length of the peptides. The generated structures were ranked in two steps: firstly a cluster with the best models with lowest free energy (below -4.0 kcal.mol−1), and secondly with a root-mean-square deviation (RMSD), for all atoms docked in the serine proteinase catalytic pocket, showing tolerance of 4Å, as recommended for blind docking. The program PyMOL was used to characterize peptide-protein interactions. [...] The inhibition of β-lactamase activity degree by dBLIP-1 and dBLIP-2 was determined spectrophotometrically by the hydrolysis of nitrocefin as substrate. Assay mixture contained 83 mg of nitrocefin, 167 mg of BSA, 10% glycerol and 0.30 mL (0.5 μg.mL−1 of β-lactamase, obtained from Bacillus cereus 569) in a final volume of 1.5 mL of 50 mM phosphate buffer. β-lactamase activity was checked by measuring the absorbance reduction at 340 nm. Inhibitors, dBLIP-1 and dBLIP-2 at various concentrations (10 to 500 μM) were pre-incubated with the enzyme for 10 min at 30°C before addition of the substrate. Percent inhibition was calculated as 100 × [(c-r)/c], where c is the activity in control samples incubated without inhibitor and r is the remaining activity in samples incubated with inhibitor. IC50 values are calculated to inhibit 50% of enzyme activity from the plot of percent inhibition versus the logarithm value of inhibitor concentration. Kinetic parameters were derived from the initial velocity using SIGMAPLOT version 10.0. […]

Pipeline specifications

Software tools AutoDock, PyMOL, SigmaPlot
Applications Miscellaneous, Protein interaction analysis
Organisms Mus musculus, Escherichia coli, Staphylococcus aureus, Homo sapiens
Chemicals Amoxicillin, Ampicillin, Cefotaxime