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. 2025 Sep 2;10(36):41113–41123. doi: 10.1021/acsomega.5c03658

Rational Design of PCR12mod6, a Cathelicidin-HR-Derived Peptide with Enhanced Dual-Action Antimicrobial and Antioxidant Activity

Anupong Tankrathok †,‡, Chutima Karnmongkol †, Pavarit Kamlaiwong †, Nattapong Srisamoot †, Piyachat Wiriyaampaiwong †, Nisachon Jangpromma ‡,§,*
PMCID: PMC12444544  PMID: 40978391

Abstract

The escalating threat of antibiotic resistance demands novel antimicrobial strategies. This study introduces PCR12mod6, a rationally designed peptide derived from the amphibian Hoplobatrachus rugulosus cathelicidin-HR, engineered for both antimicrobial and antioxidant functions. PCR12mod6 was optimized through computational modeling and sequence modifications, incorporating lysine and tryptophan to enhance positive charge, amphipathicity, and membrane interactions. In vitro assays corroborated improved broad-spectrum antimicrobial activity and radical scavenging ability in ABTS and DPPH assays compared to those of its parent peptide, PCR12. Furthermore, PCR12mod6 demonstrated significant cytoprotective effects against H2O2-induced oxidative stress in RAW 264.7 macrophages and exhibited anti-inflammatory properties by reducing nitric oxide production. Molecular docking suggests that PCR12mod6 interacts with key proteins involved in oxidative stress and inflammation, including Keap1, MD-2, and iNOS. These findings position PCR12mod6 as a promising dual-function therapeutic candidate for combating both antimicrobial resistance and oxidative stress-related disorders.

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Introduction

The escalating crisis of antibiotic resistance represents a dire global health threat, demanding the urgent development of novel antimicrobial strategies. The rise of multidrug-resistant organisms presents a significant challenge. Adding to this, oxidative stress has been increasingly recognized as a factor that exacerbates infectious diseases and reduces the efficacy of conventional antibiotics. Given this interplay, there is a pressing need for therapeutic interventions capable of simultaneously combating microbial infections and mitigating oxidative damage. Antimicrobial peptides (AMPs), key components of the innate immune system across diverse species, have emerged as potential candidates due to their broad-spectrum antimicrobial activity.

Among AMPs, cathelicidins play a crucial role due to their unique ability to disrupt bacterial membranes. Their amphipathic nature, characterized by both hydrophobic and hydrophilic regions, facilitates interaction with microbial lipid bilayers, leading to membrane destabilization and cell death. Additionally, some AMPs can induce oxidative stress in bacteria, further contributing to their demise. However, the clinical utility of various AMPs is limited by factors such as susceptibility to proteolysis and, in some cases, toxicity to host cells. Rational design offers a powerful approach to overcome these limitations and enhance the therapeutic potential of AMPs.

Rational design strategies involve the deliberate modification of peptide sequences based on a thorough understanding of structure–function relationships. Optimizing parameters such as amphipathicity, net charge, and secondary structure can improve peptide stability, enhance antimicrobial potency and specificity, and reduce host cell toxicity. Within the AMP superfamily, cathelicidins stand out due to their potent antimicrobial, antioxidant, and anti-inflammatory properties. Their ability to modulate multiple biological pathways makes them particularly attractive for addressing the complex challenges posed by infectious diseases and associated inflammation. Several amphibian-derived cathelicidins, such as PN-CATH1 and PN-CATH2 from Pelophylax nigromaculata, Nv-CATH from Nanorana ventripunctata, and OL-CATH2 from Odorrana livida, exemplify this dual-functionality.

Hoplobatrachus rugulosus, a Southeast Asian amphibian, expresses two cathelicidin peptides: HR-CATH, which exhibits antimicrobial and immunomodulatory activities, and Cathelicidin-HR, known for its antioxidant activity and ability to protect DNA against UV/H2O2-induced damage, despite its relatively weak antimicrobial potency. In this study, we aimed to enhance the antimicrobial activity of Cathelicidin-HR from H. rugulosus through a rational design. Specifically, we focused on modifying the N-terminal region (PCR12) of Cathelicidin-HR. Our design incorporated lysine (Lys) residues to increase the overall positive charge of the peptide. This is crucial because the electrostatic attraction between cationic peptides and the negatively charged bacterial membrane is a primary driver of peptide-membrane interaction. Gram-negative bacteria, for example, possess lipopolysaccharide (LPS) on their outer membrane, while Gram-positive bacteria have phosphatidylglycerol in their cell membranes, both of which present negatively charged surfaces. The incorporation of tryptophan (Trp) residues was intended to enhance membrane insertion and peptide helicity. Tryptophan’s bulky, hydrophobic side chain acts as an “anchor”, facilitating the peptide’s insertion into the hydrophobic core of the lipid bilayer.

Computational modeling and targeted sequence modifications were employed to optimize interactions of peptides with microbial membranes. The redesigned peptide, PCR12mod6, was evaluated for its antimicrobial efficacy against various bacterial strains. PCR12mod6 showed superior antioxidant activity in 2,2′-azino-bis­(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. In addition, it increased cell viability in hydrogen peroxide-treated RAW 264.7 macrophages and exhibited anti-inflammatory properties by reducing nitric oxide (NO) production, consistently outperforming the parent peptide, PCR12. These results indicate that rationally modifying the PCR12 peptide to increase its positive charge and amphipathicity could markedly enhance both its antimicrobial and antioxidant activities, thereby creating a novel dual-function beneficial candidate.

Results and Discussion

Bifunctional Peptide Design for Enhancement of Antimicrobial and Antioxidant Activities

The PCR12 peptide (PCRGIFCRTGSR) represents the first 12 amino acids of the cathelicidin-HR peptide, identified as the active antimicrobial region through AMPA server analysis. This peptide served as the template for designing a series of modified peptides (PCR12modXs), with the goal of enhancing antimicrobial and antioxidant activities.

To improve antimicrobial potency, the primary peptide sequence was modified by incorporating Lys to increase the total positive net charge and Trp to enhance peptide helicity by promoting hydrophobic packing and hydrogen bonding. These modifications were designed based on the helical wheel diagram, optimizing amphipathicity by reinforcing both the cationic and hydrophobic faces of the peptides (Figure ). Secondary structure prediction using the PEP-FOLD3 server revealed that while the original PCR12 peptide exhibited a predominantly random coil conformation, most of the designed PCR12modX derivatives transitioned to an α-helical structure (Figure ), a hallmark of many potent antimicrobial peptides. In contrast, PCR12mod2, similar to the template peptide, was predicted to retain a random coil conformation, which may be attributed to the suboptimal positioning of helix-promoting residues and an uneven distribution of hydrophobic amino acids, thereby limiting its ability to adopt a stable α-helical structure. Furthermore, all predicted structures showed both cysteine residues in their reduced (free thiol) form, with no evidence of disulfide bond formation, indicating that the designed peptides likely exist in a monomeric, linear conformation under the conditions modeled (Supplement Figure S1). The physicochemical properties and membrane positioning parameters of the designed peptides are summarized in Table . The parent peptide PCR12 expressed moderate hydrophobicity (33%) and a net charge of +3, with a membrane penetration depth of 0.7 Å and a relatively weak ΔG transfer (−2.6 kcal/mol). In contrast, modified derivatives displayed notable changes in these parameters, particularly in their hydrophobicity, net charge, and membrane interactions.

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Helical wheel projection, linear sequence, and predicted secondary structure of PCR12 and its derivatives. Hydrophobic moment vectors are shown as arrows in the helical wheel projections. Color code: red (polar/basic), blue (polar/acidic), green (polar/uncharged), and yellow (nonpolar) residues. Peptide structures were generated using PEP-FOLD and visualized with PyMOL.

1. Physicochemical Properties, Peptide Positioning on Membrane Parameters, and Bioactivity Prediction of Designed Peptides.

name
 peptide sequence
 physicochemical properties
 peptide positioning
 bioactivity prediction
    hydrophobicity (%) net charge depth (Å) ΔG transfer (kcal/mol) peptide ranker antioxidative peptide predict
PCR12 PCRGIFCRTGSR 33% +3 0.7 ± 1.5 –2.6 0.7474 AnOxP
PCR12mod1 PCRKIFCRTGKR 33% +5 2.7 ± 0.5 –5.4 0.8152 AnOxP
PCR12mod2 PCRKIFCRTWKR 41% +5 1.5 ± 0.4 –3.1 0.8970 AnOxP
PCR12mod3 PCRKIFCRWGKR 41% +5 4.8 ± 1.2 –4.3 0.9291 AnOxP
PCR12mod4 PCRKIFKRWGKR 33% +6 2.4 ± 0.4 –5.1 0.9134 AnOxP
PCR12mod5 KCRKIFCRWGKR 41% +6 3.2 ± 1.3 –4.1 0.9580 Non-AnOxP
PCR12mod6 PCRKIFCRWKKR 41% +6 5.0 ± 0.2 –5.5 0.9321 AnOxP
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Increasing the net positive charge has been well established as a strategy to improve antimicrobial efficacy, given the electrostatic attraction between cationic peptides and negatively charged bacterial membranes. Modifications in PCR12mod1 to PCR12mod6 resulted in an elevated net charge (+5 to +6), thereby enhancing their electrostatic interactions with negatively charged bacterial membranes. Specifically, the incorporation of lysine (Lys) played a crucial role in strengthening electrostatic interactions, as it contributes a +1 charge while exhibiting lower mammalian cell cytotoxicity compared to other cationic residues. Hydrophobicity and helicity are critical factors for antimicrobial activity, which was strengthened by introducing Trp residues. The addition of Trp residues to the nonpolar face of the peptide increased the helical content and improved the membrane interaction through amplified hydrogen bonding and hydrophobic packing. In addition, the incorporation of Trp facilitates hydrogen bonding with lipid bilayers due to its indole side chain. Therefore, this modification promoted membrane penetration, as evidenced by the higher hydrophobicity (41%) and more favorable ΔG transfer values (−3.1 to −4.3 kcal/mol) observed in PCR12mod2 and PCR12mod3. The predicted secondary structure of all modified peptides was adopted from a random coil to the α-helix structure except for PCR12mod2 (Figure ). However, among the modified peptides, PCR12mod6 showed superior membrane penetration depth (5.0 ± 0.2 Å) and the most negative ΔG transfer (−5.5 kcal/mol), suggesting the strongest affinity for bacterial membranes. These attributes are indicative of elevated antimicrobial potential, further supported by its high Peptide Ranker score (0.9321), surpassing both PCR12 (0.7474) and other derivatives.

Peptide-ligand docking was employed to assess the antioxidant potential of the peptide. PCR12 and its derivatives were investigated for their binding affinities with reactive oxygen species (ROS), including ABTS, DPPH, hydrogen peroxide (H2O2), and hydroxyl radicals (OH•) (Table and Figure ). Lower (more negative) affinity values correlate with stronger antioxidant interactions, while total energy values indicate binding stability. Among all peptides, PCR12mod6 exhibited the strongest predicted interaction with ABTS, as reflected by its lowest binding affinity (−7.757 kcal/mol). In contrast, PCR12mod2 (−7.186 kcal/mol) and PCR12mod3 (−7.190 kcal/mol) showed more favorable affinities toward DPPH than did PCR12mod6 (−7.139 kcal/mol). Nonetheless, PCR12mod6 maintained a consistently high antioxidant potential across multiple radical species. This peptide illustrated competitive binding to H2O2 (−5.667 kcal/mol) and OH• (−5.59 kcal/mol), further reinforcing its antioxidant efficacy. PCR12 displayed comparatively moderate antioxidant activity with weaker affinities for ABTS (−7.567 kcal/mol) and DPPH (−6.976 kcal/mol), suggesting that the parent peptide scavenges oxidative agents less effectively than PCR12mod6. Although PCR12mod5 displayed a slightly higher Peptide Ranker score (0.9580) and favorable docking affinities toward ROS, it was not predicted to exhibit antioxidant activity by AnOxPePred. This discrepancy highlights that strong binding energy is insufficient to guarantee antioxidant function, which depends on specific sequence motifs and physicochemical properties, criteria that are more effectively satisfied by the PCR12mod6 peptide.

2. Molecular Docking Analysis of Designed Peptides with ABTS, DPPH, Hydrogen Peroxide, and the Hydroxyl Radical.

Peptide name
 ABTS
 DPPH
 hydrogen peroxide (H2O2)
 hydroxyl radical (OH•)
  affinity (kcal/mol) total energy (kcal/mol) affinity (kcal/mol) total energy (kcal/mol) affinity (kcal/mol) total energy (kcal/mol) affinity (kcal/mol) total energy (kcal/mol)
PCR12 –7.567 32.608 –6.976 92.463 –5.597 –7.534 –5.693 –9.478
PCR12mod1 –6.584 27.63 –6.828 93.259 –5.726 –7.422 –5.661 –13.323
PCR12mod2 –7.311 26.533 –7.186 89.861 –5.605 –6.763 –5.678 –9.2
PCR12mod3 –6.822 32.397 –7.19 93.956 –5.625 –5.771 –5.665 –12.081
PCR12mod4 –7.402 24.107 –6.854 92.421 –5.645 –6.347 –5.631 –12.994
PCR12mod5 –7.176 28.228 –6.988 93.174 –5.653 –6.074 –5.645 –12.724
PCR12mod6 –7.757 29.477 –7.139 90.372 –5.667 –7.058 –5.59 –9.34
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Peptide–radical interaction analysis. PCR12 (A) and PCR12mod6 (B) peptides were docked against ABTS, DPPH, hydrogen peroxide, and the hydroxyl radical. Identification of amino acid residues involved in peptide–ligand interactions was conducted using the Protein–Ligand Interaction Profiler web server (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index), and the interacting amino acid residues were visualized in stick representation.

Among the designed derivatives, PCR12mod6 was selected for experimental validation based on a systematic in silico evaluation incorporating physicochemical profiling, membrane interaction analysis, and bioactivity prediction. This peptide demonstrated a combination of favorable features, including the highest net charge, deepest predicted membrane insertion, the most favorable free energy of membrane transfer, and a high Peptide Ranker score, along with a positive prediction for antioxidant potential. These integrated criteria informed its prioritization as a lead candidate for an in vitro study. Nonetheless, other variants, notably PCR12mod3, exhibited comparable biophysical properties. This peptide shares the same hydrophobicity (41%) as PCR12mod6, a similar membrane insertion depth (4.8 ± 1.2 Å), and a strong ΔG transfer (−4.3 kcal/mol) alongside competitive antioxidant docking affinities. While these results suggest the therapeutic potential of PCR12mod3, the present study focused on PCR12mod6 as a proof-of-concept to validate the utility of rational design principles in enhancing peptide bifunctionality.

Secondary Structure Analysis of the Peptides by CD Spectroscopy

Circular dichroism (CD) spectroscopy was carried out to evaluate the secondary structure of PCR12 and PCR12mod6 peptides in aqueous (PBS) and membrane-mimicking (80% TFE) environments (Figure ). In PBS, PCR12 predominantly displayed a random coil conformation, while PCR12mod6 displayed partial α-helical characteristics. In 80% TFE, both peptides adopted a more pronounced α-helical structure, as indicated by the characteristic negative bands at 208 and 222 nm, with PCR12mod6 demonstrating greater helical content than PCR12. This structural transition aligns with previous reports that AMPs adopt α-helical conformations in hydrophobic environments, facilitating membrane insertion and antimicrobial activity. The augmented helicity of PCR12mod6 suggests improved membrane interaction and stability, which may contribute to its superior bioactivity compared to that of the parent peptide.

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Circular dichroism (CD) spectra of PCR12 and PCR12mod6 peptides. The spectra were recorded from 180 to 260 nm using CD spectroscopy, with data presented as mean residue ellipticity.

Antimicrobial Activity of the PCR12mod6 Peptide

The growth inhibition results of PCR12 and its modified version, PCR12mod6, against various bacterial strains are shown in Figure . PCR12mod6 denoted substantially reinforced growth inhibition across a broad spectrum of pathogens compared with PCR12, which exhibited limited or negligible activity against most strains. Especially, PCR12mod6 achieved strong inhibition rates against Bacillus megaterium (64.60%), Aeromonas hydrophila (82.36%), and Salmonella derby (94.35%). These results underscore the importance of increasing the net positive charge (+6) through the incorporation of Lys residues, which strengthen electrostatic interactions with the negatively charged bacterial membranes, a well-established mechanism for AMPs. The inclusion of Trp residues further augmented membrane penetration by promoting hydrophobic interactions and hydrogen bonding with lipid bilayers, leading to more efficient bacterial disruption. This is consistent with previous studies highlighting the role of Trp in enhancing the membrane-disruptive capabilities of AMPs. In addition to its efficacy against Gram-negative bacteria, PCR12mod6 also presented increased activity against Gram-positive strains, including Bacillus subtilis (14.05%) and Bacillus cereus (29.08%), where PCR12 showed minimal or no inhibition. This broad-spectrum activity highlights the success of the rational design approach in optimizing peptide properties for escalated antimicrobial performance.

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Antimicrobial activity of PCR12 and PCR12mod6 peptides assessed using the broth microdilution assay. Bars with the same letter are not significantly different at p < 0.05 (n = 3) as determined by one-way ANOVA followed by Duncan’s multiple range test.

To further validate the antimicrobial enhancement, minimum inhibitory concentration (MIC) assays were conducted to quantitatively compare the efficacy of PCR12mod6 against parent peptide PCR12. As shown in Table , PCR12mod6 exhibited MIC values ranging from 25 to 50 μg/mL against multiple Gram-positive bacteria, including Bacillus subtilis, B. cereus, and Enterococcus faecalis, and also showed efficacy against certain Gram-negative strains such as Edwardsiella tarda and S. typhimurium. In contrast, PCR12 showed no inhibitory effect at concentrations of up to 400 μg/mL across all tested strains. These results showed that PCR12mod6 exhibits superior antimicrobial potency in both qualitative growth inhibition assays and quantitative MIC evaluations. While other PCR12modX variants such as PCR12mod3 possess similar hydrophobicity and ΔG transfer values, these derivatives were not subjected to MIC testing in this study. Therefore, it cannot be conclusively determined that PCR12mod6 is superior to all other derivatives or that Lys/Trp substitutions universally enhance activity. However, the data clearly demonstrate improved antimicrobial activity compared to the template peptide, thereby validating the efficacy of the rational design methodology employed in this investigation.

3. Minimum Inhibition Concentration of PCR12 and PCR12mod6 Peptides.

microorganism
 MIC (μg/mL)
  PCR12 PCR12mod6 Melittin
Gram-positive bacteria      
Bacillus megaterium TISTR067 >400 50 25
Bacillus subtilis TISTR1248 >400 25 100
Bacillus cereus TISTR1449 >400 25 25
Enterococcus faecalis TISTR927 >400 25 25
Staphylococcus aureus ATCC25923 >400 >400 25
Gram-negative bacteria      
Aeromonas hydrophila ATCC7966 >400 >400 50
Edwardsiella tarda DMST38217 >400 25 12.5
Salmonella derby DMST16881 >400 >400 25
Salmonella typhimurium TISTR1472 >400 25 12.5
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Antioxidant Activity of the PCR12mod6 Peptide

The antioxidant activity of the designed peptides, PCR12 and its modified derivative PCR12mod6, was evaluated using ABTS and DPPH radical scavenging assays, with vitamin C serving as a positive control due to its well-established capacity to donate electrons and neutralize free radicals, as depicted in Figure . The results demonstrate that PCR12mod6 exhibits a greatly enlarged antioxidant capacity compared to PCR12 across all tested concentrations. In the ABTS assay (Figure A), PCR12mod6 displayed a strong dose-dependent scavenging activity, starting at 7.91% at 25 μg/mL and reaching 98.95% at 400 μg/mL. In contrast, PCR12 showed weaker activity, with scavenging rates of 17.18% at 200 μg/mL and 74.88% at 400 μg/mL. Similarly, in the DPPH assay (Figure B), PCR12mod6 outperformed PCR12, scavenging 3.85% of radicals at 25 μg/mL compared to 1.13% by PCR12. At the highest concentration (400 μg/mL), PCR12mod6 achieved 52.96% scavenging, while PCR12 reached only 27.70%. These results indicate the superior antioxidant potential of PCR12mod6, which can be attributed to strategic modifications in its amino acid sequence. The heightened antioxidant activity of PCR12mod6 might be driven by both structural and physicochemical factors. The rational design of PCR12mod6 incorporated strategic modifications to enhance hydrophobicity, a critical factor influencing antioxidant activity. Notably, a key modification was the introduction of Trp residues, which play a pivotal role in antioxidant activity due to the indole side chain’s high susceptibility to oxidation. The indole ring of Trp can stabilize free radicals through electron donation, making it highly effective in scavenging ROS. Additionally, the substitution of Lys residues further contributed to the peptide’s antioxidant capacity. The lone electron pair on the nitrogen atom of Lys is readily oxidized, allowing this amino acid to be directly targeted by oxidants, thereby enhancing ROS scavenging.

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Antioxidant activity of PCR12 and PCR12mod6 peptides. ABTS (A) and DPPH (B) scavenging activity of PCR12 and PCR12mod6 peptides. Bars with the same letter are not significantly different at p < 0.05 (n = 3) as determined by one-way ANOVA followed by Duncan’s multiple range test.

Cytoprotective Effects of PCR12mod6 Peptide under Normal and Oxidative Stress Conditions

The cytoprotective effects of PCR12mod6, a modified derivative of the parent peptide PCR12, were evaluated in RAW 264.7 macrophage cells under both normal conditions and oxidative stress induced by hydrogen peroxide (H2O2), as illustrated in Figure . Under normal conditions (Figure A), both peptides revealed minimal cytotoxicity at concentrations up to 500 μg/mL, with PCR12mod6 consistently demonstrating higher cell viability compared to that of PCR12. At lower concentrations (15.625–250 μg/mL), both peptides slightly boosted cell viability, with PCR12mod6 showing a more pronounced effect (107.86% at 15.625 μg/mL) compared to PCR12 (106.86%). This increase in viability may be attributed to the peptides’ potential role in promoting cell proliferation, a phenomenon observed with certain bioactive peptides. Remarkably, at 500 μg/mL, PCR12mod6 maintained cell viability at 103.09%, while PCR12 caused a reduction to 90.77%. At the highest concentration tested (1000 μg/mL), PCR12mod6 preserved viability at 95.14%, whereas PCR12 represented significant cytotoxicity, reducing viability to 22.39%. These results suggest that rational modifications to PCR12mod6, such as optimized hydrophobicity and increased charge distribution, significantly ameliorated its biocompatibility at higher concentrations.

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Cytotoxicity and cytoprotective effects of PCR12 and PCR12mod6 peptides in RAW 264.7 cells assessed by the MTT assay. (A) Cell viability of RAW 264.7 cells under normal conditions after peptides treatment. (B) Cytoprotective activity of peptides against H2O2-induced oxidative stress. Bars with the same letter are not significantly different at p < 0.05 (n = 3) as determined by one-way ANOVA followed by Duncan’s multiple range test.

The cytoprotective capacity of PCR12mod6 was further evaluated under oxidative stress conditions (Figure B) induced by 250 μM H2O2. Untreated cells experienced a substantial reduction in viability (36.51%) due to oxidative damage. In contrast, both PCR12 and PCR12mod6 performed with concentration-dependent protective effects, with PCR12mod6 consistently outperforming PCR12. At 500 μg/mL, PCR12mod6 restored cell viability to 78.80%, compared to 65.66% for PCR12. Meanwhile, at 250 μg/mL, PCR12mod6 and PCR12 restored viability to 42.61% and 42.09%, respectively. Even at lower concentrations (15.625 to 31.25 μg/mL), PCR12mod6 maintained greater cell viability, highlighting its accumulated capacity to mitigate oxidative damage.

Anti-Inflammatory Activity of the PCR12mod6 Peptide

The anti-inflammatory properties of PCR12 and its modified counterpart, PCR12mod6, were evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells, with a focus on nitric oxide (NO) production and cell viability, as shown in Figure A,B. LPS stimulation triggers an inflammatory response characterized by the overproduction of NO and other pro-inflammatory mediators, which can compromise cell viability. , This study demonstrates the anti-inflammatory activity of PCR12mod6 through its concentration-dependent suppression of NO production. At lower concentrations, PCR12mod6 expressed tiny margins of greater efficacy in reducing NO levels compared to PCR12. The differences in efficacy became more pronounced at higher concentrations. At 250 μg/mL, PCR12mod6 markedly reduced NO production to 69.11% of the control, whereas PCR12 reduced it to 90.54%. At the maximum concentration of 500 μg/mL, PCR12mod6 further reduced NO production to 41.66% of the control compared to 74.25% for PCR12. %. These results suggest that the anti-inflammatory activity of PCR12mod6 is mediated, at least in part, by the suppression of NO production in LPS-stimulated cells. The MTT assay indicated that both peptides had low cytotoxic effects on LPS-treated RAW 264.7 cells, with PCR12mod6 showing superior cytoprotective effects compared to PCR12. Particularly at higher concentrations (500 μg/mL), PCR12mod6 preserved cell viability at 81.67%, substantially outperforming PCR12, which showed a reduction to 71.30%. Previously, Amphibian-derived cathelicidin peptides, such as cathelicidin-PY from Paa yunnanensis and cathelicidin-PP from Polypedates puerensis, have been found to exhibit robust antimicrobial and anti-inflammatory activities. These peptides act by disrupting microbial cell membranes. Moreover, they can inhibit the production of NO and pro-inflammatory cytokines in LPS-stimulated macrophage cells. , This anti-inflammatory effect is mediated by the suppression of key signaling pathways, including TLR4-mediated TRIF-dependent signaling, MAPKs (ERK, JNK, and p38), and NF-κB pathway. ,

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Anti-inflammatory effects of PCR12 and PCR12mod6 peptides in RAW 264.7 cells. Cells were treated with 100 ng/mL LPS alone or with test peptides. (A) Nitric oxide (NO) production was assessed using the Griess reagent. (B) Viability of RAW 264.7 cells after NO assessment, determined via the MTT assay. Bars with the same letter are not significantly different at p < 0.05 (n = 3) as determined by one-way ANOVA followed by Duncan’s multiple range test.

Investigation of Peptides–Protein Interaction Using Molecular Docking

Molecular docking analysis was carried out to investigate the interaction of PCR12mod6 with key molecular targets involved in oxidative stress and immune responses. The docking results reveal that PCR12mod6 exhibits binding affinities with these targets. In the case of the Kelch domain of Keap1 (Figure A), PCR12mod6 demonstrated a stable interaction, suggesting its potential role in modulating the Nrf2-Keap1 signaling pathway. This pathway is crucial for cellular defense against oxidative stress, as Nrf2 regulates the expression of antioxidant proteins. The binding of PCR12mod6 to Keap1 may competitively inhibit the interaction between Keap1 and Nrf2, thereby promoting Nrf2 translocation to the nucleus and enhancing the antioxidant gene expression. This mechanism aligns with the observed antioxidant activity of PCR12mod6 in ABTS and DPPH radical scavenging assays. Furthermore, the interaction of PCR12mod6 with the TLR4-MD-2 complex (Figure B) highlights its potential anti-inflammatory properties. The TLR4-MD-2 complex is a key mediator of the innate immune response, particularly in the recognition of LPS from Gram-negative bacteria. By binding to this complex, PCR12mod6 may interfere with LPS-induced TLR4 activation, thereby suppressing downstream pro-inflammatory signaling pathways, such as NF-κB and MAPK. Moreover, the docking of PCR12mod6 with iNOS (Figure C) further supports its anti-inflammatory and antioxidant capabilities. iNOS is responsible for the production of NO, a key mediator of inflammation and oxidative stress. The binding of PCR12mod6 to iNOS elucidated that PCR12mod6 occupied a similar binding pocket as the iNOS inhibitor AR-C118901. This binding suggests its potential to inhibit NO overproduction, which is often associated with chronic inflammation and tissue damage. This is consistent with the observed reduction in the level of NO production in LPS-stimulated RAW 264.7 macrophages, indicating that PCR12mod6 can mitigate inflammatory responses by targeting TLR4 signaling.

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Peptide–protein docking complex of the PCR12mod6 peptide. The PCR12mod6 structure was docked with the Kelch domain of Keap1 (A), TLR4-MD-2 complex (B), and iNOS (C) using the HPEPDOCK web server. Protein structures were presented on the surface, the PCR12mod6 peptide was shown in cartoon, and ligands were illustrated in white, orange, and green sticks for the inhibitor of Nrf2-keap1 complex formation (S,R,S)-1a), Lipopolysaccharide (LPS), and iNOS inhibitor (AR-C118901), respectively.

Hemolytic Activity of the PCR12mod6 Peptide

The hemolytic activity of the PCR12mod6 peptide was evaluated against sheep red blood cells (shRBCs) at different concentrations, ranging from 25 to 400 μg/mL, with the results summarized in Figure . Triton X-100 was used as the positive control, showing 100% hemolysis, while DW served as the negative control, showing a hemolysis rate of 18.63%. PCR12mod6 exhibited minimal hemolytic activity across all tested concentrations. At 25 μg/mL, the hemolysis rate was 19.39%, only slightly above the negative control, and hemolysis slightly decreased at higher concentrations, with 15.95% hemolysis observed at 400 μg/mL. These results indicate that PCR12mod6 has a low tendency to disrupt red blood cell membranes, even at high peptide concentrations, suggesting it possesses minimal toxicity to red blood cells. The relatively low hemolytic activity of PCR12mod6 can be linked to its hydrophobicity and net charge properties. It is well-known that a balance between hydrophobicity and charge is critical in determining a peptide’s ability to selectively target bacterial membranes over mammalian cells.

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Hemolytic activity of the PCR12mod6 peptide against sheep red blood cells. Hemolytic activity was assessed after a 1 h incubation with PCR12mod6. 1% Triton X-100 was used as the positive control and double-distilled water (DW) was used as the negative control. Bars with the same letter are not significantly different at p < 0.05 (n = 3) as determined by one-way ANOVA followed by Duncan’s multiple range test.

Conclusions

In summary, this study successfully demonstrated the rational design and in vitro characterization of PCR12mod6, a novel cathelicidin-HR-derived peptide with reinforced dual-action antimicrobial and antioxidant properties. By strategically incorporating lysine and tryptophan residues, we significantly boosted the peptide’s antimicrobial efficacy and its antioxidant activity in ABTS and DPPH radical scavenging assays. Besides, PCR12mod6 manifested significant cytoprotective effects against H2O2-induced oxidative stress in RAW 264.7 macrophages and exerted anti-inflammatory properties by reducing nitric oxide production. Meanwhile, molecular docking studies suggest potential interactions with key proteins involved in oxidative stress and inflammation. These findings indicate that PCR12mod6 holds considerable promise as a candidate therapeutic lead for addressing the growing challenges of antibiotic resistance and oxidative stress-related disorders.

Materials and Methods

Design Peptide, Bioinformatic Analysis, and Molecular Docking

The antimicrobial peptide was designed using a template-based approach combined with physicochemical property analysis. The amino acid sequence of the cathelicidin-HR peptide from H. rugulosus (GenBank: MW725232) served as the design template. The antimicrobial activity region was predicted using the Antimicrobial Sequence Scanning System (AMPA) server (http://tcoffee.crg.cat/apps/ampa/). To visualize the peptide’s helical structure, a helical wheel diagram was generated using the PEPWHEEL program (http://www.tcdb.org/cgi-bin/projectv/pepwheel.py). The physicochemical properties of the peptides were calculated using the Antimicrobial Peptide Database (APD) (http://aps.unmc.edu/AP/main.php). The bioactivity probability of the designed peptides was predicted using the PeptideRanker server (http://distilldeep.ucd.ie/PeptideRanker/). To assess the antioxidant potential of the peptide derivatives (Antioxidant peptide: AnOxP), sequences were analyzed using the Antioxidative Peptide Predictor (AnOxPP) web server (http://www.cqudfbp.net/AnOxPP/index.jsp). Secondary structure prediction was performed using the PEP-FOLD server. The positioning of the peptides within membranes was modeled using the PPM (Positioning of Proteins in Membranes) web server (http://opm.phar.umich.edu/server.php), which uses an anisotropic solvent model with a DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayer to estimate peptide penetration depth and transfer free energy (ΔG transfer).

For the peptide-ligand interaction analysis, the PDB file of the designed peptide secondary structures, obtained from PEP-FOLD3, was used as the receptor molecule. The Simplified Molecular Input Line Entry System (SMILES) representations of the ligandsABTS, DPPH, hydrogen peroxide (H2O2), and hydroxyl radical (OH•)were retrieved from the PubChem database. These were then converted into 3D PDB format using the NovoPro online tool (https://www.novoprolabs.com/tools/smiles2pdb). Peptide-ligand docking simulations were created using the DockThor web server. Peptide–protein docking was conducted using the HPEPDOCK web server, where PCR12mod6 was docked against key targets: the Kelch domain of Keap1 (PDB: 7Q6S), the Toll-like receptor 4 (TLR-4) and myeloid differentiation factor 2 (MD-2) complex (PDB: 2Z64), and inducible nitric oxide synthase (iNOS) (PDB: 3E6T). Visualization of peptide structures and docking results was carried out using PyMOL (Schrödinger LLC).

Peptide Synthesis

Peptides were synthesized according to prior research. PCR12 (PCRGIFCRTGSR) and PCR12mod6 peptides (PCRKIFCRWKKR), with molecular weights of 1352.60 and 1621.04 Da, respectively, were produced by GenScript (Piscataway, NJ, USA). Reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry were utilized to determine the peptide purity (95%) and sequence, respectively.

Peptide Circular Dichroism

The peptides of interest were prepared at a concentration of 0.4 μg/μL in either 1× phosphate-buffered saline (PBS), pH 7.4, or 80% aqueous trifluoroethanol (TFE), with a final volume of 100 μL. Circular dichroism (CD) spectra were acquired using a Jasco spectrometer (Easton, MD, USA) at a scanning rate of 20 nm/min across the wavelength range of 180260 nm. The resulting data were expressed as the mean residue ellipticities.

Antimicrobial Activity Assay

To evaluate the antimicrobial activity of the peptides, a broth microdilution method was employed. In brief, bacterial cells were grown in a nutrient broth at 37 °C until they reached the mid logarithmic phase. The cultured cells were then diluted to a concentration of 104 CFU/mL with PBS, at pH 7.4. A 50 μL aliquot of the diluted cells was added to microcentrifuge tubes, followed by the addition of 50 μL of 4 mg/mL peptide solution or double-distilled water (DW) (negative control). The mixture was incubated at 37 °C for 16–18 h. Bacterial growth was monitored by measuring the optical density at 600 nm (OD600) using a spectrophotometer. A reduction in OD600 indicated the antimicrobial activity of the peptide. The percentage of bacterial growth inhibition was calculated using the formula: [(OD600 nm, controlOD600 nm, peptide)/OD600 nm, control] × 100.

The MIC was assayed as described previously. Briefly, bacterial strains were cultured in Mueller–Hinton Broth (MHB) at 37 °C until reaching mid logarithmic phase. Cultures were then diluted in PBS, pH 7.4 to a final concentration of 104 CFU/mL. Subsequently, 25 μL of the diluted bacterial suspension was transferred into each well of a 96-well microtiter plate, followed by the addition of 25 μL of peptide solution prepared as a 2-fold serial dilution (ranging from 400 to 1.5625 μg/mL). Melittin was used as a positive control. The plates were incubated at 37 °C for 3 h, after which 50 μL of fresh MHB was added to each well and incubated for an additional 18 h. Bacterial viability was assessed by adding 30 μL of 0.015% (w/v) resazurin solution to each well, followed by incubation at 37 °C for 2 h. The MIC was defined as the lowest peptide concentration that prevented a color change from blue to pink, indicating complete inhibition of bacterial growth.

Antioxidant Activity Assay

The ABTS radical scavenging activity was assessed following the method described by. Briefly, the ABTS radical solution was prepared by mixing 2.8 mM potassium persulfate with 7 mM ABTS in water, followed by a 6 h incubation in the dark. This solution was then diluted 50-fold with DW. Samples, dissolved in water, were added to the diluted ABTS solution, while the same volume of solvent served as the negative control. Ascorbic acid was used as a positive control. The reactions were shielded from light for 30 min. A reduction in the absorbance at 415 nm indicated the antioxidant activity of the samples. The percentage of free radical scavenging was calculated using the formula: [(A415 nm, blankA415 nm, sample) /A415 nm, blank] × 100.

The DPPH radical scavenging activity was evaluated as described in previous studies. The reaction mixture consisted of 190 μL of 50 μM DPPH solution in ethanol and 10 μL of a 2-fold-diluted peptide sample. The mixture was incubated in the dark at room temperature for 30 min. After incubation, the absorbance was measured at 517 nm using a blank as the reference. Ascorbic acid was used as a positive control. The percentage of DPPH radical scavenging activity was calculated using the formula: [(A517 nm, blankA517 nm, sample) /A517 nm, blank] × 100.

Cell Viability

Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 cells were seeded in 96-well plates at a density of 2 × 104 cells/well and incubated for 24 h at 37 °C in a 5% CO2 atmosphere in Dulbecco’s Modified Eagle Medium (DMEM). Subsequently, cells were treated with 2-fold serial dilutions of the peptides for an additional 24 h. The medium was then replaced with 100 μL of an MTT solution (0.5 mg/mL) and incubated for 30 min under the same conditions. The resulting formazan crystals were solubilized in 100 μL of dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using a Varioskan LUX microplate reader (USA). Cell viability was expressed as a percentage with the viability of untreated cells set as 100%.

Cytoprotective Effect Assay

To evaluate the protective effect of the peptide against H2O2-induced toxicity, RAW 264.7 cells were seeded at a density of 2 × 104 cells per well in 96-well plates and incubated for 24 h. Following incubation, the cells were treated with varying concentrations of peptides prepared in 2-fold serial dilutions, either in the presence or absence of 250 μM H2O2, for 24 h. Cell viability was then assessed by using the previously described method.

Anti-Inflammatory Assay

The anti-inflammatory activity was evaluated following the methodology described by. RAW 264.7 macrophage cells were seeded in 96-well plates at a density of 2 × 104 cells/well in a total volume of 100 μL/well. Cells were then treated with 100 ng/mL of LPS either alone or in combination with various test peptides. After 24 h of incubation at 37 °C in a 5% CO2 atmosphere with 95% relative humidity, 100 μL of cell culture medium from each treatment was combined with an equal volume of Griess reagent and incubated at room temperature for 10 min. The absorbance was subsequently measured at 540 nm, and NO production was calculated as a percentage relative to the no-peptide control. Cell viability was assessed following each treatment using the MTT assay, with the viability of LPS-treated cells serving as the 100% viability reference.

Determination of Hemolytic Activity

The hemolytic activity of the peptides was evaluated using defibrinated sheep red blood cells (shRBCs). The shRBCs were washed with PBS at pH 7.4 and diluted to a concentration of 0.5% (v/v) in PBS. A 100 μL aliquot of the shRBC solution was added to microcentrifuge tubes, followed by the addition of 10 μL of a 2-fold serial dilution of peptides. The mixtures were incubated at 37 °C for 1 h. After incubation, the samples were centrifuged at 1000g for 5 min, and the absorbance of 100 μL of the supernatants was measured at 415 nm using a spectrophotometer. Triton X-100 (1% v/v) and DW were used as positive and negative controls, respectively. The percentage of hemolysis was calculated using the formula: [(A415 nm, peptide)/(A415 nm, 1% (v/v) Triton X-100)] × 100.

Statistical Analysis

Statistical analysis was accomplished using ANOVA for a completely randomized design (CRD) with the Statistical Tool for Agricultural Research (STAR). Results were expressed as the mean ± standard deviation (SD). A p-value of less than 0.05 was considered statistically significant. To identify differences among treatment means, Duncan’s Multiple Range Test (DMRT) was employed following ANOVA. In graphical representations, bars annotated with the same letter indicate no significant difference, whereas bars with different letters denote statistically significant differences at p < 0.05 (n = 3).

Supplementary Material

ao5c03658_si_001.pdf (246.9KB, pdf)

Acknowledgments

This research was conducted at Kalasin University and Khon Kaen University. The authors sincerely acknowledge the Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, for its valuable collaboration and technical support, which were instrumental in the successful completion of this study.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03658.

  • Predicted secondary structures of PCR12 and PCR12modX peptides highlighting cysteine thiol groups and mass spectrum and HPLC chromatogram of synthetic PCR12 and PCR12mod6 peptides (PDF)

AT: Conceptualization, Funding acquisition, Investigation, Formal Analysis, Validation, and Writing-original draft. CK and PK: Investigation and Formal Analysis. NS and PW: Formal Analysis, Validation, and Writing-review and editing. NJ: Conceptualization, Funding acquisition, Supervision, Validation, and Writing-review and editing.

This work was financially supported by Thailand Research Fund and Office of the Higher Education Commission (Grant No. MRG 6280081) and supported by Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University

The authors declare no competing financial interest.

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Supplementary Materials

ao5c03658_si_001.pdf (246.9KB, pdf)

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