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. 2018 Dec 7;1:221.
doi: 10.1038/s42003-018-0224-2. eCollection 2018.

Structure-function-guided Exploration of the Antimicrobial Peptide polybia-CP Identifies Activity Determinants and Generates Synthetic Therapeutic Candidates

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Free PMC article

Structure-function-guided Exploration of the Antimicrobial Peptide polybia-CP Identifies Activity Determinants and Generates Synthetic Therapeutic Candidates

Marcelo D T Torres et al. Commun Biol. .
Free PMC article

Abstract

Antimicrobial peptides (AMPs) constitute promising alternatives to classical antibiotics for the treatment of drug-resistant infections, which are a rapidly emerging global health challenge. However, our understanding of the structure-function relationships of AMPs is limited, and we are just beginning to rationally engineer peptides in order to develop them as therapeutics. Here, we leverage a physicochemical-guided peptide design strategy to identify specific functional hotspots in the wasp-derived AMP polybia-CP and turn this toxic peptide into a viable antimicrobial. Helical fraction, hydrophobicity, and hydrophobic moment are identified as key structural and physicochemical determinants of antimicrobial activity, utilized in combination with rational engineering to generate synthetic AMPs with therapeutic activity in a mouse model. We demonstrate that, by tuning these physicochemical parameters, it is possible to design nontoxic synthetic peptides with enhanced sub-micromolar antimicrobial potency in vitro and anti-infective activity in vivo. We present a physicochemical-guided rational design strategy to generate peptide antibiotics.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Schematic of the structure-function-guided exploration approach leveraged to generate peptide antibiotics. a The wasp venom derived antimicrobial peptide Polybia-CP was subjected to structure-function analysis to elucidate determinants responsible for biological activity. b Data from antimicrobial activity, physicochemical properties, and structure analyses was harnessed to c identify functional determinants and generate enhanced synthetic variants with therapeutic potential
Fig. 2
Fig. 2
Design, physicochemical features and activity of Pol-CP-NH2 and Ala-scan analogs. a Schematic of the in vitro biological activity experimental design. Briefly, 104 bacterial cells and serially diluted peptides (0–128 μmol L−1) were added to a 96-well plate and incubated at 37 oC. One day after the exposure, the solution in each well was measured in a microplate reader (600 nm) to check inhibition of bacteria compared to the untreated controls and presented as heat maps of antimicrobial activities (μmol L−1) against four bacteria strains: E. coli strain BL21, S. aureus strain ATCC12600 and P. aeruginosa strains PA01 and PA14. Assays were performed in three independent replicates and heat map OD600 values are the arithmetic mean of the replicates in each condition. b Graph correlating MIC (μmol L−1) averages vs. H and c MIC (μmol L1) mean vs. μH, where blue boxes represent peptides with lower activity and red boxes show peptides with higher activity compared to the wild-type, in which we can observe ranges of optimal activity in determined intervals of H and μH values. d Bi-dimensional helical wheels representations of the wild-type indicating positions where Ala-substitution decreased (blue arrows) and enhanced activity (red arrows) and three-dimensional representation from molecular modeling showing substitution positions in which the residues are arranged in two defined faces (hydrophobic and hydrophilic)
Fig. 3
Fig. 3
Physicochemical features and structure of Pol-CP-NH2 and Ala-scan analogs. a Circular dichroism spectra of Pol-CP-NH2 and Ala-scan derivatives at 50 µmol L−1 in water, PBS (pH 7.4) and TFE/Water (3:2, v/v) showing peptides transition from unstructured in water to helically structured in TFE/water. Circular dichroism spectra were recorded after four accumulations at 20 oC, using a 1 mm path length quartz cell, between 260 and 190 nm at 50 nm min-1, with a bandwidth of 0.5 nm. b MIC (µmol L−1) average for each peptide against the first set of bacteria (E. coli BL21, P. aeruginosa PA01 and PA14, and S. aureus ATCC12600) in three independent replicates vs. fH in TFE/Water solution, where blue boxes represent peptides with lower activity and red boxes show peptides with higher activity compared to the wild-type. Optimal activity is reached in most of the cases for fH values higher than the wild-type
Fig. 4
Fig. 4
Physicochemical features and structure of Pol-CP-NH2 and second-generation analogs. Circular dichroism spectra of the peptides at 50 µmol L−1 in water, MeOH/Water (1:1, v/v), PBS (pH 7.4), POPC (10 mmol L−1), POPC:DOPE (3:1, 10 mmol L−1), POPC:POPG (3:1, 10 mmol L−1), SDS (20 mmol L−1), TFE/Water (2:3, 3:2, 4:1, v/v) showing peptides transition from unstructured in water to helically structured in TFE/water. Circular dichroism spectra were recorded after four accumulations at 20 oC, using a 1 mm path length quartz cell, between 260 and 190 nm at 50 nm min−1, with a bandwidth of 0.5 nm
Fig. 5
Fig. 5
Antimicrobial activity of second-generation library of synthetic peptides. a In vitro activity of Pol-CP-NH2 and second-generation of analogs against Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Bacillus megaterium), Fungi (Candida albicans and Candida tropicalis) and Gram-negative bacteria (Escherichia coli, Enterobacter cloacae and Serratia marcescens). Assays were performed in three independent replicates and heat map OD600 values are the arithmetic mean of the replicates in each condition. b MIC (µmol L−1) average vs. fH in TFE/Water solution. c Graph correlating MIC (μmol L−1) averages vs. H and d MIC (μmol L−1) averages vs. μH, where blue boxes represent peptides with lower activity and red boxes show peptides with higher activity compared to the wild-type, in which we can observe ranges of optimal activity in determined intervals of H and μH values
Fig. 6
Fig. 6
Hemolysis and resistance to protease-mediated degradation of engineered peptides. a Schematic of experimental design and hemolytic assay results of Pol-CP-NH2 and derivatives, where hemolytic activity was evaluated by incubating the peptides (0.1–100 μmol L−1) with human red blood cells in PBS at room temperature for 1 h. Experiments were performed in three independent replicates. b Resistance to degradation of Pol-CP-NH2 and analogs exposed to fetal bovine serum (FBS) proteases for 6 h. Experiments were done in three independent replicates (statistical significance was determined using one-way ANOVA, *p < 0.039, error bars represent standard deviation values)
Fig. 7
Fig. 7
Cytotoxicity of engineered peptides. a Schematic of the experimental design for cytotoxicity assays of Pol-CP-NH2 and derivatives against HEK293 human embryonic kidney cells. Briefly, cells were cultured in DMEM medium supplemented with FBS and antibiotics at 37 oC and 5% CO2 and b results obtained by seeding HEK293 50,000 cells and incubating with peptides’ solution (0–64 μmol L−1) at 37 oC for 48 h. Cell viability was measured by MTS assay. All experiments were performed in independent triplicates (heat map values represent mean values of three independent replicates)
Fig. 8
Fig. 8
In vivo activity of Pol-CP-NH2 and its analogs. a Schematic of the experimental design. Briefly, the back of mice was shaved and an abrasion was generated to damage the stratum corneum and the upper layer of the epidermis. Subsequently, an aliquot of 50 μL containing 5 × 107 CFU of P. aeruginosa in PBS was inoculated over each defined area. One day after the infection, peptides (4 μmol L−1) were administered to the infected area. Four animals per group were euthanized and the area of scarified skin was excised two days post-infection b homogenized using a bead beater for 20 min (25 Hz), and serially diluted for CFU quantification (statistical significance was determined using two-way ANOVA followed by Dunnett’s test, ****p < 0.0001). c Mouse body weight measurements throughout the experiment normalized by the body weight of non-infected mice. The wild-type peptide and the most active analog ([Lys]7-Pol-CP-NH2) were used at 64 μmol L−1, where infection and CFU quantification were performed as described in b, the body weight of mice treated with peptide did not change abruptly compared to untreated mice. d Longer experiment (four days) using a higher concentration (64 μmol L−1) of peptides Pol-CP-NH2 and [Lys]7-Pol-CP-NH2 (four mice per group and statistical significance was determined using two-way ANOVA followed by Dunnett’s test, ****p < 0.0001)

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