Introduction

The rapid emergence and widespread distribution of antibacterial resistance is recognized as one of the most serious global threats to human health1,2,3,4. Most antibiotics in clinical use are becoming sometimes ineffective in treating infections caused by either Gram-positive or Gram-negative superbugs5,6. Hence new antibiotics or alternative therapeutics are of a great importance for clinical treatments.

As an essential part of the innate immune system of nearly all living organisms, antimicrobial peptides (AMPs) are considered a promising therapeutic strategy in fighting bacterial infections7. With the rise of antimicrobial resistance and an urgent need for new antibiotics, AMPs have attracted more attention recently, resulting in 36 different AMPs undergoing clinical trials against various infectious diseases8. However, AMPs display a somewhat limited practical application due to rapid in vivo degradation and issues with systemic toxicity, as well as high production costs9. Poly-N-substituted glycines (peptoids), on the other hand, comprise a class of peptidomimetics where the side chains are attached to the backbone amide nitrogen rather than to the α-carbon10. Antimicrobial peptoids were first developed in 200311. Over the past two decades, various antimicrobial peptoid designs have proven to efficiently retain their antimicrobial activity, without displaying any apparent stability downsides12.

Over 4,000 halogenated compounds isolated from natural sources constitute a diverse group of natural products that display a wide range of biological activities, including anticancer and antimicrobial properties13,14. However, until recently, only limited attention has been given to the identification of antimicrobial properties of peptides and peptidomimetics containing halogen atoms. So far, fluorination has received the most interest, though the studies tackling the link between fluorination and antimicrobial activity have led to somewhat inconclusive results. For example, introduction of hexafluoroleucine into magainin and buforin conferred enhanced antimicrobial activity and retained low hemolytic properties, while the presence of fluorine atoms and trifluoromethyl groups improved the potency of short cationic peptides15,16,17. By contrast, incorporation of hexafluoroleucine into protegrin analogs led to decreased potency, while lipopeptides with fluorinated tails demonstrated moderate antibacterial activity combined with pronounced hemolytic properties18,19. Bolt et al. have also demonstrated that substitution of fluorine in peptoids led to enhanced antimicrobial activity against both Gram-positive and Gram-negative bacteria with no to low toxicity towards mammalian cells20. Recently, Molchanova et.al. reported the link between introduction of fluorine atoms into the peptidomimetic sequences and increased antimicrobial activity against Gram-positive bacteria without significantly enhancing hemolytic potential21,22. Importantly, both vancomycin and salinosporamid A require the presence of one to two chlorine substituents to achieve their antimicrobial activity23,24. Jia et al. introduced fluorine, chlorine, bromine and iodine atoms into the honeybee peptide Jelleine-1 via halogen-substituted phenylalanine which led to improved protease stability; a fluorinated analogue showed similar antimicrobial activity to the parent peptide while chlorinated, brominated and iodinated analogues displayed two to eightfold increase in their activities in vitro25. Interestingly, the in vitro antimicrobial activity of the iodine analogue was the highest, while the chlorinated and brominated versions displayed a more potent efficacy in vivo. Both chlorinated and brominated variants of antibiotic NAI-107 have demonstrated higher antimicrobial activity, where the brominated one displayed slightly higher potency26. Peptoid 1 is an example of a well-studied promising antimicrobial peptoid with a wide spectrum of antimicrobial activity, however it exhibits relatively high cytotoxicity in vitro (although it has been tested and was reasonably well tolerated intraperitoneally in vivo against S. aureus)27. A recent attempt to enhance the antimicrobial potency of Peptoid 1 has also been reported, however the incorporation of fluorine or chlorine atoms via Nspe units did not lead to any sufficient improvement in the antimicrobial profile28.

Introduction of halogens in the chemical structure of peptides or peptoids is known to generally increase the hydrophobicity of the molecules29. This may lead to conformational changes and self-assembly into supramolecular nanostructures, driven by increased hydrophobic interactions. Correlation between antimicrobial activity and self-assembly has been extensively discussed in the literature, where the impact on the antimicrobial properties and overall toxicity can trend in both directions30,31. While, for instance, Xu and co-workers found a link between increased antimicrobial activity and the self-assembly of defined supramolecular nanofibers, Chu-Kung and co-workers on the other hand found a clear tendency for their fatty acid conjugated peptides to show reduced antimicrobial activity32,33. Antimicrobial peptoids are significantly less prone to fold and form secondary structures, and to the authors’ knowledge, there have been no studies directly aimed to explore on the effect of self-assembly on the antimicrobial efficacy of peptoids.

In this study, we present the first structure–activity study of halogenated peptoids. The aim of the study is to investigate the link between the nature of the halogen, the amount of halogen substitution, their ability to self-assemble into nanostructures and their antimicrobial activity. Next, we proceeded to incorporate halogen atoms into the scaffold of the well-known Barron lab compound, Peptoid 134,35, in an attempt to increase its antimicrobial activity, or to modulate its cytotoxicity.

Results and discussion

Probing the link between halogenation and antimicrobial activity

We synthesized 36 peptoids using a scaffold containing alternating NLys and Npm units which vary by length (6-, 8-, 10-, 12-mers), and the level of halogen substitution (full or alternate). Halogen atoms (fluorine, chlorine, bromine or iodine) were introduced via phenyl rings in position 4 and synthesized using submonomer approach (Fig. 1).

Figure 1
figure 1

Structures of first-generation peptoids containing halogen atoms.

All 36 peptoids were tested against seven bacterial strains: five Gram-positive (Staphylococcus aureus ATCC 25923 and ATCC 29213, methicillin-resistant Staphylococcus aureus USA 300, methicillin-resistant Staphylococcus epidermidis ET-024 and ATCC 51625) and two Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa PA01) (Table 1). The non-halogenated peptoids demonstrated no potency against the selected bacterial strains. Thus, compounds 1, 10, 19, 28 (a 6-mer, 8-mer, 10-mer, and 12-mer respectively) represent the inactive controls.

Table 1 Minimal inhibitory concentrations (μg/mL) of the first generation peptoids containing halogen atoms.

The 36 compounds are divided in four sets according to their length. Each set contains a non-substituted control, four fully substituted peptoids with fluorine, chlorine, bromine or iodine, and four “half substituted” peptoids where every second phenyl ring is substituted with a halogen atom.

Halogenation had no effect on the activity of the peptoids against either E. coli and P. aeruginosa (Table 1). However, a clear correlation was observed between antimicrobial activity against Gram-positive strains, the level of substitution, and the nature of a halogen, among all sets. The fully halogenated peptoids demonstrated drastically enhanced activity against wild type and resistant strains of both S. aureus and S. epidermidis. Interestingly, for 6- (25) and 8-mers (1114) the activity rose from fluorine to iodine, where the latter was most potent. For the 6-mers, addition of just three iodine atoms led to up to > 64-fold increase against S. aureus (MIC for 1 =  > 512 μg/mL; for 5 = 8 μg/mL) and 256-fold increase against MRSE (MIC for 1 = 512 μg/mL; for 5 = 2 μg/mL), while 8-mer 14 exhibited the same activity against S. aureus, and 128-fold increase against MRSE (MIC for 10 = 256 μg/mL; for 14 = 2 μg/mL). However, moving to fully substituted 10-mer (2023) and 12-mer (2932) sets, the antimicrobial trend is lost as compounds bearing iodine atoms displayed lower potency against both S. aureus and multidrug resistant S. epidermidis compared to their chlorinated and brominated analogues. Bromination of the 10-mer led to > 256-fold and 128-fold increase of activity against S. aureus and MRSE compared to the unsubstituted control peptoid (S. aureus: MIC for 19 =  > 512 μg/mL; for 22 = 2 μg/mL; MRSE: MIC for 19 = 128 μg/mL; for 22 = 1 μg/mL), while a brominated 12-mer analogue displayed similar or lower activity against both S. aureus and S. epidermidis.

The “half-substituted” sets fell under a similar trend, though generally displaying similar or lower potency. Interestingly, the half-substituted peptoids bearing bromine exhibited comparable activity to their fully substituted analogues. For example, compound 26 showed MICs between 1–8 μg/mL versus 1–4 μg/mL for the fully substituted analogue 22.

As expected, the peptoids’ hydrophobicity increased with the addition of halogen atoms, where fluorine displayed a less pronounced effect while incorporation of iodine led to noticeably higher hydrophobicity profiles. In parallel, the peptoids’ antimicrobial activity fell into a well-established correlation, where increase in hydrophobicity led to an increased antimicrobial activity. However, when a certain hydrophobicity threshold was met (e.g. for compounds 23 and 32), the activity was lost, the phenomena that has been previously observed in other peptide/peptoid studies36,37. All in all, we could see that introduction of halogen atoms led to an increase of hydrophobicity that accompanies an increase of antimicrobial activity against Gram-positive bacteria. However, when a certain level of hydrophobicity was met, the activity of peptoids started decreasing.

Studying the effects of halogen substitutions on peptoid self-assembly in solution

To further understand the impacts of variation in length, size of halogen groups and degree of substitution, the nanostructures of these compounds were studied in detail using Small Angle X-ray Scattering (SAXS).

SAXS allows for the determination of whether these peptoids self-assemble into nanostructures or instead exist as single molecules in aqueous solution38,39,40,41. Furthermore, through detailed theoretical modelling, the techniques allow for an accurate estimation of molecular weight, shape and the overall physical structures of peptoid assemblies. The results revealed that the observed structures depend on the length and hydrophobicity of the various peptoids; and self-assembly into defined nanostructures was observed for a few of them. Scattering intensity is plotted as a function of the modulus of the scattering vector, q = 4πsin(θ/2)/λ, where λ is the wavelength of the X-rays and θ is the scattering angle (Fig. 2). It should be noted that 1/q has the dimension of length and the quantity represents a sort of ‘measuring stick’; at low q, large structures are probed, while at high q, SAXS is sensitive to more local structures.

Figure 2
figure 2

SAXS data showing the scattered intensity plotted towards the modulus of the scattering vector, q for 10-mer peptoids at 5 mg/mL obtained at BM29, ESRF at 37 °C. (A) displays the fully halogenated and (B) includes half-halogenated peptoids. The non-halogenated peptoid 19 is included in both graphs as a reference. Results indicate predominantly free unstructured peptoid chains with a small fraction of sheet-like filaments for compounds 19–21 and 24–27, and defined bundles for compounds 22 and 23, seen as an increase in the scattering intensity and a change in shape of the curve.

For the 10-mers (compounds 1927), the scattering curve for the fully brominated 22 and fully iodinated 23 peptoids exhibited significantly higher intensity and a different shape as compared to the rest of the 10-mers (Fig. 2A,B). The latter exhibited typical polymer-like scattering pattern for random (Gaussian) chains although an upturn at low q revealed a small fraction of larger structures or aggregates. The upturn follows a power law of ~ q−2 indicating plate-like fibers, and no larger aggregates that would typically follow the power law of ~ q−442. Through fit analysis of these data using a model with a combination of free chains and rectangular fibers, we obtained a radius of gyration (Rg) of the free chains of 7–9 Å, and a small mole fraction of only 0.001–0.0005% fiber-like sheets (see supporting information Table S1 for full list of fit parameters).

The scattering for the fully brominated 22 and all iodinated 23 did not exhibit the same upturn at low q and overall shape at intermediate and high q could therefore not be explained with the fit model described above. Instead, the scattering intensity exhibited a flatter q-dependence at low q indicating discrete, smaller nanostructures. The data from both compounds could be analyzed using a bundle model where cylinders representing folded/helical units are assembled into trimeric/tetrameric bundles39. The scattering of a concentration range from 5–0.6 mg/mL of both systems was analyzed simultaneously and the obtained fit parameters are listed in Table S1. The fit analysis also revealed the critical aggregation concentration (CAC) for the self-assembled structures and indicated a CAC value of 2.3 mg/mL and 0.4 mg/mL for the fully brominated 22 and iodinated 23, respectively (see supporting information Figure S1). The lower CAC value of 23 is a result of the high hydrophobicity as reported in Table 1. The increased hydrophobicity might provide an explanation of the observed loss of antimicrobial activity for the fully iodinated 10- and 12-mer (c.f. Table 1). These results suggest that a clear correlation between self-assembly and activity as seen by Chu-Kung and co-workers in the past32, cannot be drawn for these compounds as the estimated lowered CAC from SAXS is higher than the MIC values.

To further investigate if the self-assembly properties of the iodinated peptoids, we proceeded with the 6-, 8- and 12-mer of the fully iodinated peptoids (5, 14, 23, 32). The results are shown in Fig. 3 (see Table S2 for fit parameters). From the fit analysis, we found a CAC of 2.8 mg/mL for the fully iodinated 6-mer compound 5, 1.4 mg/mL for the 8-mer 14 and 0.4 mg/mL for the 12-mer 32.

Figure 3
figure 3

Full SAXS results for the fully iodinated peptoids measured with Bruker NANOSTAR instrumentation (except the 10-mer 23 measured at ESRF) in the indicated concentrations together with model fits (fitted using the bundle model as described in the supplementary information). Panels A, B, C and D shows the 6-mer (5), 8-mer (14), 10-mer (23) and 12-mer (32) respectively.

These results show that even at very short lengths, these peptoids self-assemble in the probed concentration range due to their high hydrophobicity, which is evident from the retention times in Table 1. The detected CAC is highly correlated with the length of the peptoid. However, as seen from the MIC values these shorter peptoids are highly active compared to the 10-mer and 12-mer, indicating a potential threshold of hydrophobicity. Furthermore, the detected CAC is still highly correlated with the length of the peptoid, but a link between CAC and MIC could not be drawn. This is in contrast with the fatty acid conjugated peptides studied by Chu-Kung and co-workers, who found a correlation32. The reduction of activity seen for the 10-mer and 12-mer is therefore likely related to the increased hydrophobicity, indicating that there is a threshold to stay within than the self-assembly properties themselves. However, further studies into the consequences of hydrophobicity and self-assembly with the activity and toxicity for peptoids are needed to fully explain the observed trends.

Halogenation as a tool to improve antimicrobial activity

In order to investigate whether halogens can be used as a tool to improve the antimicrobial potency of a known peptoid, we chose the well-studied Peptoid 1 and synthesized two small libraries of chlorinated and brominated analogues as these halogens demonstrated overall higher potency compared to the fluorinated ones, and iodine variants raised the concern of aggregation and loss of activity. Both halogen atoms were introduced via Nspe units in position 4 on the phenyl rings. The level of substitution varied between full substitution (all phenyl rings bear a halogen atom) and “half” substitution (every other phenyl ring bears a halogen atom). This strategy yielded six chloro- and six bromo- modified versions of Peptoid 1 (Fig. 4).

Figure 4
figure 4

Chlorinated and brominated variants of peptoid 1 and shortened brominated peptoid 1 analogues.

The library of 12 compounds (3748) was tested against the same panel of bacterial strains as the first generation of peptoids. However, none of the modifications led to increased potency, while most caused a loss of activity (Table 2). Judging from long HPLC retention times, this could be explained by the fact that the critical hydrophobicity level was reached, which causes aggregation and loss of activity similar to what we have seen for compounds 23 and 32, as the highest activity was observed for the compounds with lower retention times. Interestingly, compounds 38, 4042 have the same number of chlorine atoms, but their hydrophobicity differ. For the brominated derivatives 44, 4648, this effect was even more pronounced. It shows that even distribution of halogen atoms across the peptoid chain led to higher hydrophobicity compared to positioning chlorine or bromine atoms at the ends or the middle of the peptoid sequence.

Table 2 MICs (μg/mL) of the chlorinated and brominated analogues of Peptoid 1.

Short brominated Peptoid 1 analogues

As seen from the increased values of retention times, we have hit the hydrophobicity ceiling during the introduction of halogens into the sequence of Peptoid 1 (Table 2), hence we decided to shorten the length of Peptoid 1 from twelve to six residues, cutting its length in half, and synthesized a small library of four analogues with different levels of halogenation (Fig. 4). As seen from Tables 1 and 2, introduction of fluorine didn’t result in the desired increase of activity, while bromination had a more pronounced effect on the hydrophobicity than chlorination and similar to one displayed by the iodine-containing peptoids. Hence, three brominated short Peptoid 1 analogues (4951) and one non-halogenated peptoid Pep1-6mer were synthesized and tested against the same bacterial strains as the previous peptoids. Data are shown in Table 3 where the new data set is compared with the original Peptoid 1 data27.

Table 3 MICs (μg/mL) and IC50 (μg/mL) towards HaCaT cell line of short brominated Peptoid 1 analogues.

Compound 49 has only one terminal bromine, 50 has two bromine atoms and for 51—all four phenyl rings are substituted with a bromine atom. The addition of just two bromine atoms was enough to improve the activity of a non-active short analogue, the Pep1-6mer, 16–32-fold against both S. aureus and S. epidermidis. Incorporation of two extra bromines yielding four in total failed to significantly affect the activity. Addition of one terminal halogen atom, on the other hand, was already enough to see a several-fold increase in antimicrobial activity. Despite the previous data, where introduction of halogens did not improve the activity against Gram-negative bacteria, in this case addition of two bromines was enough to reach sufficient hydrophobicity in order to obtain an MIC against P. aeruginosa that is close to the original Peptoid 1 and reach 32-fold increase of activity against E. coli compared to the Pep1-6mer.

As we have shown that the incorporation of halogens can improve the activity of inactive peptoids, we decided to examine their cytotoxicity profiles. Peptoid 1, Pep1-6mer and the three brominated analogues were tested towards a HaCaT cell line for 1 h. The results were obtained using MTS/PMS assay (Fig. 5, Table 3).

Figure 5
figure 5

IC50 curves of the selected peptoids toward HaCaT cell line.

We have observed a clear trend between the number of halogen atoms and corresponding increased cytotoxicity, however while compound 50 demonstrated the same antimicrobial activity profile to Peptoid 1 it was less cytotoxic with IC50 146.9 μg/mL versus only 35.0 μg/mL for the latter. All in all, initial results indicate reduced cytotoxicity of the brominated analogues when compared to Peptoid 1, though compounds 50 and 51 displayed similar activity and hydrophobicity profiles.

Conclusions

In this study, we have investigated the effect of fluorine, chlorine, bromine and iodine on the antimicrobial activity of peptoids. First, using an inactive model (Nlys-Npm)n peptoid scaffold, we have identified that incorporation of chlorine or bromine led to an improvement of antimicrobial activity against Gram-positive bacteria, while fluorination did not display any pronounced effect. Introduction of iodine in 6- and 8-mers analogues dramatically increased the activity, but led to loss of activity due to aggregation in 10- and 12-mers. Afterwards, we attempted to improve the antibacterial potency of Peptoid 1 by incorporating chlorine or bromine atoms via Nspe units, which led to overall loss of activity. We interpret this oberservation as sign of the hydrophobicity limits being reached. However, bromination of a shorter inactive 6-mer analogue of Peptoid 1 resulted in the same activity as the 12-mer Peptoid 1 against some bacteria, while noticeably improving its cytotoxic profile.

Therefore, here we have shown that halogenation, and particularly bromination, can be used to readily modify and alter the physicochemical and antibacterial properties of peptoids but the effect strongly depends on the choice of the halogen. In addition, the effect is quite sequence- and length- specific, and inclusion of halogens can also lower antimicrobial activity.

Methods

General information

Solvents, amines, and resins were purchased from Iris Biotech, Sigma‐Aldrich, and Merck and used without further purification. For compounds 1–48: purity was determined as described previously49, by analytical HPLC using a Waters 717 plus Autosampler, In-line Degasser AF, 600 Controller, and 2,996 Photodiode Array Detector; the column used was a Waters Symmetry C18, 5 µm, 4.6 mm × 250 mm. An aqueous acetonitrile (MeCN) gradient with 0.1% trifluoroacetic acid (TFA) added (eluent A: 5:95 MeCN‐H2O + 0.1% TFA, eluent B: 95:5 MeCN‐H2O + 0.1% TFA) was employed using water filtered through a 0.22‐μm Millipore membrane. All tested compounds had a purity of at least 95%. Preparative HPLC was performed by using a Waters XSelect Peptide CSH C18 OBDTM, 5 µm, 19 mm × 250 mm column and the same eluents as for analytical HPLC. High‐resolution mass spectrometry (HRMS) spectra were obtained by using a Waters QTOF premier mass spectrometer equipped with an electrospray ionization source and a Quadruple and time of flight MS detector. For compounds 49–51, pep1-6mer and peptoid 1: Product purity was determined by means of analytical UPLC/MS using a Water Acquity UPLC system, equipped with an Acquity Diode Array UV detector and a Waters SQD2 mass spectrometer. As stationary phase, a Waters Acquity UPLC Peptide BEH C18 Column (300 Å pore size, 1.7 µm particle size, 2.1 mm × 100 mm) with an Acquity UPLC BEH C18 VanGuard pre‐column (1.7 μm, 2.1 mm × 5 mm) was employed. Purification by means of preparative HPLC was carried out using a Waters Prep150LC system, equipped with a Waters 2,489 UV/Visable detector and a Waters Fraction Collector III collector. As stationary phase, a Waters XBridge BEH300 Prep C18 column (5 μm particle size, 19 mm × 100 mm) with a Waters XBridge Peptide BEH300 C18 guard column (5 μm particle size, 19 mm × 10 mm) was employed.

General synthesis of peptoids

All peptoids have been synthesized manually on Rink amide resin (Novabiochem, 0.65 mmol/g) according to the submonomer method10. After synthesis, oligomers were cleaved and deprotected in trifluoroacetic acid (TFA)/triisopropylsilane/water (95:2.5:2.5 by vol.) for 30 min.

Determination of minimal inhibitory concentration

Bacterial growth inhibition was determined by using broth microdilution according to the Clinical Laboratory Standards Institute43. The antibacterial activity of peptoids was tested against S. aureus ATCC 25923, ATCC29213, and methicillin resistant S. aureus USA 300, methicillin resistant S. epidermidis ATCC 51625, a biofilm producing methicillin resistant S. epidermidis ET-02444, P. aeruginosa Pa01 (H103), and E. coli ATCC 25922. Bacteria, grown on agar plates for 18 h at 37 °C, were diluted to 1 × 108 CFU/mL in Mueller‐Hinton Broth II (MHB II). Twofold serial dilutions of peptoids in MHB II were inoculated with bacteria to achieve a final concentration of 5 × 105 CFU/mL in polypropylene 96 U‐well microtiter plates (Corning 3,897; ThermoFisher Scientific, Roskilde), followed by incubation at 37 °C in ambient air for 18 h. The MIC values were determined as the lowest concentration showing no visible bacterial growth. Experiments were performed twice (in technical triplicates) on 2 different days.

Small angle X-ray scattering

SAXS experiments of the 10-mer peptoids were performed at the automated BM29 bioSAXS beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France45. The data was obtained using an energy of 12.5 keV and a detector distance of 2.87 m, covering a q range (\(q=4\pi \mathrm{sin}(\theta /2)/\lambda\)), where \(\theta\) is the scattering angle and \(\lambda\) is the X-ray wavelength) of about 0.0047 to 0.5 Å−1. The data set was calibrated to an absolute intensity scale using water as a primary standard. 40 µL samples were run through a capillary using the flow mode of the automated sample changer46. SAXS data was collected in ten successive frames of 0.5 s each to monitor radiation damage and the data reduction was done using the standard tool at BM2947.

The SAXS experiments on the fully iodinated 6-, 8-, and 12-mers to determine the CAC of the compounds were performed using a Bruker NANOSTAR equipped with a microfocus X-ray source (IμS Cu, Incoatec, Germany) and a VÅNTEC-2000 detector. Raw scattering data was calibrated to absolute intensity scale using water as a primary standard and radially averaged in order to obtain the 1D scattered intensity profile as a function of the scattering vector, with a wavelength of 1.54 Å. Two concentrations of compound 23 and 19 was also run on the NANOSTAR to verify that the results were comparable with the results from synchrotron SAXS at ESRF.

The modelling fit analysis of the scattering data is explained in detail in the supplementary information.

Cell culturing

An immortalized human keratinocytes (HaCaT) cell line (Gift from David G. Naym at Bispebjerg Hospital) were cultured to 90% confluence after 21–25 h of growth at 5% CO2 and 37 °C as previously48. In brief, the cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 μg/mL), purchased from Sigma-Aldrich (St. Louis, MO, United States).

Cell viability assay

Cell viability assessment was performed on cell monolayers grown to 90% confluence in 96-well plates Corning Costar (Sigma-Aldrich, Brøndby, Denmark) by using the MTS/PMS assay as previously described48. Briefly, the adhered cells were washed with 37 °C PBS solution (ThermoFisher Scientific, Roskilde) and exposed for 1 h at 37 °C to 100 μL of peptoids dissolved in the medium also used for culturing of the cell line (at concentrations in the range 0–1,000 μg/mL). The precise exposure-time were selected to enable comparison with related peptoid compounds21,35. Then the cells were washed twice with 37 °C PBS and then 100 μL of an MTS/PMS solution in media, consisting of 240 μg/mL MTS (Promega, Madison, WI, United States) and 2.4 mg/mL PMS (Promega, Madison, WI, United States), were added to the cells, which then were incubated for 1 h at 37 °C protected from light. A plate reader (SpectraMax i3X; Molecular devices, San Jose, CA) was used to measure the absorbance at 492 nm. The relative viability was calculated by using 0.2% (w/v) sodium dodecyl sulfate (SDS) as the positive control, while cells exposed to medium without test compound were used as the negative control. Data were obtained in three independent biological replicates performed on separate passages of cells and on separate days with a total number of six replicates.