Engineering of antimicrobial peptide Brevinin-1pl: arginine, lysine, and histidine substitutions enhance antimicrobial-anticancer efficacy with reduced cytotoxicity

Abstract

Introduction: Antimicrobial peptides (AMPs) are promising candidates for combating multidrug-resistant infections, but their clinical application is often limited by challenges such as poor selectivity and high cytotoxicity. This study aimed to optimize the therapeutic potential of brevinin-1pl, a frog-derived AMP with broad-spectrum antimicrobial and anticancer activities.

Methods: Major experimental approaches encompassed antibacterial activity evaluation, hemolytic potential assessment, bactericidal rate determination via time-kill kinetics, SYTOX Green-based membrane integrity analysis, and MTT assays for anti-proliferative effects.

Results: Substitutions with arginine (brevinin-1pl-2R and brevinin-1pl-5R) enhanced activity against Gram-positive bacteria but reduced efficacy against Gram-negative strains. Lysine substitution (brevinin-1pl-6K) decreased activity against Gram-positive bacteria due to reduced hydrophobicity. In contrast, histidine substitution (brevinin-1pl-3H) showed diminished activity against Gram-negative bacteria (e.g., MRSA MIC increased from 2 µM to 4 µM) but reduced hemolysis, indicating improved selectivity. Mechanistic studies using SYTOX green assays confirmed membrane disruption as a primary mode of action, while suggesting alternative mechanisms for Gram-positive Enterococcus faecium and Gram-negative Escherichia coli. The brevinin-1pl and its analogues demonstrated significant inhibitory efficacy against both MCF-7 breast cancer cells and H838 non-small cell lung cancer cells at a concentration of 10^-4 M. Notably, brevinin-1pl-3H exhibited low cytotoxicity toward normal HaCaT cells despite its high hydrophobicity, suggesting potential for dermatological applications.

Conclusion: These findings demonstrate that strategic amino acid substitutions can optimize the therapeutic potential of AMPs, offering a promising approach to develop peptides with enhanced efficacy and reduced clinical side effects.

Keywords: amino acid substitutions; anticancer activity; antimicrobial peptide; brevinin-1; drug-resistant; selectivity and cytotoxicity.

FIGURE 1

Helical wheel projection of the 24 amino acids of brevinin-1pl (A) /brevinin-1pl-2R (B), brevinin-1pl-5R (C), brevinin-1pl-6K (D) and brevinin-1pl-3H (E) predicted by HeliQuest. In the schematic representation, the color-coded amino acids are categorized based on their physicochemical properties: yellow residues denote non-polar amino acids, while grey residues indicate polar amino acids. Green residues specifically represent proline, which serves as the initiation point of the alpha-helical structure. Blue residues correspond to amino acids bearing an electrical charge. The arrow illustrates the orientation of the hydrophobic surface, with its directionality indicating the spatial orientation of hydrophobicity and its magnitude reflecting the relative extent of hydrophobic character.

FIGURE 2

brevinin-1pl (A), brevinin-1pl-2R (B), brevinin-1pl-5R (C), brevinin-1pl-6K (D) and brevinin-1pl-3H (E) predicted by Pepfold-4.

FIGURE 3

CD spectrum of brevilin-1pl (A), brevilin-1pl-2R (B), brevilin-1pl-5R (C), brevilin-1pl-6K (D), and brevilin-1pl-3H (E). The red line represents the results of peptides in 10 mM NH4AC solution. The blue line represents the results of peptides in 50% TFE buffer.

FIGURE 4

The hemolytic activities of peptide brevinin-1pl (A), brevinin-1pl-2R (B), brevinin-1pl-5R (C), brevinin-1pl-6K (D), and brevinin-1pl-3H (E) brevinin-1pl were evaluated using horse erythrocytes at peptide concentrations from 1 μM to 128 μM. The error bars represent the means ± SEMs for each independent experiment. Asterisks denote statistical significance compared to the 0.5% DMSO control group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The P-value represents the statistical significance of the observed differences.

FIGURE 5

Time-killing kinetic curves of brevinin-1pl, brevinin-1pl-2R, and brevinin-1pl-5R against S. aureus 6538 (A–C), MRSA 12493 (D–F) and (E) faecium 12697 (G–I). In (A–I), the MIC was 2 μM, while in Figures G and H, the MIC was 4 μM. The error bars represent the means ± SEMs for each independent experiment.

FIGURE 6

Effects of brevinin-1pl and analogues of brevinin-1pl the on the membrane of MRSA (A), E. faecium (B), E. coli (C), and K. pneumoniae (D) strains. Changes in cytoplasmic membrane permeability in the presence or absence of peptides. Melittin is served as the positive control (Melittin = 100% lysis), and PBS was served as the negative control (PBS = 0% lysis). The error bars represent the means ± SEMs for each independent experiment. Asterisks represent **** P-value <0.0001, *** P-value <0.001, ** P-value <0.01, * P-value <0.5 compared to Negative control. The P-value represents the statistical significance of the observed differences.

FIGURE 7

Anti-proliferation activity of peptide brevinin-1pl (A), brevinin-1pl-2R (B), brevinin-1pl-5R (C), brevinin-1pl-6K (D) and brevinin-1pl-3H (E) on H838 cells. Cell lines treated with 0.1% Triton X-100, 0.5% DMSO and medium were used as the positive control, negative control and growth control. The blank group was treated with medium without cells. The error bars represent the means ± SEMs for each independent experiment. Asterisks represent **** P-value <0.0001 compared to 0.5% DMSO group. The P-value represents the statistical significance of the observed differences. Data were obtained from 9 replicates in three independent experiments.

FIGURE 8

Anti-proliferation activity of peptide brevinin-1pl (A), brevinin-1pl-2R (B), brevinin-1pl-5R (C), brevinin-1pl-6K (D) and brevinin-1pl-3H (E) on MCF-7 cells. Cell lines treated with 0.1% Triton X-100, 0.5% DMSO and medium, were used as the positive control, negative control and growth control. The blank group was treated with medium without cells. The error bars represent the means ± SEMs for each independent experiment. Asterisks represent **** P-value <0.0001 compared to 0.5% DMSO group. The P-value represents the statistical significance of the observed differences. Data were obtained from nine replicates in three independent experiments.

FIGURE 9

Anti-proliferation activity of peptide brevinin-1pl (A), brevinin-1pl-2R (B), brevinin-1pl-5R (C), brevinin-1pl-6K (D) and brevinin-1pl-3H (E) on HaCaT cells. Cell lines treated with 0.1% Triton X-100, 0.5% DMSO and medium, were used as the positive control, negative control and growth control. The blank group was treated with medium without cells. The error bars represent the means ± SEMs for each independent experiment. Asterisks represent **** P-value <0.0001 compared to 0.5% DMSO group. The P-value represents the statistical significance of the observed differences. Data were obtained from nine replicates in three independent experiments.