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. 2024 Sep 25;15(12):4168–4179. doi: 10.1039/d4md00612g

Synthesis and structure–activity study of the antimicrobial lipopeptide brevibacillin†

Omar Fliss a,b,c,‡, Louis-David Guay a,c,d,‡, Ismail Fliss b,c, Éric Biron a,c,d,✉
PMCID: PMC11450366  PMID: 39371433

Abstract

The antimicrobial lipopeptide brevibacillin is a non-ribosomally synthesized peptide produced by Brevibacillus laterosporus with inhibitory activity against several clinically relevant Gram-positive pathogenic bacteria such as Staphylococcus aureus, Listeria monocytogenes, and Clostridium difficile. In this study, we report the total synthesis of brevibacillin and analogues thereof as well as structure–activity relationship and cytotoxicity studies. Several novel synthetic analogues exhibited high inhibitory activities with minimal inhibitory concentration values in the low micromolar range against several bacteria including Gram-positive L. monocytogenes, S. aureus, Enterococcus faecalis, and Clostridium perfringens as well as Gram-negative Campylobacter coli and Pseudomonas aeruginosa. Of particular interest, four analogues showed a broad spectrum of action and greater antimicrobial activity versus cytotoxicity ratios than native brevibacillin. With a more accessible and efficient production process and improved pharmacological properties, these synthetic analogues are promising candidates to prevent and control the proliferation of various pathogens in the food industry as well as veterinary and human medicine.

The total synthesis of brevibacillin on solid support allowed structural exploration of the peptide scaffold and yielded N-terminal modified analogues with strong antimicrobial activity against several bacterial strains and reduced cytotoxicity.graphic file with name d4md00612g-ga.jpg

Introduction

The discovery of antibiotics was perhaps the most important scientific achievement of the 20th century in terms of human health and socioeconomic equality but their misuse in medicine and farm animals has led to the emergence of multidrug-resistant bacteria.1–5 The spread of antibiotic resistance among pathogenic bacteria has become a serious threat to public health and the development of new antimicrobials is now a global priority.6,7 Faced with this urgent need, antimicrobial peptides (AMPs) are increasingly considered promising alternatives to conventional antibiotics in several sectors.8,9 AMPs are low-molecular weight, ribosomally or non-ribosomally synthesized peptides (NRPs) with antimicrobial activity against a wide range of bacteria, viruses, and fungi.10 Moreover, these molecules can display high potency, good specificity and low toxicity, and act via a wide range of mechanisms of action, which are often different from those exploited by currently used antibiotic classes.11 Because of their attractive antimicrobial and techno-functional properties, AMPs of bacterial origin are appealing alternatives to conventional antibiotics in the food and pharmaceutical industries and their use as food preservatives, biocontrolling agents, and antimicrobials in human and veterinary medicine has been widely studied.12,13

Among AMPs produced by bacteria, the lipotridecapeptides produced by Brevibacillus laterosporus particularly attracted our attention because of their wide spectrum of activity against Gram-positive and Gram-negative pathogens.14,15 These lipopeptides are non-ribosomally synthesized and characterized by the presence of an N-terminal lipid chain and several non-proteinogenic amino acid residues such as d-amino acids and ornithine in their structure. The lipotridecapeptides from B. laterosporus can be divided into three subfamilies described as the bogorols, brevilaterins, and brevibacillins.16–21 For this study, we were particularly interested in brevibacillin 1 (FA-Dhb-Leu-d-Orn-Ile-Ile-Val-d-Lys-Val-Val-Lys-d-Tyr-Leu-valinol), a cationic linear lipopeptide of 13 amino acid residues bearing an N-terminal fatty acid (FA = (2S,3S)-2-hydroxy-3-methylpentanoic acid or l-isoleucic acid) and containing five non-proteinogenic amino acids including three d-amino acids, an α,β-dehydroaminobutyric acid (Dhb) residue, and a C-terminal valinol.22 Good antimicrobial activities with minimal inhibitory concentrations (MIC) ranging from 2 to 64 μg mL−1 against several pathogenic bacteria including resistant strains like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) have been reported.22 However, moderate hemolytic activities and cytotoxicity have been observed.23 The mechanism of action of brevibacillin has been recently studied and shown to primarily target the lipoteichoic acid (LTA) of Gram-positive bacteria to reach and disrupt the cytoplasmic membrane.16,23,24 For Gram-negative bacteria, it has been proposed that the peptide first binds the lipopolysaccharides (LPS) to enter periplasmic space and reach the cytoplasmic membrane to cause membrane disruption and ultimately cell death.25

Despite the great potential of brevibacillin, the problems associated with its production by fermentation and undesired effects on eukaryotic cells limit its applicability and delay regulatory approval. Chemical synthesis is an attractive approach to overcome these limitations and allow further pharmacological optimization. Indeed, the reduction in costs for peptide synthesis reagents and building blocks in recent years has made chemical synthesis of AMPs more attractive than fermentation in some cases.26 Moreover, it allows rapid amino acid substitution, the use of unnatural amino acids, and modifications on the side chains and peptide scaffold to improve the pharmacological profile of a peptide.27,28 To pave the way for its future use in the food, veterinary and medical sectors, we investigated the synthesis and modification of brevibacillin to improve the activity, increase stability, diminish undesired effects on eukaryotic cells, and reduce the production costs. We report here the total synthesis of brevibacillin 1 using a combination of solution- and solid-phase synthesis as well as a structure–activity relationship investigation and cytotoxicity studies. Using a panel of Gram-positive and Gram-negative bacterial strains, we show that brevibacillin 1 and many of the synthesized analogues are potent inhibitors of important pathogenic bacteria such as S. aureus, Listeria monocytogenes, Clostridium perfringens, Campylobacter coli, and Pseudomonas aeruginosa. In addition, we show that key modifications on brevibacillin can significantly reduce undesired effects on eukaryotic cells and generate analogues with a more interesting selectivity index for potential uses as alternatives to antibiotics in the food, veterinary, and biomedical sectors.

Results and discussion

Synthesis of brevibacillin and analogues thereof

The synthesis of brevibacillin 1 can be challenging because of some features in the structure such as the C-terminal valinol, the Dhb amino acid, and the N-terminal isoleucic acid residue. The Dhb residue poses significant challenges in peptide synthesis due to its enamine moiety exhibiting low nucleophilicity. This makes coupling on the amine difficult during peptide elongation.29 To overcome this problem, several strategies have been developed to incorporate dehydroamino acids in peptides and allow further elongation.29,30 While some approaches aim for direct Dhb formation in the peptide by selective dehydration of threonine, others use prior preparation of a short peptide fragment containing the Dhb residue in solution to then couple it to the growing peptide on solid support or a ligation reaction for amide formation on the N-terminal Dhb residue.31,32 For example, Yamashita and coworkers have reported the total synthesis of the structurally similar peptide bogorol A using an elegant final Staudinger ligation to incorporate the isoleucic acid on the Dhb moiety directly on solid support.31 However, this approach required the synthesis of an azide derivatives of the Dhb residue as well as the phosphinophenol ester of the isoleucic acid. For this study, our strategy was to prepare the N-terminal dipeptide fragment of brevibacillin in solution and then use it in solid-phase peptide synthesis.

The synthesis of the N-terminal dipeptide containing isoleucic acid and the Dhb residue started by generating protected isoleucic acid from l-isoleucine (Scheme 1). Briefly, diazotation of l-isoleucine gave isoleucic acid in excellent yield and is well known for retention of the stereochemistry.33 The carboxylic acid and alcohol groups were successively protected in presence of BnBr/K2CO3 followed by reaction with TBSCl/imidazole to give fully protected isoleucic acid 2.31 The benzyl ester was then cleaved by hydrogenolysis to yield the TBS-protected carboxylic acid 3. Coupling of 3 to H-l-Thr-OMe with HATU afforded dipeptide 4. It is noteworthy that significant higher yield was obtained when using HATU instead of other coupling methods such as EDAC·HCl/HOAt. The dipeptide 4 could then be dehydrated by EDAC·HCl/CuCl promoted elimination to yield protected dipeptide 5.29 It is well-known that these reaction conditions form the most thermodynamically stable product which is the 2-Z enantiomer.34 Even if minimal formation of the 2-E enantiomer occurred, the dipeptide 5 was used as is in the next step owing to earlier demonstration by Yamashita and coworkers that the stereochemistry of the Dhb alkene does not impact the activity.31 Finally, hydrolysis of the methyl ester gave the desired protected isoleucic acid-Dhb dipeptide 6 which is compatible with solid-phase peptide synthesis and will be used during the last coupling step for N-terminus capping of the anchored dodecapeptide.

Scheme 1. Synthesis of the isoleucic acid-Dhb dipeptide 6.

Scheme 1

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The next step was to assemble the peptide on solid support but the presence of the C-terminal valinol required the use of a linker compatible with an alcohol anchoring. The first valinol anchoring test was performed on 2-chlorotrityl chloride resin but sluggish results and low loadings were obtained.35 From our experimentation, the best results for N-Fmoc-l-valinol anchoring were obtained with the trichloroacetimidate Wang resin with a loading of 0.49 mmol g−1 (see ESI†).36,37 After anchoring of the valinol residue, the peptide was assembled by standard solid-phase peptide synthesis using the Fmoc/tBu strategy up to the l-leucine residue in position 2 (Scheme 2). The final coupling of the dipeptide 6 appeared to be particularly challenging by conventional approaches. Several coupling reagents were tested to couple the dipeptide 6 at room temperature, but the reaction never reached completion. It has been previously described in the literature that the carboxyl group of dehydroamino acid can be difficult to activate.38,39 Significant improvements were obtained when the coupling of the dipeptide 6 was performed at 40 °C with DIC/HOAt in DMF. These conditions allowed completion of the reaction after 16 h, as shown by negative chloranil test. After cleavage from the resin and side chain deprotection with a TFA cocktail, the peptide was purified by HPLC and characterized by MS (Fig. S4†) to afford brevibacillin 1 in 14% yield.

Scheme 2. Total synthesis of brevibacillin 1. o = d-ornithine, k = d-lysine, y = d-tyrosine.

Scheme 2

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For the structure–activity study, a series of structural analogues were generated by modifying key parts of the peptide to evaluate the impact on the activity (Table 1). A first group of three analogues, originally produced by fermentation with B. laterosporus DSM-25 and isolated by Zhao et al.,23 were synthesized according to Scheme 2 and include the substitution of one or two hydrophobic residues in position 4, 5 or 8 such as I4V 7 (brevibacillin Val4), I4,5V 8 (brevibacillin Val4,5), and V8I 9 (brevibacillin Ile8). We next aim to study the impact of C-terminus modification by replacing the terminal alcohol by a carboxylic acid or an amide. Analogues with C-terminal carboxylic acid 10 and amide 11 were assembled on a Fmoc-Val preloaded 2-chlorotrityl resin and rink-amide resin, respectively. To increase synthetic accessibility and assess the importance of the Dhb residue for activity, a third group of analogues containing substitutions at position 1 was prepared. These analogues were synthesized according to Scheme 2 but instead of the dipeptide 6 coupling, different amino acids were incorporated by standard coupling followed by the TBS-protected isoleucic acid 3 to complete the peptide. Amino acids used to substitute the Dhb residue in these analogues include Thr (12), Ser (13), Ala (14), l-2-aminobutyric acid (Abu) (15), and Val (16). Finally, with different insight on the peptide structure and activity, a fourth group of more complex analogues was generated by combining two or more modifications. The first compound in this group, analogue 17, contain a Val residue in positions 4 and 5 as well as an Abu residue in position 1 instead of the Dhb. For analogue 18, the Dhb residue was replaced by an Abu and the C-terminal valinol by a valinamide. To evaluate the importance of the N-terminal section, the isoleucic acid and Dhb were replaced by N-acetyl-isoleucine and Abu, respectively, to yield analogue 19. For the last analogue of this group, both N- and C-termini were modified to give compound 20 containing the N-terminal N-Ac-Ile-Abu dipeptide and a C-terminal valinamide. Peptides 7–20 were purified by semi-preparative RP-HPLC and obtained in yields ranging from 6 to 28%.

Structure of brevibacillin 1 and synthesized structural analogues 7–20.

graphic file with name d4md00612g-u1.jpg
# Peptide R1 R2 R3 R4 R5 R6
1 Brevibacillin (Brevi) graphic file with name d4md00612g-u2.jpg graphic file with name d4md00612g-u3.jpg graphic file with name d4md00612g-u4.jpg graphic file with name d4md00612g-u5.jpg graphic file with name d4md00612g-u6.jpg graphic file with name d4md00612g-u7.jpg
7 Brevi Val4 –a – – graphic file with name d4md00612g-u8.jpg – –
8 Brevi Val4,5 – – graphic file with name d4md00612g-u9.jpg graphic file with name d4md00612g-u10.jpg – –
9 Brevi Ile8 – – – – graphic file with name d4md00612g-u11.jpg –
10 Brevi COOH – – – – – graphic file with name d4md00612g-u12.jpg
11 Brevi CONH2 – – – – – graphic file with name d4md00612g-u13.jpg
12 Brevi Thr1 – graphic file with name d4md00612g-u14.jpg – – – –
13 Brevi Ser1 – graphic file with name d4md00612g-u15.jpg – – – –
14 Brevi Ala1 – graphic file with name d4md00612g-u16.jpg – – – –
15 Brevi Abu1 – graphic file with name d4md00612g-u17.jpg – – – –
16 Brevi Val1 – graphic file with name d4md00612g-u18.jpg – – – –
17 Brevi Abu1/Val4,5 – graphic file with name d4md00612g-u19.jpg graphic file with name d4md00612g-u20.jpg graphic file with name d4md00612g-u21.jpg – –
18 Brevi Abu1/CONH2 – graphic file with name d4md00612g-u22.jpg – – – graphic file with name d4md00612g-u23.jpg
19 Brevi N-Ac-Ile/Abu1 graphic file with name d4md00612g-u24.jpg graphic file with name d4md00612g-u25.jpg – – – –
20 Brevi N-Ac-Ile/Abu1/CONH2 graphic file with name d4md00612g-u26.jpg graphic file with name d4md00612g-u27.jpg – – – graphic file with name d4md00612g-u28.jpg
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a

No modification versus brevibacillin 1.

Antimicrobial activity

The antimicrobial activity of the synthesized peptides was first assessed using radial diffusion assays with Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 25853 (Fig. 1). While synthetic brevibacillin 1 showed important inhibition for both strains, no activity was observed with analogues 10 and 20, which contain a C-terminal carboxylic acid and a combination of N- and C-terminal modifications, respectively. Analogues 7–9 from the first group and 12–14 from the third group showed inhibition diameters like brevibacillin 1 ranging from 11 to 12 mm for S. aureus and 12 to 14 mm for P. aeruginosa. A small decrease in activity was observed with peptides 11, 15 and 17 but analogues 16, 18, and 19 showed a much greater reduction in activity with inhibition diameters ranging from 7 to 10 mm for both strains. These results were very promising and implied that brevibacillin scaffold is flexible and allow modifications towards our aims as some substitution yielded equipotent analogues while others decreased or abolished the activity.

Fig. 1. Agar diffusion assay of brevibacillin 1 and its analogues 7–20 against (A): Staphylococcus aureus ATCC 6538 in soft agar TSB (tryptic soy broth) medium and (B): Pseudomonas aeruginosa ATCC 25853 in soft agar LB (Luria–Bertani) medium with 80 μL of solution containing 128 μg mL−1. A solution with 10% of DMSO was used as negative control (T−).

Fig. 1

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The antimicrobial activity of the synthesized peptides was then evaluated in terms of minimal inhibitory concentrations (MIC) against a panel of 5 Gram-positive and 4 Gram-negative bacterial strains (Table 2). Synthetic brevibacillin 1 and analogues 7–9 from the first group showed strong inhibitory activity against Gram-positive bacteria and Gram-negative P. aeruginosa and Campylobacter coli with MIC values ranging from 1 to 8 μg mL−1. Even though the inhibitory activities of peptides 7–9 are very similar, there appears to be a decrease in activity when the hydrophobic Ile amino acids are replaced by a less hydrophobic Val residue with analogue 8 showing the highest MIC and analogue 9 the lowest. While peptides 1 and 9 showed the largest spectrum of activity, most analogues exhibited low or no activity against Escherichia coli and Salmonella enterica. As previously observed in the radial diffusion assay, substitution of the C-terminal valinol by Val in peptide 10 led to a loss of activity on tested strains except against C. coli with a MIC of 32 μg mL−1. On the other hand, an average fourfold increase in MIC values is observed against Gram-positive bacteria and P. aeruginosa with the analogue 11 containing a C-terminal amide. Surprisingly, this peptide did not exhibit activity against C. coli. Substitution of the Dhb residue in position 1 by other amino acids such as Thr, Ser, Ala, Abu or Val, led to analogues with equivalent and/or increased activity against most selected strains, except for S. enterica and E. coli (Table 2). Among analogues of this third group, compound 15 with Abu1 showed the best profile with MIC values ranging from 0.5 to 2 μg mL−1 against Gram-positive bacteria and 1 to 4 μg mL−1 against P. aeruginosa and C. coli, an average 2-fold increase in activity compared to brevibacillin 1. Surprisingly, while equivalent inhibition was observed for analogues 12–14 and 16 against strains sensitive to brevibacillin 1, the peptide 13 with Ser1 showed a fourfold increase in MIC value against C. coli and no inhibitory activity against E. faecalis. Overall, most analogues from this third group displayed a spectrum of activity like brevibacillin 1. These results complement the study reported by Yamashita et al. on bogorol A where it is shown that the stereochemistry of the alkene has no impact on the activity of the lipopeptide.31 Analogues 17–20 from the fourth group contain a combination of modifications and substitutions. While peptides 17 and 18 contain combinations of modifications from analogues 8, 11, and 15, the importance of the fatty acid isoleucic acid was explored in peptides 19 and 20 by its substitution with neutral N-acetyl-Ile. A complete loss of activity was observed for compound 20 containing the N-terminal N-Ac-Ile-Abu and C-terminal amide but other peptides from this group showed good inhibitory activity against selected Gram-positive bacteria and P. aeruginosa and moderate activity against C. coli. These results showed that some modifications can be beneficial or detrimental against specific strains and affect the spectrum of action. Results for the fourth group demonstrated that a combination of beneficial modifications does not necessarily lead to analogues with improved activity as illustrated by peptide 20. Overall, E. coli and S. enterica did not show significant sensitivity to brevibacillin 1 and its analogues 7–20, while E. faecalis was the least sensitive of the tested Gram-positive bacteria.

Minimal inhibitory concentrations of synthetic brevibacillin 1 and its analogues 7–20 against selected bacteria.

# Peptide Minimal inhibitory concentration (μg mL−1)
Gram-positive Gram-negative
S.a a L.m a B.s a E.f a C.p b P.a c S.e c E.c c C.c d
1 Brevibacillin (Brevi) 2 2 1 4 4 1 64 32 8
7 Brevi Val4 4 1 2 16 4 2 >128 32 16
8 Brevi Val4,5 4 4 8 32 16 4 >128 64 32
9 Brevi Ile8 2 1 0.5 2 2 1 64 32 8
10 Brevi COOH >128 128 >128 >128 >128 >128 >128 >128 32
11 Brevi CONH2 8 2 4 16 16 4 >128 >128 >128
12 Brevi Thr1 2 4 1 16 4 2 >128 32 16
13 Brevi Ser1 8 1 2 >128 8 4 >128 32 32
14 Brevi Ala1 2 1 2 4 2 1 >128 64 8
15 Brevi Abu1 1 0.5 1 2 2 1 >128 >128 4
16 Brevi Val1 1 1 1 4 4 1 >128 >128 8
17 Brevi Abu1/Val4,5 2 4 4 >128 8 2 >128 64 16
18 Brevi Abu1/CONH2 2 2 2 64 8 2 >128 >128 16
19 Brevi N-Ac-Ile/Abu1 2 2 4 32 2 2 >128 >128 16
20 Brevi N-Ac-Ile/Abu1/CONH2 >128 >128 >128 >128 >128 >128 >128 >128 >128
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a

Incubated in TSB (tryptic soy broth) for 20 h at 37 °C.

b

Incubated in RCM (reinforced clostridial medium) for 20 h at 37 °C.

c

Incubated in LB (Luria–Bertani) for 20 h at 37 °C.

d

Incubated in Brucella for 48 h at 37 °C.

Next, the type of inhibition of the synthesized peptides was assessed by evaluation of the minimal bactericidal concentration (MBC) (Table S2†) and determination of the MBC/MIC ratio (R) (Table S3†). The results are interpreted as follows: if R ≤ 4, the activity of the compound is considered bactericidal and if R > 4, the activity is considered bacteriostatic. Surprisingly, most synthesized analogues able to inhibit bacterial growth showed bactericidal activity with MBC/MIC ratios ranging from 1 to 4. Only analogues 8 and 15 exhibited a bacteriostatic activity with an MBC/MIC ratio of 8 against S. aureus and C. coli, respectively. Overall, these results suggest that brevibacillin and the synthesized analogues are bactericidal. In addition, the absence of bacterial growth over 48 h at the MBC demonstrates the persistence of the bactericidal effect of brevibacillin 1 and active synthetic analogues.

Effect on eukaryotic cells

It was recently reported that native brevibacillin exert considerable hemolytic activity on red blood cells and cytotoxicity on liver HepG2 cells.23 However, the same study showed that substitution of Ile at positions 4 and 5 by Val significantly reduces these undesired activities on eukaryotic cells. Based on these promising results, we decided to evaluate the impact of the modifications carried out on our analogues on the hemolytic activity and Caco-2 cells viability. First, the hemolytic activity of synthetic brevibacillin 1 and its analogues 7–20 was assessed by exposing human erythrocytes to increasing concentrations of peptides (up to 256 μg mL−1) for 1 h (Fig. S5†). In our hands, brevibacillin 1 displayed 9.6% hemolysis at 128 μg mL−1 and 25.2% at 256 μg mL−1, which is 100- to 200-fold the observed MIC values for most tested strains (Fig. 2A). Substitution of Ile4 and Ile5 by Val residues in analogues 7 and 8 decreased the hemolytic activity at 128 μg mL−1 to 1.65% and 0.65%, respectively. No increase in hemolytic activity was observed for analogue 8 at 256 μg mL−1 suggesting that it is 25-fold less hemolytic than brevibacillin 1 at this concentration. On the other hand, substitution of Val8 by Ile in analogue 9 led to significant increase in hemolytic activity with 55% and 89.9% hemolysis at 128 and 256 μg mL−1, respectively. These results support the observations reported by Zhao et al. for the analogue brevibacillin Val4,5 8 and confirmed the importance of Ile and Val residues at position 4, 5, and 8 in the hemolytic activity.23 For the second group, no hemolytic activity was observed for peptide 10 at the tested concentrations but is not relevant considering the lack of antibacterial activity. In contrast, the brevibacillin amide analogue 11 showed hemolytic activity like brevibacillin 1 with 11.8% and 30.1% hemolysis at 128 μg ml−1 and 256 μg mL−1, respectively. In the third group, substitution of the Dhb1 residue with different amino acids affected hemolytic activity in opposite directions. Analogues 12 and 13 containing respectively Thr1 and Ser1 displayed very low hemolytic activity with hemolysis percentage ranging from 1.2% to 2.5% at 128 μg mL−1 and 4.9% to 6.8% at 256 μg mL−1. While analogue 14 showed a hemolysis profile like brevibacillin 1, substitution of the Dhb1 by Abu or Val in analogues 15 and 16 led to a significant increase in hemolytic activity with 15.5% and 53.1% hemolysis at 128 μg mL−1 and 58.7% and 84% at 256 μg mL−1, respectively. The results obtained for analogues 12 and 13 are very interesting and showed that the substitution of the Dhb1 residue can not only maintain antimicrobial activity but also decrease hemolytic activity. Finally, the combination of different modifications in the last group led to an increase in hemolytic activity for most analogues except for peptide 17. In this case, low hemolytic activity was observed with 1.2% and 4.2% hemolysis at 128 μg mL−1 and 256 μg mL−1, respectively. On the other hand, analogues 18–20 caused important red blood cells damage with hemolysis ranging from 23.3% to 53.6% at 128 μg mL−1 and 38.2% to 86.2% at 256 μg mL−1. Overall, analogues 8, 12, 13 and 17 showed the best hemolytic activity profiles and caused very low hemolysis at concentrations more than 100- to 200-fold their MIC values against most tested strains.

Fig. 2. Effects of increasing concentrations of brevibacillin 1 and analogues 7–20 on eukaryotic cells. A) Hemolytic activity on human erythrocytes, positive control: 1% Tween in PBS, negative control: PBS; B) viability of Caco-2 cells, positive control: 125 μM cisplatin, negative control: 0.1% DMSO in complete medium. * mean ± standard error of the mean (SEM).

Fig. 2

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To assess the effect of the synthesized brevibacillin and analogues thereof, human colorectal epithelial Caco-2 cells were exposed to increasing concentration of peptides (Fig. S6†). The Caco-2 cell line was selected for this assay because it exhibits properties similar to the small intestine epithelium and has been widely used as model to evaluate absorption and potential toxicity of orally administered substances.40–42 The IC50 value was determined from the percentage of cell viability compared to the control. Except for the inactive peptide 10, brevibacillin 1 and all tested analogues showed moderate to high cytotoxicity (Fig. 2B). In our hands, brevibacillin 1 exhibited an IC50 of 10.9 μg mL−1 and all the Caco-2 cells were killed at a concentration of 32 μg mL−1. As observed with hemolysis assays, substitution of Ile at positions 4 and 5 with Val led to decreased cytotoxicity with IC50 of 25.7 μg mL−1 and 87.5 μg mL−1 for analogues 7 and 8, respectively. Similarly, substitution of Val8 by Ile in peptide 9 increased cytotoxicity with an IC50 at 6.5 μg mL−1. The same tendency was observed for analogues of the other groups where peptides 12, 13 and 17 displayed good cytotoxicity profiles with IC50 of 32.2, 42.2, and 32.4 μg mL−1, respectively. On the other hand, IC50 ranging from 8.3 to 17.6 μg mL−1 was observed for analogues 11, 14–16, 18–20. These results showed that simple modifications can significantly improve the cytotoxicity profile of an antimicrobial peptide. Here again, analogues 8, 12, 13, and 17 showed the most attractive profiles and caused low cell death at concentrations significantly higher than their MIC values.

Structure–activity relationships

Combination of the results from antimicrobial activity assays and evaluation of the effect on eukaryotic cells helps to identify some promising analogues. Apart from peptides 10 and 20, the synthesized analogues showed either the same activity as brevibacillin 1 or a two- to fourfold increase or decrease against most tested strains. In the first group, it was observed that substitution of Ile residues by Val slightly decreased the antimicrobial activity but even more significantly reduced the hemolytic activity and cytotoxicity. These results are consistent with the study reported by Zhao et al.23 In the second group, it was observed that the neutrality of the C-terminal is important for activity since the introduction of a carboxylic acid yielded inactive peptide 10. It has been previously suggested that this type of lipotridecapeptide could bind to negatively charged components of the bacterial membrane such as the lipoteichoic acid (LTA) of Gram-positive bacteria and the lipopolysaccharide acid (LPS) of Gram-negative bacteria.16,43 The overall cationic charge of the peptide analogues could then be an important property for binding to negatively charged bacterial components and antimicrobial activity.44,45 Accordingly, replacement of the C-terminal methyl alcohol of valinol by an amide group in analogue 11 led to a small loss of antimicrobial activity and no change in the effect on eukaryotic cells. It is also possible that the loss of activity observed for analogue 10 is due to introduction of a negative charge at the C-terminus that induces a detrimental structural change via an intramolecular ionic interaction with a positively charged side chain.

For the third group, substitution of the Dhb1 residue by different amino acids generated analogues 12–16 displaying antimicrobial activities like brevibacillin 1 but significantly reduced hemolytic activity and cytotoxicity for peptides 12 and 13 containing Thr1 and Ser1, respectively. The modifications made in this group do not appear to affect the structure of the brevibacillin scaffold as the analogues have retained high activity. These results showed that the Dhb1 residue can be replaced without losing activity and to improve the safety profile. The most important advantage of these analogues is their synthetic accessibility and improved yields of production. As no dipeptide fragment preparation and coupling are needed, the synthesis requires fewer steps and can be carried out in a straightforward manner. Finally, for the last series of peptide analogues, we strived to maintain the antibacterial potential, capitalize on synthetic accessibility, and minimize effect on eukaryotic cells by introducing multiple beneficial modifications observed in previous groups. Unfortunately, combination of different modifications in analogues 17–20 did not lead to significant improvement in antimicrobial activity and cytotoxicity. The loss of activity observed for peptide 20 could be explained by its low solubility and an adverse structural change due to the N-terminal N-acetyl isoleucine residue. In this group, the analogue 17 containing Abu1, Val4, and Val5 showed the best antimicrobial activity versus cytotoxicity ratio with MIC values ranging from 2 to 16 μg mL−1, <5% hemolysis at 256 μg mL−1, and IC50 of 32.4 μg mL−1 on Caco-2 cells.

Overall, the structural investigation done in this study highlights important features of brevibacillin and its tolerability to different modifications. While the study showed that N- and C-terminal residues can be modified to facilitate the synthesis, the antimicrobial activity and effect on eukaryotic cells were significantly affected by the level of hydrophobicity and electronic parameters. In that sense, hydrophobicity has been shown to be a key parameter in cationic antimicrobial lipopeptide that can be linked to cytotoxicity and hemolytic activity.46–48 Among the synthesized analogues, peptide 15 with Abu1 displayed the strongest activity against tested strains but also exhibited important hemolytic activity at 128 and 256 μg mL−1 and cytotoxicity with an IC50 of 10.0 μg mL−1. On the other hand, analogues 8, 12, 13 and 17 showed the lowest impact on eukaryotic cells with <7% hemolysis at 256 μg mL−1 concentration and IC50 ranging from 32.2 to 87.5 μg mL−1 on Caco-2 cells. In addition, no or only small changes (two-fold increase or decrease) in the activity of these peptides were observed against most tested bacteria compared to brevibacillin 1. Only E. faecalis showed less sensitivity to analogues 8, 12, 13 and 17 with fourfold increase in their MIC or loss of activity. Overall, these peptides showed the most promising antimicrobial activity/cytotoxicity ratios but considering synthetic accessibility and production costs, analogues 12 and 13 would be the most interesting for further development and optimization.

Conclusion

Cationic antimicrobial lipotridecapeptides like bogorol and brevibacillin have been discovered in the early 2000's. Since then, many studies have shown their potency against multi-resistant bacteria such as MRSA or VRE. However, their use in different applications has been limited by problems associated with their production and their significant effect on eukaryotic cells and potential toxicity. Through our work, the chemical synthesis of brevibacillin was successfully achieved by the prior preparation and coupling of a dipeptide fragment to incorporate the Dhb moiety. This approach allowed us to produce several brevibacillin analogues and perform a structure–activity relationship study on this lipotridecapeptide scaffold. Results from the structural investigation showed that the special features such as the C-terminal valinol, N-terminal isoleucic acid and the Dhb residue are not essential for activity and that equipotent analogues can be synthesized with suitable substitutions and modifications. It was also found that brevibacillin and most promising analogues were bactericidal. Besides strong antimicrobial activity against the tested Gram-positive pathogenic bacteria, we observed that brevibacillin and several of the synthesized analogues were also able to inhibit Gram-negative bacteria such as P. aeruginosa and C. coli. These results are very promising for uses in the food industry since three problematic foodborne pathogens, L. monocytogenes, C. perfringens, and C. coli, can be inhibited with the same peptide. The next step will be to evaluate the efficacy of the most promising analogues on different multidrug-resistant bacteria and determine the presence of cross- and co-resistance with commonly used antibiotics. Alongside, evaluation of the analogues for their effect on eukaryotic cells showed that some modifications on the brevibacillin scaffold can significantly reduce hemolytic activity and cytotoxicity without affecting antimicrobial activity. With their enhanced synthetic accessibility and improved selectivity index, these analogues are promising candidates for further optimization and development of new antimicrobials for the prevention and treatment of infections in the food and animal production industry as well as human and veterinary medicine.

Materials and methods

Materials

All the reagents and solvents were purchased from commercial suppliers and used without further purification, unless otherwise indicated. Coupling reagents and Fmoc-protected amino acids were purchased from Matrix Innovation (Quebec, QC, Canada). Wang resin was purchased from Chem-Impex (Wood Dale, IL). HPLC analyses were conducted on a Shimadzu Prominence system equipped with a Phenomenex Kinetex EVO C18 column (100 mm × 4.6 mm, 100 Å, 2.6 μm) using 0.1% TFA/H2O (A) and 0.1% TFA/CH3CN (B), with a linear gradient from 10 to 100% (B) for 10.5 min at a rate of 1.4 mL min−1 and ultraviolet detection at 220 and 254 nm. High resolution mass spectra (HRMS) were measured with an Agilent 6210 LC Time of Flight mass spectrometer in electrospray mode with Agilent HP-0921 as internal standard. Either protonated molecular ions [M + nH]n+, sodium adducts [M + Na]+ or ammonium adducts [M + NH4]+ were used for empirical formula confirmation.

Peptide synthesis

Peptides were prepared by standard solid-phase peptide synthesis (SPPS) on a Prelude peptide synthesizer from Gyros Protein Technologies (Tucson, AZ) using the Fmoc/tBu strategy on preloaded N-Fmoc-valinol-Wang resin (0.49 mmol g−1). Briefly, the Fmoc protecting group was removed from the resin by treatments with 20% piperidine in DMF (v/v) (2 × 7 min) and amino acid couplings were performed with Fmoc-Xaa-OH (3 equiv.), HATU (3 equiv.) and NMM (6 equiv.) in DMF (2 × 20 min). The resin was washed with DMF (5 × 30 s) between every deprotection and coupling steps. For peptides containing a Dhb residue, the resin was transferred in a glass vial equipped with a pressure relief septum. In a separate flask, dipeptide 6 (5 equiv.) and HOAt (5 equiv.) were dissolved in DMF followed by addition of DIC (5 equiv.). The solution was then added to the resin and the mixture was left without stirring for 16 hours at 40 °C. After completion, the resin was filtered and washed with DMF (5 × 30 s) and CH2Cl2 (5 × 30 s). Cleavage and side chain deprotection of peptides were achieved by treating the resin with a solution of TFA/TIS/H2O (95 : 2.5 : 2.5) for 90 min. The resin was filtered, and the filtrate evaporated to approximately 2 mL before precipitating the peptide with cold Et2O. The solid was washed twice with Et2O and dried under vacuum. The peptides were purified by semi-preparative RP-HPLC with a Shimadzu Prominence system on a Phenomenex Kinetex EVO C18 column (250 mm × 21.2 mm, 300 Å, 5 μm) using 0.1% TFA/H2O (A) and 0.1% TFA/CH3CN (B), with a linear gradient from 10 to 60% (B) for 20 min at a rate of 12 mL min−1 and UV detection at 220 and 254 nm. The collected fractions containing the compound with ≥95% purity were lyophilized to afford the desired peptide as a white powder. Peptide purity was determined by HPLC and composition confirmed by HRMS.

Antimicrobial activity assays

The antimicrobial activity of the synthesized peptides was evaluated qualitatively and quantitatively by agar diffusion and microdilution assays, respectively. ATCC bacteria strains were obtained from the American Type Culture Collection (Rockville, MD, USA). The strains were grown overnight in the appropriate medium and incubation temperature before being inoculated at 1% in soft agar medium (0.75 % w/v). Wells were formed and 80 μL of purified product at a concentration of 128 μg mL−1 was placed in each well. The antimicrobial activity of each peptide was characterized by the presence of an inhibition zone formed around the indicator strain after incubation under strain specific condition. The tested peptides were weighed and dissolved in MilliQ water with 10% of DMSO. The pictures of each plate were taken using ChemiDoc XRS (Bio-Rad, Hercules, CA, USA).

The minimum inhibitory concentration (MIC) of each peptide was determined by the microdilution assay. Briefly, serial dilutions were made from stock solutions at a concentration of 2 mg mL−1. The solutions to be tested were prepared from the stock solution using MilliQ water with 10% of DMSO as diluent to achieve a concentration equal to 512 μg mL−1. The MIC determination was performed in 96-well microplates (Corning Inc., Corning, NY, USA). Microplates were filled with twofold serial dilution of each peptide in the appropriate medium were seeded with targeted strain diluted in the same culture medium to attend approximately 1 × 104 cfu per well. Plates were then incubated under the conditions required for each strain, and absorbance measured at 595 nm using an Infinite® F200 PRO spectrophotometer (Tecan US Inc., Durham, NC, USA). The MIC was determined to be the lowest concentration that results in complete inhibition of visible growth of the indicator bacterial species after incubation under favorable conditions. The minimal bactericidal concentration (MBC) was determined by inoculating an agar surface with 10 μL of solutions from wells showing a complete growth inhibition and followed by incubation for 48 h under the conditions required for each strain. The MBC is defined as the lowest concentration that completely inhibits the growth of the indicator strain on the agar medium. The MIC and MBC experiments were conducted in triplicate.

Hemolysis assay

The hemolytic activity of the peptides was measured as hemoglobin release by lysis of human red blood cells. Briefly, uninfected red blood cells (RBC's) were collected from a healthy human volunteer donor and washed three times with phosphate-buffered saline pH = 7.4 (PBS) (Gibco™, Thermo Fisher, MA, USA) to finally be resuspended in PBS at 2% (v/v). After transferring 100 μL of PBS to wells 2 to 11 of 96-well polypropylene clear round bottom microplates (Corning Inc., Corning, NY, USA), 100 μL of the peptide solutions at 1024 μg mL−1 in PBS was added to forth wells of the plates and serial dilutions were performed by transferring 100 μL from one well to the next up to the twelfth well. Then, 100 μL of the washed erythrocyte suspension was deposited in the wells to obtain a total volume of 200 μL in each well containing a maximum of 1% DMSO at 1% hematocrit and final peptide concentrations between 256 μg mL−1 and 2 μg mL−1. PBS and Triton X-100 solution (1%) and were used in wells 2 and 3 as negative and positive controls, respectively. The microplates were then incubated at 37 °C for 1 h and centrifuged at 1000 × g for 5 min. After incubation, 100 μL of the supernatants were transferred into polystyrene clear flat bottom 96-well microplates (Corning Inc., Corning, NY, USA). Hemoglobin leakage from erythrocytes was quantified by measuring its absorbance at 405 nm with an Infinite® M200 PRO plate reader (Tecan US Inc., Durham, NC, USA). The experiments were performed in triplicate. Percent hemolysis was defined as follows: [(sample absorbance − phosphate buffer absorbance)/(Triton X-100 absorbance − phosphate buffer absorbance)] × 100.

Cytotoxicity on Caco-2 cells

Caco-2 (HTB-37) cells were obtained from ATCC. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 10 mM non-essential amino acids, and 100 mM sodium pyruvate. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C and the medium was changed every two or three days. Cells were seeded at a density of 25 000 cells per well in a 96-well plate. After overnight incubation to allow cell adhesion, the culture medium was replaced with complete medium containing different concentrations (2 to 256 μg mL−1) of the peptides. Cisplatin was used as a positive control at 125 μM and complete medium containing 0.1% DMSO as a negative control. After incubation for 48 h (37 °C, 5% CO2), cell viability was measured using the Cell Titer GLO kit (Promega G7571) according to the manufacturer's instructions. This kit measures the presence of ATP as an indicator of the metabolic activity of living and active cells. Intracellular ATP is released into the supernatant after cell lysis and this ATP is then measured using a reaction with luciferase. A luminescent signal, proportional to the quantity of ATP, is then measured using a plate reader (Spark20, Tecan) and obtained as relative luminescence units (RLU). Results were expressed as the percent viability compared to non-treated cells according to the following formula: % viability = [(RLU treated with peptide − RLU background)/(RLU untreated − RLU background)] × 100.

Statistical analysis

Dose–response curves and bar charts for hemolytic activity and cells viability were fitted using GraphPad Prism 10.2.3 (San Diego, CA, USA). The results are expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments.

Ethics approval

The study was approved by the Canadian Blood Services (CBS) research ethics board, project number 2020.010 and by the CHU de Québec IRB, project number 2015-2230, B14-12-2230, SIRUL 104595. Written consent was obtained by the CBS for all study participants. Participants were informed about the study before providing consent. All experiments were performed in accordance with relevant guidelines and regulations.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

OF and LDG have contributed equally. LDG, OF, IF, and EB designed experiments, LDG and OF performed experiments and wrote the first draft of the manuscript, IF and EB provided guidance and direction and reviewed and edited the manuscript. All authors have read and agreed to the submitted version of the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Supplementary Material

MD-015-D4MD00612G-s001

Acknowledgments

This work was supported by the Fonds de recherche du Québec-Nature et Technologie (FRQNT) (2022-PR-191869) and Natural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN-2020-07217). LDG thanks the FRQNT, the Fondation du CHU de Québec, and the Fonds d'enseignement et de recherche de la Faculté de pharmacie de l'Université Laval for research scholarships. The authors are grateful to Dr. Dominic Gagnon and Prof. Dave Richard of CHU de Québec Research Center for blood samples and assistance for hemolytic activity assays. We also thank the Institute of Nutrition and Functional Foods (INAF) for financial support for the cytotoxicity assays and Dr. Yvan Boutin of TransBioTech for their implementation.

†

Electronic supplementary information (ESI) available: Experimental details for compound 6 synthesis. Experimental data of the NMR and HRMS analysis of building blocks 2–6 and HPLC and MS analysis of peptides 1, 7–20. Table of the MBC values and dose–response curves of erythrocytes lysis and Caco-2 cells survival exposed to peptides 1, 7–20. See DOI: https://doi.org/10.1039/d4md00612g

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MD-015-D4MD00612G-s001
MD-015-D4MD00612G-s001.pdf (4.6MB, pdf)

Data Availability Statement

The data supporting this article have been included as part of the ESI.†

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