Real‐Time Monitoring of Multitarget Antimicrobial Mechanisms of Peptoids Using Label‐Free Imaging with Optical Diffraction Tomography

Abstract Antimicrobial peptides (AMPs) are promising therapeutics in the fight against multidrug‐resistant bacteria. As a mimic of AMPs, peptoids with N‐substituted glycine backbone have been utilized for antimicrobials with resistance against proteolytic degradation. Antimicrobial peptoids are known to kill bacteria by membrane disruption; however, the nonspecific aggregation of intracellular contents is also suggested as an important bactericidal mechanism. Here,structure‐activity relationship (SAR) of a library of indole side chain‐containing peptoids resulting in peptoid 29 as a hit compound is investigated. Then, quantitative morphological analyses of live bacteria treated with AMPs and peptoid 29 in a label‐free manner using optical diffraction tomography (ODT) are performed. It is unambiguously demonstrated that both membrane disruption and intracellular biomass flocculation are primary mechanisms of bacterial killing by monitoring real‐time morphological changes of bacteria. These multitarget mechanisms and rapid action can be a merit for the discovery of a resistance‐breaking novel antibiotic drug.

6. Effect of counter-ion exchange on antimicrobial, hemolytic, and cytotoxic activity Real-time monitoring of E. coli treated with peptoid 29 using 3D optical diffraction tomography 10. Representative fluorescent images and RI-based 3D rendered images of E. coli treated with melittin, buforin-II, and peptoid 1 in the presence of thioflavin

List of Figures and Tables
Peptoid synthesis and monomer structures . HPLC chromatograms of counter-ion exchanged peptoids with UV detection at Real-time monitoring of E. coli treated with 29 (1 MIC) using 3D optical Real-time changes in oxygen consumption rate in response to treatment with peptoid 29, ampicillin (Amp), and chloramphenicol (Cam) in E. coli measured on a Seahorse XFe 96 extracellular flux analyzer.
When the cell density reached approximately 70% confluency, the medium was replaced with serial dilutions of the peptoid stock in DMEM and incubated for an additional 24 h.At this point, 20 µL of the CellTiter 96 aqueous non-radioactive cell proliferation assay reagent (Promega, Madison, WI, USA), which contains the tetrazolium compound, was added to each well.Plates were incubated for 4 h at 37 °C to allow time for metabolism.The MTSformazan products in viable cells were measured at 490 nm using a microplate reader, and the percentage of cell viability (%) was calculated by comparing readings to the values of untreated wells.Percentage values of cell viability were calculated as A/(A control)×100, where A is the absorbance of the test well and A control is the average absorbance of wells containing untreated cells.

Structure-activity relationship (SAR) analysis
Peptoids 1 and 2 with known α-helical or non-helical structures, respectively, were used as references. [5]Using the helical peptoid 1 as a lead sequence, NTrp was either added

S15
Previously, we observed that an increased cationic charge in antimicrobial peptoids generally led to selective interaction with the negatively charged bacterial membrane and simultaneously reduced toxicity against eukaryotic cells. [6]Peptoids 24-27 had one additional NLys due to the sequential replacement of an Nspe in peptoid 9.While these substituted compounds maintained antimicrobial activity against S. aureus, their antimicrobial activity against E. coli decreased.At the same time these compounds showed reduces hemolysis.
Peptoids 11 and 14 exhibited potent antimicrobial activity.The cationic charge of these compounds was increased by replacing one Nspe with an NLys residue, providing peptoids 28-31 and 32-34, respectively.These seven peptoids had an increased cationic charge and contained a WW motif.Among these, peptoids 29 and 32 retained antimicrobial activity while significantly increasing selectivity (i.e., selectivity index of >15.9 for 29 and 12. Notably, the peptoids in this library were mostly active against gram-positive S. aureus with MIC values in the range of 0.8 -3.1 μM.However, we initially focused on the activity against gram-negative E. coli and selectivity (i.e., lack of hemolytic activity) to select the best peptoid.These considerations resulted in the selection of peptoids 29 and 32 as hit compounds in this library screening.
To elucidate the role of the indole side chain, peptoids 42-47 were synthesized as controls of peptoids 5, 6, 11, 29, 14, and 32, respectively, where NTrp(s) was (or were) substituted with Npm(s).Interestingly, the two best peptoids in the library, 29 and 32, lost the antimicrobial activity when the WW motif was replaced by two phenyls (two Npm's) as demonstrated by 45 and 47, respectively.This result highlights the importance of two adjacent NTrp residues (WW motif) in the antimicrobial activity of 29 and 32.
In case of peptoids 48-64, further variations in lengths, positions, and the number of NLys or NTrp monomers were made.Finally, in peptoids 65 and 66, the NLys residues of 23 and 29 were substituted by the hydroxyl containing N4hb residues, resulting in the complete loss of antimicrobial activity.

Effect of counter-ion exchange on antimicrobial, hemolytic, and cytotoxic activity
Counter-ion exchange procedure For the counter-ion exchange, trifluoroacetate (TFA) ions were removed using a carbonate ion-exchange resin (VariPure columns, Agilent, Santa Clara, CA, USA) according to the manufacturer's instruction and following previously reported procedures. [7]gure S4.Quantitative analysis of 19 F-NMR after counter-ion exchange The effectiveness of counter-ion exchange was verified using 19 F-NMR spectroscopy by measuring the amount of fluorine in the residual TFA (Figure S4).Quantitative NMR analysis was performed for peptoid 1 (1 (as a TFA salt), 1-HCl, and 1-AcOH), and the amount of fluorine in 1-HCl and 1-AcOH was quantified by comparing the peak area of 19 F of TFA.In the prepared NMR solution, the fluorine molarity ratios were 2.88, 0.031, and 0.014 in 1 (TFA), 1-HCl, and 1-AcOH, respectively.These tests confirmed that ~99% of

HPLC chromatogram, retention time (t R ), and LC-MS data
HPLC characterization HPLC analysis of peptoids was conducted using a Waters HPLC system equipped with a Waters 2489 UV/Visible Detector, 1525 Binary HPLC Pump, 2707 Autosampler, 5CH column oven, and a C18 column (SunFire C18, 4.6 × 250 mm, 5 μm; Waters Corp., Milford, MA, USA).The mobile phases were deionized water (A, with + 0.1% TFA) and acetonitrile (ACN; B, with + 0.1% TFA).Before sample injection, the column was conditioned with 5% B for 10 min.Then, the mobile phase was maintained at 5% B for 2 min, followed by increasing B to 100% using a linear gradient over 30 min.At this point, 100% B was maintained for 5 additional min.The flow rate of the mobile phase was 1 mL/min.The sample was detected by measuring absorbance at 220 nm.The chromatograms of the peptoids used in this study are presented in Figure S5.
The absorbance was measured at 220 and 254 nm to monitor sample elution.Analytical HPLC confirmed the purity of the peptoid products in each fraction.Fractions containing the pure product (>96 % purity) were collected, lyophilized and stored at -80 °C.
Reversed-phase HPLC chromatograms of purified peptoids and their retention times are shown in Figure S6.Changes in HPLC retention times allowed the approximate comparison of the hydrophobicity of peptoids (Table S1).Starting from peptoid 1, the addition of a NTrp residue resulted in increased retention time (3-6)  S1), which leads to poor aqueous solubility of the two peptoids.
The retention time of peptoid 1 (t R = 54.5 min) was similar to that of melittin (t R = 55.4 min), interestingly both causing severe membrane lysis in eukaryotic cells.The retention time of peptoid 29 was comparable to that of omiganan (t R = 45.7 min) and both of these were relatively selective.

S21
The identity of the synthesized peptoids was confirmed by electrospray ionization-mass spectrometry (Table S4).

Bacterial respiration assays
Cellular oxygen consumption rate (OCR) of E. coli ATCC 25922 was measured using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent) as published previously. [9]Briefly, after overnight culture in LB, bacterial cells were diluted 1:200 into fresh M9 media (M6030, Sigma) and incubated for an additional 3 h at 37 °C.To conduct the measurements, bacteria were diluted to OD 600 of 0.02 in M9 supplemented with 10 mM glucose.90 μL of this cell suspension was loaded into a 96-well microplate precoated with poly-D-lysine (100 μg/mL, Sigma).Microplates were centrifuged for 10 min at 2,200 rpm to attach the cells, and 90 μL additional media was added to each well.To monitor uniform cell seeding, OCR baseline measurements were taken for two cycles before the automated injection of the indicated peptoid or antibiotics.OCR values were quantified every 2.5 min (mix and measure for 30 sec and 2 min, respectively) for an additional 48 cycles, and normalized to bacterial density measured at OD600.

Killing kinetics
The kinetics of antimicrobial activity against E. coli (ATCC 25922) were assessed at a peptoid concentration corresponding to 1×, 2× and 4 × MIC.Briefly, an overnight culture of bacteria was diluted at mid log-phase bacteria (2-5 × 10 5 cfu/mL) and incubated with peptoid with desired concentration in MHB2.The bacterial suspension was added to a 96-well polypropylene u-bottomed plate containing the peptoid.The plate was incubated without shaking at 37 °C for 4 h.Samples (20 μl) were taken at time 0.5, 1, 2 and 4 h and diluted in ice PBS buffer from which 100 μl was plated on LB agar plates.The plates were incubated for 18-24 hours at 37 °C and colony forming units (CFU) were counted.The experiment was conducted triplicates, and a curve was plotted between CFU and time (h).

NPN uptake assay
The ability of peptoids to increase the permeability of the outer membrane of Gram-

Figure S10 .Figure S11 .
Figure S10.Quantitative analysis of time-lapse monitoring for a) volume, b) mass, and c) biomolecular density of control or 29 (0.5 MIC)-treated E. coli at 1.5 min intervals over

1 .
(3-6) or substituted(7-23).The number(1, 2, 4, and 8 NTrps) and positions (N-or C-termini or central) of the added or substituted NTrp varied to determine the correlation between NTrp and antimicrobial activity.In peptoids 3 and 4, one NTrp was added to the N-terminus or Cterminus, respectively.In 5 and 6, two NTrp's were added to both termini or to the Nterminus.In peptoids 7 to 10, a single NTrp substituted an Nspe in peptoid 1 in a sequential order from the C-to N-terminus.Four peptoids, from 11 to 14, contained two adjacent NTrp residues by substituting two Nspe's in the same sequence of substitutions.In 15-17, two non-adjacent Nspe's were substituted by two NTrp's.As the compound number increases, the distance between the two NTrp's becomes greater.Five peptoids, 18-22, contained four NTrp's.Peptoid 22 had four repeats of NLys-Nspe-NTrp unit and did not contain adjacent NTrp residues.Finally, all Nspe's were substituted with eight NTrp residues in peptoid 23.When the NTrp residue was added at the N-or C-terminus (3-6) of peptoid 1, MIC values for E. coli generally increased (except for 3), but these peptoids showed decreased selectivity due to severe hemolysis.Peptoids 9 and 10, with one NTrp substitution, exhibited increased antimicrobial activity (MIC = 3.1 μM) with a marginal increase in selectivity over peptoid 1.Interestingly, peptoid 14, containing two adjacent NTrp's (WW motif) as an N-terminal substitution, showed increased antimicrobial activity and selectivity, but peptoid 11 containing two NTrp's at the C-terminus had a very similar MIC and selectivity index as peptoid Peptoids containing two separate NTrp's without a WW motif (15-17) exhibited the same MIC values as peptoid 1 with slight gains in selectivity.Substitutions containing more than two NTrp residues (18-23) did not show any increase in activity or selectivity.Peptoids 3-23 contained four NLys residues (i.e., fixed cationic charges) and showed HPLC elution at 53~58% MeCN indicating increased hydrophobic character of these peptoids.This resulted in strong hemolytic activity and poor selectivity.The MIC of the 12mer peptoids 7-23 (with the same CTLR value of 0.33) was analyzed to determine the effect of the number of NTrp monomer(s) on the antibacterial action against E. coli (FigureS3).This series of experiments showed that peptoids containing one or two NTrp monomer(s) exhibited more potent antimicrobial activity against E. coli.

Figure S3 .
Figure S3.Effect of the number of NTrp monomer(s) on the E. coli MIC value.Peptoids 7 -23 were analyzed, representing sequence variants of peptoid 1 with the same length (CTLR = 0.33).

Figure S5 .
Figure S5.HPLC chromatograms of counter-ion exchanged peptoids with UV detection at 220 nm . Substitution of Nspe with NTrp did not result in a definite trend in retention time change.As expected, additional cationic charge by the incorporation of NLys residue or the substitution of Nspe with NLys always led to a decreased retention time.For example, peptoid 29 (t R = 48.6 min) had decreased retention time compared to peptoid 11 (t R = 54.0 min).Notably, substitution of NLys amine groups with N4hb hydroxyl groups resulted in a significant increase in retention time as shown in peptoid 63 (t R = 66.7 min) and 64 (t R = 64.7 min) (Table

Figure S10 .
Figure S10.Quantitative analysis of time-lapse monitoring for a) volume, b) mass, and c) biomolecular density of control or 29 (0.5 MIC)-treated E. coli at 1.5 min intervals over

Figure S15 .
Figure S15.SAXS data revealing the concentration dependence of the self-assembled structure of peptoids.SAXS data on peptoids in aqueous environment at different peptoid concentrations (indicated in legends), plotted together with best fit.a) 11 b) 29.
negative bacteria was determined by measuring the incorporation of the NPN fluorescent dye into the outer membrane of E. coli ATCC 25922.Bacterial cells were suspended to a final concentration of OD 600 = 0.05 in 5 mM HEPES buffer, pH 7.2, containing 5 mM KCN.Then S36 200 μL NPN dye was added to wells of a 96 well plate to produce a final concentration of 10 μM, and the background fluorescence was recorded (λ ex = 350 nm, λ em = 420 nm).Aliquots of peptoids were added to the well, and fluorescence was recorded as a function of time until there was no further increase in fluorescence.Increases of the permeability of the outer membrane after the addition of peptoids was reflected by increased fluorescence values as a result of NPN incorporation into the membrane.ONPG hydrolysis assayThe permeability of the inner membrane was determined using E. coli ML-35 cells exhibiting β-galactosidase activity.The intact bacterial inner membrane is non-permeable to o-nitrophenyl-β-D-galactopyranoside (OPNG), a chromogenic substrate of the β-galactosidase.Following damage to the inner membrane, OPNG enters the cells and is metabolized by βgalactosidase creating a chromogenic substrate.E. coli cells were washed in 10 mM sodium phosphate (pH 7.4) containing 100 mM NaCl and resuspended in the same buffer at a final concentration of OD 600 = 0.5 containing 1.5 mM ONPG.The hydrolysis of ONPG and generation of o-nitrophenol over time were monitored as absorbance change at 405 nm following the addition of peptoids.S9 fraction assayThe resistance of peptoids against metabolic enzyme degradation was measured by an S9 fraction assay.Human liver S9 fraction is the 9000 g supernatant of a human liver homogenate containing mainly cytochromes P450 (CYPs 450), uridine 5'diphosphoglucuronosyltransferase (UGTs), and various cytosolic enzymes.The peptoid was dissolved in autoclaved water, added to 100 mM pH 7.4 Tris-HCl buffer solution containing 3.3 mM magnesium chloride, S9 fractions, and nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor stimulating metabolism by CYPs 450.The prepared solution was incubated at 37 o C overnight.The desired amount of this solution was transferred into a quenching solution (MeCN + 0.1% TFA) at each specific time point.Quenched samples were centrifuged for 5 minutes at 1200 rpm and analyzed by analytical HPLC or LC-MS.Protein extraction and quantification using Bradford assay Bacteria were incubated overnight and sub-cultured when the optical density of the culture at λ = 600 nm reached at 0.5.The cultures were transferred to 50 mL conical tubes, and S37 bacteria were pelleted at 4,000 rpm for 10 minutes at 8 o C. The supernatant was removed and the pellet was suspended in 2 mL of PBS buffer.This suspension was transferred to a 15 mL conical tube and cooled on ice.The culture was sonicated for 5 sec and cooled again for 55 sec.This cycle was repeated 15 times.The lysate was centrifuged at 4,000 rpm for 20 minutes at 8 o C. The supernatant was transferred into a fresh 2 mL Eppendorf tube and stored at -80 o C. Protein quantification was performed using TaKaRa Bradford Protein Assay Kit according to the manufacturer's instructions.DNA extraction and quantificationDNA extraction was performed using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 according to the manufacturer's instructions.Protein and DNA aggregation assayThe flocculation of the proteins and DNA caused by peptoids was confirmed by the change in the fluorescent intensity of thioflavin T (Th T) that detects aggregated proteins and double stranded DNA.The appropriate combination of 500 μg/mL of protein solution, 50 ng/mL of genomic DNA, 25 μM of Th T, and 25 μM of peptoid 29 were added into a black 96-well plate in triplicate and incubated at 37 °C for an hour.After incubation, the fluorescence was measured using a microplate reader (excitation wavelength = 450 nm, emission wavelength = 485 nm).

Table S2 .
Antimicrobial and hemolytic activities of 1 − 38 a These concentrations represent mean values in triplicate.b HC 10 and HC 50 are the concentrations of compounds causing 10% and 50% hemolysis in rat erythrocytes, respectively.These concentrations represent mean values in triplicate.c H max is the percentage (%) of hemolysis at the highest concentration tested (100 µM).d The selectivity index was calculated as HC 10 divided by the minimum inhibitory concentration (MIC) values in E. coli ATCC 25922.e Not determined.

Table S2 (
continued).Antimicrobial and hemolytic activities of 39-66 a These concentrations represent mean values in triplicate.b HC 10 and HC 50 are the concentrations of compounds causing 10% and 50% hemolysis in rat erythrocytes, respectively.These concentrations represent mean values in triplicate.c H max is the percentage (%) of hemolysis at the highest concentration tested (100 µM).d The selectivity index was calculated as HC 10 divided by the minimum inhibitory concentration (MIC) values in E. coli ATCC 25922.e Not determined.

Table S3 .
Antimicrobial, hemolytic, and cytotoxic activities of counter-ion-exchanged These concentrations represent mean values in triplicate.b HC 10 and HC 50 are the concentrations of compounds causing 10% and 50% hemolysis in rat erythrocytes, respectively.These concentrations represent mean values in triplicate.c H max is the percentage (%) of hemolysis at the highest concentration tested (100 µM).d The selectivity index was calculated as HC 10 divided by the minimum inhibitory concentration (MIC) values in E. coli ATCC 25922.e LC 50 are the concentrations of compounds causing 50% lethality in the cells.
f nd f a f Not determined.