Synthetic Random Copolymers as a Molecular Platform To Mimic Host

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Review

Synthetic random copolymers as a molecular platform to mimic host-defense antimicrobial peptides Haruko Takahashi, Gregory A Caputo, Satyavani Vemparala, and Kenichi Kuroda Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00114 • Publication Date (Web): 05 Apr 2017 Downloaded from http://pubs.acs.org on April 9, 2017

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Bioconjugate Chemistry

Bioconjugate Chemistry Topical review

Synthetic random copolymers as a molecular platform to mimic hostdefense antimicrobial peptides

Haruko Takahashi,a Gregory A. Caputo,b,c Satyavani Vemparala,d and Kenichi Kurodae,*

a

Center for International Research on Integrative Biomedical Systems, Institute of Industrial Science,

The University of Tokyo, Tokyo, Japan. b

Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ, USA.

c

Department of Biomedical and Translational Sciences, Rowan University, Glassboro, NJ, USA.

d

The Institute of Mathematical Sciences, C.I.T. Campus, Taramani, Chennai, India.

e

Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann

Arbor, MI, USA. * E-mail: [email protected], Tel: (+1) 734-936-1440, Fax: (+1) 734-647-2805.

Abstract Synthetic polymers have been used as a molecular platform to develop host-defense antimicrobial peptide (AMP)-mimetics which are effective in killing drug-resistant bacteria. In this review, we will discuss the AMP-mimetic design and chemical optimization strategies as well as the biological and biophysical implications of AMP mimicry by synthetic polymers. Traditionally, synthetic polymers have been used as a chemical means to replicate the chemical functionalities and physicochemical properties of AMPs (e.g. cationic charge, hydrophobicity) to recapitulate their mode of action. However, we propose a new perception that AMP-mimetic polymers are an inherently bioactive platform as whole molecules, which mimic more than the side chain functionalities of AMPs. The tunable nature and chemical simplicity of synthetic random polymers facilitate the development of potent, cost-effective,

ACS Paragon Plus Environment

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broad-spectrum antimicrobials. The polymer-based approach offers the potential for many antimicrobial applications to be used directly in solution or attached to surfaces to fight against drug-resistant bacteria. 1. Introduction Novel antimicrobial compounds are urgently needed to address the increasing number of drugresistant bacterial infections.1 In the effort to discover new antibiotics, agents that target bacterial cell membranes have been of interest owing to their efficacy against drug-resistant bacteria.2 However, it has been difficult to design such molecules that can selectively discriminate between bacterial cell membranes and host cell membranes. In addition, the molecular design of membrane-targeting agents is also a challenge because cellular membranes are dynamic and diverse, lacking defined active sites to be targeted. Therefore, the traditional “lock-and-key” drug design, in which the shape of molecule is designed to fit well into the target active site, is not viable. One strategy is to learn from nature. Host-defense antimicrobial peptides (AMPs) are small peptides produced as part of the innate immune system for the clearance of bacterial pathogens. These peptides directly act on bacteria by disrupting bacterial membranes, but some also show immunomodulatory properties.3-6 AMPs are generally amphiphilic, rich in cationic and hydrophobic residues. The cationic side chains of AMPs preferentially bind to anionic bacterial membranes over zwitterionic human cell membranes. Upon binding to bacterial membranes, AMPs form cationic amphipathic α-helices with the hydrophobic face of the α-helix inserted into the cell membrane, causing membrane disruption and ultimately cell death.3, 6 Because AMPs exhibit a low propensity for resistance development in bacteria, and their unique structures are considered evolutionarily optimized to selectively attack bacterial cell membranes, the therapeutic potential of AMPs, including their synthetic derivatives, has been explored for several decades. However, clinical implementation of AMPs has suffered from low bioavailability, susceptibility to proteolytic degradation, and high manufacturing cost.7 To address these issues, a new strategy was developed to create synthetic mimics of AMPs. Peptide derivatives such as β-peptides8-11 and peptoids11 have been developed to replicate the AMP helical structure. Recently designed sulfono-γ-AApeptides are a new entry in this class of AMP mimetics.12 While these AMP-mimics have shown potent activity and stability in in vitro physiological conditions, the biomedical applications of these agents are hampered due to the cost- and labor-intensive synthesis.


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More recently, synthetic polymers have been used as a molecular platform, which contributes to the development of robust, cost-effective, potent AMP-mimetic polymers. Synthetic polymers are generally resistant to enzymatic degradation, which should improve pharmacokinetics and/or pharmacodynamics when systemically or topically administered. Specifically, these AMP-mimetic polymers of interest are typically, short synthetic polymers with random sequences of monomers containing either cationic or hydrophobic side chains.

This design mimics the overall cationic

amphiphilicity and small molecular size of AMPs, rather than the traditional secondary structures of AMPs. Such antimicrobial polymers have demonstrated the hallmarks of AMP activities: a broad spectrum of activity, rapid biocidal kinetics, and low propensity for bacterial resistance development.13 These results indicate that the traditional, well defined facially-amphiphilic helical structure may not necessarily be required for membrane activity. Then, what are the polymers mimicking in AMPs? Conventionally, synthetic polymers have been used as a simple chemical means to replicate the chemical functionalities and physicochemical properties of AMPs (e.g. cationic charge and hydrophobicity) to recapitulate AMPs mode of action. However, our molecular dynamics simulations have demonstrated that though the AMP-mimetic polymers are unstructured (random coil) in solution, they adopt amphiphilic conformations capable of membrane disruption when bound to bacterial membranes.14-16 Here, we propose a new perception that our AMP-mimetic strategy has effectively captured some aspects of the folding process of α-helical AMPs, and therefore, the AMP-mimetic copolymers are an inherently bioactive platform which can be used to further develop membrane-active antimicrobials. In this review, we will first re-visit the currently accepted AMP-mimetic design hypothesis and then re-evaluate chemical optimization strategies. We will also review the antimicrobial activity of several polymers and highlight the approach to using functionalized polymers as an antimicrobial platform. The biological and biophysical implications of AMP mimicry by synthetic polymers will be discussed based on our findings. This review is intended to discuss the current problems and challenges in the field through new interpretations of previous data, primarily from our laboratories, over the last decade. Therefore, the scope of this review is focused on only a few classes of AMP-mimetic polymers. Many excellent comprehensive review articles on antimicrobial polymers developed in other laboratories and recent


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AMP mimetics are available,17-30 and the readers are recommended to refer to them in order to learn the state of art in the general area of antimicrobial polymers.

2. AMP-mimetic polymer design and challenges AMPs and mimicry by synthetic polymers. Naturally occurring AMPs are small peptides (typically 2 to 5 kDa) which are rich in cationic (lysine and arginine) and hydrophobic (leucine, valine, and isoleucine) residues (Fig. 1A).3, 4, 6, 7, 31-33 The current consensus is that the cationic side chains of AMPs preferentially bind to anionic bacterial membranes over zwitterionic human cell membranes, imparting selective activity against bacteria. While unstructured (random coil) in solution, upon binding to bacterial membranes many AMPs adopt the bioactive amphipathic α-helical structure with the cationic and hydrophobic side chains segregated to opposite faces of the helix. The hydrophobic face of the αhelix is inserted into the nonpolar core of the membrane, causing pore formation or non-specific membrane disruption and ultimately, cell death (Fig. 1B). Several molecular models have been proposed for the AMP-induced pore formation and membrane disruption.3 Some peptides are reported to form discrete ion-channel like pores in which the cationic domains of helices are facing the interior of the channel, with the hydrophobic domains of helices interact with the hydrophobic core of membrane. The peptide chains are aligned with lipids (barrel-stave model) or form complexation with lipid head groups (toroidal model) (Fig. 1B). Some AMPs also accumulate on the membrane surface and non-specifically disrupt membrane integrity, compromising the permeability barrier function of membranes (carpet model) (Fig. 1B). Accordingly, the cationic and hydrophobic properties are thought to be the essential and minimal functionalities to impart AMP-like functionality. Based on this hypothesis of the minimal AMP functionalities, many AMP-mimetic polymers currently studied in the field are random copolymers consisting of binary mixtures monomers with cationic and hydrophobic side chains. If acting through an AMP-mimetic mechanism, the cationic side chains of polymers bind to anionic bacterial cell membranes through electrostatic interactions (Fig. 2) followed by insertion of hydrophobic groups into the membrane, and subsequent membrane disruption and bacterial cell death.


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A

90° view from N-terminus

Cationic

B Bacterial cell membrane

Toroidal-pore model

Barrel-stave model

Pore formation

Carpet Model

Non-specific disruption

Figure 1. Structure and antimicrobial mechanism of human α-helical cationic host-defense antimicrobial peptide LL-37. (A) α-Helical structure of LL-37 (PDBID 2K6O, 37aa, MW 4.49 kDa). Cationic residues (blue) and hydrophobic residues (yellow) are segregated into the opposite sides of helix. The backbone structure was colored gray. Image was produced using PyMol. (B) Membrane disruption models of α-helical AMPs: pore formation (left: toroidal and barrel-stave models) and nonspecific disruption (right: carpet model). In the pore formation models, AMP helices form channel like structures with peptide-lined pores (barrel-stave model) or peptides in complexes with lipids (toroidalpore model). Reprinted with permission.16 Copyright 2013 American Chemical Society.


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AMP-mimetic polymer

Membrane Insertion

Surface binding ++

+

+ ++

+ + + +++++

Membrane disruption

++ + + + + +

++ + + + + +

Membrane

Bacteria

Cell death

Figure 2. Antimicrobial mechanism of cationic amphiphilic random copolymers. Our laboratories selected synthetic methacrylate random copolymers to develop AMP-mimetic copolymers (Fig. 3) while others used nylon-3,34-36 norbornene,18, 37, 38 maleimide,39 quaternary vinyl pyridine,40 oxetane,41 and (meth)acrylate42-45. Our selection of methacrylate was originally inspired by the pioneering work on membrane-active poly(alkylacrylic acid)s by Tirrell, Stayton, and Hoffman.46, 47 As poly(alkylacrylic acid)s disrupt human cell membranes at low pH, the polymers escape endosomes and deliver cargo to the cytosol.48 Our initial intent was to synthesize a cationic version of poly(alkylacrylic acid)s. Our methacrylate polymers were prepared by free radical polymerization with thiol agents as chain transfer agents49 or Reversible Addition–Fragmentation chain Transfer (RAFT) controlled radical polymerization.50 The polymer length (the degree of polymerization) was controlled by altering ratios of chain transfer agents relative to monomers, to give low molecular weight (MW = 23 kDa) polymers with high yields (>80%) to mimic the size of α-helical AMPs.


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R1 O

cationic + + + + + + +

O m

l

O

O

= hydrophobic

R2

R1: cationic +

R2: hydrophobic CH3

NH 3 NH 3 NH 3 NH N NH 3

Figure 3. Chemical structure of cationic amphiphilic methacrylate random copolymers. Basic design rule of AMP-mimetic copolymers: binary monomer composition. In the early stage of our study, we investigated the effect of monomer compositions and chemical structures of hydrophobic groups on the antimicrobial activity and toxicity to human cells in the effort to identify potent, non-toxic antimicrobial copolymers.51 We found that both the antimicrobial and hemolytic activities of copolymers are enhanced with the increase in the hydrophobic side chain content. However, the copolymers with the high hydrophobic character are also highly hemolytic and therefore, not selective to bacteria. Also, the antimicrobial and hemolytic activities of copolymers were enhanced as the length of alkyl chains in the hydrophobic side chains was increased. Similarly, the copolymers with longer alkyl side chains are highly hemolytic. These findings suggest that the hydrophobicity of copolymers is a driving force in antimicrobial activity, though the excess of the same causes hemolytic activity. These efforts led to the basic design rule that the cationic-hydrophobic balance of polymers should be optimized to maximize their antimicrobial activity and minimize hemolytic activity, which is also common in other polymers reported in literature37, 43, 52 and AMP sequences.53, 54 This basic design rule can be rationalized by the functional roles of cationic and hydrophobic side chains in the polymer-membrane interactions (Fig. 4). The cationic homopolymers without any hydrophobic side chains are likely to selectively bind to anionic bacterial cell membranes over


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zwitterionic human cell membranes by electrostatic interactions, but not capable of partitioning (inserting) into and subsequently disrupting membranes. However, if the overall copolymer hydrophobicity is too high, hemolysis occurs through non-specific binding to all membranes including host cells, driven by hydrophobic interactions. Therefore, the optimal monomer composition for potent antimicrobial activity with bacterial selectivity is the balancing point between the ability of polymers to disrupt membranes by hydrophobic interactions and the selective binding to bacteria by electrostatic interactions.

Cationic – Hydrophobic Balance

Cationic

Active Non-hemolytic

Inactive Non-hemolytic + + + + + + + + +

++ ++ + +

Outer

+

Hydrophobic Active Hemolytic

+ + + + + + +

+

+

++

+ ++ + +

+ +

+

+

+

+

+

+

+ +

+

++

+

+

Inner Human cell membrane

Bacterial cell membrane

Human cell membrane


Human cell membrane


Figure 4. Cationic-hydrophobic balance in cationic amphiphilic random copolymers. It should be noted that most anionic lipids such as PS are regulated to be asymmetrically distributed in the inner leaflet (the plasmic side) of human cell membranes.55, 56 Antimicrobial and anti-biofilm efficacy. We here review the antimicrobial activities of the methacrylate copolymers, which are linked to their polymer structures. The copolymers exhibit antimicrobial activity against both Gram-positive and Gram-negative bacteria (Table 1). Importantly, clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin intermediate S. aureus (VISA) are susceptible to the copolymers.57 At twice the MIC of copolymer, more than 99.8% of bacteria including Escherichia coli, Staphylococcus aureus and Streptococcus mutans were killed within


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2 hours.13, 58 The result also indicates that the copolymers exert their antimicrobial effect by killing bacteria (bactericidal), while many conventional antibiotics act by stopping bacterial growth (bacteriostatic)59. In addition, the copolymers did not result in the development of measurable resistance in E. coli when the bacteria were cultured in the sub-lethal concentrations of the copolymers.13 Finally, the copolymers exhibited various degrees of lytic activity against human red blood cells (hemolysis), with several lysing RBCs only at the highest polymer concentrations tested.15 Taken together all these data, the methacrylate copolymers show a broad spectrum of activity, quick bactericidal effects, and low propensity of resistance development in bacteria in similar to AMPs. Recently, we also demonstrated that a set of copolymers PE0 and PE31 could eradicate biofilms of cariogenic bacterium S. mutans when the biofilm was vigorously mixed with the copolymer solution for 30 seconds (Fig. 5).58 This study suggests a potential for the use of another class of copolymers as an active ingredient in oral care products such as mouthwash or toothpaste, ultimately aimed toward the development of a “liquid toothbrush". Membrane-disrupting mechanism of methacrylate copolymers. The membrane disruption by the methacrylate copolymers has been investigated using E. coli13, 60 and lipid vesicles (liposomes)61. In liposome systems, these copolymers disrupted the lipid bilayer to induce the leakage of entrapped fluorescent dye molecules.61 The ability of methacrylate polymers to disrupt the cytoplasmic membrane of E. coli appears to be dependent largely on the polymer structure/composition.13,

60

However, it

remains unclear whether the methacrylate polymers form defined membrane pores or not, as proposed for the mechanistic models of AMPs (Fig. 1B). Although the study is not directly related to bacterial membranes, it is interesting that the methacrylate copolymers have been reported to form nano-sized pores in the cell membrane of human red blood cells.62 Similarly, high molecular weight random copolymers were shown to form pores in a human cell membrane-mimetic lipid bilayer.63 These studies suggest that random copolymers are capable of forming membrane pores. Alternatively, the copolymers may act by non-specifically disrupting bacterial cell membranes, similar to a detergent or carpet model. In addition, it is not yet clear how the actual bacterial environment affects the activity and mechanism of methacrylate copolymers. Specifically, for example, bacteria have been shown to have esterases on or within their cell membranes.64, 65 While no experimental evidence is available, it is conceivable that these esterases may hydrolyze the ester groups of the polymer side chains. The liberated cationic or alkyl side chain groups may contribute to membrane disruption or provide a secondary effect on the


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bacterial metabolic activity, resulting in enhanced or modulated antimicrobial activity. Alternatively, the methacrylate polymers with hydrolyzed side chains would result in a backbone rich in carboxylic acids, unlikely to be active due to lack of both cationic and hydrophobic groups, lowering the susceptibility of bacteria to the polymers. More detailed mechanistic studies on how the copolymers act in bacterial membranes would be a subject of future studies to develop more stringent rational design rules for the synthesis of potent antimicrobial polymers. Table 1. Antimicrobial activity of copolymers. a

Bacteria (Gram stain)

Clinical relevance

Bacillus subtilis (+)

Associated with food-borne illness.

Enterococcus faecalis (+)

66, 67

2–8

63

Urinary tract infections, bacteremia, bacterial endocarditis, diverticulitis, 68 meningitis.

4–31

>500

Staphylococcus aureus (+)

Skin infection, bacteremia, endocarditis (infection of heart valves), and 69 osteomyelitis bone infection).

16–63

>500

Methicillin-resistant staphylococcus aureus b (MRSA) (+)

Nosocomial (hospital-acquired) and 70 community-acquired infections.

15–20

n.d.

c

Vancomycin intermediate staphylococcus aureus b (VISA) (+)

Nosocomial (hospital-acquired) and 71 community-acquired infections.

15

n.d.

c

8

n.d.

c

4–10

n.d.

c

8–21

125

8–16

500

Streptococcus mutans (+)

Acinetobacter baumannii (-)

Escherichia coli (-)

Pseudomonas aeruginosa (-)

MIC (μg/mL) Copolymers 13 Magainin-2 13, 15, 50, 58

Major cariogenic (acid-producing) 72 organisms to cause tooth decay. Bacteremia, pneumonia, meningitis, urinary tract infection, and wound infection. Multidrug-resistant infections among military personnel with traumatic injuries during the conflicts in Iraq and 73 Afghanistan. Some strains can cause food-bone illness; E. coli O157:H7 can causes severe food poising. Others can cause urinary tract infections, respiratory illness and 74 pneumonia. Opportunistic, cross-infections in hospitals, endocarditis, sepsis. ~51,000 healthcare-associated P. aeruginosa 75 infections occur in the U.S. each year.


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Estimated 1.2 million illnesses each year in the U.S. The leading cause of hospitalizations and deaths from 76, 77 foodborne disease

Salmonella enterica (-) a

16

500

Minimum inhibitory concentration, The MIC values of copolymers and magaian-2 were measured

under the same assay condition in the authors’ laboratory. b

Clinical isolates57

c

Not determined

A

S

S S n

NC O

NH 3 CF 3COO

B

S NC

PE0

n

m O

O

NH 3 CF3COO

O

O

O

PE31

Polymers S. mutans biofilm

Swishing for 30 sec.

Figure 5. Anti-biofilm activity of antimicrobial copolymers. Anti-biofilm activity of copolymers against cariogenic bacterium S. mutans. The polymer removed a one-day matured S. mutans biofilm by vigorously swishing treatment using a pipet for 30 seconds. Reprinted with permission.58 Copyright 2017 American Chemical Society.

Molecular design approaches beyond the side chain modulation and the minimalist design. Recently, we have developed a new approach to modulate the hydrophobicity/hydrophilicity of copolymer end groups prepared by RAFT polymerization (Fig. 6).50 The radical-mediated end-group


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alternation using free radical initiators transformed the ω-end group of the parent copolymer (Boc-P1 in Fig. 6) from dithiobenzoate (RAFT agent) to a cyanoisobutyl or aminoethyl cyanopentanoate group to reduce the hydrophobicity of the end-group. This is important as the hydrophobic/hydrophilic balance is a key component in the activity of these polymers and is directly linked to cytotoxicity. The initial results demonstrate a complex relationship between end-group hydrophobicity and antimicrobial activity, but it appears to be bacterial species dependent. Additionally, this end-group approach will be useful to investigate the role of polymer end groups in antimicrobial activity and mechanism. Modified end-groups

+ + + + + + + End-group + + + + + + + modification

S

S NC O

O

+

O

O

S AIBN

Acetonitrile, 70°C

NHBoc

S NC O

NHBoc

O

AIBN or Boc-amine AZO

n

m O

O

R NC O

Acetonitrile, 70°C

Boc-P1

n

m O

O

O

NHBoc

TFA

R NC

n

m O

O

O

O

R:

CN P1a CN

O O

NH3+CF3COO-

P1b

NH3+CF3COO-

Figure 6. End-group alteration of copolymers by radical-mediated modification using free radical initiators. Reprinted with permission.50 Copyright 2017 Wiley Periodicals, Inc.

While the basic design rule of cationic-hydrophobic balance facilitates polymer synthesis, the sequences of AMPs are more complex than cationic and hydrophobic residues.33 These functional groups are known to provide specific molecular interactions with membranes to control, for example, the depth of helix insertion into membranes and selective binding to anionic membranes.78-80 Therefore, it is possible that the above mentioned simple binary monomer design might not take full advantage of evolutionarily optimized AMPs functions, which are involved in the specificity toward bacterial cell membranes. Recently, the functional side chains of amino acids are incorporated into synthetic polymer chains. Gellman and Wong extend the design strategy to a tertiary monomer system with cationic


12

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ammonium groups, hydrophobic groups, and neutral hydroxyl groups.34 Yang designed amphiphilic methacrylate copolymers with carboxylic acid for low pH activation of polymers.81 The same laboratory also exchanged hydrophobic groups with hydroxyl groups to control the amphiphilicity of polymers.42 Locock developed polymers with tryptophan groups to mimic the Trp-rich class of AMPs.82 These approaches using the key side-chain moieties found in natural AMPs will more closely mimic the natural biological functionalities of AMPs and potentially recapture the more specific interactions with membranes which could improve the polymer activity and selectivity.

3. Synthetic polymers as an inherently bioactive platform: beyond compositional optimization The basic design rules from the chemical optimization of binary compositions supports the hypothesis that the mechanism of AMP-mimetic polymers is via targeting membranes. However, this rule also implies that, in principle, any given molecule with cationic and hydrophobic groups could act as membrane-active antimicrobials and show AMP-like activity. We wondered if our AMP mimic approach could more broadly prove that synthetic random polymers could be designed to be inherently antimicrobial without stringent restrictions on higher order structure? Polymer conformation in membranes. Molecular dynamics (MD) simulations provided a clue to find the answer to the question. When the model copolymer E4 chain is inserted into the hydrophobic region of the lipid bilayer, the polymer backbone is relatively extended (Fig. 7).14-16 Interestingly, the cationic and hydrophobic ethyl side chains are spontaneously segregated to opposite sides of the polymer backbone, adopting amphipathic conformations capable of membrane disruption. This polymer conformation mimics the facially amphiphilic structure of AMP α-helices, as previously postulated by Gellman52. Critically, the polymer chain maintained the facially amphiphilic structure throughout the simulations (100 ns). These results indicate that the facially amphiphilicity is not necessary to be built into the polymer structures. Yethiraj also showed in computational simulations on random copolymeric β-peptides that neither global amphiphilicity nor regular secondary structure is required for short peptides to effectively interact with bilayers.83 These results support the notion that our AMP-mimetic design is actually meant to identify synthetic polymers which adopt membrane-active conformations in bacterial membranes similar to AMPs.


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A

B

C

CH 3

H O

O

O O

O

O O

O

O

O

O

O

O

O

O

O

O

O

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O

O

NH 3

NH 3

Z NH 3

D

NH 3

NH 3

AMP

NH 3

NH 3

X

AMP-mimetic polymer

Surfactant

+ + +

High-MW polymer + + + + + + + +

+ + Bacteria cell membrane

Figure 7. Molecular dynamics (MD) simulations of copolymer conformation in a bacterial-mimetic bilayers. (A) Representative monomer sequence and structures of model copolymer E4. (B) Atomistic snapshot of the polymer chain at the end of 100 ns simulations. Water is not shown for clarity. The cationic side chain groups of aminobutyl methacrylate and hydrophobic ethyl groups of ethyl methacrylate (EMA) comonomers are colored orange and green, respectively. (C) Average conformation of the copolymers at the end of 100 ns simulation; molecules were oriented parallel to the membrane normal (in the XZ plane). The cationic and EMA ethyl side chains are colored red and blue, respectively. The polymer backbones are colored green. Reprinted with permission.15 Copyright 2012 American Chemical Society. (D) Membrane binding (insertion) of antimicrobial agents. AMPs and AMP-mimetic polymers fold to amphiphilic conformations upon binding to membranes. Thermodynamic considerations of polymer-membrane interactions. How can the copolymers adopt the amphiphilic conformations without any programed secondary structures? To explore this, first the thermodynamics of copolymers binding to membranes/bilayers should be understood. First, the total free energy for binding should be favorable (ΔGbinding = ΔH – TΔS < 0). Second, there is likely large entropic cost when the polymer backbone is extended and the side chain groups are segregated, and this entropic penalty must be compensated by the energy gain from the polymer-membrane interaction to reduce the


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total free energy of binding. When the polymer chains are extended in the membrane, the hydrophobic surface area of polymer chains is increased such that the contact area with the hydrophobic core of the membrane is increased, contributing to the favorable van der Waals energy gain. The electrostatic interactions between the cationic side chains and anionic lipid head groups also contribute to the energy (enthalpy) gain. This energy gain was indeed observed in our previous MD simulations.14 Therefore, the membrane binding (insertion) and conformational change of polymer chains to extended amphiphilic forms are coupled. If the loss of entropy of the polymer chain in solution is not compensated by the energy gain (ΔGbinding > 0), the polymers would not be able to insert into the membranes and therefore, they could not form active conformations. Critically, this polymer mechanism recapitulates the thermodynamics of folding process of α-helical AMPs on membranes; AMPs are unstructured in solution, but they adopt α-helices upon binding to membranes. The formation of α-helices costs a significant entropic penalty, which should be outweighed by the energy gain from electrostatic and hydrophobic interactions with membranes.84-86 Our AMP-mimetic polymers may exploit the same biophysical principle with AMPs in their membrane-active mechanism. While the stretching of polymer chains upon binding has only been demonstrated through molecular dynamics simulations, the direct observation of polymer conformations in membranes using spectroscopic methods would be of future interest to support the proposed mechanism. Our previous investigations using sum frequency generation spectroscopy (SFG) have shown the insertion of hydrophobic alkyl side chains of the polymers into the hydrophobic core of the lipid bilayer, clearly demonstrating the ability of SFG technique to probe bound polymer conformations.87

AMP-mimetic polymers and other amphiphilic antimicrobial molecules. One critical point in our findings is that the small molecular size (2-3 kDa) of AMP-mimetic polymers enables the entire polymer chain to be inserted into bacterial membranes, and therefore, we consider the polymer chains are bioactive as whole molecules (Fig. 7D). This new view may contrast with traditional antimicrobial polymers, which are generally high-molecular-weight cationic polymers, which also act by disrupting membranes.17 However, polymers with high molecular weight probably interact primarily with membrane surfaces and will likely remain unstructured or possibly collapsed because the extended states of such long polymers cost significant entropic penalties. For example, Wynne and Yadavalli demonstrated that antimicrobial polycation copolyoxetane (~7.7 kDa) accumulates on the bacterial


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surface and cause membrane disruption through the carpet mechanism.41 On the other hand, low molecular weight surfactants such as CTAB bind to membranes and are readily inserted into membranes, which typically cause membrane disruption or solubilization.88 However, this detergent mechanism is not likely to be associated with significant molecular conformational changes. Because of the relatively small molecular size, but still possessing structurally responsive polymeric chain structures in the membrane environment, AMP-mimetic polymers can be designed as a bioactive platform for membrane active agents as whole molecules, similar to peptides. Polymers as AMP-mimetic platforms. Traditionally, the role of molecular rearrangement is not typically viewed as an important characteristic for functionality, but rather a mechanism to simply orient the appropriate functional groups in space. The molecular design of AMPs suffers the lack of simple correlations between the amphiphilic structure and antimicrobial activity of AMPs, which is an obstacle to advancing further development of clinically useful AMPs.89 This is particularly due to the strong inter-relationships between structural and physicochemical parameters (i.e. net cationic charges, helicity, etc.), so that slight modifications can result in significant changes to peptide properties.90 Simply put, peptides are more complicated than they appear. By contrast, the low molecular-weight methacrylate copolymers that we developed are compositionally simple but can functionally imitate the folding of AMPs into active amphiphilic helices. Their simple copolymer structures can be beneficial to molecular design, providing a simple model that may allow more independent tuning of functionality. Therefore, we may be able to translate the functionality of AMPs required for membrane activity to simple polymer platforms. 4. Other polymer platforms beyond random copolymers. In this review articles, the focus in our discussion was short linear polymers with random sequences and flexible backbones. In the field, many different polymer platforms have been reported, including ones with different chemical structures (rigid backbones),23, alternating, gradient, telechelic)

93-96

91, 92

amphiphilic patterns (random, block,

, morphologies (micelles, single-chain nanoparticle)97, 98 topologies

(branched, dendrimers, star-shaped)99-101 and functionalities (biodegradable, hydrogels)26, 102, 103. These examples are likely to have different mechanisms in their membrane-active antimicrobial actions. Recently, polymer nanoparticles have been of interest in the field.21 Antimicrobial nanoparticles were prepared by self-assembly of individual polymer chains102 or by surface-initiating polymerization from


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nano-sized cores104. These nanoparticles are likely to have different interactions with bacterial membranes from linear analogues, due to the localization of polymer chains, which may work in a cooperative manner. The nanoparticle platform can provide greater chemical space than shorter polymers for additional functionalities and ligands targeting specific bacteria. In addition, we also recently exploited a non-covalent molecular assembly between the copolymer and a hydrogel-like nanoparticle (nanogel) to control their antimicrobial activity (Fig. 8).105 The nanogel is capable of capturing and release of copolymer chains in response to addition of cyclodextrin, providing a new means to modulate their bactericidal activity.

B

A

120 100

CHP nanogel

Capture Antimicrobial Copolymer

Copolymer–CHP complex

Release

Bacteria viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40 20 0

0 CD CHP-CD complex

50

100 Time (min)

150

200

PM43 or CD PM43–CHP

Figure 8. Controlled activity of antimicrobial copolymers. (A) Capturing and releasing of amphiphilic antimicrobial copolymers using macromolecular assemble system for controlling their antimicrobial function. (B) Suppression and recovery of antimicrobial activity of copolymers against E. coli. At the time of 10 min, the copolymer (red rombus) or copolymer–nanogel complex (blue square) were added. At 70 min, cyclodextrin (CD) was added to copolymer–nanogel complex (green round) to dissociate the nanogel for release of copolymer. Reproduced from Ref.105 with permission from the Royal Society of Chemistry.


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5. Conclusion In this review, we propose a new interpretation of our AMP-mimetic design of antimicrobial polymers. The previous work has largely focused on compositional optimization of binary cationic and hydrophobic monomers. However, the polymer chains which are unstructured in solution spontaneously adopt extended amphipathic conformations capable of membrane disruption.14-16 This mechanism directly mimics the folding process of AMPs. Our thermodynamic consideration suggests that the membrane binding (insertion) and conformational change of polymer chains to extended amphiphilic forms are coupled and are an integral part of the activity. These discussions led us to propose our new, unique perception that AMP-mimetic polymers are an inherently bioactive platform as whole molecules to mimic more than the side chain functionalities of AMPs. This new concept will provide us with new challenges in the polymer design to capture the molecular and functional traits of AMPs, beyond the overall physicochemical (amphiphilic) properties. In addition, many polymer platforms with different chemical structures, morphologies, and functionalities have been utilized to develop membrane-active antimicrobials with potent activity. Taken together, the tunable nature and chemical simplicity of polymers provide a new platform to study structure-activity relationships, which can be implemented to develop potent, cost-effective, broad spectrum antimicrobials. Owing to the facile synthesis and diverse chemical spaces, the polymer-based approach offers great potential for antimicrobial applications directly in solution or attached to surfaces in the fight against drug-resistant bacteria.

Author Information Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. Acknowledgments We thank financial support from the Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan (to K. K.) and JSPS Postdoctoral Fellowship for Research Abroad (No. 26-774 to H.T.).


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TOC Antimicrobial Polymer

+

+

Antimicrobial Peptide

+ + + ++ + ++ + Peptide-mimetic design +

+ + +++++

++ + + + + +

++ + + + + +


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Synthetic Random Copolymers as a Molecular Platform To Mimic Host

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