Hydrophobicity and helicity regulate the antifungal activity of 14-helical β-peptides

Article abstract of DOI:10.1021/cb500203e

Candida albicans is one of the most prevalent fungal pathogens, causing both mucosal candidiasis and invasive candidemia. Antimicrobial peptides (AMPs), part of the human innate immune system, have been shown to exhibit antifungal activity but have not be

Full text of DOI:10.1021/cb500203e

Articles
pubs.acs.org/acschemicalbiology
Open Access on 05/16/2015
Hydrophobicity and Helicity Regulate the Antifungal Activity of
14-Helical β‑Peptides
Myung-Ryul Lee,†,§ Namrata Raman,†,§ Samuel H. Gellman,‡ David M. Lynn,*,†,‡ and Sean P. Palecek*,†
‡
†Department of Chemical and Biological Engineering and Department of Chemistry, University of Wisconsin−Madison, Madison,
Wisconsin 53706, United States
S
* Supporting Information
ABSTRACT: Candida albicans is one of the most prevalent fungal pathogens, causing both mucosal candidiasis and invasive
candidemia. Antimicrobial peptides (AMPs), part of the human innate immune system, have been shown to exhibit antifungal
activity but have not been effective as pharmaceuticals because of low activity and selectivity in physiologically relevant
environments. Nevertheless, studies on α-peptide AMPs have revealed key features that can be designed into more stable
structures, such as the 14-helix of β-peptide-based oligomers. Here, we report on the ways in which two of those features,
hydrophobicity and helicity, govern the activity and selectivity of 14-helical β-peptides against C. albicans and human red blood
cells. Our results reveal both antifungal activity and hemolysis to correlate to hydrophobicity, with intermediate levels of
hydrophobicity leading to high antifungal activity and high selectivity toward C. albicans. Helical structure-forming propensity
further influenced this window of selective antifungal activity, with more stable helical structures eliciting specificity for C. albicans
over a broader range of hydrophobicity. Our findings also reveal cooperativity between hydrophobicity and helicity in regulating
antifungal activity and specificity. The results of this study provide critical insight into the ways in which hydrophobicity and
helicity govern the activity and specificity of AMPs and identify criteria that may be useful for the design of potent and selective
antifungal agents.
bacteria, fungi, viruses, and tumors.18−20 More than 2,000
Candida albicans is a commensal organism and the most
AMPs are listed in the Antimicrobial Peptide Database,21 and
common fungal pathogen in humans, causing both mucosal
candidiasis and invasive candidemia.1 As an opportunistic
35% of these have been reported to possess some degree of
antifungal activity. However, AMPs have several limitations as
therapeutics, including low stability and activity in physiological
media, low specificity toward fungal cells, and susceptibility to
proteolysis in vivo.22−26 While naturally occurring AMPs may
not be well suited for use as antifungal therapeutics for these
and other reasons, they have nevertheless provided key
molecular-level insights into structural features and functional
behaviors that confer antimicrobial activity. For example, AMPs
have been demonstrated to induce membrane lysis in target
cells via carpet or pore formation.27−29 As a result, the
development of target cell resistance to AMPs, a problem that
is understood to occur upon the use of conventional antifungal
drugs, has been suggested to be low.30−32 Insights and key
principles gleaned from studies investigating physicochemical
interactions between AMPs and cell targets may facilitate
pathogen, C. albicans can cause life-threatening invasive
infections in immunocompromised individuals such as organ
recipients, cancer patients, and human immunodeficiency virus
(HIV)-infected patients.1−10 The mortality rate of systemic
Candida infection is approximately 30−50%.11,12 Candidemia, a
disease in which Candida spp. are detected in the bloodstream,
is also often associated with biofilm formation on indwelling
medical devices such as central venous or urinary catheters,
joint prostheses, dialysis access, cardiovascular devices, and
central nervous system devices.13 The resistance of C. albicans
within biofilms to antifungal drugs such as fluconazole,
amphotericin B, flucytosine, itraconazole, and ketoconazole
has been reported to be 30−2000 times greater than in
planktonic cells.14 Clearly, more active and specific classes of
antifungal compounds are needed to reduce the severity of
antifungal infection, develop effective treatment protocols, and
reduce mortality in affected patients.
Received: March 17, 2014
Accepted: May 16, 2014
Published: May 16, 2014
Antimicrobial peptides (AMPs) are components of the
innate host defense system15−17 and possess activity against
© 2014 American Chemical Society
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Figure 1. 14-Helical β-peptide design and chemical structures. 3D structures (a−c) were generated on the basis of available crystal structure data85
and then geometry was optimized using Gaussian 03 at the B3LYP/6-31G level. (a) Stick view of β-peptide 4. The N-terminus (green), hydrophobic
side chains (blue), and cationic side chains (red) are indicated in color. (b and c) Surface views of β-peptide 4. Surface colors represent atom type H
(gray), C (green), O (red), and N (blue). (d) N-Terminus (X) and side chains (Y and Z) were altered as indicated to vary peptide hydrophobicity.
(e) Chemical structure of β-peptides containing a helix-stabilizing ACHC residue. (f and g) Chemical structures of β-peptides lacking an ACHC
residue. β3-hVal (f) and β3-hIle (g) were incorporated in place of the ACHC residue.
design of chemical compounds that display antifungal activity
and may possess more suitable pharmacokinetic and
pharmacodynamic properties than AMPs.
therapeutics. Antibacterial53−56 and antifungal57,58 activity of β-
peptides adopting 14-helical structures and the antibacterial
activity of β-peptides adopting 12-helical59−61 and 10/12-
helical62 structures have been reported. However, the
contributions of different β-peptide structural features to
biological activity, including antibacterial and antifungal proper-
ties, are not well understood. Thus, we sought to determine
how hydrophobicity and helicity govern, either alone or in
concert, the antifungal activity and specificity of 14-helical β-
peptides.
We synthesized 25 globally amphiphilic 14-helical β-peptides
that contain approximately three helical turns. In designing
these β-peptides, we varied hydrophobicity and helicity by
altering side chain composition and the presence or absence of
a helix-stabilizing cyclic aminocyclohexane carboxylic acid
(ACHC) side chain. To quantify the hydrophobicity of the
β-peptides, we characterized retention times using RP-HPLC, a
measure that has been used previously to assess the
hydrophobicity of peptides.63−65 Correlations have been
reported between antimicrobial peptide activity and retention
time of RP-HPLC.66−68 The helicity of β-peptides was
characterized using circular dichroism. Circular dichroism
titration showed that the helicity differences depended on the
presence or absence of a helix-stabilizing cyclic ACHC side
chain. Our results also indicate that, at constant helicity, β-
Naturally occurring AMPs, also known as host defense
peptides, can be categorized into 5 major classes: linear cationic
α-helical peptides, anionic peptides, specific amino acid-
enriched peptides, anionic and cationic disulfide bond-
containing peptides, and peptide fragments of large proteins.33
These AMPs display direct antimicrobial activity but often lose
antimicrobial cytotoxicity at physiologic pH and ionic strength.
Recent studies have demonstrated AMP host immunomodu-
latory activity, which complements their antimicrobial
activity.34
One of the predominant classes of antimicrobial peptide is
linear and α-helical in structure, and key properties that confer
antimicrobial activity include hydrophobicity, facial amphiphi-
licity, and helical propensity.27 These structural features have
also proven to be important in conferring antimicrobial activity
on peptidomimetic oligomers35−41 and polymers,42−47 includ-
ing β-peptide-based structures composed, either entirely or in
part, of β-amino acid residues.48,49 Owing to their folding
principles, structural diversity, secondary structure stability,50,51
and resistance to proteolysis,52 β-peptides represent a
promising class of compounds to elucidate mechanisms of
AMP activity and to template development of new antifungal
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peptide hydrophobicity directly correlates with both antifungal
and hemolytic activity, but that a window of hydrophobicity
exists over which these structures exhibit both high antifungal
activity and high selectivity (i.e., low hemolysis at the antifungal
minimum inhibitory concentration, MIC). We also demon-
strate that β-peptide helicity governs antifungal activity, with
more stable β-peptides possessing antifungal activity at lower
concentrations than less stable molecules at the same
hydrophobicity. Taken together, these results reveal both
hydrophobicity and helicity to regulate antifungal activity and
hemolysis, and provide design parameters for constructing
active and selective antifungal compounds.
Table 1. β-Peptide Retention Time, Minimum Inhibitory
Concentrations (MIC) against C. albicans, and % Hemolysis
at the Antifungal MIC
a
b
c
β-peptide tR (min SD) MIC (μg/mL) % hemolysis at MIC
SD
1
19.3 0.1
22.5 0.2
23.2 0.1
24.5 0.2
25.4 0.1
23.1 0.2
23.8 0.1
26.2 0.2
20.4 0.2
23.5 0.1
24.2 0.1
25.7 0.1
26.5 0.2
24.0 0.2
24.6 0.2
27.4 0.2
22.5 0.1
23.5 0.1
22.7 0.2
24.3 0.2
25.2 0.2
22.8 0.2
23.8 0.1
24.6 0.1
25.7 0.2
>128
64
32
8
2.6 0.9*
3.0 2.4
1.1 2.7
2.3 0.7
1.6 0.3
0.3 1.7
3.0 2.3
36.4 6.0
1.4 0.6
9.4 9.3
7.5 5.2
37 15
2
3
4
5
8
6
16
16
8
7
8
9
128
16
16
8
RESULTS AND DISCUSSION
■
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Design and Synthesis of 14-Helical β-Peptides. In
order to ascertain the structural features of AMPs that confer
antifungal and hemolytic activity, we synthesized a set of 25 14-
helical β-peptides (peptides 1−25, Figure 1), each 9 or 10
residues long, having a net charge of +4 and designed to adopt
globally amphiphilic structures (see Figure 1a−c). These
peptides have approximately three residues per helical
turn50,51 and were designed to contain two hydrophobic
residues and one cationic residue in a repeated trimer, thus
generating localized hydrophobic and cationic faces of the helix.
In this study, we systematically investigated the influence of
two AMP properties, hydrophobicity and helicity, on the
antifungal activity and selectivity of 14-helical β-peptides. To
probe the influence of these properties, we synthesized peptides
having different structural features that influence hydro-
phobicity and stability, including (i) the presence (peptides
1−16, Figure 1e) or absence (peptides 17−25, Figure 1f,g) of
the helix-stabilizing ACHC residue, (ii) addition of an N-
terminal β3-hTyr residue (Figure 1d), and (iii) variations in the
hydrophobic Y and cationic Z position side chains in the helical
repeat (Figure 1d).
8
39.8 2.7
9.5 2.2
11.6 2.1
72 14
16
16
4
128
64
128
32
16
>128
128
32
16
2.8 0.1
0.9 1.9
3.2 2.9
8.8 3.6
4.2 2.0
3.1 4.4*
4.5 3.2
7.2 5.0
7.2 3.4
a
The average value obtained from three independent analytical RP-
b
HPLC measurements. The value obtained from an average of three
independent experiments with triplicate measurements. The average
c
value obtained from three independent experiments with duplicate
*
measurements. Hemolysis at MIC measured at 128 μg/mL β-
peptide. Active (MIC ≤ 16 μg/mL) and selective (hemolysis at MIC
≤ 20%) β-peptides are in bold font.
β-Peptides were produced by microwave-assisted Fmoc
synthesis at 20−40 μmol scales and purified by RP-HPLC
using a C18 column. MALDI mass spectrometry was used to
validate the mass of each peptide (Supplementary Table S1).
Retention times determined by C18 RP-HPLC were used as a
measure of the relative hydrophobicity of the peptides (Table 1,
Supplementary Figure S1). To quantify the helicity of the β-
peptides, we characterized CD in solvents containing different
fractions of PBS and methanol.
mammalian cells is of particular interest. To provide a measure
of this specificity, we also compared percent hemolysis at the
MIC for each peptide (Table 1). The percent hemolysis at the
MIC ranged from <1% to ∼72% for the various peptides
investigated in this study. The most active and selective β-
peptides were peptides 4 and 5, each of which had an MIC of 8
μg/mL and less than 5% hemolysis at the MIC.
Characterization of Antifungal and Hemolytic Activ-
ities. The antifungal activities of the β-peptides were
determined by measuring their minimum inhibitory concen-
trations (MICs) against C. albicans in vitro. The peptides
exhibited a wide range of MIC values, ranging from 4 to >128
μg/mL. As an example, a plot of the concentration-dependent
growth inhibition of one of the most active peptides, peptide 5,
is shown in Figure 2a, with the MIC of 8 μg/mL indicated by
the arrow. The MICs of all of the peptides synthesized in this
study are provided in Table 1 and the growth inhibition plots
used to identify the MICs are shown in Supplementary Figure
S2.
To evaluate β-peptide specificity toward fungal cells, we
compared their ability to inhibit C. albicans growth with their
induction of human red blood cell lysis. As an example, the
percent hemolysis of peptide 5 as a function of peptide
concentration is indicated in Figure 2b, and results for all other
peptides tested are provided in Supplementary Figure S3. The
specificity of β-peptides against target fungal cells versus
β-Peptide Hydrophobicity Directly Correlates to
Antifungal and Hemolytic Activity. To explore the
relationship between β-peptide hydrophobicity and antifungal
activity and selectivity in compounds with similar helicity, we
varied (i) the N-terminal residue (Figure 1a, green; Figure 1d,
X), (ii) the composition of the hydrophobic side groups in the
repeating unit (Figure 1a, blue; Figure 1d, Y), and (iii) the
composition of the cationic residue in the repeating unit
(Figure 1a, red; Figure 1d, Z). All of the β-peptides used in
these studies (peptides 1−16) contained an ACHC residue as
one of the hydrophobic residues in the repeating unit.
We also added a β3-hTyr residue to the N-terminus, (Figure
1a and 1d, X) to increase the hydrophobicity of β-peptides 1−
8. Peptides containing the N-terminal β3-hTyr (peptides 9−16)
exhibited a RP-HPLC retention time that was approximately
1.0
0.15 min longer, and are thus regarded as more
hydrophobic, than those that did not contain the N-terminal β3-
hTyr (peptides 1−8) (Table 1). We also observed that β-
peptides with RP-HPLC retention times of approximately 23
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ACHC > β3-Et > β3-hAla) in β-peptides both containing and
lacking an N-terminal β3-hTyr residue (Table 1). The
antifungal activity and selectivity upon varying side chain
carbon number exhibited the same trends as observed upon N-
terminal β3-hTyr modification. The antifungal MICs increased
as retention times dropped below 23 min (Supplementary
Figure S4c), and the percent hemolysis at the MIC generally
increased as β-peptide retention time surpassed 25 min
(Supplementary Figure S4d). These trends were observed for
β-peptides with different N-termini and cationic side chains,
including (i) X = H, Z = β3-hLys (peptides 1, 2, 4, 6, 8), (ii) X
= β3-hTyr, Z = β3-hLys (peptides 9, 10, 12, 14, 16), (iii) X = H,
Z = β3-hArg (peptides 3, 5, 7), and (iv) X = β3-hTyr, Z = β3-
hArg (peptides 11, 13, 15) (Supplementary Figure S4c,d).
We also varied β-peptide hydrophobicity by changing the
identity of the cationic residue in the repeating trimer.
Naturally occurring AMPs, including magainin 2,69 cecropin
2,70 dermaseptin B2,71 mellitin,72 SMAP-29,73 MBP-1,74 and
melamine75 contain lysines and arginines as cationic residues.
Thus, we compared the antifungal activity and hemolysis of β-
peptides containing β3-hLys and β3-hArg. Peptides containing
β3-hArg exhibited RP-HPLC retention times 0.7
0.14 min
longer than those of analogous peptides containing β3-hLys
(Table 1). The changes in antifungal activity upon varying the
cationic side chain exhibited similar trends as were observed
upon adding an N-terminal β3-hTyr and varying the hydro-
phobic side chain carbon number (discussed above). The MICs
increased as β-peptide retention time decreased below 23 min
and did not change upon cationic side chain modification for
more hydrophobic β-peptides (Supplementary Figure S4e).
However, the hemolysis at the MIC did not change upon
substitution of β3-hArg for β3-hLys at any of the retention
times. All peptides, regardless of substitution of β3-hArg for β3-
hLys, with retention times greater than 25.5 min exhibited
elevated hemolysis at the MIC (Supplementary Figure S4f).
To visualize the relationships between peptide hydro-
phobicity and antifungal and hemolytic activity, we plotted
the MIC and the percent hemolysis at the MIC as a function of
peptide retention time for the series of ACHC-containing β-
peptides (peptides 1−16). Figure 3a reveals a strong
correlation between β-peptide hydrophobicity and β-peptide
antifungal and hemolytic activity as the N-terminus, hydro-
phobic side chain, and cationic side chain residues are varied.
The MIC decreased, and hemolysis at the MIC increased, as
the retention time increased. β-Peptides with retention times
between 23 and 25.5 min, indicated in the box in Figure 3a and
in bold text in Table 1, exhibited an MIC of 16 μg/mL or less
and less than 20% hemolysis at the MIC. At retention times
below 23 min, antifungal activity was low but specificity was
high, while at retention times above 25.5 min antifungal activity
was high but specificity was low. These key results reveal a
window of hydrophobicity, with a retention time from 23 and
25.5 min under the conditions used in this study, that results in
high β-peptide antifungal activity and selectivity, and suggest
that variations in the N-terminus, hydrophobic side chain
structure, and cationic side chain structure strongly influence
MIC and hemolysis through their influence on β-peptide
hydrophobicity.
Figure 2. Examples of measurement of antifungal MIC (a) and
hemolysis at the MIC (b) of β-peptide 5. (a) C. albicans cells (103
cells/mL) were incubated with β-peptides for 48 h, and β-peptide
susceptibility was assessed using an XTT reduction assay to compare
the absorbance at 490 nm for β-peptide-treated samples and untreated
samples. Data points are the averages of three independent
experiments of three replicates each. (b) β-peptides were incubated
with human red blood cells for 1 h, and the absorbance of the
supernatant was measured at 405 nm to calculate the percent of red
blood cells lysed; 100% hemolysis was determined using a melittin
control. Error bars denote standard deviation (n = 3).
min or less exhibited a significant decrease in MIC upon
addition of an N-terminal β3-hTyr, but that the addition of an
N-terminal β3-hTyr had no effect on the retention times of
more hydrophobic β-peptides (Supplementary Figure S4a;
Table 1). Addition of an N-terminal β3-hTyr to relatively
hydrophobic β-peptides with RP-HPLC retention times of 25
min or more increased hemolysis at MIC but had no effect on
hemolysis at the MIC of more hydrophilic β-peptides
(Supplementary Figure S4b; Table 1).
To broaden the spectrum of hydrophobicity in the β-peptide
series, we introduced aliphatic β-amino acids of varying side
chain carbon number and also incorporated an aromatic β-
amino acid (β3-hPhe). The RP-HPLC retention times of 14-
helical β-peptides with different residues in the middle position
of the repeat exhibited the same trend (β3-hPhe > β3-hVal >
We next investigated the influence of hydrophobicity on the
activity and specificity of β-peptides 17−25 lacking the helix-
stabilizing ACHC side chain (Figure 1f and 1g). The ACHC-
containing β-peptides adopted a helical structure at physiologic
pH and ionic strength. However, ACHC-lacking β-peptides
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min (Figure 3b). These results indicate that hydrophobicity
alone does not govern antifungal activity and suggest that
helicity also plays a key role. β-Peptides lacking ACHC did not
exhibit the increase in hemolysis at retention times greater than
25 min that β-peptides containing an ACHC demonstrated
(Supplementary Figure S5). Thus, the hydrophobic (tR >25
min) β-peptides lacking an ACHC possessed both antifungal
activity (MIC of 16 μg/mL or lower) and specificity (<10%
hemolysis at the MIC).
Effect of Cooperativity of Peptide Helicity and
Hydrophobicity on Antifungal Activity. Our results
demonstrate a strong correlation between β-peptide hydro-
phobicity and antifungal activity and hemolysis. To elucidate
the relationship between helicity and antifungal activity and
selectivity, we replaced ACHC with β3-hVal (peptides 17−21,
Figure 1f) or β3-hIle (peptides 22−25, Figure 1g) in the first
residue of the repeating trimer. Figure 3b illustrates the nature
of this correlation and the ranges of retention times that
provide antifungal activity, namely, that antifungal activity also
depends on β-peptide properties other than hydrophobicity,
including the presence of an ACHC side chain. We
hypothesized that helicity also affects antifungal activity. To
investigate this possibility, we compared the helical stabilities of
three β-peptides with high antifungal activities (MIC = 16 μg/
mL; β-peptides 11, 21, 25), three β-peptides with intermediate
antifungal activities (MIC = 32 μg/mL; β-peptides 3, 20, 24),
and three β-peptides with low antifungal activities (MIC = 128
μg/mL; β-peptides 9, 17, 23) in solvents containing various
ratios of methanol and water. As expected, β-peptides
containing the ACHC side chain (3, 9, 11) maintained helicity
as the methanol fraction decreased to zero, while the β-peptides
lacking an ACHC lost 14-helical structure (Supplementary
Figure S6). β-Peptides containing β3-hVal in place of ACHC
(17, 20, 21) exhibited slightly greater helicity than β-peptides
containing β3-hIle (23, 24, 25).
Figure 3. (a) Antifungal and hemolytic activities correlate with β-
peptide RP-HPLC retention times in β-peptides containing an ACHC.
The active (MIC ≤ 16 μg/mL) and selective (hemolysis at MIC ≤
20%) β-peptides are indicated in the box. MIC was determined by
incubating C. albicans cells (103 cells/mL) with β-peptides for 48 h,
and β-peptide susceptibility was assessed using an XTT reduction
assay to compare the absorbance at 490 nm for β-peptide-treated
samples and untreated samples. Data points are the averages of three
independent experiments of three replicates each. Hemolysis at the
MIC was determined by incubating β-peptides with human red blood
cells for 1 h, and the absorbance of the supernatant was measured at
405 nm to calculate the percent of red blood cells lysed; 100%
hemolysis was determined using a melittin control. (b) The
relationships between antifungal activity and RP-HPLC retention
time in β-peptides containing (1−16) and lacking (17−25) an ACHC
residue. The ACHC was replaced with β3-hVal in peptides 17−21 and
β3-hIle in peptides 22−25. Error bars denote standard deviation (n =
3).
Figure 4 illustrates the relationship between the antifungal
MIC and β-peptide helicity and hydrophobicity. In comparing
β-peptides with the same MIC, greater helicity corresponded to
exhibited 10−20% helicity at physiological pH and ionic
strength (Supplementary Figure S6). To vary the hydro-
phobicity of β-peptides lacking ACHC residues, we incorpo-
rated β3-hVal or the less hydrophobic β3-Et in the middle (Y)
position of each repeating trimer. β-Peptides lacking an ACHC
exhibited increased antifungal activity as hydrophobicity
increased, similar to β-peptides containing the ACHC residue.
However, the β-peptides lacking an ACHC required a greater
degree of hydrophobicity to exhibit significant antifungal
activity. For example, whereas β-peptides containing an
ACHC possessed MICs of 16 μg/mL or lower at a retention
times above 23 min, the β-peptides lacking an ACHC did not
exhibit MICs of 16 μg/mL until retention times exceeded 25
Figure 4. Relationship between helicity and HPLC retention times of
β-peptides exhibiting antifungal MICs of 16, 32, and 128 ug/mL. β-
Peptides 11, 3, and 9 contain an ACHC residue, while β-peptides 21,
20, and 17 contain a β3-hVal and β-peptides 25, 24, and 23 contain a
β3-hIle in place of the ACHC. Percent helicity in 100% PBS
normalized to helicity in 100% methanol was determined by circular
dichroism. Error bars denote standard deviation (n = 3).
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a lower retention time. For example, β-peptides 11, 21, and 25
all exhibited an MIC of 16 μg/mL. β-Peptide 11 demonstrated
the greatest helicity and lowest retention time, while β-peptide
25 formed the least stable helix and possessed the greatest
retention time. Similar trends existed at the 32 and 128 μg/mL
MIC values. Thus, helicity and hydrophobicity collectively
govern antifungal activity.
Comparing β-peptides with similar helical stabilities,
increasing retention time correlated with increasing antifungal
activity (Supplementary Figure S7). The antifungal MIC of β-
peptides possessing similarly high levels of helicity (e.g., Figure
1e series: 3, 9, 11) decreased with increasing retention time.
Similar trends existed in the β-peptides with lower helicity in
PBS (Figure 1f series: 17, 20, 21; and Figure 1g series: 23, 24,
25).
Comparing β-peptides with similar retention times (e.g.,
peptides 11, 20, 23) illustrates that antifungal activity increased
as 14-helicity increased (Supplementary Figure S8). The
ACHC-containing β-peptide 11 had an MIC of 16 μg/mL,
while the β3-hVal series peptide 20 and β3-hIle series peptide
23 exhibited reduced antifungal activity. Thus, helicity appears
to regulate β-peptide antifungal activity independently of
retention time. Taken together, these results indicate that
helicity and hydrophobicity cooperatively regulate β-peptide
antifungal activity.
Structural Features That Govern Antifungal Activity
and Specificity of AMPs and Their Analogues. The
finding that helicity and hydrophobicity collectively control β-
peptide antifungal activity is consistent with reports of α-
peptide activity against microbes and mammalian cells. For
example, Dathe and co-workers studied antibacterial activity of
magainin 2 amide (M2a).76 They reported that the most
hydrophobic analogue of M2a exhibited the greatest activity
against E. coli (MIC 2.4 μg/mL). The activity of that analogue
(I6A8L15I17) was 16-fold greater than the activity of M2a, and
hemolytic activity also increased by about 13-fold. In addition,
Hodges and co-workers68 studied the effects of hydrophobicity
on the antimicrobial activity of analogues of D-V13K77 derived
from V681.78 In Gram-negative bacteria and zygomycota fungi,
increasing hydrophobicity decreased activity, but in Gram-
positive bacteria, ascomycota fungi, and red blood cells activity
increased with hydrophobicity. These results are consistent
with the general view that the cell lytic activity of AMPs
increases with hydrophobicity but suggest that effects also
depend on the specific target organism, perhaps as a result of
differences in cell membrane composition.
specificity and provide design parameters for developing activity
and selective antifungal agents.
Summary. In conclusion, our results demonstrate that 14-
helical β-peptide antifungal activity and hemolysis are regulated
by hydrophobicity and helicity. We also identified a hydro-
phobicity range that confers selective antifungal activity to β-
peptides. Finally, we identified a cooperative relationship
between hydrophobicity and helicity in controlling antifungal
and hemolytic activities of β-peptides. These results provide
insight into mechanisms of action of AMP mimetics and
provide guidelines for designing active and specific compounds
for antifungal applications.
METHODS
■
Materials. Fmoc-β-amino acids, including Fmoc-L-β-homoalanine,
Fmoc-L-β-homovaline, Fmoc-L-β-homoisoleucine, Fmoc-(1S,2S)-2-
aminocyclohexane carboxylic acid, Fmoc-L-β-homophenylalanine,
Fmoc-O-tert-butyl-L-β-homotyrosine, Nβ-Fmoc-Nω-Boc-L-β-homoly-
sine, and Fmoc-Nω-(2,2,5,7,8-pentamethyl-chromane-6-sulfonyl)-L-β-
homoarginine were purchased from Chem-Impex International, Inc.
TentaGel S RAM Fmoc, HBTU (O-(benzotriaole-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate), and HOBt·H2O (N-
hydroxybenzotrizole monohydrate) were purchased from Advanced
ChemTech. (S)-3-Aminopentanoic acid was purchased from Sigma-
Aldrich for synthesis of Fmoc-(S)-3-aminopentanoic acid (Fmoc-β3-
Et-OH).82 RPMI powder (with L-glutamine and phenol red, without
HEPES and sodium bicarbonate) and 2,3-bis(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) were purchased
from Invitrogen. 3-(N-Morpholino) propanesulfonic acid (MOPS)
was purchased from Fisher Scientific. Phosphate-buffered saline (PBS)
liquid concentrate (10X) was purchased from OmniPur. Menadione
and melittin were purchased from Sigma. Freshly expired human red
blood cells were obtained from the blood bank at University of
Wisconsin-Madison Hospital.
β-Peptide Synthesis. β-Peptides were synthesized using TentaGel
(20−40 μmol) microwave-assisted solid phase peptide synthesis
procedures similar to those reported previously.83 Briefly, Fmoc-β-
amino acid, coupling reagent (HBTU, HOBt), and base (DIEA) were
dissolved in DMF individually and then mixed before coupling.
Microwave (CEM Discover) irradiation was used for coupling of
Fmoc-β-amino acid (600 W maximum power, 70 °C, ramp 2 min, hold
12 min) and deprotection of Fmoc (600 W maximum power, 80 °C,
ramp 2 min, hold 6 min). After coupling and deprotection, the resin
was washed with DMF (5 times), CH2Cl2 (5 times), and DMF (5
times), and then the peptide was cleaved from the resin by TFA-
containing H2O (2.5% v/v) and triisopropylsilane (2.5% v/v) for 1−2
h. The crude product was purified by preparative RP-HPLC with a
gradient of 25−73% (v/v) CH3CN in water containing 0.1% (v/v)
TFA.
Characterization of β-Peptide Hydrophobicity. To character-
ize the hydrophobicity of the 14-helical β-peptides, we measured
retention times by analytical RP-HPLC using a C18 column (Waters,
X-bridge). The β-peptides (dissolved to a concentration of 0.5−1 mg/
mL using deionized H2O containing 20−30% ACN and 0.1% TFA(v/
v)) were injected (50 μL) into the HPLC. Retention time was
characterized in triplicate with a gradient of 20−80% CH3CN in water
containing 0.1% TFA (v/v) over 5−35 min.
Characterization of β-Peptide Helicity. β-Peptides were
dissolved at 1 mg/mL in deionized H2O, divided into desired amounts
of solution using a gastight syringe (Hamilton), and then lyophilized.
Peptides were then dissolved in either MeOH (0.1 mM), PBS, or a
mixture of MeOH/PBS (20 to 80% (v/v)). Circular dichroism (CD)
was measured using an AVIV spectrometer at 20 °C with a 1 mm path
length cell and 5 s averaging times. The CD signal in 100% methanol
was assumed to be 100% helical. The retained helicity in 100% PBS
compared with 100% MeOH was calculated by using the following
equation:
Helicity has also been shown to affect antibacterial activity
and selectivity of α-peptide AMPs. For example, a Gly to Ala
substitution in magainin II was reported to increase the helicity,
antimicrobial activity, and hemolysis compared to magainin I
and II.79 In addition, Pro, which is generally considered to be a
helix-disrupting amino acid, has been introduced into
antimicrobial peptides. The Pro-free antibacterial peptide
V681 exhibited superior antibacterial activity against S.
typhimurium and higher hemolytic activity than peptides
containing one or two proline residues.80 Similarly, the helicity
of temporin L, an antimicrobial peptide that exhibits high
hemolytic activity, was varied by incorporating Pro and
corresponding D-isomers.81 Increasing temporin L helicity
increased both antibacterial and antifungal activity. Thus, our
results suggest that helical β-peptides represent structural
models for understanding mechanisms of AMP activity and
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ACS Chemical Biology
([θ]MeOH − ([θ]MeOH − [θ]PBS ))
Articles
AUTHOR INFORMATION
■
helicity (%) =
× 100
[θ]MeOH
Corresponding Authors
*E-mail: dlynn@engr.wisc.edu.
*E-mail: palecek@engr.wisc.edu.
Characterization of Antifungal Minimum Inhibitory Con-
centration (MIC). The antifungal activities of the β-peptides against
C. albicans were assayed in 96-well plates according to the planktonic
susceptibility testing guidelines provided by the Clinical and
Laboratory Standards Institute (formally, National Committee for
Clinical Laboratory Standards) broth microdilution assay84 modified
to include a quantitative XTT assessment of cell viability. Two-fold
serial dilutions (100 μL) of β-peptides in RPMI (pH adjusted to 7.2
with MOPS) were mixed with 100 μL of a C. albicans strain SC5314
cell suspension (grown for 24 h at 35 °C and concentration adjusted
to (1−5) × 103 cells/mL based on absorbance at 600 nm), and the
plates were incubated at 35 °C for 48 h. Wells lacking β-peptide (cell
controls) and wells lacking both peptide and cells (medium sterility
controls) were included in every plate that was assayed. After 48 h, 100
μL of XTT solution (0.5 g L−1 in PBS, pH 7.4, containing 3 μM
menadione in acetone) was added to all wells, and plates were
incubated at 37 °C in the dark for 1.5 h. The supernatants (75 μL)
from all wells were transferred to a fresh plate, and absorbance
measurements at 490 nm were recorded using a plate reader (EL800
Universal Microplate Reader, Bio-Tek instruments, Inc.). The cell
viability was plotted as a function of β-peptide concentration. Percent
cell viability was calculated as
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH grant 1R01 AI092225. The
peptide quantum calculations in Figure 1 were supported in
part by National Science Foundation Grant CHE-0840494.
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positive lysis control, and TBS was used as a negative lysis control.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed synthetic procedures of Fmoc-β3-Et-OH, HPLC
profiles, MIC analyses, hemolysis data, MALDI-TOF data,
circular dichroism titration curves, and the plots illustrating
relationships between hydrophobicity, helicity, and activity.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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medical devices. Clin. Microbiol. Rev. 17, 255−267.
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