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. 2023 Jul 19;4(7):101459.
doi: 10.1016/j.xcrp.2023.101459.

Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

Andreia Boaro  1   2   3   4   5 , Lucía Ageitos  1   2   3   6   5 , Marcelo Der Torossian Torres  1   2   3 , Esther Broset Blasco  1   2   3 , Sebahat Oztekin  1   2   3   7 , Cesar de la Fuente-Nunez  1   2   3   8
Affiliations

Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

Andreia Boaro et al. Cell Rep Phys Sci. .

Abstract

Antimicrobial peptides (AMPs) derived from natural toxins and venoms offer a promising alternative source of antibiotics. Here, through structure-function-guided design, we convert two natural AMPs derived from the venom of the solitary eumenine wasp Eumenes micado into α-helical AMPs with reduced toxicity that kill Gram-negative bacteria in vitro and in a preclinical mouse model. To identify the sequence determinants conferring antimicrobial activity, an alanine scan screen and strategic single lysine substitutions are made to the amino acid sequence of these natural peptides. These efforts yield a total of 34 synthetic derivatives, including alanine substituted and lysine-substituted sequences with stabilized α-helical structures and increased net positive charge. The resulting lead synthetic peptides kill the Gram-negative pathogens Escherichia coli and Pseudomonas aeruginosa (PAO1 and PA14) by rapidly permeabilizing both their outer and cytoplasmic membranes, exhibit anti-infective efficacy in a mouse model by reducing bacterial loads by up to three orders of magnitude, and do not readily select for bacterial resistance.

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Conflict of interest statement

DECLARATION OF INTERESTS A provisional patent application has been filed on the de la Fuente Lab’s related work (ID number 23-10379). C.d.l.F.-N. provides consulting services to Invaio Sciences and is a member of the scientific advisory boards of Nowture S.L. and Phare Bio. The de la Fuente Lab has received research funding or in-kind donations from United Therapeutics, Strata Manufacturing PJSC, and Procter & Gamble, none of which were used in support of this work. C.d.l.F.-N. is on the advisory board of Cell Reports Physical Science.

Figures

Figure 1.
Figure 1.. Design, antimicrobial activity, and secondary structure elucidation of peptides from wasp venom
(A) Schematic representation of the structure-function relationship studies, from the selection of the templates (EMP-EM1 [WT1] and EMP-EM2 [WT2]), isolated from the venom of the solitary wasp Eumenes micado, to the design of an optimized second-generation peptide. (B) Antimicrobial activity of WT1 and WT2 and Ala-Scan analogs for the four pathogenic bacterial strains tested in this study. The red color represents bacterial growth inhibition, and the blue color represents bacterial growth. (C and D) CD spectra of WT1 (C) and WT2 (D) and their respective Ala-Scan derivatives at 50 μmol L−1 in TFE:water 3:2 v/v and water showing the conformational transition of the peptides from random coil in water to α helix in TFE:water. CD spectra were recorded in three replicates at 25°C, using a quartz cuvette with 1-mm path length, between 260 and 190 nm at 50 nm min−1, with a bandwidth of 0.5 nm. (E) Bidimensional helical wheel representations of the wild-type peptides WT1 and WT2, indicating positions where Ala-substitution decreased (blue arrows) or enhanced (red arrows) activity.
Figure 2.
Figure 2.. Antimicrobial activity and elucidation of secondary structure
(A) Antimicrobial activity of WT1, WT2, and second-generation analogs for all tested pathogenic bacteria. The red color represents bacterial growth inhibition, and the blue color represents bacterial growth. Heat maps obtained directly from OD measurements of 96-well plates after treatment are shown in Figure S4. (B) CD spectra of WT1 and WT2 and their respective second-generation derivatives at 50 μmol L−1 in TFE:water 3:2 v/v, showing α helix conformation, and in water, showing random-coil conformation. CD spectra were recorded in three replicates at 25°C, using a quartz cuvette with 1-mm path length, between 260 and 190 nm at 50 nm min−1, with a bandwidth of 0.5 nm.
Figure 3.
Figure 3.. Mechanism of action, synergy, and bacterial resistance
(A) Schematic representation of the NPN assay, in which molecules of NPN (represented by gray spheres) present weak fluorescence emission intensity in an aqueous environment. When the outer membranes are permeabilized by peptides, the NPN molecules interact with the lipidic environment of damaged outer membranes and the intensity of blue fluorescence emission increases (represented by blue spheres). (B) NPN graph for outer membrane permeabilization of Pseudomonas aeruginosa PAO1 by polymyxin B (PMB), WT1, K12-1, K13-1, WT2, and K13-2 peptides. Profiles with a rapid increase in fluorescence emission intensity, followed by a slow decay, were obtained after measurement of white 96-well plates on a Thermo Scientific Varioskan LUX fluorescence spectrophotometer, with the excitation wavelength set to 350 nm and the emission wavelength set to 420 nm, according to the experimental procedure described in the section “experimental procedures.” All NPN assays were done in three replicates, including the controls, which consisted of only HEPES solution, HEPES solution and NPN (not shown), HEPES solution and P. aeruginosa PAO1 (not shown), and HEPES solution with both P. aeruginosa PAO1 and NPN. Data are represented as mean ± SD. (C) Schematic representation of the DiSC3-5 assay, in which molecules of DiSC3-5 (represented by gray spheres) accumulate in cytoplasmic membranes and aggregate at high concentrations, causing fluorescence quenching. When the cytoplasmic membrane is destabilized by peptides, DiSC3-5 migrates to the cytoplasm or to the external environment, and red fluorescence emission intensity (represented by red spheres) increases. (D) DiSC3-5 graph for cytoplasmic membrane depolarization of P. aeruginosa PAO1 by PMB, WT1, K12-1, K13-1, WT2, and K13-2 peptides. Profiles with increases and decreases in fluorescence emission intensity were obtained after measurement of black 96-well plates on a Thermo Scientific Varioskan LUX fluorescence spectrophotometer, with the excitation wavelength set to 622 nm and emission wavelength set to 670 nm as described in the section “experimental procedures.” DiSC3-5 graph obtained after the addition of triton solution is shown in Figure S5. All DiSC3-5 assays were done in three replicates, including the controls, which consisted of only HEPES solution, and HEPES solution containing PAO1 and DiSC3-5. Data are represented as mean ± SD. (E) Synergy assay for activity against P. aeruginosa PAO1 between ciprofloxacin, ofloxacin, gentamicin, polymyxin B, or erythromycin, and each of four peptides: WT1, WT2, K12-1, and K13-1. The Fractional Inhibitory Concentration Index (FICI) values, which indicate the degree of synergy between two antimicrobial agents against a target microorganism, were calculated based on the MICs of WT1, WT2, K12-1, and K13-1 and the commercial antibiotics used alone and in combination. FICI values <0.5 indicate synergy; 0.5 < FICI < 1 indicates additive effects; 1 < FICI < 4 indicates indifference; and FICI > 4 indicates antagonism (not represented in the graph). (F) Resistance assay: development of resistance to ciprofloxacin, PMB, WT1, K12-1, and K13-1 in Escherichia coli ΔmutS. The experiment was performed for 20 days as described in detail in the section “experimental procedures.” Data are represented as mean ± SD.
Figure 4.
Figure 4.. Cytotoxicity and in vivo studies
(A and B) Cytotoxic activity of WT1, A14-1, K12-1, K13-1, WT2, K10-2, and K13-2 against (A) human embryonic kidney cells (HEK293T) and (B) primary human keratinocytes. (C) Schematic representation of the in vivo assay procedure. The mice were anesthetized with isoflurane and weighed; their backs were shaved, and a superficial linear skin abrasion was made using a needle to damage the stratum corneum and upper layer of the epidermis. Then 50 μL of 107 CFU mL−1 in phosphate-buffered saline (PBS) of P. aeruginosa PAO1 was inoculated over the scratch in the back of the mice. After 1 h, peptide solutions in PBS at 32 μmol L−1 for K13-2 and 16 μmol L−1 for K12-1 and K13-1 were added to the infected area. This procedure was done for four mice per peptide tested. After 2 days, mice from each group were killed and weighed, and the area of scarified skin was cut, homogenized using a bead beater for 20 min (25 Hz), and serially diluted for CFU quantification. This procedure was repeated after 4 days with the mice from each group. Two technical replicates were performed for each sample to ensure accuracy. (D) Mice weight monitoring for potential in vivo toxicity assessment. The body weight of infected mice was normalized by the body weight of uninfected mice. Data are represented as mean ± SD. (E) Anti-infective activity of K12-1, K13-1, and K13-2 in vivo compared with control groups. Statistical significance was determined using one-way ANOVA followed by Dunnett’s test; p values are shown in the graph.
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