# Substitution of a proline residue in the frog skin host-defense peptide, figainin-2PL by D-lysine generates an analog with potent activity against antibiotic-resistant ESKAPE pathogens

Laure Guilhaudis, Taylor Cunning, Jack Delaney, Nigel Ternan, Milena Mechkarska, Samir Attoub, J. Michael Conlon

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Laure Guilhaudis, Taylor Cunning, Jack Delaney, Nigel Ternan, Milena Mechkarska, et al. Substitution of a proline residue in the frog skin host-defense peptide, figainin-2PL by D-lysine generates an analog with potent activity against antibiotic-resistant ESKAPE pathogens. Peptides, 2025, 192, pp.171430. 10.1016/j.peptides.2025.171430. hal-05456680

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# Substitution of a proline residue in the frog skin host-defense peptide, figainin-2PL by D-lysine generates an analog with potent activity against antibiotic-resistant ESKAPE pathogens

Laure Guilhuonis<sup>a</sup>, Taylor S. Cunning<sup>b</sup>, Jack J. Delaney<sup>b</sup>, Nigel G. Ternan<sup>b</sup>, Milena Mechkarska<sup>c</sup>, Samir Attoub<sup>d</sup>, J. Michael Conlon<sup>e,*</sup>

## ARTICLE INFO

### Keywords:

Frog skin

Figainin-2PL: Antimicrobial peptide

Cytotoxicity

Circular dichroism

## ABSTRACT

Figainin-2PL (FLGTVLKLGKIAKTVVPMLTNAMQPKQ.NH₂) is active against a range of antibiotic-resistant bacteria as well as human tumor-derived cells. The study aimed to determine the effects of a proline-substitution on the biological potency of the peptide. Secondary structure predictions indicate that figainin-2PL can adopt a helix-turn-helix conformation in membrane-mimetic environments. Circular dichroism spectra are consistent with these predictions. Replacement of the Pro¹⁸ residue by either L-Ala, L-Lys or D-Lys increased the extent and stability of the helical domains. All substitutions increased cytotoxic activity against a range of human tumor-derived cells (between 1.5- and 3-fold) but major (between 4- and 10-fold) increases in hemolytic activity against mouse erythrocytes were also observed. While the substitutions Pro¹⁸ → L-Ala and Pro¹⁸ → L-Ala produced only minor and inconsistent changes in antibacterial activity, the [P18k] analog displayed a 2- to 4-fold greater (MIC and MBC in the range 1–8 µM) against the ESKAPE pathogens *Enterococcus faecalis*, *Enterococcus faecium*, *Klebsiella pneumoniae* and *Pseudomonas aeruginosa* compared with figainin-2PL and the peptide retained high potency against *Acinetobacter baumannii*, *Escherichia coli*, *Staphylococcus aureus* and *Clostridium difficile* (MIC and MBC = 2 µM). Consequently, the [P18k] peptide shows therapeutic potential for development into a broad-spectrum, topical antimicrobial agent.

## 1. Introduction

Skin secretions from several species of Anura (frogs and toads) contain host-defense peptides (HDPs) with the ability to inhibit the growth of bacteria and fungi with varying degrees of potency [1,2]. Such peptides are believed to be a component of the frog's system of innate immunity protecting the organism against pathogenic microorganisms in the environment [3]. They may also possess the ability to permeabilize eukaryotic cells and it has been proposed that they may act synergistically with other toxins in the secretions providing a defense against predators [4]. It is now appreciated that many frog skin peptides that were first identified on the basis of their antibacterial properties are in fact multifunctional and may display a range of biological activities

with potential clinical applications including the abilities to produce tumor regression in animal models of cancer [5] and lower circulating glucose and lipid concentrations and stimulate β-cell proliferation in murine models of Type 2 diabetes [6].

With few exceptions, frog skin HDPs are to a varying extent cationic due to the presence of one or more lysine residues (less commonly arginine) and have the propensity to adopt a stable amphipathic α-helical conformation in a membrane-mimetic solvent or in the presence of a (phospho)lipid micelle [7,8]. The occurrence of one or more proline residues in HDPs from invertebrates is quite common and the Pro-Arg-Pro motif is present in numerous compounds isolated from annelids, crustaceans, insects, and molluscs [9,10]. The frequency of proline in HDPs isolated from amphibian skin secretions is more limited

<footnote><sup>a</sup> Univ Rouen Normandie, INSA Rouen Normandie, Univ Caen Normandie, ENSICAEN, CNRS, Institut CARMeN UMR 6064, Rouen F-76000, France</footnote>

<footnote><sup>b</sup> Nutrition Innovation Centre for Food and Health (NICHE), School of Biomedical Sciences, Ulster University, Coleraine BT52 1SA, UK</footnote>

<footnote><sup>c</sup> Department of Life Sciences, Faculty of Science and Technology, University of the West Indies, St. Augustine Campus, Trinidad and Tobago</footnote>

<footnote><sup>d</sup> Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates</footnote>

<footnote><sup>e</sup> Diabetes Research Centre, School of Biomedical Sciences, Ulster University, Coleraine BT52 1SA, UK</footnote>

<footnote>* Correspondence to: Diabetes Research Centre, School of Biomedical Sciences, Ulster University, Cromore Rd, Coleraine BT52 1SA, UK.
E-mail address: jmconlon1@gmail.com (J.M. Conlon).</footnote>
but the amino acid is generally found in HDPs from the extensive tem-
porin [11] and brevinin-1 families [12] in frogs belonging to the Rani-
dae and in several peptide families from Australian frogs belonging to
the genus *Litoria* [13]. A Pro residue is known to destabilize the α-helical
conformation of frog skin HDPs and may contribute to the formation of a
bend or kink in the molecule [14,15] and/or promote ‘helix-turn-helix’
structures [16–18]. It is not possible to predict with any degree of cer-
tainty the effect on antimicrobial or cytotoxic activities of such
proline-induced perturbations of helicity and substitution of the residue
can lead to either increased or decreased activities (see Discussion
section).

A previous study identified a range of peptides with cytotoxic ac-
tivity against bacteria and human tumor-derived cells in
norepinephrine-stimulated skin secretion from the Caribbean banana
tree dwelling frog *Boana platanera* [19]. In particular, one such HDP,
figainin-2PL (FLGTVLKLGKIAAKTVVPMLTNAMQPKQ.NH₂) displayed
rapid and potent cytotoxicity against a range of Gram-positive and
Gram-negative bacteria including ESKAPE pathogens and the anaerobe
*Clostridioides difficile*. The term ESKAPE refers to a group of virulent and
nosocomial pathogens with a pronounced ability to develop resistance
to commonly used antibiotics [20,21]. In addition, figainin-2PL showed
potent *in vitro* cytotoxic activity against a range of human tumor-derived
cell lines but appreciably lower hemolytic activity against mouse
erythrocytes [19].

The immediate aim of the present study was to investigate the effects
on antibacterial and cytotoxic activities of substituting the proline res-
idue at position 18 in the central region of figainin-2PL by either L-
alanine, L-lysine or D-lysine. In the wider context, the study is a
component of a program to assess the possibility of using figainin-2PL
and other HDPs from *B. platanera* as templates for the development of
a therapeutically valuable agent with anti-infective and/or anti-cancer
properties.

## 2. Materials and methods

2.1. Synthetic peptides

Figainin-2PL, [P18A]figainin-2PL, [P18K]figainin-2PL and [P18k]figainin-2PL were supplied in crude form by Anlian Peptide Co. (Shanghai, China) and purified to near homogeneity (>98 % purity) by reversed-phase HPLC on a (2.2 cm×25 cm) Vydac 218TP1022 (C-18) preparative column under the conditions previously described [19]. The identities of the peptides were confirmed by MALDI-TOF mass spectrometry and their purities by their elution from a (0.46 cm×25 cm) Vydac 218TP54 (C-18) analytical column as a single, symmetrical peak. Full details are provided in Supplementary Material. The presence of helical domains in these peptides was predicted using the AGADIR [22] and PEP-FOLD3 [23] programs. Analyses were performed at pH 7 and 293 K.

## 2.2. Circular dichroism studies

Circular dichroism spectra were recorded on a MOS-500 Circular
Dichroism Spectrometer (BioLogic, Seyssinet-Pariset, France) as previ-
ously described [24]. Full details are provided in Supplementary Ma-
terial. The spectra of figainin-2PL and its [P18A], [P18K] and [P18k]
analogs were determined in water, 25 % (v/v) trifluoroethanol
(TFE)/water, and methanol and also in aqueous solution containing
10 mM dodecylphosphocholine (DPC) and 10 mM sodium dodecyl sul-
fate (SDS). Since the [P18K] analog was incompletely soluble in meth-
anol and the [P18A] analog in micellar environments (10 mM DPC and
10 mM SDS), only the interpretable spectra were analysed. Peptide
secondary structure was estimated using the on-line CD spectra decon-
volution server DichroWeb [25]. The secondary structure content was
determined by averaging the results given by CONTINLL, CDSSTR and
SELCON3 [26] deconvolution programs. Peptide α-helical content was

also calculated by using the Forood formula: 100 × ([θ]222/max[θ]222) with max[θ]222 = -40,000 [1 - (2.5/n)], where n = number of amino acid residues [27].

2.3. Antimicrobial assays

Antimicrobial activities (minimum inhibitory concentration, MIC and minimum bactericidal concentration, MBC) of synthetic replicates of the peptides were determined against the following strains of ESKAPE⁺ bacterial pathogens: the Gram-negative aerobes multidrug-resistant *Acinetobacter baumannii* DSM 30008, *Escherichia coli* DSM 787, carbapenem-resistant *Klebsiella pneumoniae* (CRE) ATCC BAA 1705 and multidrug-resistant *Pseudomonas aeruginosa* PA01 DSM 50071, the Gram-positive aerobes vancomycin-resistant *Enterococcus faecalis* (VRE) MF06036, vancomycin-resistant *Enterococcus faecium* (VRE) NCTC 12201 and methicillin-resistant *Staphylococcus aureus* (MRSA) ATCC 43300, and against two strains of the anaerobic pathogen *Clostridioides difficile* - R20291 DSM 27147 and biofilm-producing 630 ATCC BAA 1382. Assays were performed according to procedures mandated by CLSI [28] as previously described [19]. Full details are provided in Supplementary Material.

2.4. Cytotoxicity assays

Synthetic peptides in the concentration range 4–256 µM were incu-
bated for 60 min at 37 °C with washed erythrocytes (2 × 10⁷ cells) taken
from male NIH male Swiss mice (Harlan Ltd, Bicester, UK) as previously
described [29]. The LC₅₀ value was taken as the mean concentration of
peptide producing 50 % hemolysis in three independent incubations. All
procedures involving animals were carried out in accordance with the
UK Animals (Scientific Procedures) Act 1986 and EU Directive
2010/63EU for animal experiments

Cytotoxicities against human non-small cell lung adenocarcinoma A549 cells (RRID:CVCL_0023), human breast adenocarcinoma MDA-MB-231 cells (RRID:CVCL_0062), human colorectal adenocarcinoma HT-29 cells (RRID:CVCL_0320) and human umbilical vein endothelial (HUVEC) cells (RRID:CVCL_2959) were measured as previously described [29]. The effects of the peptides (1–100 µM) on cell viability following a 24 h incubation at 37 °C were determined by measurement of ATP concentrations using a CellTiter-Glo Luminescent Cell Viability assay (Promega Corporation, Madison, WI, USA). The LC₅₀ value was taken as the mean concentration of peptide producing 50 % cell death in a minimum of three independent experiments. Values are presented as mean ± standard error of mean (SEM).

2.5. Statistical analysis

Data were compared using unpaired Student's t test (non-parametric, with two-tailed P values and 95 % confidence interval) and one-way ANOVA with Bonferroni post-hoc test wherever applicable. Values are considered to be significantly different if P < 0.05.

3. Results

3.1. Circular dichroism studies

Secondary structure predictions indicate that figainin-2PL has the
propensity to adopt a stable helix-turn-helix conformation in an
appropriate membrane-mimetic environment. The AGADIR program
[22] predicts helical domains in the 2–13 and 19–24 regions of the
peptide. The replacement of Pro¹⁸ by L-Ala or L-Lys increased the hel-
icity in the 14–24 region (Supplementary material Fig. S1). The
PEP-FOLD3 program [23] predicts helical domains in the 1–12 and
18–22 regions for the native peptide and a single helical domain from
residues 1–22 in the L-Ala¹⁸ and L-Lys¹⁸ analogs.

As shown in Fig. 1, CD spectra of figainin-2PL are consistent with
<figure><img src="image_3.png" /><figcaption>Fig. 1. Circular dichroism spectra of figainin-2PL at room temperature and at a concentration of 0.25 mg.mL⁻¹ in water (pH 7), 25 % (v/v) trifluoroethanol (TFE)-water, methanol, 10 mM dodecylphosphocholine (DPC) aqueous solution and 10 mM sodium dodecyl sulfate (SDS) aqueous solution.</figcaption></figure>

these conclusions. In common with other frog skin HDPs, the peptide adopts a random coil confirmation in water but in methanol, 25 % (v/v) TFE, 10 mM DPC and 10 mM SDS the peptide adopts a predominantly α-helical secondary structure. In these four environments, the CD spectra display two characteristic negative minima at approximately 208 and 222 nm, and a positive maximum near 192 nm, indicative of a α-helical structure. In membrane-mimetic environments, anionic sodium dodecyl sulfate (SDS) micelles and zwitterionic dodecylphosphocholine (DPC) micelles, these three dichroic signals are more intense, suggesting that the helical conformation is stabilized through interactions with detergent micelles. The wavelengths of both the positive and negative peaks are slightly shifted from the canonical α-helical values (191 nm in micelles vs. the typical 192 nm, and 207 nm vs. 208 nm). These shifts may reflect the contribution of minor additional secondary structure elements, such as turns. For analogs in which a lysine residue (D- or L-) replaces the native proline at position 18 (Fig. 2), the most significant change observed in the CD spectra in micellar media is an increase in intensity of the maximum near 191 nm, indicating an enhanced degree of structural organization. Table 1 summarizes the estimated secondary structure content derived from DichroWeb analyses [25,26], as well as the α-helical content calculated from the mean residue ellipticity at 222 nm [27]. For the native peptide, the helical contents in micellar conditions (59 % in DPC and 66 % in SDS) correspond to approximately 18 out of 28 amino acids, consistent with predictions based on secondary structure modeling. Substitution of the Pro¹⁸ residue by L-lysine increased the helical content from 59 % in the native peptide (calculated using the DichroWeb deconvolution program) to 68 % in the presence of zwitterionic DPC micelles and to 77 % in the presence of anionic SDS micelles (Table 1). Similarly, the substitution of Pro¹⁸ by D-lysine increased the helical content to 75 % in a solution containing DPC micelles and to 72 % in a solution containing SDS micelles (Table 1). The [P18A] analog was not sufficiently soluble in water containing DPC- or SDS-micelles to permit recording CD spectra at the same concentration used for the other peptides but, the helical content of the peptide in methanol (67 %) and 25 % TFE (57 %) is about 10 % higher than that of the native peptide in these solvents (Fig. 3 & Table 1). This increase in helical content is of the same order of magnitude as that observed for the [P18K] and [P18k] analogs in the presence of micelles indicating that the substitution of proline by alanine similarly promotes helix stabilization.

## 3.2. Antibacterial activities

Consistent with previously obtained data involving the peptide [19], figainin-2PL displayed antimicrobial activity against a range of clinically relevant Gram-positive and Gram-negative aerobic pathogens and the anaerobic pathogen *C. difficile*. Potency was relatively high (MIC and MBC between 6.25 and 12.5 µg/mL; 2–4 µM) except in the case of *E. faecalis* (MIC and MBC 50 µg/mL; 16 µM) and *P. aeruginosa* (MIC and MBC 100 µg/mL; 32 µM) (Table 2). Replacement of the Pro¹⁸ residue by L-Ala and L-Lys produced relatively minor and inconsistent effects on antimicrobial activity with both increases and decreases in potency being observed. However, replacement of Pro¹⁸ by D-Lys resulted in 2- to 4-fold greater activity (MIC and MBC in the range 3.13–25 µg/mL; 1–8 µM) against the ESKAPE pathogens *E. faecalis*, *E. faecium*, *K. pneumoniae* and *P. aeruginosa* compared with figainin-2PL. In addition, the analog retained the high potency against *A. baumannii*, *E. coli*, *S. aureus* and *C. difficile* (MIC and MBC = 6.25 µg/mL; 2 µM) exhibited by the native peptide.

## 3.3. Cytotoxic activities

The effects of increasing concentrations of figainin-2PL and its analogs on the viability of A549 human non-small cell lung adenocarcinoma cells, MDA-MB-231 human breast adenocarcinoma cells, HT-29 human colorectal adenocarcinoma cells, human umbilical vein endothelial cells (HUVEC) and mouse erythrocytes are summarized in Table 3. All three analogs showed significantly (P < 0.05) greater cytotoxic potency against the tumor-derived cells compared with figainin-2PL with LC₅₀ values between 1.5- and 3-fold less. Cell death was observed to be extremely rapid (< 5 min) at the highest concentration tested (100 µM). However, the analogs were even more strongly cytotoxic to non-neoplastic HUVEC cells (LC₅₀ values 12 and 24-fold less) and mouse erythrocytes (between 4- and 10-fold less) compared with the naturally occurring peptide.

# 4. Discussion

The therapeutic potential of HDPs, and peptide-based drugs in general, is limited by their often relatively low potency and rapid clearance from the circulation compared with non-peptidic agents. Several
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Circular dichroism spectra of (A) [P18K]figainin-2PL and (B) [P18k]figainin-2PLat room temperature and at a concentration of 0.25 mg.mL⁻¹ in water (pH 7), 25 % (v/v) trifluoroethanol (TFE-water), 10 mM dodecylphosphocholine (DPC) aqueous solution and 10 mM sodium dodecyl sulfate (SDS) aqueous solution.</figcaption></figure>

strategies have been adopted to increase activity and prolong the half-lives of antimicrobial peptides with a view to increasing their utility for systemic use (reviewed in [30]). The antimicrobial activities of peptides are determined by complex interactions between charge, hydrophobicity, helicity and amphipathicity and factors such as susceptibility to enzymatic degradation as well as resistance to oligomerization and inactivation at high salt concentrations [31,32]. In the case of frog skin HDPs, approaches to increasing activity include increasing cationicity while preserving amphipathicity by replacement of acidic (Asp and Glu) and neutral residues by L-Lys and/or L-Arg, increasing stability by modifications to N- and C-termini, replacement of appropriate L-amino acids by their D-stereoisomer and modification of the peptide backbone to generate peptoids [33]. Attempts to modulate helicity involve the substitution of helix-disrupting residues such as Pro by other amino acids while preserving amphipathicity and incorporation of helix-promoting non-proteinogenic amino acids such as

2-aminoisobutyric acid (Aib) [34].

CD studies have shown that figainin-2PL adopts a predominantly helical conformation in a range of membrane-mimetic environments with a helix-turn-helix conformation predicted by the AGADIR [22] and PEP-FOLD3 [23] programs. The helical proportions derived from deconvolution using DichroWeb [25] are consistent with these secondary structure predictions. Substitution of Pro¹⁸ by L-Ala produced an increase in helicity determined in 25 % TFE-water and methanol compared with figainin-2PL but the peptide was incompletely soluble in micellar media. The relative effect on the % helical content produced by substitution of the Pro¹⁸ residue by L-Lys compared with D-Lys was dependent on the surface charge of the micelles present in solution. For these two analogs, the degree of stabilization differs between zwitterionic and negatively charged micelles. This is likely due to changes in the nature of the peptide–micelle interactions. The [P18K] analog displayed a maximum degree of helicity in SDS micelles whereas the
<table><caption>**Table 1**

Prediction of secondary structure content of figainin-2PL and its analogs from CD spectra.</caption><thead><tr><th>Peptide</th><th>Medium</th><th>Helix</th><th>β-sheet</th><th>Turns</th><th>Random</th></tr></thead><tbody><tr><td rowspan="5">Figainin-2PL</td><td>Water</td><td>1 (6)</td><td>4</td><td>4</td><td>90</td></tr><tr><td>10 mM DPC</td><td>59 (64)</td><td>4</td><td>9</td><td>19</td></tr><tr><td>10 mM SDS</td><td>66 (63)</td><td>3</td><td>9</td><td>22</td></tr><tr><td>TFE 25 %</td><td>48 (41)</td><td>10</td><td>14</td><td>28</td></tr><tr><td>MeOH</td><td>58 (42)</td><td>3</td><td>16</td><td>23</td></tr><tr><td rowspan="3">[P18K]figainin-2PL</td><td>Water</td><td>3 (7)</td><td>19</td><td>10</td><td>67</td></tr><tr><td>10 mM DPC</td><td>68 (63)</td><td>4</td><td>8</td><td>21</td></tr><tr><td>10 mM SDS</td><td>77 (72)</td><td>4</td><td>5</td><td>17</td></tr><tr><td rowspan="3">[P18k]figainin-2PL</td><td>Water</td><td>2 (5)</td><td>10</td><td>6</td><td>79</td></tr><tr><td>10 mM DPC</td><td>75 (72)</td><td>4</td><td>7</td><td>19</td></tr><tr><td>10 mM SDS</td><td>72 (63)</td><td>4</td><td>6</td><td>19</td></tr><tr><td rowspan="3">[P18A]figainin-2PL</td><td>Water</td><td>7 (14)</td><td>13</td><td>9</td><td>73</td></tr><tr><td>TFE 25 %</td><td>57 (43)</td><td>11</td><td>13</td><td>20</td></tr><tr><td>MeOH</td><td>67 (65)</td><td>11</td><td>6</td><td>16</td></tr></tbody></table>

The values given for DichroWeb are the average values of the % provided by the CONTIN-LL, SELCON3 and CDSSTR except for figainin-2PL in methanol and 10 mM SDS, for [P18K]figainin-2PL and [P18k]figainin-2PL in 10 mM SDS and 10 mM DPC for which CDSSTR failed to deconvolute the CD spectrum or gave values which were excluded as too divergent. Values in parentheses correspond to the % helicity calculated using the Forood formula. DPC: dodecylphosphocholine, SDS: sodium dodecyl sulfate.

[P18k] analog displayed greatest % helicity in DPC micelles (Table 1). A major difference between D- and L-lysine substituted peptides lies in the position of the side chain. Since the lysine side chain carries a positive charge and the micelles used are either anionic (SDS) or zwitterionic (DPC), the position of the side chain will influence the interaction with the micellar surface and stabilization of the helix. Although detergent micelles are mimetics of membrane-like environments, they do not reflect the complexity of either prokaryotic or eukaryotic membranes. Consequently, additional studies in environments that more closely mimic the composition and architecture of bacterial or mammalian membranes [35,36] will be necessary to provide further insights into the nature of the interactions between the peptide analogs and the membranes.

It has been shown that zwitterionic micelles formed from phosphatidylethanolamine (PE) may be superior to those from DPC in terms of stability and their ability to solubilize peptides and other therapeutic

agents [35]. In addition, mixed micelles formed from PE and polyethylene glycol (PEG) are particularly useful for drug delivery, especially for poorly soluble agents such as [P18A]figainin-2PL [37]. Future studies involving the design of “second generation” analogs based upon the structure of [P18k]figainin-2PL will investigate the properties of the peptides in this environment. Similarly, the therapeutic potential of anionic mixed micelles formed from phosphatidylserine (PS) and phosphatidylglycerol (PG) as a drug-delivery vehicle may be superior to micelles formed from SDS as the former displays anti-inflammatory properties [38].

Evidence has been obtained that the Pro¹⁸ residue plays an important role in determining the selectivity against bacteria and eukaryotic cells. Figainin-2PL displays relatively high potency against both Gram-negative and Gram-positive aerobes and against the anaerobe *C. difficile* but appreciably less hemolytic activity against mouse erythrocytes (Table 2 & 3). Substitution of Pro¹⁸ by L-Ala had the effect of increasing the helicity in the non-helical central region of the molecule which results in minor and inconsistent changes in anti-bacterial activity but major increases in hemolytic activity and cytotoxicity against non-neoplastic umbilical vein endothelial cells. The substitution also increased cytotoxic activity against human tumor-derived cells but to a lesser extent.

Replacing Pro¹⁸ by either L-Lys or D-Lys not only modulates helicity but increases the molecular charge at pH 7 from +5 to +6. The substitution by L-Lys again produced relatively minor and variable changes in antibacterial potency but major increases in hemolytic activity. In contrast, the substitution by D-Lys produced a consistent increase in antibacterial activity against the microorganisms tested but this effect was also accompanied by a concomitant major increase in hemolytic activity. Resistance to proteolytic degradation by bacterial enzymes may contribute to the increase antimicrobial potency of the D-Lys analog relative to the L-Lys analog [39].

Previous studies with the frog skin HDP pseudin-2 (GLNALKKVFQ-GIHEAIKLINNH VQ) from the frog *Pseudis paradoxa* showed that increasing the molecular charge from +2 in the native peptide to +5 by substituting residues at positions 3, 10 and 14 by D-Lys increased activity against clinical isolates of antibiotic-resistant *E. coli* by 4-fold. Further increases in cationicity up to a charge of +8 did not increase antibacterial activity further but enhanced hemolytic activity [40]. While the very high cytotoxicity against human cells precludes

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Circular dichroism spectra of [P18A]figainin 2PL at room temperature and at a concentration of 0.25 mg.mL⁻¹ in water (pH 7), 25 % (v/v) TFE-water and methanol.</figcaption></figure>
<caption>**Table 2**</caption>

Minimum Inhibitory Concentrations (µg/mL) and Minimum Bactericidal Concentrations (µg/mL) for figainin-2PL analogues against a range of clinically relevant Gram-positive and Gram-negative aerobic pathogens and the anaerobe C. difficile.

<table><thead><tr><th>Pathogen</th><th>Figainin-2PL</th><th>[P18A]</th><th>[P18K]</th><th>[P18k]</th></tr></thead><tbody><tr><td><i>E. coli</i><br>DSM 787</td><td>6.25 (6.25)</td><td>6.25 (6.25)</td><td>6.25 (12.5)</td><td>6.25 (6.25)</td></tr><tr><td><i>S. aureus</i><br>ATCC 43300</td><td>6.25 (6.25)</td><td>12.5 (25)</td><td>12.5 (12.5)</td><td>6.25 (6.25)</td></tr><tr><td><i>P. aeruginosa</i><br>DSM 50071</td><td>100 (100)</td><td>50 (100)</td><td>100 (&gt;100)</td><td>25 (50)</td></tr><tr><td><i>K. pneumoniae</i><br>ATCC BAA 1705</td><td>25 (25)</td><td>25 (25)</td><td>25 (100)</td><td>12.5 (12.5)</td></tr><tr><td><i>A. baumannii</i><br>PA01 DSM 30008</td><td>6.25 (6.25)</td><td>6.25 (6.25)</td><td>6.25 (6.25)</td><td>6.25 (6.25)</td></tr><tr><td><i>E. faecalis</i><br>MF 06036</td><td>50 (50)</td><td>12.5 (12.5)</td><td>12.5 (12.5)</td><td>12.5 (12.5)</td></tr><tr><td><i>E. faecium</i><br>NCTC 12201</td><td>12.5 (12.5)</td><td>6.25 (6.25)</td><td>6.25 (6.25)</td><td>3.13 (3.13)</td></tr><tr><td><i>C. difficile</i> R20291<br>DSM 27147</td><td>6.25 (6.25)</td><td>12.5 (12.5)</td><td>12.5 (12.5)</td><td>6.25 (6.25)</td></tr><tr><td><i>C. difficile</i> 630<br>ATCC BAA 1382</td><td>6.25 (6.25)</td><td>6.25 (6.25)</td><td>6.25 (6.25)</td><td>6.25 (6.25)</td></tr></tbody></table>

Data show Minimum Inhibitory Concentration (MIC) and the values in parentheses show Minimum Bactericidal Concentration (MBC).

<caption>**Table 3**</caption>

Cytotoxicities of figainin-2PL and its analogs against lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, colorectal adenocarcinoma HT-29 cells, human umbilical vein endothelial (HUVEC) cells and mouse red blood cells (RBC).

<table><thead><tr><th></th><th>A549</th><th>MDA-MB-231</th><th>HT29</th><th>HUVEC</th><th>RBC</th></tr></thead><tbody><tr><td>Figainin-2PL</td><td>6.4<br>± 0.3</td><td>7.0 ± 0.5</td><td>19.2<br>± 0.8</td><td>16.6<br>± 0.3</td><td>105<br>± 11</td></tr><tr><td>[P18A]</td><td>4.9</td><td>4.5 ± 0.1</td><td>5.9</td><td>1.1 ± 0.4</td><td>10 ± 2</td></tr><tr><td>figainin-2PL</td><td>± 0.3</td><td></td><td>± 0.1</td><td></td><td></td></tr><tr><td>[P18K]</td><td>4.1</td><td>5.1 ± 0.1</td><td>6.6</td><td>0.7 ± 0.1</td><td>12 ± 3</td></tr><tr><td>figainin-2PL</td><td>± 0.1</td><td></td><td>± 1.0</td><td></td><td></td></tr><tr><td>[P18k]</td><td>4.8</td><td>4.5 ± 0.1</td><td>6.1</td><td>1.4 ± 0.2</td><td>28 ± 5</td></tr><tr><td>figainin-2PL</td><td>± 0.1</td><td></td><td>± 0.4</td><td></td><td></td></tr></tbody></table>

Data show mean LC₅₀ values (µM) ± S.E.M.

development of [P18k]figainin-2PL into an anti-infective agent for systemic use, the possibility of using the peptide as a template for topical application is worthy of consideration.

It is well established from several published examples that a proline residue in a frog skin HDP results in the formation of a bend or kink in the molecule but it is not possible to generalize regarding the effect of this structural feature on biological activity. In gaegurin 5 (FLGALFKVA SKVLPSVFCAITKKC), a brevinin-1 from the Korean frog, *Glandirana rugosa*, an amphipathic α-helical domain spanning residues 3–20 is kinked around Pro¹⁴ [41]. Replacement of the proline residue with alanine produces a straight and rigid helix that was more stable than the kinked helix and resulted in an increase in both antimicrobial and hemolytic activities. In contrast, in temporin-PE (FLPIVAKLLSGLL.NH₂) from the hybridogenic frog *Pelophylax lessonae* x *Pelophylax ridibundus* substitution of Pro³ by Tyr resulted in decrease in hemolytic activity and activity against Gram-negative bacteria but an increase in potency against Gram-positive bacteria [42]. Substitution of Gly¹¹ with Pro in pseudin-2 transformed the secondary structure of the peptide from a linear α-helix from Leu² to Glu²⁴ to one with a bend at Pro¹¹ between two short α-helices. The proline-substituted analog exhibited lower cytotoxicity and antimicrobial activity despite an increased ability to penetrate the bacterial cell membranes [43]. In caerin 1.1 (GLLSVLGSVAKHVLPHVVPVIAEHL.NH₂), isolated from a number of Australian frogs of the *Litoria* genus, two α-helices, are separated by a

hinge region bounded by two prolines. Replacing these residues with either Ala or Gly resulted in general decrease antimicrobial activity suggesting the importance of the hinge region in caerin 1.1 and related peptides [17].

Introduction of a kink into the hydrophobic face of the synthetic amphipathic α-helical model peptide, KLW (KWKKLLKKLLKKLLKKLLK. NH₂) by replacement of Leu⁹ by either L-Pro or D-Pro resulted in a reduction in the α-helicity of the peptide and incorporation of D-Pro produced a significantly greater disruption than L-Pro. The [L9p] analog displayed a more significant improvement in antibacterial activity and less hemolytic activity than the [L9P] analog leading to the general suggestion that D-Pro incorporation into amphipathic α-helical peptides may be useful for the design of novel antimicrobial peptides possessing high bacterial cell selectivity coupled with very low hemolytic activity [44].

## 5. Conclusion

The study has demonstrated that a central proline residue in the frog skin HDP, figainin-2PL has an important role in determining the conformation of the peptide in a membrane-mimetic environment. Consistent with theoretical predictions provided by the AGADIR and PEP-FOLD3 programs, CD spectra suggest that in the presence of both zwitterionic DPC micelles and anionic SDS micelles, Pro¹⁸ introduces a turn or kink in the predominantly α-helical conformation of the peptide. This structural feature is involved in determining the cytotoxic selectivity of the peptide for bacteria and eukaryotic cells. Substitution of Pro¹⁸ by either L-Ala, L-Lys or D-Lys increased cytotoxic potency against a range of human tumor-derived cells but an even greater increase in activity against non-neoplastic HUVEC cells and mouse erythrocyte was observed thereby severely limiting the therapeutic potential of the peptides for development into anti-cancer agents.

While the substitution of Pro¹⁸ by L-Ala and L-Lys had relatively minor effects on the activity against ESKAPE⁺ pathogens, the [P18k] analog showed significantly increased potency against both antibiotic-resistant Gram-positive and Gram-negative bacteria. The initial excitement arising from the discovery of peptides with antibacterial activity in skin secretions of *Xenopus laevis* [45] has waned somewhat. Although pexiganan acetate (MSI 78), an analog of magainin-2, showed promise as an agent for topical treatment of infected diabetic foot ulcers [46], it failed to gain FDA approval and no peptide derived from a frog skin HDP is currently in clinical practice. Nevertheless, in view of the high potency of [P18k]figainin-2PL against methicillin-resistant *S. aureus*, vancomycin-resistant *Enterococcus* sp., *A. baumannii* and *P. aeruginosa*, bacteria commonly associated with skin and wound infections [47], further studies involving the peptide as a template for development of an antimicrobial agent for topical applications are warranted.

### CRediT authorship contribution statement

**Taylor S. Cunning:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Jack J. Delaney:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Nigel G. Ternan:** Writing – review & editing, Supervision, Formal analysis, Conceptualization. **Milena Mechkarska:** Writing – review & editing, Methodology, Conceptualization. **Samir Attoub:** Writing – review & editing, Supervision, Formal analysis, Conceptualization. **J. Michael Conlon:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Formal analysis, Conceptualization. **Laure Guilhaudis:** Writing – review & editing, Methodology, Funding acquisition, Formal analysis.

### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence
the work reported in this paper.

## Acknowledgments

Support for this study was provided by Ulster University Strategic Funding, Northern Ireland Department of Education and Learning. This work was also partially supported by the Université de Rouen Normandie (URN), INSA Rouen Normandie, the Centre National de la Recherche Scientifique (CNRS), European Regional Development Fund (ERDF), Labex SynOrg (ANR-11-LABX-0029), Carnot Institute (I2C), and the Graduate School of Research XL-Chem (ANR-18-EURE-0020 XL CHEM). The authors thank Ms. Shahrazad Sulaiman and Ms. Aya Al-Azawi (UAEU) for cytotoxicity assays.

## Conflict of interest

The authors declare that they have no conflict of interest.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.peptides.2025.171430.

## Data availability

Data will be made available on request.

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