Ternary nylon-3 copolymers as host-defense peptide mimics: Beyond hydrophobic and cationic subunits

Article abstract of DOI:10.1021/ja507576a

Host-defense peptides (HDPs) are produced by eukaryotes to defend against bacterial infection, and diverse synthetic polymers have recently been explored as mimics of these natural peptides. HDPs are rich in both hydrophobic and cationic amino acid residu

Full text of DOI:10.1021/ja507576a

Article
pubs.acs.org/JACS
Ternary Nylon‑3 Copolymers as Host-Defense Peptide Mimics:
Beyond Hydrophobic and Cationic Subunits
†
,⊥
†,‡,⊥
†,#
†
†
†
Saswata Chakraborty, Runhui Liu,
Zvi Hayouka, Xinyu Chen, Jeffrey Ehrhardt, Qin Lu,
†
†
§
∥
‡
Eileen Burke, Yiqing Yang, Bernard Weisblum, Gerard C. L. Wong, Kristyn S. Masters,
,†
and Samuel H. Gellman*
†
‡
§
Department of Chemistry, Department of Biomedical Engineering, and Department of Medicine, University of Wisconsin,
Madison, Wisconsin 53706, United States
∥
Department of Bioengineering, University of California, Los Angeles, California 90095, United States
*
S Supporting Information
ABSTRACT: Host-defense peptides (HDPs) are produced by
eukaryotes to defend against bacterial infection, and diverse
synthetic polymers have recently been explored as mimics of
these natural peptides. HDPs are rich in both hydrophobic and
cationic amino acid residues, and most HDP-mimetic polymers
have therefore contained binary combinations of hydrophobic
and cationic subunits. However, HDP-mimetic polymers rarely
duplicate the hydrophobic surface and cationic charge density found among HDPs (Hu, K.; et al. Macromolecules 2013, 46,
1908); the charge and hydrophobicity are generally higher among the polymers. Statistical analysis of HDP sequences (Wang, G.;
et al. Nucleic Acids Res. 2009, 37, D933) has revealed that serine (polar but uncharged) is a very common HDP constituent and
that glycine is more prevalent among HDPs than among proteins in general. These observations prompted us to prepare and
evaluate ternary nylon-3 copolymers that contain a modestly polar but uncharged subunit, either serine-like or glycine-like, along
with a hydrophobic subunit and a cationic subunit. Starting from binary hydrophobic−cationic copolymers that were previously
shown to be highly active against bacteria but also highly hemolytic, we found that replacing a small proportion of the
hydrophobic subunit with either of the polar, uncharged subunits can diminish the hemolytic activity with minimal impact on the
antibacterial activity. These results indicate that the incorporation of polar, uncharged subunits may be generally useful for
optimizing the biological activity profiles of antimicrobial polymers. In the context of HDP evolution, our findings suggest that
there is a selective advantage to retaining polar, uncharged residues in natural antimicrobial peptides.
1
b
INTRODUCTION
of molecule in real-world applications. HDP-mimetic
copolymers are interesting from a fundamental perspective
because their properties pose a question: why would evolution
have elicited sequence-specific polypeptides for a task that can
be accomplished in the absence of sequence control? One
possible answer to this question is that HDPs perform
functions in addition to bacterial membrane disruption.
■
The innate immune response to bacterial infection includes the
release of relatively short peptides that attack prokaryotic cells
in preference to eukaryotic cells. These “host-defense
peptides” (HDPs) often have a net positive charge near neutral
pH; Coulombic forces attract HDPs to bacterial cell surfaces,
which generally bear a substantial net negative charge. Once
HDPs are localized on a cell surface, they are thought to disrupt
membrane barrier function. These disruptions appear to be
associated with induction of membrane curvature, and the type
of curvature involved requires certain proportions of lysines,
arginines, and hydrophobic residues, which sets a specific range
of cationic charge density and hydrophobicity for HDPs. Many
HDPs adopt α-helical conformations in interfacial environ-
ments, while others form small β-sheets or discrete tertiary
1
2
−4
The HDP-mimetic copolymers studied to date have been
largely binary, containing one subunit that displays a positive
charge on a side chain and another subunit that displays a
2
,3
6
−9
hydrophobic side chain.
In addition, antimicrobial homo-
polymers have been constructed from subunits that contain
10
5
both cationic and hydrophobic moieties. This focus on
hydrophobic and cationic components, however, is somewhat
at odds with the considerable sequence variation among HDPs.
Indeed, HDP-mimetic polymers typically display higher charge
density and more extensive hydrophobic surface than do
4
structures enforced by internal disulfides.
The variation in conformation, composition, and sequence
evident among HDPs has led a number of groups to explore
11
HDPs. A high-fidelity mimic must simultaneously have lower
cationic charge density and lower levels of hydrophobicity,
6
−8
sequence-random copolymers as functional HDP mimics.
Such efforts are appealing from a practical perspective because
the high cost of synthesizing peptides or other oligomers with
discrete sequences would make it very difficult to use this type
Received: July 24, 2014
©
XXXX American Chemical Society
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Article
which is difficult to achieve with most binary cationic and
hydrophobic subunit combinations. These considerations
prompted us to examine three-component polymers containing
cationic, hydrophobic, and “neutral” subunits as potential HDP
mimics. Glycine is the single most common residue among
known HDPs, even though Gly ranks behind Leu and Ala
trityl-protected to generate HSβ. Each ternary polymer
contains a hydrophobic subunit, CO or CH, and a cationic
subunit, MM or DM. The latter subunits bear a peripheral
amino group that should be protonated and therefore positively
charged at neutral pH. The necessary β-lactams, COβ, CHβ,
MMβ, and DMβ, have been used previously to generate
12
8
among known proteins. Serine is quite common among
HDPs, with a prevalence comparable to that found among
biologically active binary nylon-3 copolymers. All of the chiral
β-lactams were used in racemic form, and all of the ternary
copolymers discussed here are therefore heterochiral mixtures.
Nylon-3 copolymers were synthesized via base-initiated
1
2
proteins. We synthesized and evaluated the biological activity
profiles of ternary nylon-3 copolymers in which previously
described hydrophobic and cationic subunits are supplemented
by either a glycine-like or serine-like subunit.
15
anionic ring-opening polymerization (AROP) of β-lactams.
Reactions involving HGβ were conducted in N,N-dimethyla-
cetamide (DMAc), and reactions involving HSβ were
conducted in tetrahydrofuran (THF). p-tert-Butylbenzoyl
chloride was included in all of the polymerizations; this acid
chloride presumably reacts rapidly under AROP conditions to
RESULTS AND DISCUSSION
■
Polymer Synthesis and Characterization. The ternary
nylon-3 polymers discussed here contain subunits from one of
two β-lactams that have not previously been employed to
generate antimicrobial materials (Scheme 1). One β-lactam
8
,16,17
generate a mixture of N-acyl-β-lactams.
These imides then
serve as co-initiators for the ring-opening polymerization
process, leaving a p-tert-butylbenzoyl group at the N-terminus
16,17
Scheme 1. β-Lactams and Corresponding Subunits within
the Nylon-3 Polymer Chain
of each chain.
An imide derived from one of the three β-
lactam starting materials remains at the C-terminus of each
chain.
Acid chloride:β-lactam ratios were chosen to generate
average chain lengths of 18−35 subunits. The resulting
materials were analyzed via gel-permeation chromatography
(
GPC) prior to removal of side-chain protecting groups.
Polydispersity index (PDI) values were in the range 1.05−1.43
according to data from multiangle light scattering (MALS) and
a refractive index detector. Boc and trityl side-chain
deprotection was accomplished by treatment with trifluoro-
acetic acid (Figure 1). The structures of the ternary polymers
are shown in Figure 2.
Biological Activities. A panel of four bacteria, including
18
one Gram-negative species (Escherichia coli ) and three Gram-
1
9
20
positive species (Bacillus subtilis, Staphylococcus aureus, and
21
Enterococcus faecium ), was used to gauge the antibiotic
properties of the ternary copolymers. Antibacterial activity was
measured in terms of minimum inhibitory concentration
(
MIC), i.e., the lowest concentration of a polymer that fully
inhibits bacterial growth. Evaluation of HDPs and synthetic
analogues typically involves an assessment of prokaryotic cell
versus eukaryotic cell selectivity based on a comparison of MIC
values with the extent to which an agent disrupts red blood cell
(
RBC) membranes. The latter property was measured by
monitoring the release of hemoglobin from RBCs as a function
of agent concentration. We used HC , the concentration of a
10
polymer that causes release of 10% of the hemoglobin from
human RBCs, as our index of hemolytic activity.
The studies reported here are based largely on two previously
reported binary nylon-3 copolymers, MM CO and
6
0
40
8c
DM CH . Both polymers are active against the bacteria in
5
0
50
our test panel (low MIC values), but both are also quite
hemolytic (low HC10 values). These two binary polymers
provide an excellent opportunity to determine whether
incorporation of a third nylon-3 subunit, one that is neither
cationic nor hydrophobic, can enable decoupling of the
antibacterial activity from the hemolytic activity. In all previous
studies by our group and others involving binary cationic−
hydrophobic copolymers, diminishing the hydrophobic content
necessarily meant increasing the cationic content and vice versa.
Use of an HG or HS subunit, however, allows the hydrophobic
content to be changed without affecting the overall cationic
bears no substituent and gives rise to a glycine-like subunit in
polymer chains. This simplest of β-lactams is a derivative of β-
13
alanine, which can also be designated β-homoglycine. We
designate this β-lactam HGβ and the resulting subunit HG.
3
The other β-lactam, designated HSβ, gives rise to a β -
homoserine subunit (HS) in polymer chains. The hydrox-
ymethyl β-lactam precursor to HSβ has previously been
14
synthesized in enantiopure form, and we followed the
reported route to generate racemic material, which was then
B
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Journal of the American Chemical Society
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Figure 1. Synthesis of DM + CH + HS ternary copolymers. All of the other ternary copolymers discussed here were synthesized analogously.
Figure 2. Structures of the ternary nylon-3 copolymers examined in this work. R′ represents the side chain from one of the starting β-lactams. All of
the polymers are sequence-random and heterochiral.
Table 1. Biological Activities of MM + CO + HG and MM + CO + HS Ternary Copolymers versus MM CO
6
0
40
d
MIC (μg/mL)
a
b
c
e
polymer
MM CO
Mn
DP
PDI
E. coli
B. subtilis
S. aureus
E. faecium
HC10 (μg/mL)
4402
5352
5704
5670
5441
6296
8873
22
29
31
33
25
28
35
1.06
1.29
1.31
1.43
1.07
1.27
1.05
25
25
0.78
3.13
3.13
3.13
1.6
3.13
6.25
12.5
25
3.13
3.13
6.25
50
6.25−12.5
3.13
6
0
40
MM CO HG
6
0
30
10
15
30
MM CO HG
25
12.5
6
0
25
MM CO HG
200
25
>400
6
0
10
MM CO HS
10
6.25
6.25
100
3.13
6.25
100
50−100
100−200
>400
6
0
30
MM CO HS
200
1.6
6
0
25
15
30
MM CO HS
>200
6.25
6
0
10
a
b
c
Number-average molecular weight. Average degree of polymerization. Polydispersity index based on GPC data obtained for polymers bearing Boc
d
protecting groups on MM unit side chains and trityl protecting groups on HS unit side chains. Minimum inhibitory concentration for bacterial
growth. Polymer concentration for 10% lysis of hRBCs.
e
content or the overall charge to be altered without changing the
net hydrophobicity.
general, the β-lactam starting materials were fully consumed in
the polymerization reactions. The data in Table 1 show that
replacing a small proportion of the hydrophobic CO subunits
with “neutral” HG units has relatively little effect on the
biological activity profile: the ternary copolymers
MM CO HG and MM CO HG are very similar to the
Table 1 compares the biological activity profile of
MM CO with the profiles of six new ternary copolymers,
60
40
three in which varying proportions of the hydrophobic CO
subunits have been replaced with HG subunits and three in
which varying proportions of the CO subunits have been
replaced with HS subunits. The HC10 values listed in Table 1
are supplemented by the full hemolysis data sets presented in
Figure 3. The proportions indicated in the polymer designation
are based on the β-lactam proportions used for the synthesis of
that polymer (i.e., the material designated MM CO HS was
6
0
30
10
60
25
15
binary polymer MM CO in terms of MIC and HC10 values.
6
0
40
Replacing most of the hydrophobic CO units with HG, as in
MM CO HG , causes a stark decline in the hemolytic
6
0
10
30
activity, but the antibacterial activity declines as well (i.e., the
MIC value becomes higher). This observation is consistent with
the general view that a minimum level of hydrophobicity is
necessary for membrane-disruption activity.
6
0
30
10
prepared by copolymerization of a β-lactam mixture containing
0 mol % MMβ, 30 mol % COβ, and 10 mol % HSβ). In
6
C
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Journal of the American Chemical Society
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Figure 3. Hemolytic profiles of (left) MM + CO + HG and (right) MM + CO + HS ternary copolymers vs MM CO .
60
40
Table 2. Biological Activities of DM + CH + HG and DM + CH + HS Ternary Copolymers versus DM CH
50
5
0
d
MIC (μg/mL)
a
b
c
e
polymer
DM CH
Mn
DP
PDI
E. coli
B. subtilis
S. aureus
E. faecium
HC10 (μg/mL)
4766
4341
5539
5731
7482
4383
5443
26
24
33
28
32
26
28
1.05
1.25
1.25
1.09
1.08
1.21
1.09
12.5
12.5
50
≤1.6
3.13
3.13
1.6
6.25
12.5
25
6.25
12.5
50
6.25
25−50
200
5
0
50
DM CH HG
5
0
40
10
25
DM CH HG
5
0
25
DM CH HS
10
25
12.5
50
6.25
25
50
5
0
40
DM CH HS
25
100
6.25
3.13
3.13
3.13
200
5
0
25
DM CH HG
10
12.5
12.5
12.5
12.5
12.5
100
4
0
50
DM CH HS
10
12.5
4
0
50
a
b
c
Number-average molecular weight. Average degree of polymerization. Polydispersity index based on GPC data obtained for polymers bearing Boc
d
protecting groups on DM unit side chains and trityl protecting groups on HS unit side chains. Minimum inhibitory concentration for bacterial
growth. Polymer concentration for 10% lysis of hRBCs.
e
Figure 4. Hemolytic profiles of (left) DM + CH + HG and (right) DM + CH + HS ternary copolymers vs DM CH .
50
50
The HS subunit is expected to be more hydrophilic than the
HG subunit because of the pendant hydroxyl group in the
former, and replacement of CO with HS exerts more
interesting effects on the biological activity profile relative to
replacement with HG. Thus, the ternary copolymer
MM CO HS is generally similar to MM CO in terms
antibacterial activity erodes, however, with further replacement
of CO subunits by HS subunits.
Table 2 presents the biological activity profiles for ternary
copolymers related to the binary copolymer DM CH ;
5
0
50
hemolytic assay results are shown in Figure 4. Replacing a
small proportion of the hydrophobic CH units with either HG
or HS has a similar impact: relative to the binary starting point,
DM CH HG and DM CH HS display comparable
60
30
10
60
40
of antibacterial activity, but the ternary copolymer is
considerably less hemolytic than the binary prototype. The
5
0
40
10
50
40
10
antibacterial activities but significantly diminished hemolytic
D
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Table 3. Biological Activities of Representative Binary versus Ternary Nylon-3 Copolymers Having the Same
Hydrophobic:Cationic Molar Ratio and Varied Content of the Third (Hydrophilic) Subunit HG or HS
d
MIC (μg/mL)
a
b
c
e
polymer
DM CH
Mn
DP
PDI
E. coli
B. subtilis
S. aureus
E. faecium
HC10 (μg/mL)
4766
3558
3174
4943
5539
26
20
18
25
26
1.05
1.20
1.24
1.12
1.05
12.5
12.5
25
≤1.6
≤1.6
≤1.6
1.6
6.25
12.5
12.5
12.5
25
6.25
12.5
25
6.25
5
0
50
DM47.5CH47.5HG5
DM CH HG
400
400
4
5
45
10
DM CH HS
10
25
12.5
50
200−400
200−400
4
5
45
DM CH HS
20
50−100
3.13
4
0
40
a
b
c
Number-average molecular weight. Average degree of polymerization. Polydispersity index based on GPC data obtained for polymers bearing Boc
protecting groups on DM unit side chains and trityl protecting groups on HS unit side chains. Minimum inhibitory concentration for bacterial
growth. Polymer concentration for 10% lysis of hRBCs.
d
e
Figure 5. Hemolytic profiles of (left) DM + CH + HS and (right) DM + CH + HG ternary copolymers vs DM CH .
50
50
activities. Increasing the extent of hydrophobic unit replace-
hydrophobic and cationic components are augmented by
“neutral” components, i.e., components that are neither charged
nor hydrophobic. On the basis of amino acid residue prevalence
among HDPs, we selected two new subunits, one glycine-like
(HG) and one serine-like (HS). Starting from previously
known binary hydrophobic−cationic nylon-3 copolymers that
display strong antibacterial activities but are also highly
hemolytic, we have shown that partial replacement of
hydrophobic subunits, cationic subunits, or both can lead to a
decline in an unfavorable property (hemolysis) while a
desirable property (antibacterial activity) is maintained. These
findings suggest that the exploration of ternary copolymers may
be useful for other efforts to optimize synthetic biomaterial
performance.
ment to generate DM CH HG or DM CH HS causes a
5
0
25
25
50
25
25
substantial decline in antibacterial activity. In this series, we
explored ternary copolymers in which a subset of the cationic
units is replaced with either HG or HS. This effort was
motivated by the prior observation that the DM homopolymer
8
c
is quite hemolytic. For these modifications, HS provides a
more interesting outcome than HG. Both DM CH HG and
4
0
50
10
DM CH HS are relatively similar to DM CH in terms of
40
50
10
50
50
antibacterial profile, but DM CH HS is significantly less
4
0
50
10
hemolytic than the binary copolymer, while DM CH HG is
4
0
50
10
comparable to the binary copolymer in hemolytic activity.
The results summarized in Table 2 suggest that improve-
ments in the biological activity profile (i.e., increases in HC10
without major changes in MIC) can be achieved by replacing
small amounts of either the hydrophobic or the cationic subunit
ASSOCIATED CONTENT
Supporting Information
Materials and instrumentation, polymer synthesis, bioassays of
polymers, dose−response curves, H NMR spectra, and GPC
■
in DM CH with neutral HG or HS subunits; therefore, we
50
50
S
*
examined another set of ternary copolymers in which small
amounts of both DM and CH subunits were replaced (Table 3
and Figure 5). This approach proved to be effective; the
1
chromatograms. This material is available free of charge via the
Internet at http://pubs.acs.org.
polymer DM47.5CH47.5HG appears to have the most favorable
5
profile among all of the ternary polymers examined in this
study.
AUTHOR INFORMATION
Corresponding Author
gellman@chem.wisc.edu
■
CONCLUSIONS
■
We have explored a new, biologically inspired approach to
creating sequence-random copolymers that mimic the anti-
bacterial/hemolytic activity profile characteristic of host-
defense peptides. In contrast to previous efforts focused on
binary copolymers in which one subunit is hydrophobic and the
other cationic, we have examined ternary copolymers in which
Present Address
Z.H.: Institute of Biochemistry, Food Science and Nutrition,
The Hebrew University of Jerusalem, Rehovot, Israel.
#
Author Contributions
S.C. and R.L. contributed equally.
⊥
E
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Journal of the American Chemical Society
Article
Notes
acidic side chain in addition to subunits with hydrophobic and basic
side chains. This design was intended to render the antibacterial
activity pH-dependent. The acidic side chains are protonated and
therefore neutral at lower pH, which gives the copolymers a net
cationic charge and makes them active. The acidic side chains are
deprotonated and therefore anionic at higher pH, which makes them
zwitterionic and therefore inactive.
The authors declare the following competing financial
interest(s): S.H.G. and B.W. are co-inventors on a patent
covering nylon-3 copolymers.
ACKNOWLEDGMENTS
■
We thank Chris Lacriola for assistance with antibacterial assays.
This research was supported in part by NIH Grants
R21EB013259 and R01GM093265. The content is solely the
responsibility of the authors and does not necessarily represent
the official views of the National Institutes of Health. Z.H. was
supported in part by the Nanoscale Science and Engineering
Center at UW-Madison (DMR-0425880) and in part by a
Fulbright Fellowship. E.B. was supported by the Nanoscale
Science and Engineering Center at UW-Madison (DMR-
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