Antimicrobial peptides (AMPs) are a promising class of molecules found in various organisms, from bacteria and fungi to plants and animals, including humans [1, 2]. These peptides play a crucial role in the innate immune system, serving as a frontline defense against a wide range of pathogens, including bacteria, viruses, and fungi [3–5]. AMPs exhibit potent antimicrobial properties, disrupting the membranes or cellular processes of these invaders. What makes AMPs particularly intriguing is their versatility and potential for therapeutic applications [4]. Researchers are exploring their use in developing novel antibiotics, antiviral drugs, and even as a defense against drug-resistant microbes, which pose a growing global health threat. In addition to the naturally occurring AMPs, a considerable number of engineered antimicrobial peptides have been prepared thus far [6]. These molecules were designed based on the SARs observed in natural AMPs. This artificial synthesis allows for the customization of AMPs to target microbial pathogens or to enhance their stability and effectiveness. Therefore, each source will have its own specific advantages and drawbacks. In an effort to gain a deeper understanding of these issues, we have taken the first step by conducting a comparative study involving two representative AMPs from each group, MPC and BP52.
Accordingly, MPC was isolated from the venom of social wasp Vespa crabro [7–9] whereas BP52 was designed based on the structure of two natural AMPs named Melittin M and Cecropin A [10–12]. Despite their different origins, both compounds share similar structural properties, including 1. An alpha helical conformation - the most prevalent secondary structure found in natural AMPs. 2. The cationic amphipathic properties of two AMPs were established with multiple cationic residues, thus resulting in a net positive charge and multiple hydrophobic residues (see Table 1 and Fig. 1); 3. Amidated C-terminal and free amino group at N-terminal; 4. None of the helix breakers (proline and glycine) or anionic residues was found in their peptide sequences. Nonetheless, the MPC sequence is longer but lower net charge than BP52 (see Table 1). Besides, BP52 has a “perfect” amphipathic helical structure as indicated by the helical wheel projection in Fig. 1B. In contrast, MPC has a hydrophilic residue asparagine in the middle of the hydrophobic face whereas the hydrophilic face is interfered with two hydrophobic residues - leucine and alanine at position 1 and 8, respectively. Hence, though the percentage of hydrophobic residues and hydrophilic residues in the two AMPs were relatively equal, their amphipathic styles were different.
Table 1
Some structural properties of MPC and its derivatives
Properties
|
MPC
|
BP52
|
Origin
|
Natural
|
Artificial
|
Structure
|
Linear, α-helix
|
Linear, α-helix
|
Molecular weight
|
1506.98
|
1443.94
|
Length of sequence
|
14
|
11
|
Hydrophobic residue
|
8 (57%)a
|
6 (55%)
|
Hydrophilic residue
|
6 (43%)
|
5 (45%)
|
Cationic residue
|
3 (21%)
|
5 (45%)
|
Anionic residue
|
0
|
0
|
Helix breakers
|
0
|
0
|
N-terminal
|
Free
|
Free
|
C-terminal
|
Amidation
|
Amidation
|
Net charge
|
+ 4
|
+ 6
|
Hydrophobicityb
|
0.634
|
0.545
|
Hydrophobic moment (µH)b
|
0.399
|
0.876
|
Retention time (min)
|
6.939
|
5.796
|
a The content within the parentheses is the percentage of each residue type relative to the total number of residues.
b Hydrophobicity (H) and Hydrophobic moment (µH) were predicted by HeliQuest server for standard α-helix [13].
In the antimicrobial assay on two Gram-positive species Bacillus cereus and Enterococcus faecalis, both BP52 and MPC inhibited more than 90% of bacteria growth at 4µM (see Table 2). Besides, BP52 was more potent than MPC on Listeria monocytogenes with the percentage of cell inhibition at 4µM were 100% and 72%, respectively. Regarding Gram-negative bacteria, BP52 showed higher potency against Escherichia coli and Salmonella enterica whereas slightly less active on Pseudomonas aeruginosa than MPC. Moreover, BP52 also exhibited a lower inhibition percentage on Candida albicans than MPC as indicated in Table 2.
Furthermore, MPC and BP52 showed almost no hemolysis at the low concentration (8µM), thus demonstrating a high level of selectivity. Nonetheless, at a high concentration (128µM), MPC caused significantly higher toxicity on red blood cells than BP52 (see Fig. 2). Interestingly, the anticancer activity of MPC on the A427 cell line was notably less potent than BP52 (see Fig. 3). Of note, it can be estimated that the IC50 value of MPC in the range from 4µM to 8µM whereas this value of BP52 is less than 4µM.
Table 2
Antimicrobial activity of B52 and MPC
Microbial pathogens
|
Percentage of cell growth inhibition
|
BP52a
|
MPCa
|
BP52b
|
MPCb
|
Gram positive
|
Bacillus cereus
|
100,03 ± 0,78
|
99,45 ± 0,36
|
100,28 ± 0,96
|
99,63 ± 0,58
|
Enterococcus faecalis
|
95,04 ± 1,23
|
94,8 ± 1,08
|
98,87 ± 1,51
|
96,64 ± 1,97
|
Listeria monocytogenes
|
100,34 ± 0,52
|
72,14 ± 5,44
|
101,49 ± 0,93
|
81,4 ± 1,57
|
Gram negative
|
Escherichia coli
|
99,58 ± 1,03
|
12,1 ± 5,27
|
100,89 ± 1,76
|
98,54 ± 0,72
|
Pseudomonas aeruginosa
|
16,92 ± 3,45
|
17,5 ± 7.84
|
23,12 ± 7,35
|
44,9 ± 7,86
|
Salmonella enterica
|
98,57 ± 2.17
|
24,97 ± 3,70
|
97,64 ± 1,54
|
96,07 ± 1,53
|
Fungi
|
Candida albicans
|
19,92 ± 5,91
|
40,15 ± 7,65
|
39,16 ± 7,51
|
98,35 ± 1,17
|
a: The percentage of cell growth inhibition was examined at 4µM concentration.
b: The percentage of cell growth inhibition was examined at 8µM concentration.
Taken together, the two peptides MPC and BP52 have similar ratios of hydrophobic-hydrophilic residues. Besides, the length of the MPC sequence is longer than BP52, thus it is expected that MPC would have superior potency due to the higher ability to interact and span the hydrophobic thicknesses of bacteria membranes [14]. However, there was no big difference in the antimicrobial profiles of two AMPs whereas the hemolytic activity was increased. These results can be explained as:
-
First, though BP52 has a shorter sequence and a smaller hydrophobicity, the hydrophobic moment (µH) is significantly higher (see Table 1). Such enhanced amphipathicity can facilitate the interaction with hydrophobic interiors in targeted membranes.
-
Secondly, BP52 also has a higher net charge than MPC, which can improve the selective interaction with negatively charged components in the bacterial membranes. Moreover, MPC has a narrower hydrophilic face while having a broader hydrophobic face, thus having a higher tendency to cause hemolysis than BP52.
-
Thirdly, BP52, as indicated by the primary structure in Fig. 1A, has a hydrophobic segment at the C-terminal with five out of six residues being hydrophobic. Hence, this hydrophobic area may play an important role in the initial step of binding and insertion into the hydrophobic interior of the targeted membrane before disrupting it.
-
Lastly, tryptophan is commonly located within the transmembrane regions of membrane-active proteins, where it serves vital anchoring roles [15–18]. Notably, the appropriate arrangement of tryptophan residue in the interface between hydrophobic and hydrophilic faces is considered to promote interaction with bacteria membranes [19–21]. Therefore, the presence of tryptophan may further improve the membrane action of BP52.
Furthermore, BP52 also showed markedly more potent than MPC on the A427 cancer cell line (see Fig. 3). This data suggested that the enhanced positive charge and hydrophobic moment could be a key factor for the improved potency toward cancer cells.
Overall, tt is demonstrated that both the artificial design peptide BP52 and natural peptide MPC exhibited comparable antimicrobial activity to MPC. Nonetheless, the former displayed remarkably higher anticancer activity against the A427 cell line while exhibiting significantly less hemolysis than the latter one. Remarkably, BP52 accomplished all this with a shorter sequence than MPC. The preliminary observation in this study indicated the major advantage of the rational design of AMPs over natural design - the capability to “learn and apply” the common SARs to achieve shorter, more selective and highly potent membrane-active peptides. Through rational design and advanced molecular techniques, the power of AMPs can be explored, not only as a defense mechanism borrowed from nature but also as a promising avenue for the development of innovative antimicrobial therapies.