†These authors contributed equally.
Academic Editor: Melanie R. Power Coombs
Background: Antimicrobial peptides (AMPs) are short, cationic, amphipathic molecules that have gained tremendous popularity as alternatives to traditional antibiotics due to their lower propensity to develop bacterial resistance. However, the clinical developability of AMPs remains impeded due to shortcomings such as proteolytic instability and poor penetration leading to low bioavailability. Aims: To improve the access of AMPs to cells and subsequent bacteria killing, we evaluated the cell-penetrating and antimicrobial properties of three novel libraries of synthetic peptoids using Minimum Inhibitory Concentration, killing efficacy and membrane permeabilization assays against mycobacteria and Staphylococcus aureus. In addition, we investigated cell selectivity using mammalian cells to assess peptoid toxicity. Results: We showed that short tetrameric Rhodamine B-labeled peptoids composed of a balance of aromatic and lipophilic residues have potent selective antimicrobial activity against a range of microorganisms. The most potent candidates were active against drug-resistant S. aureus isolates as well as mycobacterial strains, with cell penetrating capabilities reported in HeLa and RAW 264.7 macrophage cells. Conclusions: These data suggest that peptoids with novel dual functionalities may potentially be an interesting class of therapeutics and/or molecular delivery agents for anti-infective purposes.
The unrestricted use of antibiotics over the past 50–60 years has created multidrug-resistant (MDR) strains of microorganisms for which currently only limited treatment options are available [1, 2, 3, 4, 5]. Compounding the problem, the development and approval of new antibiotics has not kept pace with the progression of this global health threat [6, 7]. Based on reports from the U.S. Centers for Disease Control and Prevention (CDC), approximately 119,000 cases of S. aureus bloodstream infections occurred in 2017, resulting in significant number of deaths. In tandem, cases of both hospital and community-acquired methicillin-resistant S. aureus (MRSA) infections have not decreased in the past decade . Other Gram-positive bacteria such as mycobacteria also account for a large number of community cases, especially in third world countries . In 2018, 10 million people were diagnosed with tuberculosis (TB), with at least half a million of these new cases being rifampicin-resistant. Caused by the Mycobacterium tuberculosis bacteria, treatment regimens for drug-sensitive TB are complex involving long-term therapy over a period of 6 months with a cocktail of several drugs [7, 9]. Hence, there is an urgent need for novel antimicrobial agents with new mechanisms of action that could replace or complement current conventional therapies.
Cationic antimicrobial peptides (AMPs) are a class of short, cationic, amphipathic molecules that constitute a major part of the innate host defense system in many organisms [10, 11, 12, 13]. Their mechanism of action primarily involves the disruption or displacement of the bacterial membrane, giving rise to a lower likelihood of resistance development as compared to traditional antibiotics . However, AMPs have several drawbacks including proteolytic instability and poor penetration leading to low bioavailability . These shortcomings make the translation of AMPs as therapeutics extremely challenging. Oligo-N-substituted glycines, also known as antimicrobial peptoids (AMPos), are sequence-specific synthetic peptidomimetics with a peptide backbone but differ from AMPs in that the side chains are attached to the backbone amide nitrogen instead of the alpha-carbon (Fig. 1B) [14, 15, 16]. This structural difference means that there are no known proteases that will recognize and degrade the peptoid structure making them more stable. Peptoids also do not face similar conformational constraints as the corresponding peptides, due to the loss of chirality of the alpha-carbon. They are highly modifiable with diverse side chains, thus offering a toolkit for the development of artificial functional compounds [17, 18]. There is increasing interest in peptoid development due to their antimicrobial activity against a broad spectrum of pathogens, non-specific mode of action, decreased susceptibility to enzymatic degradation, and the relative ease of synthesis (Fig. 1A). The latter is also bolstered by the low costs of combinatorial solid phase synthesis . More interestingly, peptoids have been shown to display better membrane permeability when compared to peptides, or by interacting with intracellular targets such as bacterial DNA.
Peptoid Synthesis. (A) Submonomer synthesis of peptoids. (B) General chemical structure of peptoids. Abbreviations: DIC, Diisopropylcarbodiimide; DMF, Dimethylformamide; RhodB, Rhodamine B; TFA, Trifluoroacetic acid. Sub-bullets a) and b) describe sub-sequential synthesis steps.
As most AMPs resemble the amphiphilic properties of cell penetrating peptides or CPPs, their peptoid counterparts called Cell Penetrating Peptoids (CPPos) have been good mimics, despite the lack of chirality and absence of hydrogen bond formation by amide nitrogens. Multiple cationic charges favour endosomal accumulation, while lipophilicity promotes mitochondrial localization . By using a radiofrequency tag supported combinatorial split and mix approach (IRORI), Kölmel et al.  showed that an increase in lipophilicity in tetrameric peptoids promoted their localization in mitochondria. By learning from mitochondrial CPPs and CPPos as described by Horton et al.  and Kölmel et al. , we developed a library of tetrameric cell penetrating antimicrobial peptoids with the suitable chemical properties for localization in mitochondria.
Three libraries comprising a total of 401 peptoids with varying side chains were designed and synthesized to study whether peptoids can have dual functionality of being both cell penetrating and antimicrobial. Our hypothesis was to examine whether the modulation of the lipophilicity of tetrameric peptoids would continue to allow for cell penetration without disrupting the cell membranes. These modifications would enable us to develop novel therapeutic peptoids with good biocompatibility and ease of synthesis.
Peptoid synthesis was performed using IRORI technology at room temperature
reported by Kölmel et al.  using permutation of four different
side chains (Fig. 2). Library 1 was synthesized using N-(2-prop-2-yn
1-yl) glycine (Nprg),
N-(p-chlorobenzyl)glycine (Npcb), N-(4-aminobutyl)glycine,
(Nlys), and N-(benzyl)glycine (Nphe) for side chains.
Library 2 was performed with N-(benzyl)glycine (Nphe),
N-(4-Hydroxybenyl)glycine (Nphb) and
N-(4-fluorobenzyl) glycine (Npfb) side chains. Library 3 was
performed with N-(2-prop-2-yn-1-yl)glycine (Nprg),
N-(4-Hydroxybenyl)glycine (Nphb) and
N-(1-aminohexanyl)glycine (Nhe) side chains. All CPPos were
labeled with Rhodamine B at the C-terminus of the peptoid as described in
Kölmel et al. . All products were verified to have
Peptoid monomer side chain structures.
Minimum inhibitory concentration or MIC is the concentration at which a compound
is able to inhibit the growth of bacteria. All experiments were conducted in
microtiter plates. 75
MIC for mycobacteria was determined similar to S. aureus in a
microtiter plate with minor modifications. 100
Molar attenuation coefficient of Rhodamine B in water and octanol at I = 550 nm
was determined by diluting Rhodamine B in octanol and water to a final
concentration of 20
Cell culture was carried out under sterile conditions. 1
HEK 293T cells were cultured in DMEM media. A peptoid solution plate (100
where A is the absorbance of the test well and A
Peptoid solution (100
Mid-log phase culture of S. aureus ATCC 29737 was diluted in fresh MHB
to achieve an OD600 of 0.15. It was then washed twice with PBS and re-suspended
in PBS. The suspension was incubated with 1
We synthesized libraries of AMPos to determine whether the antimicrobial activities of peptoids are affected by structural and sequence modifications in a similar fashion as AMPs [21, 22]. The design of the peptoids in these libraries was derived from changes in lipophilicity and amphiphilicity. Sequence-specific N-substituted glycine oligomers can be efficiently synthesized via primary amines as submonomers to incorporate a large number of diverse side chain functionalities . This method iterates sequential steps of bromoacylation and nucleophilic displacement of bromide using any kind of amine primary amine submonomer synthons. N-acetylated linear oligomers were synthesized on the Rink amide resin to obtain C-terminal amides. Thereafter, the N- termini were acetylated with acetic anhydride prior to TFA cleavage (Fig. 1A). In total, 401 peptoids were synthesized using split mix combinatorial solid-phase synthesis. Amines used for synthesis in Library 1 were 4-aminobutylglycine (Nlys), prop-2-yn-1-amine (Nprg), benzylamine (Nphe) and 4 chlorobenzylamine (Npcb). Amines used for synthesis in Library 2 are tetradecylglycine (Ntetradec), 4 fluorobenzylglycine (Npfb), benzylamine (Nphe) and 4-hydroxybenzylglycine (Nphb). Amines used for synthesis in Library 3 are hexylamine (Nhe), prop-2-yn-1-amine (Nprg), 4-hydroxybenzylglycine (Nphb) and 4-chlorobenzylamine (Npcb). For microscopic analysis, all peptoids have been labelled with Rhodamine B. All other experiments have also been performed with labelled peptoids. The monomers used for the synthesis are shown in Fig. 2. The positive charge was introduced by incorporating lysine-like (Nlys) monomers, while the overall lipophilicity was altered using a variety of short and aromatic monomers. The chemical structures for the side chains used in creating the libraries were mimics of natural amino acid side chains, in addition to a broader selection of bulky hydrophobic side chains such as Nphb, Npfb, and Npcb. To vary the amphiphilicity of the peptoids, we used different permutations of the monomers in the peptoid sequence.
401 compounds were synthesized, of which 362 were screened for MIC
Schematic of screening of hits and workup of lead compounds.
362 peptoids were screened for their antimicrobial activity against S. aureus ATCC 29737 (Fig. 4). The highest tested concentration was 200
Scatter plot of primary screen of peptoids against S. aureus ATCC 29737. Three libraries, comprising a total of 362 compounds were
screened for potency. A cut-off of 10
|S. aureus||S. aureus||S. aureus||S. aureus||S. aureus||M. bovis||M. chelonae|
For the initial MIC screening, S. aureus was incubated with different
concentrations of peptoids for 18 hours. MICs were determined by measuring
The lowest MICs
Cell membranes are known to be selectively permeable and hence limit the penetration of large molecules or non-lipophilic molecules such as hydrophilic and large molecular weight drugs [17, 29]. As a result, antimicrobials are unable to reach intracellular targets or intracellular microbes. This in turn restricts the arsenal of drugs that can be employed to efficiently target intracellular microbes . Existing studies have shown the use of viral vectors as shuttle for AMPs or membrane disruption for delivery, but this can potentially result in unintended cytotoxicity. Since peptoids have previously been shown to overcome barriers for cell penetration without membrane destruction, our libraries of peptoids were similarly tested for these abilities. Hela cells have been widely used in several studies to determine the cell penetrative ability of peptides [31, 32]. Due to their robust proliferative capability, we have chosen them as hosts for large scale screening of our peptoids in this study. It has also been established that most of the peptide entry mechanisms are endocytic in nature and do not involve specific cell receptors, hence HeLa cells though not perfect will act as good surrogates for determining the cell penetrating ability of our peptoids. Here, we showed that all our peptoids except for peptoid 6, localized in the mitochondria. We used the Pearson correlation coefficient (Rr) to estimate the co-localization in the mitochondria, using the ImageJ Plugin JACoP (ImageJ bundled with 64-bit Java, ImageJ, https://imagej.nih.gov/ij/download.html, https://imagej.net/plugins/jacop). All peptoids showed a coefficient of 0.75–0.95, except for peptoid 6 (0.43) which in this case could indicate endosomal localization rather than mitochondrial entry. The Rr for the peptoids were as follows: Rr (1) = 0.75, Rr (2) = 0.824, Rr (3) = 0.917, Rr (4) = 0.957, Rr (5) = 0.869, Rr (6) = 0.438, Rr (7) = 0.911, Rr (8) = 0.922, Rr (9) = 0.916, Rr (10) = 0.944, Rr (11) = 0.909. This is confirmed by the confocal images in Fig. 5 which showed yellow colour in the merged column except for peptoid 6. This indicates that all the peptoids except 6 localized in the mitochondria making them ideal for targeting intracellular pathogens or even as delivery agents to transport antimicrobial agents into cells.
Cellular uptake of library 1 (peptoid 1, 2), library 2
(peptoid 3, 4, 5, 6, 7, 8) and library 3 (8, 9, 10, 11) compounds in HeLa cells.
We evaluated the biocompatibility of selected oligomers in HEK 293T cells by
using the MTS assay. CC
|S. aureus ATCC 29737|
We then investigated the ability of the peptoids 1, 2, 5, 6, 10, and 11 to
effectively kill S. aureus ATCC 29737 as shown in Fig. 6. Peptoid 1 had
a killing efficiency of 99.99% (
Time-concentration kill kinetics of peptoids and Vancomycin
against S. aureus ATCC 29737. Vancomycin was used as a positive
control. The CFU/mL values represent the means
To examine whether the selected peptoids caused membrane permeabilization of
S. aureus ATCC 29737, cultures were treated with 1
Membrane permeabilization of S. aureus ATCC 29737 induced by the peptoids, as reflected by the percentage of cells in the culture positive for SYTOX green uptake.
Granuloma, which are compact aggregates of immune cells such as macrophages, are the hallmark structures of tuberculosis. It is historically regarded as a host-protective structure that shields off the infected area from healthy tissue . This is problematic as most drugs cannot penetrate the granuloma, and thus cannot reach the inside-residing bacteria. As a result, the bacteria can become dormant with the potential of future reactivation, especially when patients become immune-compromised . Thus, it is important that our peptoids are able to penetrate into macrophages to exert their antimicrobial function. We incubated RAW 264.7 cells with peptoid solution (1 representative peptoid from each library) (labelled with Rhodamine B) and imaged the localization of the peptoids in living cells using confocal microscopy (Fig. 8). Cellular uptake in macrophages was detected for the selected peptoids. The confocal microscopy images showed, that the peptoids accumulated in the mitochondria of the cells. This finding provides a strong justification to the design of cell penetrating peptoids which can enter and localize within macrophages. This suggests that our peptoids could be a good starting point for development of new antimycobacterial drugs or as adjunctive therapy in combination with existing regimens.
Cellular uptake of peptoid 1, 3, 11 RAW 264.7 cells. 2
Here we have demonstrated that short tetrameric Rhodamine B-labeled peptoids composed of a mix of aromatic and lipophilic residues have potent antimicrobial activity and are selective towards bacterial cells. The peptoids demonstrated activity against drug resistant S. aureus isolates as well as mycobacterial strains. Further, we demonstrated the cell penetrating capability of potent leads in HeLa and RAW 264.7 cells with their localization in the mitochondria. These data suggest that these labelled peptoids with novel dual functionalities at its best will act as interesting tools for the further study of short tetrameric antimicrobial peptoids as a novel class of therapeutics and/or molecular delivery agents for anti-infective purposes. Further studies are underway to examine the peptoids at their pristine states for in vivo efficacy.
BSF and DM contributed equally to this work. BSF, DM and NDTT conducted the experiments. BSF, DM, US and PLRE conceptualized this work and designed the experiments. BSF and DM analysed the data and prepared the figures. BSF, DM, US and PLRE wrote the manuscript. All authors have approved the final version of the manuscript.
We would like to thank Dr Lakshminarayanan Rajamani, Singapore Eye Research Institute for the drug resistant clinical isolates of S. aureus. B.S.F. acknowledges the support of the Jürgen Manchot Stiftung and the German Academic Exchange Service (DAAD). N.T. was awarded the NUS Research Scholarship. We acknowledge support by the KIT-Publication Fund of the Karlsruhe Institute of Technology.
This work was supported by the Deutsche Forschungsgemeinschaft (DFG), within the Research Training Group 2039 (B.S.F. and U.S.) and the Helmholtz Program Biointerfaces in Techology and Medicine (BIF-TM). U.S. and S.B. acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy via the Excellence Cluster 3D Matter Made to Order (EXC-2082/1 – 390761711). This research is supported by the Singapore Ministry of Health’s National Medical Research Council under its Individual Research Grant Scheme (NMRC/OFIRG/0026/2016 awarded to E.P.L.R.).
The authors declare no conflict of interest.