Molecular hybridization strategy for tuning bioactive peptide function

Abstract

The physicochemical and structural properties of antimicrobial peptides (AMPs) determine their mechanism of action and biological function. However, the development of AMPs as therapeutic drugs has been traditionally limited by their toxicity for human cells. Tuning the physicochemical properties of such molecules may abolish toxicity and yield synthetic molecules displaying optimal safety profiles and enhanced antimicrobial activity. Here, natural peptides were modified to improve their activity by the hybridization of sequences from two different active peptide sequences. Hybrid AMPs (hAMPs) were generated by combining the amphipathic faces of the highly toxic peptide VmCT1, derived from scorpion venom, with parts of four other naturally occurring peptides having high antimicrobial activity and low toxicity against human cells. This strategy led to the design of seven synthetic bioactive variants, all of which preserved their structure and presented increased antimicrobial activity (3.1-128 μmol L^-1). Five of the peptides (three being hAMPs) presented high antiplasmodial at 0.8 μmol L^-1, and virtually no undesired toxic effects against red blood cells. In sum, we demonstrate that peptide hybridization is an effective strategy for redirecting biological activity to generate novel bioactive molecules with desired properties.

Fig. 1. Schematic figure of the design used to generate hybrid peptides and to evaluate their biological activity.

The hydrophilic and hydrophobic faces of the helical structure of the AMPs temporin A, protonectin anoplin, and decoralin were combined with the hydrophilic and hydrophobic face of the AMP VmCT1 to generate hybrid peptides. The hybrid peptides were characterized to determine their secondary structure, stability in the presence of proteases and their in vitro and in vivo antimicrobial, hemolytic, and antiplasmodial activities, and for selected AMPs, their anticancer activities.

Fig. 2. Hybrid peptide design.

Helical wheel projections of VmCT1, anoplin, protonectin, decoralin, temporin A, and the hybrid peptides described in this study. The yellow circles indicate aromatic and aliphatic hydrophobic residues; gray circles correspond to residues with hydrophobicity close to zero; blue circles point to basic positively charged residues; purple circles represent polar uncharged residues; green circles represent the restricted pseudo amino acid proline; and pink circles represent polar uncharged amino acid residues. Black arrows indicate the direction and intensity of the hydrophobic moment. P and N indicate the hydrophilic and hydrophobic faces of the amphipathic projection, respectively.

Fig. 3. Antimicrobial, antiplasmodial, cytotoxicity, anticancer, and hemolytic activities of hAMPs and their parent AMPs.

a Peptides in Peptone, Potato Dextrose, or Luria-Bertani broths were exposed to a fixed number of pathogenic (fungi, Gram-positive, and Gram-negative bacteria) cells (10⁶ CFU mL⁻¹) in a gradient of concentrations (0–64 μmol L⁻¹). Medium with no peptides and no bacteria was used as the control for microbial growth. Absorbance values colored in red represent maximal inhibition of microbial growth. On the contrary, blue corresponds to absorbance values reflective of growth (Anoplin-VmCT1: AV, Protonectin-VmVT1: PV, VmCT1-Protonectin: VP, Decoralin-VmCT1: DV, VmCT1-Decoralin: VD, Temporin A-VmCT1: TV, and VmCT1-Temporin A: VT). Anoplin, AV, Decoralin, DV, and VD were not tested against the ESKAPE pathogens. MTT assay results for peptides after (b) 4 h (upper left panel) and (c) 24 h (upper right panel) using MCF-7 cancer cell lines. Dulbecco’s Modified Eagle Medium was used as medium for the growth of MCF-7 and MCF-10A cell lines. Absorbance values in red are reflective of a decrease in viable MCF-7 cells, whereas blue correspond to cells that preserved viability. Experiments were performed in three independent replicates. d Values are expressed as the percentage of fluorescent mature sporozoites. Absorbance values in red represent active compounds against Plasmodium gallinaceum (fluorescent sporozoites), whereas absorbance values in blue indicate lack of antiplasmodial activity. Digitonin/PBS solution was used as the positive control and PBS solution alone was used as the negative control. e MTT assay results for healthy human breast epithelial cells MCF-10A lines exposed to the peptides for 24 h. Dulbecco’s Modified Eagle Medium was used as the control for cell maximum growth. Again, in these experiments, blue represented viable MCF-10A cells, whereas red corresponded to cell death. Experiments were performed in three independent replicates. f Red blood cells were exposed for 1 h and at room temperature to peptides at concentrations ranging from 0.1 to 100 μmol L⁻¹, in PBS. Surfactant (1% SDS in PBS) was used to ensure complete hemolysis and PBS was used as a control to preserve erythrocyte integrity. A PBS solution and the surfactant SDS were used as negative and positive controls, respectively. The MHC (maximal non-hemolytic concentration) was determined as the maximal concentration at which 100% of the erythrocytes were viable. All assays were done in three independent replicates. The graph in f shows mean and standard deviation.

Fig. 4. Structural analysis of hAMPs.

Circular dichroism (CD) spectra of the parent peptides and the hAMPs designed in this study in (a) water, (b) PBS (pH 7.4), (c) SDS (20 mmol L⁻¹), (d) POPC (10 mmol L⁻¹), (e) POPC:POPG (3:1, mol:mol - 10 mmol L⁻¹), (f) POPC:DOPE (3:1, mol:mol - 10 mmol L⁻¹), and (g) TFE/Water (3:2, v-v). CD spectra were recorded after four accumulations at 20 °C, using a 1 mm path length quartz cell, between 195 and 260 nm at 100 nm min⁻¹, with a band width of 0.5 nm. All assays were performed with peptides at 50 μmol L⁻¹. h Helical fractions (f_H) values were calculated based on the Lifson-Roig model for each of the conditions analyzed (a–g).

Fig. 5. Resistance to enzymatic degradation and mechanism of action of hAMPs.

a Peptides were exposed to fetal bovine serum for 6 h. Aliquots were collected and analyzed by liquid chromatography. Three independent replicates were performed. b Damage and permeabilization of the bacterial outer membrane by the peptides using the NPN assay. c Effect of the peptides on the depolarization of the cytoplasmic membrane using the DiSC₃(5) assay. The graphs in a–c show both the mean and standard deviation.

Fig. 6. Anti-infective activity in a skin abscess infection mouse model.

a Anti-infective activity of VmCT1, Temporin A, VT, and TV peptides in a mouse model of A. baumannii skin infection as detailed in the methodology section (n = 8). Statistical significance was determined using two-way ANOVA followed by Dunnett’s test, **p < 0.01 and ***p < 0.001. b The toxicity of the peptide was monitored by mouse body weight measurements obtained during the experiment. Mouse weight changes were normalized to the body weight of each mouse at the beginning of the experiment, as a proxy for potential toxicity. The graph in b shows mean and standard deviation.