Cyclization Improves Membrane Permeation by Antimicrobial Peptoids

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Cyclization Improves Membrane Permeation by Antimicrobial Peptoids Konstantin Andreev, Michael W. Martynowycz, Andrey Ivankin, Mia L. Huang, Ivan Kuzmenko, Mati Meron, Binhua Lin, Kent Kirshenbaum, and David Gidalevitz Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03477 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 29, 2016

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Cyclization Improves Membrane Permeation by Antimicrobial Peptoids Konstantin Andreev†, Michael W. Martynowycz† § ∆, Andrey Ivankin†#, Mia L. Huang‡ǁ, Ivan Kuzmenko§, Mati Meron┴, Binhua Lin┴, Kent Kirshenbaum‡, and David Gidalevitz†* †

Department of Physics, Center for Molecular Study of Condensed Soft Matter (μCoSM),

Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology, 3440 South Dearborn Street, Chicago, Illinois 60616, United States ‡

Department of Chemistry, New York University, 100 Washington Square East, New York,

New York 10003, United States §

Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Lemont,

Illinois 60439, United States ┴

The Center for Advanced Radiation Sources (CARS), University of Chicago, Chicago, Illinois

60637, United States Antimicrobial peptoids • bacterial membranes • cyclization • lipid monolayers • surface X-ray scattering

ABSTRACT: The peptidomimetic approach has emerged as a powerful tool for overcoming the inherent limitations of natural antimicrobial peptides, where the therapeutic potential can be 1 ACS Paragon Plus Environment

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improved by increasing selectivity and bioavailability. Restraining conformational flexibility of a molecule may reduce the entropy loss upon its binding to the membrane. Experimental findings demonstrate that cyclization of linear antimicrobial peptoids increases their bactericidal activity against Staphylococcus aureus, while maintaining high hemolytic concentrations. Surface X-ray scattering shows that macrocyclic peptoids intercalate into Langmuir monolayers of anionic lipids with greater efficacy than their linear analogues. It is suggested that cyclization may increase peptoid activity by allowing the macrocycle to better penetrate through bacterial cell membrane.

1. INTRODUCTION The development of new antimicrobial therapeutics is of paramount clinical importance. 1 Host defense peptides (HDPs) of the innate immune system have been shown to exhibit broad spectrum antimicrobial activity.2,

3

However, HDPs are highly susceptible to proteolytic

degradation and potentially toxic against mammalian cells. 4,

5

Emerging research avenues are

focusing on de novo designed molecules that can mimic the structure and function of HDPs and may serve as a viable alternative to conventional antibiotics. 6, approaches,8,

9

7

Among multiple synthetic

peptoids, (oligo-N-substituted glycines) are among the most prominent

compounds with improved antimicrobial activity and minimal cytotoxicity,10, 11, 12 which have been shown to maintain their effectiveness in vivo.13, 14 In order to rationally design and then optimize these new candidates for future pharmaceutical applications, a better understanding of their structure-function relationships is required.15 The antibacterial activity of peptides displayed in vivo and in vitro is often governed by their interaction with bacterial membranes. 16, 17 The cellular membrane of pathogens acts as a primary barrier for antimicrobial molecules, which must be either disrupted or traversed. 18,

19

Unlike 2

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mammalian cells, bacterial membranes contain a high amount of negatively charged components such as lipopolysaccharides (Gram-negative) and teichoic acids (Gram-positive). Cationic charge of antimicrobial agents facilitates their selective initial binding to bacterial cell surface, while the amphipathicity enables their incorporation into the membrane. 20, 21, 22 Insertion of antimicrobials requires overcoming entropic and enthalpic energy barriers associated with the loss of molecular conformational freedom and creation of space within the lipid bilayer required to accommodate the molecules.23 Membrane activity of molecules can therefore be modulated via tweaking their conformational rigidity, thereby altering the depth of entropic barrier well. 24, 25 A straightforward and simple way to restrict the flexibility of a linear molecule is to constrain the termini via formation of a cyclic backbone.26 Importantly, the cyclization strategy allows modifying the flexibility of a molecule in an orthogonal manner such that the charge, amphipathicity, and molecular volume remain unchanged. Recently, cyclic antimicrobial compounds mimicking natural HDPs such as: γ-AApeptides,27 D,L-α-peptides28 and peptoids29,

30

have been designed and synthesized. Cyclization has been

shown to enhance activity of Arg- and Trp-rich hexapeptides by improving their ability to permeate through the outer and inner membranes of E. coli.31 Oren et al. reported that cyclization of amphipathic α-helical peptides increases their capability to insert into negatively charged phospholipid bilayers.32

Molecular dynamics simulations have also suggested that cyclic

peptides may embed deeper into the membrane bilayer and form toroidal pores while their linear counterparts remain at the surface demonstrating reduced antimicrobial activity. 33, 34 Similarly, peptoids have been shown to benefit from cyclization displaying superior in vitro bacterial growth inhibition to their linear analogues. 35 However, the underlying molecular mechanisms of this phenomenon remain poorly understood. Here, in order to enable the rational

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use of cyclization in the design of peptoid-based antimicrobials, we explore how this parameter impacts membrane activity of peptoids by comparing two pairs of linear and cyclic peptoid molecules (Figure 1), hereafter referred to as L1/C1 and L2/C2, respectively. Membrane interactions are investigated using scanning electron microscopy on bacterial cells, and highresolution X-ray scattering alongside epifluorescence microscopy on Langmuir monolayers.

Figure 1. (A) Molecular structures of cyclic (left) and linear (right) peptoids used in this study. (B) Peptoid monomer subunits. 4 ACS Paragon Plus Environment

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2. MATERIALS AND METHODS 2.1. Peptoids synthesis and purification. Peptoid sequences were chosen to have an uncharged C-terminal amide both to give the best comparison to the peptoid macrocycles, which do not bear a charged carboxylate, and to form the most likely molecules to display potent antimicrobial activity. This was done such that Sequence-specific peptoids were synthesized via “sub-monomer chemistry” method, applying iterative sequential steps of bromoacylation and nucleophilic displacement to construct each monomer unit. 35 Briefly, the amine on the initial sub-monomer is bromoacetylated and the resin-bound acyl bromide is displaced by a primary amine that affords to form the side chain of interest. In the case of linear peptoid synthesis, the final step is acetylation of oligomer with acetic anhydride followed by cleavage from the resin with trifluoroacetic acid, forming a C-terminal amide. For cyclization, linear precursors are synthesized on 2-chlorotrityl chloride resin to generate free N-terminal amino and C-terminal carboxylic acid groups, cleaved with 20% hexafluoroisopropanol and dichloromethane 30 and cyclized

using

(benzotriazol-1-yl-oxytripyrrolidinophosphonium

hexafluorophosphate

(PyBOP).29 All compounds were purified to >95% homogeneity by reversed phase high-performance liquid chromatography (RP-HPLC), characterized by mass spectrometry to confirm the molecular weight of the purified product, and stored as dry lyophilized powders at −20°C. 2.2 Antibacterial activity in vitro. Peptoid-susceptibility assays for all bacterial strains were conducted in 96-well plates using the broth-macrodilution procedure outlined in documentM07A7 of the CLSI.36 After incubation at 37°C for 18 to 24 hours, the turbidity of each sample was analyzed by visual inspection, and the minimum inhibitory concentration (MIC) was defined as the lowest concentration of peptoid resulting in an optically clear bacterial culture. Experiments


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were conducted in three independent replicates of three parallel trials to ensure statistical significance.

2.3.

Constant pressure insertion assays. Using Langmuir technique to evaluate insertion of

membrane active molecules into lipid monolayers may be done in two ways: by keeping either area or surface pressure invariable. Although the constant area approach has been widely used for Langmuir insertion assays, the latter one appears to be more relevant as confirmed by dynamic light scattering (DLS) on unilamellar liposomes of the same lipid composition. After adding peptoids to solution the average surface area of liposome sphere expanded in the same way as for planar monolayer (Figure S1). Since X-ray reflectivity gives the overall average density, experiments conducted at constant pressure may be used to monitor the total amount of excess material intercalated into the monolayer once equilibrated, and these densities may be normalized to the change in area available per lipid molecule. The instrumental setup consisted of a custom-made Teflon Langmuir trough equipped with two Teflon barriers whose motions were precisely controlled by motors for symmetric compression or expansion of monolayers at the air-liquid interface. The monolayer surface pressure was measured by a stationary Wilhelmy plate and kept at a constant pressure of 30 mNm-1 by a pressure-area feedback loop throughout the duration of experiment. Dulbecco’s phosphate buffered saline (DPBS) (Invitrogen, Carlsbad, CA) without calcium and magnesium ions was used as the subphase, with the temperature being maintained within 0.5°C from a pre-assigned temperature of 23°C. To reduce fluctuations and maintain stability, the entire equipment was mounted on a vibration isolation stage. DPPG and kdo2-lipid A were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Lipids were dissolved in chloroform and chloroform/methanol/water 75:15:10 v/v% solutions correspondingly, prior to depositing onto the aqueous surface.


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Deposited lipids self-assemble such that their hydrophobic acyl chains, or ‘tails,’ align perpendicular to the interface into the air, and their hydrophilic polar regions, or ‘heads,’ are oriented towards the liquid sub phase. The peptoid solutions were introduced into the subphase underneath the monolayer at 20% of their MICs against S. aureus. Incorporation of membrane-active compounds into lipid monolayer generally caused an increase in surface pressure. In order to counterbalance the rising surface pressure, the barriers expanded and the effective relative change in area per lipid molecule (ΔA/A) was monitored. The maximum value of ΔA/A was taken to characterize the membrane insertion capability of studied compounds. 2.4. Epifluorescence microscopy. Fluorescence image contrast arises due to different phase densities and partitioning characteristics of the dye molecules in coexisting phases. Therefore, it is possible to gain insight into the structure of the lipid layer by imaging its lateral fluorescence distribution. Experiments were performed as previously described. 37 The Langmuir trough used for insertion experiments was equipped with an epifluorescence microscope mounted to observe the phase morphology of the lipid monolayers. Lipid-linked Texas red dye (DHPE Texas Red; Invitrogen, Carlsbad, CA; 1 mol %) was incorporated into the stock phospholipid solutions prior to spreading. Data for excitation between 530 and 590 nm and emission between 610 and 690 nm were gathered through the use of a HYQ Texas red filter cube. Due to steric hindrance, the dye is localized in the disordered areas, rendering it bright, whereas the ordered loci remain dark.38 Images from the fluorescence microscope were collected sequentially at the intervals of 30 sec during initial 20 minutes after peptoids administration using a silicon intensified target camera and recorded on HD by MetaMorph Microscopy Automation & Image Analysis Software 7.0 (Molecular Devices, LLC, Sunnyvale, CA). This technique permits the monolayer


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morphology to be observed over a large lateral area (0.16 mm2) at certain time points while isotherm data are obtained concurrently and provides information about its evolution over time. A resistively heated indium tin oxide-coated glass plate (Delta Technologies, Ltd., Loveland, CO) was placed over the trough to minimize dust contamination, air convection, and evaporative losses as well as to prevent condensation of water on the microscope objective. The condensed domains on the surface of kdo2-lipid A monolayer were not observable with the resolution of microscope (~1μm). 2.5. Scanning electron microscopy in vitro. SEM experiments were performed following a previously published procedure.39 Samples of community-associated Methicillin-resistant Staphylococcus aureus USA300 (Los Angeles Country clone, LAC) were prepared by inoculating an overnight cell culture into fresh LB medium till >OD 600~0.4 (approximately 3 hours at 37 °C). After then, bacteria were treated with the antimicrobial compound at the Minimum Inhibition Concentration (MIC) as well as two-fold higher (supra-MIC) and lower (sub-MIC) concentrations (previously determined for cell density at OD600~0.4) for 1 hour or 24 hours at 37 °C. Untreated controls were kept in standard LB medium. After centrifugation (6000 x g, 20 min. at RT), washing and re-suspension in PBS, cells were deposited onto a 1 cm2 piece of a 0.45-μm-pore-size membrane filter (Millipore, Billerica, MA), fixed with 8% (v/v) glutaraldehyde/PBS (Alfa Aesar, Ward Hill, MA), and post-fixed with 0.5% (w/v) OsO4 in PBS (Electron Microscopy Sciences, Hatfield, PA) at 4 °C overnight. The samples were sequentially dehydrated with a graded ethanol series, including en bloc staining with 3% uranyl acetate in 30% ethanol. Finally, membrane pieces were air dried at room temperature, and attached to an aluminum pin stub with a carbon conductive adhesive (PELCO ®, Tedd Pella Inc, Redding, CA). A 5 nm layer of gold was sputtered on the samples to avoid charging in the microscope.


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Microscopy was performed using a Zeiss MERLIN

®

Field Emission Scanning Electron

Microscope (Oberkochen, Germany). Secondary electron images were taken at low electron energies between 2 keV and 4.0 keV. 2.6. X-Ray reflectivity. Specular X-ray reflectivity (XR) measurements were performed using liquid surface diffractometer at the 9-ID and 15-ID beam lines at the Advanced Photon Source, Argonne National Laboratory (Argonne, IL). The temperature-controlled Langmuir trough for the insertion experiments was mounted in a hermetic helium-filled canister, where the oxygen level was constantly monitored to be at 250

>16

C1/ C(NapNdp)3

C63H81N9O6

3.9

7.8

7.8

>250

>64

L2/ Ac(NapNnm)3

C56H68N10O7

125

125

125

>250

>2

C2/ C(NapNnm)3

C54H63N9O6

31.3

31.3

31.3

>250

>8

SR[c]

[a]

Minimum inhibitory concentrations against Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. [b]Hemolytic concentrations at which 50% hemolysis is observed. [c]Selectivity ratio: HC50 /MIC against S. aureus.

The damaging effects of peptoids upon S. aureus cells are imaged via scanning electron microscopy (SEM).43 SEM micrographs of bacteria after treatment with L1 or C1 at their MICs show large pores and deep craters in their cellular envelope (Figure 2B-C). In the control samples of untreated S. aureus, the surface of cells appears smooth and undamaged (Figure 2A). This observation suggests that peptoid treatment leads to cell death by compromising membrane integrity. This suggests that the bacteriostatic properties measured by MIC assays are bactericidal. However, no specific differences between L1 and C1 modes of action are

⃰

causing 50% hemolysis 11 ACS Paragon Plus Environment

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deciphered by SEM, which itself cannot elucidate the improved bactericidal properties of the cyclic peptoid in vitro.

Figure 2. SEM micrographs of untreated CA-MRSA cells on membrane filters at 60 kX magnification (A) and cells after 1hour treatment with C-1 (B) and L-1 (C) at their MICs. The samples incubated with peptoids overnight (18 hrs) are represented in the upper boxes of corresponding micrographs. The basic physicochemical properties of bacterial membranes, determined primarily by their lipid constituents, are likely to be the critical determinants of antimicrobial efficacy. 37, 44 In order to better understand the mechanism of interactions between the antimicrobial peptoids and bacterial cell membranes we study model Langmuir monolayers45, 46 that consist of either 1,2dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) or a truncated lipopolysaccharide, Di[3-deoxy-D-manno-octulosonyl]-lipid A, (kdo2-lipid A).42 The rationale behind this choice is as follows: kdo2-lipid A constitutes the core region of outer membrane in most Gram-negative bacteria, while phosphatidylglycerol is the most abundant anionic phospholipid of bacterial cytoplasmic membranes. The peptoids are injected into the aqueous subphase to enable interaction with the artificial membrane at the air-water interface, thus simulating interactions with the bacterial surface in the aqueous cell environment. 47, 48 The surface pressure of 30 mNm-


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1

, which approximates the average packing density of lipids in bilayers, 49 is held constant via

proportional-integral-derivative feedback control, while the interactions between lipid monolayers and antimicrobials are observed as a change in area over time. Area changes are recorded continually until the equilibrium is reached (approximately 15 min, Figure 3) and the relative increase in area per lipid molecule is calculated. Morphological changes on the lateral surface are simultaneously captured by epifluorescence microscopy (EFM).

Figure 3. Changes in area per lipid molecule after incorporation of peptoids into DPPG as a function of time (top left) and DPPG and kdo2-lipid A monolayers after equilibration (top right). Epifluorescence images of DPPG monolayer after C1 and L1 injection at concentrations 13 ACS Paragon Plus Environment

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corresponding to 20% of their MIC against S. aureus (bottom). Lipid-linked Texas Red-DHPE fluorescence probe (1 mol%) is added to the phospholipid solutions. Because of steric hindrance, the dye is located in the liquid-disordered phase, rendering it bright whereas the liquid-ordered phase remains dark. The immediate increase in area upon introduction of peptoids shows that they readily incorporate into DPPG monolayers (Figure 3, top left). The insertion isotherm of C1 comes to a plateau in 5 min after peptoid injection, while for L1 it takes slightly longer time (about 8 min) to reach the equilibrium. The overall increase in area per lipid molecule does not show significant excellence of cyclic compounds over their linear analogues. However, for kdo2-lipid A this difference is more pronounced and is about 15-20% (Figure 3, top right). Interestingly, the timing for morphological changes of the DPPG imaged by EFM agrees well with the insertion kinetics. Domains of liquid-ordered phase are completely obliterated by C1 and L1 after reaching the maximum area increase (Figure 3, bottom). Nearly identical behavior is observed for L2 and C2. Cyclic peptoids destroy the lateral order of DPPG monolayer faster than their linear counterparts. However, the mechanisms of their action are indistinguishable at the micrometer scale, although both potentially fluidize the membrane. This assertion agrees with previous Langmuir studies on the arenicin-derived peptides.50 High-resolution surface X-ray scattering offers further insight into the impact of antimicrobials on the molecular integrity of lipid membranes.44, 51, 52 X-ray reflectivity (XR) specifically yields the electron density profile along the surface normal (Z) of a film.53,

54

Figure 4 shows the

derived density profiles of DPPG and kdo2-lipid A monolayers before and after their interactions with C1 and L1 normalized to the area available per lipid molecule. The data for C2/L2 show similar trend and are presented in Figure S2 (see Supporting Information). All X-ray reflectivity 14 ACS Paragon Plus Environment

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experiments were conducted after the films had reached an equilibrium state where no further changes in film area were detected.

* normalized to the electron density of subphase and the average area available per lipid molecule ** region thickness and distance from air-water interface for the initial lipid monolayer only Figure 4. Electron density profiles of DPPG (A) and kdo2-lipid A (B) before and after linear (L1) and cyclic (C1) peptoids (left). Corresponding Fresnel-divided Reflectivity curves of at 30 mNm-1 (right). For XR curves the scatter plots are experimental values and solid lines are the best fits of the models to the experimental data. Molecular cartoons show general correlation between lipid structure and electron density maps. 15 ACS Paragon Plus Environment

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In order to facilitate the interpretation of electron density profiles, a lipid monolayer can be approximated with a number of slabs with a fixed electron density and thickness. Each slab corresponds to a distinct region within the lipid molecule, e.g. hydrocarbon chains or head groups. Changes in the number of electrons in each slab and in the monolayer as a whole, upon introduction of an antimicrobial, are directly related to the equilibrium lipid-to-drug ratio and localization of the drug within the membrane. Complete details for parameters derived from XR are summarized in Table 2. After injection of C1 and C2 to DPPG, the modeled electron density of the hydrocarbon chains -

3

-

3

is considerably higher as compared to pure lipid (0.381/0.324 e /Å ), while insertion of L1 and L2 reduces the electron density in this region (0.292 and 0.270 e /Å ). This observation suggests that linear peptoids are less apt to permeate far into the hydrophobic core of DPPG monolayer. The thinning of the upper slab (from 16.5 Å to 11-13 Å) upon their insertion is likely caused by molecular tilt of lipid tails with the headgroups shifted closer to the interface. The lipid-to-drug ratio with DPPG is also lower for cyclic peptoid (2.4 against 3.3); although for C2/L2 pair this difference is less pronounced (2.4/2.6). Extra electrons contributed by C1 and C2 are present in both slabs, demonstrating their ability to span the entirety of the monolayer. Considering that 2/3 of the electrons in a C1 molecule belongs to the diphenylethyl groups and the backbone ring we suggest that the hydrophobic moieties of peptoid macrocycle might be located within the upper part of DPPG monolayer (167 electrons out 249 per each lipid molecule). The distribution of extra electrons corresponding to the L1 molecules is within the bottom slab, with an additional region of excess electron density underneath the lipid monolayer, indicating that some L1 molecules accumulate on the outer


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surface of the lipid film. L2, in turn, does not contribute any electron density to the modeled internal membrane region. Cyclic peptoids also show an increased affinity for monolayers of truncated lipopolysaccharide mimicking Gram-negative bacteria outer membrane. XR modeling implies that cyclic compounds are present within the upper slab the kdo2-lipid A monolayer by contributing 76 and 74 additional electrons per lipid molecule, whereas both linear peptoids stay in the lower region only. Figure 5 shows a schematic diagram suggesting the potentially different modes of action for linear and cyclic peptoids with a focus on the outer portion of the bilayer system modeled by our Langmuir study. As monolayers used in this study are composed of saturated lipids exclusively it is notable to mention that the disruptive activity of peptoids on Langmuir model system reflects their interaction with the gel phase of natural membranes. The penetration depth and/or partitioning of inserted molecules within the fluid phase of lipid bilayer could vary based on membrane fluidity.


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Table-2: X-ray reflectivity modeling results

Experiment

N drug e-

lT (Å)

-

3

-

ρT (e /Å )

extra e T

lH (Å)

-

3

ρH (e /Å )

extra e

-

lOL (Å)

H

-

3

-

ρOL (e /Å ) extra e OL

Lipid-todrug ratio

47

-

87

3.3

DPPG

N/A

16.5

0.312

-

8.3

0.477

-

DPPG / L1

622

12.9

0.292

72±5

6.2

0.454

86±5

DPPG / C1

585

12.1

0.381

167±8

6.0

0.438

82±5

N/A

92

2.4

DPPG / L2

550

11.2

0.270

~0

9.2

0.471

208±8

N/A

85

2.6

DPPG / C2

513

11.8

0.324

72±5

7.3

0.475

140±7

N/A

86

2.4

Kdo2-lipid A

N/A

13.1

0.332

3.4

0.539

Kdo2-lipid A / L1

622

9.8

0.299

~0

7.4

0.483

Kdo2-lipid A / C1

585

9.0

0.362

76±5

7.4

Kdo2-lipid A / L2

550

10.2

0.241

~0

Kdo2-lipid A / C2

513

9.8

0.267

74±5

-

-

N/A

A lipid + ΔA lipid 2 (Å )

8.9

0.375

32±3

-

-

7.6

0.469

125

396±12

6.1

0.406

77±5

175

1.4

0.445

397±12

6.3

0.376

50±4

190

1.4

5.3

0.537

372±11

8.5

0.407

131±7

211

1.1

5.7

0.518

467±13

8.1

0.412

149±7

236

0.7

Subscripts: T – tails, H- heads, OL – outer layer Area per lipid molecule (A) = N lipid molecules / A total


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Figure 5. Suggested structural changes in outer leaflet of bacterial lipid membrane modeled by DPPG monolayers (A) after insertion of linear (B) and cyclic (C) peptoids. Cyclization allows antimicrobial molecules to intercalate better with the lipid film characterized by the molecular tilt of alkyl chains and membrane thinning. 4. DISCUSSION Charge, hydrophobicity, amphipathicity, and size define the design space for small membraneactive antimicrobial molecules. It has been recently suggested that the conformational rigidity


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could be an additional critical parameter. 24, 55, 56 Cyclization increases molecular rigidity without large changes to other physiochemical properties. Cyclic molecules should endure lower entropy loss upon incorporating into a lipid membrane as compared to their more flexible linear analogues. Inserting a molecule into a lipid membrane is associated with the packing perturbations in the lipid matrix. Energy gains in the system come from maximizing electrostatic and hydrophobic interactions between lipids and antimicrobial molecules. The cumulative change in energy defines the membrane activity of a molecule. The previously defined free energy associated with the transfer of a peptide from an aqueous solution into a lipid membrane (ΔGo) is contributed by a solvation free energy ΔGosolv, a lipid perturbation free energy ΔGolip and immobilization of the peptide ΔGoimm,. According to the model employed by Jähnig the "immobilization" term in ΔGo can be defined by

-ΔGoimm /RT ≈

(1)

The first term on the right-hand side of Eq. 1 is due to the loss of translational entropy, whereas the second term results from the loss of rotational entropy of the peptide in the membrane, as compared to the solution. 57 For the insertion of a 25-mer polyalanine α-helix with the length of dL=30 Å into a lipid membrane the ΔGoimm was calculated as ΔGoimm,trans + ΔGoimm,rot ≈0.9+2.8 ≈ 3.7 kcal/mol. This equals to 1/3 of negative contribution to ΔGo by ΔGosolv (≈-11 kcal/mol), which is the driving force for the peptide insertion into the membrane. 58 Considering this value to be similar or slightly lower for the less rigid peptoid hexamers we estimate the entropy balance for their linear and cyclic configurations. The increase of conformational rigidity upon cyclization can be transferred to the molecular level as the loss of


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rotational degrees of freedom within the peptoid backbone. Cyclization halves the rotational degrees of freedom, and the entropy change accompanying the restriction of a single one is approximately 3 cal K-1mol-1.59 Multiplied by 6 we define the entropy penalties associated with membrane insertion of cyclic peptoid to be reduced by 18 cal K-1 mol-1 (~4 kcal/mol). This roughly equals to ΔGoimm of linear α-helical molecules and is sufficient to positively shift the cumulative energy balance for peptoid macrocycle. It is noteworthy that peptoid hexamers behave experimentally similar to hexapeptides studied by MD simulations that further emphasizes the similarity in their structure-function relationships.33, 34 5. SUMMARY AND CONCLUSIONS Cyclic peptoids are found to inhibit bacterial growth better than their linear analogues. Electron microscopy on S. aureus cells demonstrates that both cyclic and linear peptoids disrupt the integrity of bacterial cellular envelopes. X-ray scattering on Langmuir monolayers shows that cyclic peptoids demonstrate higher affinity toward anionic lipids than their linear analogues. Our data provide evidence that cyclization increases the membrane activity of antimicrobial peptoid oligomers and shed the light on their underlying mechanism of action. We suggest that reduced conformational flexibility of cyclic antimicrobial molecules may bolster membrane penetrating activity, potentially leading to a superior membrane disruptive behavior. We anticipate that these findings, combined with the simplicity of the cyclization approach, will facilitate the rational design of new families of oligomeric antimicrobial agents.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Fax: (+1) 312-567-8856. E-mail: [email protected] Author Present Addresses ∆

Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147 (USA)

#

International Institute for Nanotechnology, Northwestern University, Evanston, IL 60208 (USA)

ǁ

Department of Chemistry and Biochemistry, University of California-San Diego, La Jolla, CA,

92093-0358 (USA) Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported by the NIH (R01 AI073892, D.G.), NSF (CHE-1507946, K.K.) and DARPA (W911NF-09-1-378 D.G.). ChemMatCARS Sector 15 is supported by the National Science Foundation under grant number NSF/CHE-1346572. This research used resources of the 22 ACS Paragon Plus Environment

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Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. M. W. Martynowycz was partially supported by the NSF via a fellowship through the Adler Planetary & Astronomy Museum. REFERENCES 1. 2. 3. 4. 5. 6.

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Table of Contents Graphic


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Figure 1. (A) Molecular structures of cyclic (left) and linear (right) peptoids used in this study. (B) Peptoid monomer subunits. Figure 1 1167x1585mm (96 x 96 DPI)


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Figure 2. SEM micrographs of untreated CA-MRSA cells on membrane filters at 60 kX magnification (A) and cells after 1hour treatment with C-1 (B) and L-1 (C) at their MICs. The samples incubated with peptoids overnight (18 hrs) are represented in the upper boxes of corresponding micrographs. Figure 2 161x52mm (150 x 150 DPI)


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Figure 3. Changes in area per lipid molecule after incorporation of peptoids into DPPG as a function of time (top left) and DPPG and kdo2-lipid A monolayers after equilibration (top right). Epifluorescence images of DPPG monolayer after C1 and L1 injection at concentrations corresponding to 20% of their MIC against S. aureus (bottom). Lipid-linked Texas Red-DHPE fluorescence probe (1 mol%) is added to the phospholipid solutions. Because of steric hindrance, the dye is located in the liquid-disordered phase, rendering it bright whereas the liquid-ordered phase remains dark. Figure 3 167x135mm (150 x 150 DPI)


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* normalized to the electron density of subphase and the average area available per lipid molecule ** region thickness and distance from air-water interface for the initial lipid monolayer only Figure 4. Electron density profiles of DPPG (A) and kdo2-lipid A (B) before and after linear (L1) and cyclic (C1) peptoids (left). Corresponding Fresnel-divided Reflectivity curves of at 30 mNm-1 (right). For XR curves the scatter plots are experimental values and solid lines are the best fits of the models to the experimental data. Molecular cartoons show general correlation between lipid structure and electron density maps. Figure 4 252x200mm (150 x 150 DPI)


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Figure 5. Suggested structural changes in outer leaflet of bacterial lipid membrane modeled by DPPG monolayers (A) after insertion of linear (B) and cyclic (C) peptoids. Cyclization allows antimicrobial molecules to intercalate better with the lipid film characterized by the molecular tilt of alkyl chains and membrane thinning. Figure 5 74x69mm (300 x 300 DPI)


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Table of Contents Graphic Table of Contents Graphic 82x27mm (150 x 150 DPI)


Cyclization Improves Membrane Permeation by Antimicrobial Peptoids

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