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. 2023 Jun 26;66(13):8498–8509. doi: 10.1021/acs.jmedchem.3c00140

Stapled β-Hairpin Antimicrobial Peptides with Improved Stability and Activity against Drug-Resistant Gram-Negative Bacteria

Vanitha Selvarajan †, Nhan D T Tram †, Jian Xu †, Sarah T Y Ngen †, Jun-Jie Koh †, Jeanette W P Teo ‡, Tsz-Ying Yuen §, Pui Lai Rachel Ee †,*
PMCID: PMC10350921  PMID: 37357499

Abstract

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Different stapling techniques have been used recently to address the subpar performance of antimicrobial peptides (AMPs) in clinical trials with ample focus on α-helical AMPs. In comparison, a systematic evaluation of such strategies on β-hairpin AMPs is lacking. Herein, we report the design, synthesis, and evaluation of a library of all-hydrocarbon-stapled β-hairpin AMPs with variation in key parameters intended as potent therapeutics against drug-resistant pathogens. We observed an interesting interplay between the activity, stability, and structural strength. Single-stapled peptides with a 6-carbon staple at peptide termini such as 5(c6) displayed the most potent activity against colistin-resistant clinical isolates. Using imaging techniques, we observed translocation of 5(c6) across bacterial membranes without causing extensive damage. Overall, we have engineered novel all-hydrocarbon-stapled β-hairpin AMPs with structural and functional proficiency that can effectively combat resistant pathogens, with findings from this study a point of reference for future interests in developing novel β-hairpin AMPs.

Introduction

There is an urgent need to identify alternate sources of antimicrobials to treat infections by multidrug-resistant (MDR) pathogens.1−5 Antimicrobial peptides (AMPs) are evolutionarily conserved short peptides that can be isolated from diverse species and exhibit a broad spectrum of antimicrobial activity against bacteria, viruses, and fungi.6,7 Although multiple de novo-designed synthetic AMPs with enhanced potency have been reported in the last decade,8−13 they have performed poorly in clinical trials largely due to their proteolytic instability and toxic side effects.14,15 There are ongoing attempts to improve clinical translation by addressing these limitations of AMPs.16

AMPs mostly adopt random-coiled structure in solution, which renders them highly susceptible to degradation by proteolytic enzymes in the human body before reaching the infection site to exert an antimicrobial effect.17 Various peptidomimetic strategies such as end-to-end cyclization18−21 using hydrogen-bond surrogates22 or lactam bridges23,24 and substitution of d-amino acids25,26 or N-methylated amino acids27,28 have been reported to improve the proteolytic instability of peptides by altering their conformations in solution. One such well-characterized chemical restraining strategy is the all-hydrocarbon stapling technique. It involves a ruthenium-catalyzed olefin metathesis reaction between two alkene side chains of systematically inserted amino acids in a peptide sequence.29,30 Hydrocarbon stapling serves to actively conserve the peptide secondary structure and minimize its access to proteolytic enzymes.31

Several hydrocarbon-stapled α-helical peptides have been reported as inhibitors or ligands for protein interaction studies in anticancer therapeutics.32,33 For antimicrobial applications, hydrocarbon stapling has been applied to modify both natural (e.g., esculentin-2EM,34 polybia-MP1,35 magainin II36) and synthetic α-helical AMPs.37−41 In contrast, a systematic evaluation of the effect of this strategy on novel bioactive β-hairpin AMPs has not yet been reported to our knowledge. Our lab previously developed a series of synthetic β-hairpin AMPs with selective antimicrobial activity against Gram-negative pathogens.42 In this work, we selected a potent peptide BTT3 (LKLKLKpGKLKLKL-NH2) from our earlier library for systematic installations of hydrocarbon stapling to study the impact of this modification on the antimicrobial activity and structural and functional stability of the peptide. Herein, we designed a library of BTT3 analogues bearing all-hydrocarbon staples with variation in the position, number, and chain length of the staples. These staple parameters were observed to impact the functional proficiency of the AMPs, with the 5(c6) bearing a single 6-carbon staple at the terminus identified to be the most potent candidate. Structure–activity analysis revealed that a degree of conformational flexibility is essential to facilitate membranolytic interactions, with double-stapled peptides displaying a generally poorer antimicrobial profile.

Results and Discussion

Design and Synthesis of Stapled β-Hairpin Peptide Library

A series of hydrocarbon-stapled β-hairpin peptides were synthesized using conventional Fmoc-solid-phase peptide synthesis (SPPS),43 followed by a temperature-assisted ring-closing metathesis (RCM) reaction in the presence of a ruthenium-based Grubbs catalyst (Figure 1A).44 In this library, we incorporated different combinations of Fmoc-protected alkenyl glycine residues at specific positions on the sequence of the template peptide BTT3 to enable olefin metathesis. When the peptides were subjected to standard on-resin RCM reaction conditions using the Grubbs II catalyst (20 mol %, DCE, RT, 2 h),29,45 the conversion efficiency of the linear peptide to the cyclized product was observed to be <50% after at least three rounds of treatment, as assessed by the crude mass spectrum (data not shown). Chapman et al. reported a variant of solution-phase RCM of a heptapeptide using a Hoveyda–Grubbs catalyst (5 mol %) at 50 °C in DCE for 16 h and achieved the stapled product with 55% yield.46 Accordingly, when the metathesis reaction was performed at 55 °C with the Grubbs II catalyst (20 mol %) in DCE for 50 min using a microwave synthesizer (150 W, 250 psi) and after at least two rounds of treatment, more than 90% of the linear peptide underwent cyclization (data not shown). It was also observed that either an increase in reaction temperature beyond 60 °C or prolonged reaction time (>1 h) resulted in significant formation of unwanted byproducts due to olefin-isomerization,47 which were difficult to remove during RP-HPLC purification.

Figure 1.

Figure 1

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(A) Fmoc-based SPPS and on-resin RCM of BTT3 analogues. (B) Library of 12 stapled β-hairpin peptides synthesized using SPPS and RCM based on the unstapled parent peptide, BTT3.

After optimization of synthesis, we proceeded to insert all-hydrocarbon alkene staple(s) in the side arms of BTT3, which consist of three alternating leucine and lysine residues (Figure 1B). Hydrocarbon chains were then inserted in the arm region of the peptide only at the leucine positions, leaving positively charged lysine residues intact for antimicrobial activity. Three parameters of the alkene staples were systematically varied in the library: (1) length of the hydrocarbon chain (6C or 7C), (2) position of the staple along with the β-hairpin structure (first (a), middle (b), or terminal (c) leucine pair position), and (3) number of staples (single or double). We manipulated the length of the hydrocarbon chain by incorporating the alkenyl amino acids, Fmoc-(3′-butenyl) glycine and Fmoc-(4′-pentenyl) glycine at specified positions in the sequence. RCM reaction between two (3′-butenyl) glycine residues resulted in a 6C staple, and the reaction between a (3′-butenyl) glycine and a (4′-pentenyl) glycine resulted in a 7C staple. With variations in the staple length, position, and number, a panel of 12 conformationally constrained β-hairpin AMPs (Figures 1B, S1–S24, and Table S1) was constructed. The notation for each peptide depicts a numerical annotation, the staple position, and the staple length. For example, a peptide with a 6C and a single staple at the terminal leucine position (c) is denoted as #(c6) and a peptide with a 6C and a double staple at the first (a) and the terminal (c) leucine positions is denoted as #(a6c6), all prefixed with their assigned numerical annotation.

In Vitro Antimicrobial Activity and Safety Profile

Using a broth microdilution method,48 we first evaluated the antimicrobial profile of the synthesized peptides by screening for their minimum inhibitory concentration (MIC) values against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa and Escherichia coli. All tested peptides displayed poor activity against the Gram-positive S. aureus (Figure 2). This can be attributed to the low hydrophobicity of the peptides represented as the retention time (RH) (Table S1) since AMPs with good activity against Gram-positive bacteria are comparatively hydrophobic.49 We also previously observed that BTT3 with lower hydrophobicity displayed poor activity against Gram-positive bacteria.42 Against Gram-negative bacteria, double-stapled peptides were relatively inactive, but all single-stapled peptides, except 2(a7), displayed potent antimicrobial activity (8 μM MIC). The higher conformational restriction in the structure of double-stapled peptides might have undermined their interactions with bacteria membranes, resulting in poorer killing activity. The addition of an all-hydrocarbon staple in the double-stapled analogues also caused a reduction in hydrophobicity of the peptides, as seen in a general decrease in RH (Table S1). This could have in part impaired peptide interactions with the hydrophobic components of membrane phospholipids, thus lowering antimicrobial potency.

Figure 2.

Figure 2

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Heat map depicting the MIC and HC50 of the peptides against the ATCC strains (E. coli, S. aureus, and P. aeruginosa) and colistin-resistant clinical isolates of E. coli (R1, R2, and R3). The corresponding resistance mutation in each of these isolates are listed in Table S3. Colistin was used as a positive control for E. coli, P. aeruginosa, and the clinical strains. Vancomycin was used as a positive control for S. aureus.

We then tested the antimicrobial activity of the peptides against MDR E. coli clinical isolates (Figure 2 and Table S2). The resistance genes carried by these strains are listed in Table S3, which include mobilized colistin resistance (mcr-1) genes conferring colistin resistance. All of the 6C single-stapled peptides displayed comparable antimicrobial potency against mcr-1-positive isolates versus ATCC E. coli strain, whereas only 6(c7) among the 7C single-stapled peptides retained activity against the MDR strains. All of the double-stapled peptides, except 7(a6b6) and 9(b6c6), displayed a slight improvement in antimicrobial activity against the colistin-resistant isolates, with a 2-fold reduction in MIC. Of note, the linear template peptide, BTT3 displayed clearly a much lower activity against the resistant pathogens, with at least an 8-fold increment in MICs. A typical outer membrane of Gram-negative bacteria is decorated with multiple proteases such as ompT, FtsH, protease III, and degP.50,51 These proteases are involved in multiple quality control roles within a bacterial cell such as digestion of signal peptides, degradation of damaged membrane proteins, and other vital roles in the regulatory pathways.52−55 Studies have shown that omptins, one of the most common outer membrane proteases of E. coli that specifically targets two basic residues or a basic residue and a nonpolar residue, are highly prevalent in pathogenic clinical E. coli isolates compared to the nonpathogenic ATCC strains.56−59 This prevalence is attributed to the ability of bacteria to elicit adaptive resistance in response to our body’s innate immune response. It is plausible that the decline in the antimicrobial activity of the linear peptide BTT3 may be due to the increased susceptibility to the ompT and other membrane proteases on the outer membrane of E. coli clinical isolates. On the contrary, as evident from the MIC data, all of the stapled peptides were able to endure this challenge and continued to elicit antimicrobial activity, highlighting their durability.

To evaluate the safety profile of our stapled β-hairpin peptides, we carried out human RBC hemolysis testing to determine the HC50 (Figure 2). HC50 is defined as the concentration at which up to 50% of the RBC are hemolyzed. The results showed that the stapled peptides did not lyse red blood cells (RBCs) up to the tested concentration of 128 μM, indicating the nontoxic nature of the peptides.

Stability of Stapled Peptides

A major factor that can impair the clinical competency of peptide therapeutics is their susceptibility toward physiological factors such as proteolytic enzymes, salt, and serum elements. Here, we evaluated the stability of the peptides by testing their antimicrobial activity against E. coli in the presence of trypsin, serum, and salt (Figure 3A). The lysine residues in these peptides are generally vulnerable to hydrolysis by trypsin, a serine protease enzyme known to cleave peptide bond at the carboxyl end of cationic amino acids, lysine, and arginine.60 Upon treatment with trypsin for 1 h, the linear template peptide BTT3 showed an 8-fold increase in the MIC, revealing its poor proteolytic stability (Figure 3B). Among the single-stapled peptides, 1(a6) and 5(c6) displayed excellent proteolytic stability by retaining similar MICs, whereas other peptides showed at least a 2-fold increase in MICs. Among the double-stapled peptides, all peptides, except 7(a6b6) retained their activity, which was an indication of stability. One limitation of this approach is that peptide degradation products might display antimicrobial effect themselves, thus potentially confounding the interpretation of the relationship between residual activity and proteolytic stability. To validate these findings, we employed the HPLC method by calculating the area under the curve (AUC) at different time points after the inactivation of trypsin (Figure 3E). Overall, HPLC results corroborated the findings obtained using the MIC approach. Only <10% intact peptide of BTT3, 2(a7), 4(b7), and 6(c7) was retained after 1 h of trypsin treatment, while 1(a6) and 5(c6) displayed greater trypsin stability with >40% intact peptide remaining after 1.5 h of trypsin exposure. Not every double-stapled peptide (e.g., 8(a7b7), 10(b7c7)) was more proteolytically robust than single-stapled analogues.

Figure 3.

Figure 3

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(A) Minimum inhibitory concentration (MIC) of the peptides when treated with trypsin (1:2000), 10% serum, and 150 mM NaCl against E. coli. Stability of peptides treated with (B) trypsin (1:2000), (C) 10% serum, and (D) 150 mM NaCl (represented as fold change in MIC with respect to the original MIC). (E) The stability of peptides after treatment with trypsin at a 1:2000 (trypsin/peptide) molar ratio at 37 °C for different periods (15, 30, 60, and 90 min) was analyzed using a C18 RP-HPLC column. The AUC measured from the HPLC run is displayed as % intact peptide.

In the case of serum stability, while BTT3 showed an 8-fold increase in MIC, most of the stapled peptides, except 2(a7) and 4(b7), displayed only a 2-fold or lower increase (Figure 3C). Among the single-stapled peptides, only peptides 1(a6) and 5(c6) retained the activity without any increase in MIC, indicating that their antimicrobial activity is more robust in serum conditions. All of the double-stapled peptides with restrictive conformation were also able to retain their activity. When 150 mM NaCl was added to the culture medium, 5(c6) and most of the double-stapled peptides only displayed a minimal increase in MIC, whereas BTT3 exhibited a staggering 32-fold increase (Figure 3D), which suggest that stapling modification clearly improves salt sensitivity of peptide activity. Overall, all-hydrocarbon stapling in hairpin AMPs enhanced the robustness of their antimicrobial activity in physiologically relevant media.

Secondary Structure Analysis

To understand the difference in stability and activity among the stapled peptides, we studied their secondary structures using circular dichroism (CD) spectroscopy. The structural behavior of the peptides in a bacterial microenvironment was simulated using 25 mM sodium dodecyl sulfate (SDS) in the buffer. SDS micelles with their negative charge and hydrophobic core were employed to mimic the bacterial membranes. We assessed the far-UV CD spectra (190–240 nm) of the stapled peptides collected in both aqueous buffer (Figure 4A) and in the presence of 25 mM SDS (Figure 4B). As expected, the BTT3 parent peptide displayed a random coil structure in aqueous solution indicated by a strong minimum at around 200 nm. This behavior is typically observed with many natural and synthetic AMPs in the literature. AMPs generally assume a random coil conformation in aqueous solution but readily undergo conformational change upon interactions with the bacterial membranes to either α-helices or β-sheets.42,59 All of the double-stapled peptides, except 10(b7c7) and 12(a7c7), exhibited a clear β-sheet structure, indicated by a characteristic spectrum with minima at around 217 nm and maxima below 200 nm.61,62 Remarkably, among the single-stapled peptides, only the peptides with 6C staples displayed a pronounced β-sheet behavior, whereas the CD spectra of 7C staples indicated the formation of random coil structures like BTT3. We speculate that this behavior could be due to the differential levels of flexibility imposed upon different conformers with respect to the length, position, and number of the staples. The single staples with the lowest number of carbons (1(a6), 3(b6), 5(c6)), suspected to possess a more restricted conformation, were able to retain their secondary structure to a certain level, even in the aqueous solution. With slightly lower restriction, the 7C, single-stapled peptides (2(a7), 4(b7), 6(c7)) could assume a more flexible conformation leading to a random coil structure in the aqueous solution. Similarly, the unstapled parent peptide, BTT3 with the least conformational restriction displayed the lowest mean molar ellipticity (θM) at 200 nm. In the case of double-stapled peptides, irrespective of the carbon length and position, an additional clamp ensured that the β-sheet conformation is retained in the aqueous solution.

Figure 4.

Figure 4

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Circular dichroism (CD) spectra of the peptides measured at room temperature (25 °C) in (A) aqueous buffer (10 mM Tris–Cl buffer) and (B) in the presence of 25 mM sodium dodecyl sulfate (SDS), mimicking the negatively charged bacterial microenvironment. Each spectrum represents an average of three measurements. (C) Table showing θM signals at β-sheet-specific wavelengths and % anti-parallel β-sheet of the peptides when incubated with SDS, as estimated using the web-based tool BeStSel (http://bestsel.elte.hu/index.php).

In the presence of 25 mM SDS, a bacterial membrane-mimicking environment, all of the peptides assumed a prominent β-sheet structure, which could be due to the possible interaction of the cationic peptides with the anionic SDS micelles leading to this conformational transition (Figure 4B). A previous study by Amos et al. supported that a degree of conformational flexibility is essential for exhibiting potent antimicrobial activity by facilitating better interactions with bacterial membranes.63 A study by Liu et al. also reported the importance of flexibility in driving the antimicrobial activity against E. coli.64 Their findings indicate that for cationic antimicrobial peptides, flexibility, in addition to hydrophobicity and positive charge density, is one of the major determinants of antimicrobial activity. This finding agrees with molecular dynamics (MD) simulation results from our previous work highlighting the importance of conformational flexibility.42 Koehbach et al. reported similar observations where they found that the short linear AMPs exhibited a better antimicrobial activity against the ESKAPE pathogens when compared to rigid cyclic and cyclotide-grafted variants of the AMP.65 Overall, CD spectra of several double-stapled peptides indicate a lesser extent of β-sheet conformation (Figure 4C). This might have resulted from the additional conformational rigidity imparted by the extra staple. Restrictions in folding at the surface of bacterial membranes could have interfered with their membrane-permeabilizing activity, hence the inferior antimicrobial potency of double-stapled analogues (Figure 2).

Taken together, our structural analysis data helps explain the functional difference between the single-stapled and the double-stapled peptides. The unstapled and single-stapled peptides with greater conformational flexibility exhibited better antimicrobial activity when compared to the double-stapled peptide. However, in the presence of limiting factors such as serum, salt, and trypsin, a certain level of conformational restriction promotes structural stability and helps preserve activity.32,37,66−69

Membrane Permeabilization Profile of Stapled Peptides

AMPs are known to target bacterial membranes by employing strategies such as pore formation and membrane carpeting to induce bacterial cell death.70−72 Studying the mode of interaction of AMPs is a vital step to develop more potent antimicrobials of the same class. Hence, we proceeded to evaluate the mode of cellular interaction of our most potent peptide 5(c6). First, we evaluated the outer and inner membrane permeabilization profiles of BTT3 and 5(c6) using 1-N-phenylnaphthylamine (NPN) (Figure 5A) and SYTOX green assays (Figure 5B), respectively. NPN is a highly hydrophobic fluorescent probe that fluoresces upon interaction with membrane phosphoglycolipid. However, its hydrophobic nature normally prevents the entry of the dye across the outer membrane.73 Therefore, increased fluorescence represents the disruption of the outer membrane that enables the interaction of NPN with phosphoglycolipid. Results indicate that at 1× MIC, 5(c6) was able to better permeabilize the outer membrane with around ∼70% dye uptake when compared to BTT3 which only showed ∼40% uptake (Figure 5A). SYTOX green dye is another membrane-impermeable dye that fluoresces strongly upon interaction with the nucleic acid content of the cell.74 Hence, an increased fluorescence is suggestive of a ruptured inner membrane. Here, both BTT3 and 5(c6) displayed similar uptake profiles close to 80% at 1× MIC indicating good inner membrane permeabilization (Figure 5B). Overall, it was observed that 5(c6) was able to actively penetrate both inner and the outer membrane of the bacteria.

Figure 5.

Figure 5

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Outer (A) and inner (B) membrane permeabilization profile of BTT3 and 5(c6) against E. coli evaluated using 1-N-phenylnaphthylamine (NPN) and SYTOX green uptake assay, respectively, at three different concentrations (4, 8, and 16 μM). The data is presented based on three repeats. Localization of stapled peptide in E. coli assessed using (C) confocal laser scanning microscopy (CLSM) (scale = 10 μm) and (D) three-dimensional (3D)-SIM (scale = 0.4 μm) upon treatment with FITC-tagged 5(c6) (green) at 1× MIC for 1 h. The bacterial membrane is stained with FM 4–64 dye (red). (E) Membrane permeabilization profile of different stapled peptides in E. coli studied using the degree of bacterial membrane damage upon treatment with 1× MIC of peptide 5(c6) assessed using transmission electron microscopy (TEM). (F) Killing kinetics of 5(c6) was determined against E. coli ATCC 25922. Black dotted line indicates the limit of detection (50 CFU/mL). Experiments were repeated two times.

Cellular Localization and Morphological Analysis of Cell Death

Further, we assessed the cellular localization and damage profiles as the result of antimicrobial activity. We treated E. coli cells with FITC-tagged 5(c6) at 1× MIC concentration (16 μM) for 1 h, and the fixed cells were imaged under confocal laser scanning microscopy (CLSM) and 3D-structured illumination microscopy (3D-SIM). FITC was conjugated to the N-terminal of resin-bound peptides, using an Fmoc-amino hexanoic acid linker. The CLSM images (Figure 5C) of E. coli cells stained with a membrane dye, FM 4–64 (red) clearly depicts the internalization of FITC-5(c6) peptide, indicated by the scattered green fluorescence inside the bacterial cell. Here, we observed that rather than a uniform distribution inside the bacterial cell, FITC-5(c6) peptides showed a localized accumulation only at the septa and the polar region of the rod-shaped bacteria. This localization pattern was further confirmed by imaging under 3D-SIM (Figure 5D). One known limitation of this fluorescence labeling approach is that the FITC tag might alter the physicochemical properties of the peptide molecule, thus affecting their structure and behavior to some extent. However, it remains widely employed for visualization of subcellular peptide distribution. We then performed transmission electron microscopy (TEM) imaging of E. coli cells treated with the peptide 5(c6) at 1× MIC at different time points (Figure 5E). It was observed that the morphology of the bacteria membranes remained intact after 1 h of treatment with the peptide. However, there was a slight membrane perturbation observed in the 3 and 6 h samples. Killing kinetics results showed that 5(c6) at 1× MIC caused complete bactericidal killing of E. coli within 6 h of treatment (Figure 5F), indicating that the killing mechanism of the peptide does not involve extensive rupturing of bacterial membranes. At 2× MIC, 5(c6) displayed a more rapid killing effect, causing complete killing within 3 h of treatment. Remarkably, membrane damage at 3 and 6 h was mostly localized near the polar regions of the bacteria (Figure 5E). In addition, we also observed sizeable aggregates inside the bacterial cells at both time points, which we postulate to be made of the peptides, given the similar cellular localization as the green fluorescence of FITC-labeled peptide in the 3D-SIM image (Figure 5D). Using CLSM and TEM, we demonstrated that 5(c6) could penetrate the bacterial membranes without causing extensive damages, as indicated by the localization of green FITC-5(c6) inside red-stained bacteria (Figure 5C,D) and a lack of deformation in the bacterial membrane (Figure 5E) after 1 h of treatment with the peptide. This relatively uncommon mode of interaction suggests that the presence of cationic AMPs can lead to clustering of anionic lipids on the membrane through electrostatic attractions, which in turn can lead to phase boundary defects. These defects caused by changes in lipid packaging can increase the membrane permeability in confined regions of the membrane, resulting in localized accumulation of 5(c6). When the defects are larger, it could result in the formation of pores that could collapse the entire electrochemical gradient of the cell. These defects can also result in altering the overall membrane stability which can eventually lead to its death. This mode of cellular death was previously reported in a few studies on certain cationic AMPs.75

Conclusions

In summary, a library of all-hydrocarbon-stapled β-hairpin peptides with variations in the position of stapling, the number of staples, and the length of the hydrocarbon chain was created. A detailed investigation of antimicrobial activity and stability revealed multiple pieces of insightful information. All of the peptides tested displayed a narrow spectrum of activity, selective toward Gram-negative pathogens. Despite better proteolytic and conformational stability, the double-stapled peptides exhibited poor antimicrobial activity, possibly owing to their rigid structural features. In contrast, single-stapled peptides with a longer hydrocarbon chain of 7C and more flexibility showed better activity but poor stability, suggesting the need for optimizing the chain length in peptide designs. Interestingly, it was observed that those peptides with poor stability profiles displayed a drastic reduction in activity against the clinical resistant pathogens with mcr-1 resistance genes and others. It is highly speculated that increased membrane protease activity in such clinical resistant strains could directly contribute to this effect, further emphasizing the need for better-engineered AMPs. Overall, the peptide 5(c6) with a 6-carbon-length all-hydrocarbon staple at the terminal Leu-pair position was the most potent peptide from the library with high stability and better antimicrobial activity. Our findings in this study clearly emphasize the importance of systematic installation of hydrocarbon stapling on synthetic β-hairpin AMPs and its implication in the discovery of potent AMPs with better clinical competencies.

Experimental Section

Materials

Bacteria strains E. coli ATCC 25922, P. aeruginosa ATCC 9027, and S. aureus ATCC 29737 were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Clinical isolates of E. coli (R1, R2, and R3) were a kind gift from Dr. Jeanette Teo (National University Hospital, Singapore). Rink amide resin (0.74 mmol/g, 100–200 mesh) was obtained from ChemPep (Wellington, FL). Nα-Fmoc-protected amino acids and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) were obtained from AK Scientific (California). The following amino acids with side-chain protecting groups were used: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Pro-OH, Fmoc-(3′-butenyl)Gly-OH, and Fmoc-(4′-pentenyl)Gly-OH. Nα-Fmoc-protected butenyl/pentenyl glycines were purchased from Okeanos (Beijing, China). N,N-Diisopropylethylamine (DIPEA) and piperidine were obtained from Merck. All other solvents used were obtained in the highest available purity from Aik Moh (Singapore). All solvents used were of analytical grade, purchased from Fisher Scientific, and used without further purification. Manual SPPS was performed in polypropylene tubes equipped with fitted disks, purchased from Bio-Rad (California). Fluorescein isothiocyanate (FITC) and Muller–Hinton broth (MHB) were purchased from Sigma-Aldrich (Singapore). Water was purified using a Milli-Q water purification system. The synthetic peptide BTT3 was custom-synthesized by GL Biochem (Shanghai, China). All graphs were plotted using GraphPad Prism (version 7) software.

Solid-State Peptide Synthesis

The peptides were synthesized manually using solid-phase and Fmoc chemistry at the 0.1 mmol scale using rink amide resin (0.74 mmol/g) in a vacuum manifold apparatus with three-way stopcocks as described.76 The dry resin was weighed out into a polypropylene column equipped with a fritted disk and swelled with dimethyl formamide (DMF) (3 mL) for 15 min before use. The synthesis scheme follows a repeated cycle of deprotection, washing, and coupling for each amino acid. The Fmoc protecting group was removed by treatment with 20% (v/v) piperidine in DMF (2 mL) and the mixture agitated for 10 min and drained, and this process was repeated another time. After draining the solution, the resin was washed with approximately 3 mL of DMF (3×). The coupling of individual amino acids was performed with Fmoc-protected amino acids (4.0 equiv; except for Fmoc-(alkenyl)-glycines), HATU (3.6 equiv), and DIPEA (12 equiv) in DMF (2 mL). For the coupling of Fmoc-(3′-butenyl)Gly-OH and Fmoc-(4′-pentenyl)Gly-OH, 3.2 equiv of the amino acids was used with HATU (2.9 equiv) and DIPEA (12 equiv) in DMF (2.0 mL). The coupling time was 45 min for all amino acids except for Fmoc-(alkenyl)-glycine residues, which were coupled for 1 h, after which the resin was thoroughly washed with DMF. This process is again repeated for the coupling of the next amino acid.

On-Resin Peptide RCM

Ring-closing metathesis of resin-bound Fmoc-protected peptides was performed at an elevated temperature of 55 °C in a microwave synthesizer (CEM-Discover). The solution of the Grubbs II catalyst (20 mol %, 5 mg/mL) in anhydrous 1,2-dichloroethane (DCE) was added to the resin in a microwave sealed glass tube containing the resin. This mixture was positioned in a microwave synthesizer for 50 min at 55 °C with microwave setting of 150 W and 250 psi. The solution was drained, and the resin was washed with DMSO/DMF (1:2, 2 mL, 30 min) and DMF (3 × 3 mL), followed by Boc-protection using 20% (v/v) di-tert-butyl decarbonate in DMF (2 mL) and the mixture agitated for 30 min. After draining the reaction mixture, the resin was washed with DMF (3 × 3 mL) and DCM (3 × 3 mL) and then dried under vacuum. A cycle of the metathesis reaction and Boc-protection was repeated as many times as necessary using freshly prepared solutions of the catalyst. The peptide was finally cleaved from the resin using TFA/EDTA/H2O/TIPS (94:2.5:2.5:1, v/v/v/v) at room temperature for 1 h. The crude peptide from the solution mixture was precipitated with cold diethyl ether, isolated by centrifugation, and washed further with diethyl ether. Finally, the resultant precipitate was dissolved in 50% acetonitrile in water containing 0.1% TFA and lyophilized to get the final product. The lyophilized peptides were then analyzed using mass spectrometry.

For FITC labeling, the resin-bound peptide was first coupled with an Fmoc-amino hexanoic acid linker, followed by Fmoc-deprotection 20% (v/v) piperidine in DMF (2 mL) as described in the Cellular Localization and Morphological Analysis of Cell Death section. Further, FITC was coupled to the resin in a mixture of FITC (1.1 equiv) in pyridine/DMF/DCM (12:7:5, v/v/v), and the mixture is agitated for 2 h at room temperature after which the resin was thoroughly washed with DMF. Following this, the peptide was cleaved from the resin as described above.

HPLC Analysis and Purification

Peptide purification was carried out in a reverse-phase C18 semipreparative column (XBridge Peptide BEH, 10 mm × 250 mm) in a Gilson HPLC system (GX-281) using a linear-gradient system (solvent A: 0.1% TFA in H2O, solvent B: 0.1% TFA in acetonitrile) for 50 min at a flow rate of 5 mL/min. Peptide purity analysis was carried out in a reverse-phase Waters C18 column (Symmetry, 4.6 mm × 250 mm) in a Shimadzu HPLC using a linear-gradient system (solvent A: 0.1% TFA in H2O, solvent B: 0.1% TFA in acetonitrile) for 20 min at a flow rate of 1 mL/min. The percentage purity of the peptides is assessed as the percent AUC. In both systems, the UV absorbance of the peptides was monitored at a wavelength of 220 nm. All compounds are >95% pure by HPLC (Figures S1–S24).

MIC Assay

Antimicrobial activity of the peptides was evaluated by determining its MIC, which is the lowest concentration of the antimicrobial agents, which inhibits the visible growth of microorganism. The MIC of the peptides was tested using a standard broth microdilution method.48 The peptides were also evaluated against the E. coli clinical isolates, R1, R2, and R3 with resistant genes (R1—mcr-1, R2—mcr-1 and NDM-1, R3—mcr-1 and blaKPC-2) specific to resistance against either only colistin or colistin and carbapenem antibiotics. An overnight grown culture (37 °C, 120 rpm, orbital shaking) in MHB was diluted to an optical density at 600 nm (OD600) of 0.07, which is equivalent to 108 colony-forming unit (CFU)/mL. This was further diluted 1000× to obtain 3 × 105 CFU/mL culture. Peptide stock solutions were serially diluted to different concentrations with the appropriate bacterial suspensions in triplicates using a 96-well plate. The plates were incubated at 37 °C in an orbital shaker (120 rpm) for 18 h before measuring the OD600 of each well using a microplate reader (Tecan infinite Mplex).

Hemolysis Assay

The ability of the peptides to lyse red blood cells (RBC) was assessed using the hemolysis assay. Blood was collected from healthy volunteers based on protocol approved by the Institutional Review Board (IRB) (Protocol No. H-20-025). First, the collected human blood was centrifuged to remove serum components at 1000g for 10 min with acceleration and deceleration set to 4. The pellets were then washed with 1× phosphate-buffered saline (PBS). Further, the pellets were resuspended in 10 mL of PBS and used as a stock. In the next step, 300 μL of peptide solution was prepared at different concentrations (4, 8, 16, 32, 64, and 128 μM) in PBS with 4% RBC from the stock solution. This mixture was incubated at 37 °C for 1 h. After incubation, the samples were centrifuged for 10 min at 1000g with acceleration and deceleration set to 4. About 200 μL of the supernatant from each tube was transferred to a 96-well plate, and OD540 was measured using a microplate reader (Tecan Infinite Mplex). 1% TritonX-100 was used as a positive control. The percentage hemolysis of the peptide was calculated based on the formula below:

graphic file with name jm3c00140_m001.jpg

Based on % hemolysis values, HC50 for each peptide was determined. HC50 is defined as the concentration of the peptide at which about 50% hemolysis was observed.

Stability Analysis

The stability of peptides in different media was assessed using the MIC assay described above. Here, the stability of peptides in trypsin, serum, and salt was analyzed using methods previously described.77 For trypsin stability assessment, peptides were incubated with trypsin at the molar ratio of 1:2000 (trypsin/peptide) at 37 °C for 1 h. For instance, 1 mM peptide was mixed with 0.5 μM trypsin and incubated. After 1 h treatment, the enzyme reaction was terminated by incubating the samples at 80 °C for 10 min. This peptide mixture was then serially diluted in a 96-well plate for the evaluation of antimicrobial activity against E. coli. For serum stability, peptides were assessed for their antimicrobial activity against E. coli in the presence of 10% fetal bovine serum (FBS). Similarly, for salt stability, the peptides were assessed for their antimicrobial activity against E. coli in the presence of 150 mM NaCl. The fold change in MIC with respect to the control was calculated and plotted for each peptide. To further assess the validity of this method, we also carried out trypsin stability analysis using the HPLC method, described previously.66 Here, the peptides were treated with trypsin at the same ratio and incubated at 37 °C for different time intervals up to 90 min. The reaction was terminated by the addition of an equal volume of 50% acetonitrile in water (with 1% TFA). This mixture was filtered using a 0.45 nm syringe filter and analyzed using HPLC as described above. The AUC for the peptide at different time points was evaluated and plotted.

CD Spectroscopy

The secondary structure of the synthesized peptides was analyzed using CD spectroscopy. CD spectra were acquired using a Jasco CD Spectrometer J-810 (Tokyo, Japan). Peptide solutions were prepared at a concentration of 50 μM in 10 mM Tris–Cl buffer (pH 7.5). To evaluate the secondary structure of peptides in a bacterial microenvironment, spectra of the peptides were recorded in the presence of 25 mM SDS. The negative charge of SDS micelles which emulates the bacterial membrane drives the peptide folding. CD spectra were recorded at room temperature from a wavelength of 190–260 nm and a scanning speed of 60 nm/min using a quartz cell with a path length of 1.0 mm. Measurements were averaged from 3 runs per peptide and smoothened using in-built algorithms. The acquired spectra were further converted to mean residue ellipticity using the following equation

graphic file with name jm3c00140_m002.jpg

where θM is the mean residue ellipticity (deg cm2/dmol), θobs is the observed ellipticity corrected for the blank at a given wavelength (mdeg), MRW is the residue molecular weight (MW/number of amino acids), c is the peptide concentration (mg/mL), and l is the path length (cm).

NPN Uptake Assay

The outer membrane permeabilization profile of BTT3 and the stapled peptides was evaluated using the NPN uptake assay using a method modified from Helandar and Mattila-Sandholm.73E. coli ATCC 25922 culture was harvested at an exponential growth phase. The bacterial cells were then washed twice with sterile PBS buffer (pH 7) and resuspended to yield an OD600 of 0.4. An equal volume of 40 μM NPN was added to the bacterial suspension. Colistin (100 μM) was used as a positive control in this study. Following this, an equal volume of the desired concentration of the peptides was added to the mixture. The mixtures were then incubated at 37 °C for 1 h in a shaking incubator. Fluorescence intensity was measured using a microplate reader (Tecan infinite Mplex) with an excitation wavelength of 355 nm and an emission wavelength of 405 nm. The percentage of NPN uptake in the presence of peptide was calculated based on the formula below

graphic file with name jm3c00140_m003.jpg

where F0 is the fluorescence intensity corresponding to NPN uptake in the absence of any compounds and Fmax and F are the fluorescence intensities after the addition of control peptide and test compounds, respectively.

SYTOX Green Uptake Assay

The inner membrane permeabilization profile of BTT3 and the stapled peptides was evaluated using the SYTOX green uptake assay using a method modified from Rathinakumar and co-workers.78 Briefly, bacterial culture (E. coli ATCC 25922) was harvested at an exponential growth phase. The bacterial cells were then washed twice with sterile PBS buffer (pH 7) and resuspended to yield an OD600 of 0.4. An equal volume of SYTOX green dye (4 μM) was then added and incubated for 5 min. Following this, the desired concentration of peptides (4, 8, and 16 μM) was added to this mixture and incubated for 1 h at 37 °C in a shaking incubator. BTT1, a peptide our lab previously demonstrated to permeabilize the inner membrane was used as a positive control. The fluorescence intensity (F) of the cellular mixture was measured using a microplate reader (Tecan infinite Mplex) with an excitation wavelength of 488 nm and an emission wavelength of 523 nm. The percentage of SYTOX green uptake in the presence of peptide was calculated based on the formula below

graphic file with name jm3c00140_m004.jpg

where F0 is the fluorescence intensity corresponding to SYTOX green uptake without treatment and Fmax and F are the fluorescence intensities after the addition of control peptide and test compounds, respectively.

Confocal Laser Scanning Microscopy (CLSM) and Structured Illumination Microscopy (SIM)

The overnight grown bacterial culture (E. coli ATCC 25922) was harvested using centrifugation (8000 rpm, 10 min). The bacterial cells were washed thrice in PBS (pH 7) and resuspended to an OD600 of 0.1 measured at a wavelength of 600 nm. The suspension was further incubated with FITC-5(c6) at a concentration of 1× MIC (16 μM) for 1 h at 37 °C in a shaking incubator. After incubation, the cells were washed thrice in PBS. The cells were post-stained with FM dye (FM 4–64FX) by incubating at room temperature for 30 min at a concentration of 1 μg/mL. Following this, the cells were washed thrice in PBS. The cells were then fixed using 4% paraformaldehyde. About 10 μL of the fixed bacterial sample was transferred to a poly-l-lysine-coated glass slide and allowed to dry completely. Then, the fixed bacterial sample on the glass slide was secured with a coverslip on the mounting medium. The treated bacterial samples were then imaged using CLSM (Olympus FV3000). The high-resolution single-cell imaging was achieved using 3D-SIM (DeltaVision OMX microscope).

Transmission Electron Microscopy (TEM)

E. coli ATCC 25922 was cultured overnight in MHB and then harvested using centrifugation (8000 rpm, 10 min). The cells were then washed thrice in PBS and resuspended to an OD600 of 0.2 measured at a wavelength of 600 nm. The suspension was further incubated with 5(c6) at a concentration of 1× MIC for different periods (1, 3, and 6 h) at 37 °C in a shaking incubator. After incubation, the cells were washed thrice with PBS and fixed in 500 μL of 2.5% (v/v) glutaraldehyde in PBS at 4 °C for 8 h. Thereafter, the pellets were washed again thrice with PBS and post-fixed with 1% osmium tetroxide in PBS for 2 h at room temperature. The fixed bacterial cells were washed again thrice with PBS and subsequently dehydrated in graded ethanol series (50, 70, 90, and 100%) for 15 min each. After incubating in acetone for 20 min, the samples were then transferred to a 1:1 and, subsequently, 1:3 mixture of absolute acetone and epoxy resin for 1 h in each, followed by transferring to pure epoxy resin. The epoxy resin mixed sample was then incubated for 12 h at 45 °C and 24 h at 60 °C in a cylindrical mold. The cylindrical resin was then subjected to ultrathin sectioning using Leica ultramicrotome. The ultrathin sections of 50–100 nm were then carefully transferred to a copper grid and dried overnight. These copper grids were post-stained using UranyLess (2 min) and lead citrate (1 min) and dried completely. Specimens were then observed under a JEOL 2200FS transmission electron microscope (120 kV).

Acknowledgments

The research work described in this manuscript was conducted with facilities provided by the National University of Singapore. This work was supported by the Ministry of Education (MOE) Tier 1 grants, Singapore (R-148-000240-114), awarded to P.L.R.E. The authors acknowledge the Cryo-Electron Microscopy Facility at Centre for Bioimaging Science (Department of Biological Science) and the Confocal Microscopy Unit (Yong Loo Lin School of Medicine), National University of Singapore for the scientific and technical assistance. The authors would also like to thank the A*STAR Microscopy Platform staff for the training, consultation, advice, and technical support.

Glossary

Abbreviations Used

AMPs

antimicrobial peptides

AMR

antimicrobial resistance

ATCC

American type culture collection

CD

circular dichroism

CFU

colony-forming unit

DCE

1,2-dichloroethane

DIPEA

N,N-diisopropylethylamine

DMF

dimethyl formamide

FBS

fetal bovine serum

FITC

fluorescein isothiocyanate

mcr

mobilized colistin resistance

MD

molecular dynamics

MDR

multidrug-resistant

MIC

minimum inhibitory concentration

NPN

1-N-phenylnaphthylamine

PBS

phosphate-buffered saline

RBC

red blood cells

RCM

ring-closing metathesis

RH

retention time

SDS

sodium dodecyl sulfate

SIM

structured illumination microscopy

SPPS

solid-phase peptide synthesis

TEM

transmission electron microscopy

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00140.

  • Additional HPLC methods and spectra; purified peptide assessment using RP-HPLC; mass spectra of purified peptides; characterization of hydrocarbon-stapled β-hairpin peptides; MIC and HC50 of peptides; and type of resistance genes identified in the resistant clinical isolates (PDF)

  • Molecular formula strings file (CSV)

The authors declare no competing financial interest.

Notes

By systematically transplanting all-hydrocarbon staples to a synthetic β-hairpin AMP, we present valuable knowledge to the design toolkit for enhancing proteolytic stability of novel AMPs by conferring structural rigidity. As alternative therapeutics to overcome antibiotic resistance, our de novo-designed peptides displayed potent activity against clinically relevant antibiotic-resistant strains under physiologically relevant serum conditions. The superior antimicrobial profile of single-stapled compared to double-stapled peptides indicates the importance of structural flexibility and a necessity for optimization to balance between stability and antimicrobial potency.

Supplementary Material

jm3c00140_si_001.pdf (1MB, pdf)
jm3c00140_si_002.csv (3.3KB, csv)

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