Lysine-Based α-Peptide/β-Peptoid Peptidomimetics: Influence of Hydrophobicity, Fluorination, and Distribution of Cationic Charge on Antimicrobial Activity and Cytotoxicity

Article abstract of DOI:10.1002/cmdc.201600553

Multidrug-resistant bacteria pose a serious threat to public health worldwide. Previously, α-peptide/β-peptoid hybrid oligomers were found to display activity against Gram-negative multidrug-resistant bacteria. In the present work, the influence of hydrop

Full text of DOI:10.1002/cmdc.201600553

Accepted Article
Title: Lysine-Based α-peptide/β-peptoid peptidomimetics: Influence of
hydrophobicity, fluorination and distribution of cationic charge on
antimicrobial activity and cytotoxicity
Authors: Natalia Molchanova, Paul Robert Hansen, Peter Damborg,
Hanne Mørck Nielsen, and Henrik Franzyk
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10.1002/cmdc.201600553
ChemMedChem
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Lysine-Based α-peptide/β-peptoid peptidomimetics: Influence of
hydrophobicity, fluorination and distribution of cationic charge
on antimicrobial activity and cytotoxicity
Natalia Molchanova,[a] Paul R. Hansen[a], Peter Damborg[b], Hanne M. Nielsen[c], Henrik Franzyk,*[a]
[a]
Natalia Molchanova, Assoc. Prof. Paul Robert Hansen, Assoc. Prof. Henrik Franzyk
Department of Drug Design and Pharmacology
Faculty of Health and Medical Sciences, University of Copenhagen
Universitetsparken 2, DK-2100 Copenhagen, Denmark
E-mail: henrik.franzyk@sund.ku.dk
[b]
[c]
Assoc. Prof. Peter Damborg
Department of Veterinary Disease Biology
Faculty of Health and Medical Sciences, University of Copenhagen
Stigbøjlen 4, 1870 Frederiksberg C, Denmark
Prof. Hanne Mørck Nielsen
Department of Pharmacy
Faculty of Health and Medical Sciences, University of Copenhagen
Universitetsparken 2, DK-2100 Copenhagen, Denmark
Supporting information for this article is given via a link at the end of the document.
Abstract: Multidrug-resistant bacteria pose a serious threat to public
health worldwide. Previously, α-peptide/β-peptoid hybrid oligomers
were found to display activity against Gram-negative multidrug-
resistant bacteria. In the present work, the influence of
hydrophobicity, fluorination and distribution of cationic/hydrophobic
residues on antimicrobial, hemolytic and cytotoxic properties of α-
peptide/β-peptoid hybrids were investigated. An array of 22
peptidomimetics
was
tested.
Analogues
with
enhanced
hydrophobicity exhibited increased activity against Gram-positive
bacteria. Incorporation of fluorinated residues into the
peptidomimetics conferred increased potency against Gram-positive
bacteria, while hemolytic properties and activity against Gram-
negative bacteria depended on the degree and type of fluorination.
Generally, shorter oligomers were less potent as compared to the
corresponding longer analogues. However, some short analogues
exhibited
equal
or
higher
antimicrobial
activity.
The
hydrophobic/cationic alternating design proved superior to other
distribution patterns of cationic side chains and hydrophobic moieties.
Introduction
Antimicrobial resistance (AMR) is a steadily growing threat to
public health globally[1]. In particular, continuous inappropriate
use of antibiotics in both animals and humans has accelerated
the selection of multidrug-resistant (MDR) bacterial strains,
which can be almost impossible to eradicate (e.g., recently
colistin-resistant E. coli strains have emerged)[2]. Although AMR
now receives considerable attention, the drug development
pipeline suffers from a lack of new compound classes capable of
replacing the current drugs that rapidly are becoming
3]
ineffective[2c,
. In particular, MDR Gram-negative strains
resistant to all currently used clinical antibiotics constitute a
severe health problem[2b, 4]. Notably, only five entirely new
classes of antibiotics have been launched in the period 2000-
2012, and all of these are applicable to Gram-positive infections
only[5].
Figure 1. Structures of compounds belonging to the different subclasses of
peptidomimetics investigated (suffixes a and b denote 16- and 12-mers,
respectively).
1
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Antimicrobial peptides (AMPs) play a key role in the innate
immune system of all higher organisms[6]. Since their discovery
more than three decades ago, AMPs and their synthetic
analogues have received considerable attention from academia,
and recently also by the pharmaceutical industry. Thus, in 2016
at least six synthetic AMPs were undergoing clinical trials[7].
Moreover, several AMPs have proved effective in vitro against a
wide range of pathogens[8] while exhibiting a low tendency of
resistance development[9], albeit they possess some unresolved
cytotoxicity issues[10]. The detailed antimicrobial mechanisms of
AMPs remain to be elucidated although multiple studies have
established that most cationic AMPs initially interact with the
negatively charged bacterial membrane. When a threshold
concentration or a critical degree of surface adsorption is
reached, transient or lasting pores are formed with ensuing
bacterial killing due to concomitant disturbance of the membrane
potential and/or loss of cytosolic content[11]. Yet, some AMPs
have demonstrated alternative bactericidal mechanisms, e.g.,
inhibition of cell wall synthesis as well as of nucleic acid or
protein synthesis[12]. Nevertheless, AMPs inherently possess a
number of disadvantages: (i) susceptibility to proteolytic
degradation, (ii) pH- and/or salinity-dependent activity, or (iii)
loss of activity due to binding to serum proteins[13]. However,
incorporation of unnatural amino acids or mimetic residues may
improve the bioavailability profile and metabolic stability of
fluorination further exploration of peptidomimetics was clearly
warranted. In the present work we subject the class of α-
peptide/β-peptoid hybrids to an investigation of structure-activity
relationships (SARs) with focus on the influence of
hydrophobicity (methylation or fluorination of phenyl rings and
incorporation of cyclohexane moieties), sequential distribution of
cationic residues, and side chain length on antimicrobial and
hemolytic properties.
Results and Discussion
Six different subsets of α-peptide/β-peptoid hybrids containing
cationic and hydrophobic residues in a 1:1 ratio were designed
and obtained in oligomer lengths of 12 and 16 residues (Figure
1). As guanidinium-functionalized analogues previously were
found to exhibit lower cell selectivity[19a], lysine was chosen as
the typical cationic component. The β-peptoid analogue of
phenylalanine (βNphe) was used as the typical hydrophobic
component. To investigate whether the distance between the
amino functionalities and the backbone is of importance for the
antimicrobial properties, a subset of compounds comprising
analogues with ornithine and (S)-2,4-diaminobutyric acid (Dab)
as cationic residues in addition to lysine (i.e. 1-3) was included.
In another compound series hydrophobicity was increased by
introduction of methyl, fluoro and trifluoromethyl substituents
onto the phenyl rings (i.e. 4-7) as well as by replacing the phenyl
rings with cyclohexane moieties (to give 8). In previous studies,
we have exclusively explored the subclass of α-peptide/β-
peptoid peptidomimetics with an alternating cationic/hydrophobic
design. Therefore, we also examined analogues with alternative
distributions of charged and hydrophobic side chains (i.e. 9 and
10), but with similar backbone in order to elucidate whether the
original design in fact is optimal. For comparison, also the
inversed analogues (i.e. 11) of 1 were included.
The required hydrophobic peptoid building blocks were prepared
by an aza-Michael addition approach[19b, 32], while the cationic
peptoid building block with a lysine-like side chain was obtained
via alkylation of the appropriate amine with ethyl 3-
bromopropanoate, followed by saponification and Fmoc
protection[19b, 33]. Oligomers were prepared by Fmoc-based solid-
phase synthesis on Rink amide resins using either MW-assisted
or manual protocols[19b, 32]. Acidic cleavage from the resin and
subsequent purification by preparative HPLC afforded the target
compounds.
AMPs[13a]
investigated, e.g., α-peptoids[14], β-peptoids[15], β-peptides[16], α-
peptide/β-peptide and
hybrids[17] lipo-AA-peptides[18]
peptide/peptoid hybrids[19]
Previously, α-peptide/β-peptoid
. A number of such peptidomimetics have been
,
,
.
hybrids were compared with peptidomimetics having other
backbone designs (but also displaying alternating cationic-
hydrophobic sequences) and were found to possess superior
cell selectivity[19b]. Furthermore, potency against food-related
bacteria[20]
,
anti-biofilm activity[21]
,
and immunomodulatory
properties[22] has been reported for this compound class.
Interestingly, in contrast to most AMPs these peptidomimetics
display increased potency when tested in media containing up to
25% plasma[23]
.
Structure-activity studies have revealed that antimicrobial activity
may be correlated to enhanced hydrophobicity for both AMPs
and peptidomimetics, albeit with a resulting loss of cell selectivity
and augmented hemolytic properties[17, 24]. Thus, it remains a
challenge to design peptidomimetics possessing a fine-tuned
balance between positive net charge and overall hydrophobicity
that confers a pharmacological activity profile that allows for
systemic use. A number of research groups have examined
several ways to adjust the hydrophobicity of AMPs and
Six bacterial strains representing important Gram-positive and
Gram-negative human pathogens were selected to explore the
spectrum of antimicrobial activity: Staphylococcus aureus,
Enterococcus faecium, Pseudomonas aeruginosa, Escherichia
coli, Acinetobacter baumannii, and Salmonella Typhimurium. In
antimicrobial
peptidomimetics,
e.g.,
via
end-group
or via
24b]
modification[19b,
,
lipidation with fatty acids[25]
,
introduction of bulky[26] or fluorinated amino acids in order to
achieve a higher membrane affinity. The last approach was
investigated by introducing hexafluoro-leucine residues into
magainin or buforin resulting in enhanced antimicrobial activity
and increased resistance to proteolytic degradation, while
retaining low hemolytic activity[27]. By contrast, replacement of
valine by hexafluoro-leucine in protegrin analogues led to
addition,
a
canine clinical isolate of Staphylococcus
pseudintermedius was included as an example of an important
veterinary pathogen. S. pseudintermedius is the main cause of
integumentary infections in dogs, and these infections represent
the main cause for antibiotic treatment of dogs[34]
.
Also, S. pseudintermedius poses a problem for pet owners and
veterinarians due to potential zoonosis[35]. The antimicrobial
activity of the peptidomimetics was evaluated by determination
of minimal inhibitory concentrations (MIC values; Table 1) using
reduced potency, while some analogues even lacked activity[28]
.
Incorporation of fluorine atoms[29], trifluoromethyl groups[30] or
longer fluorinated tails[31] have also been found to enhance the
antimicrobial activity, but often with concomitant increased
hemolytic activity. Due to these diverging findings of the effect of
broth
microdilution
according
to
recommendations
2
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Table 1. Antimicrobial activity, hemolytic properties, and hydrophobicity of peptidomimetics.
HD10 (µM; also
stated as
MICa (µg/ml; also stated as µM in brackets)
No.
%Bb
hemolysis
E.
P.
A.
S.
S.
observed at 150
µM in brackets)
S. aureusd
E. colih
faecium f
aeruginosag
baumanniii
Typhimuriumj
pseudintermedius
e
1a
1b
2a
2b
3a
32 (13.7)
>64 (>36.5)
16 (7.2)
2 (0.9)
4 (2.3)
4 (1.8)
4 (2.4)
8 (3.8)
32 (13.7)
>64 (>36.5)
16 (7.2)
8 (3.4)
>64 (>36.5)
4 (1.8)
4 (1.7)
8 (4.6)
4 (1.8)
16 (9.6)
8 (3.8)
8 (3.4)
64 (36.5)
8 (3.6)
4 (1.7)
64 (36.5)
2 (0.9)
34.4
33.0
33.4
30.6
35.3
>150 (5 %)
>150 (5 %)
>150 (11 %)
>150 (9 %)
18.8 (14 %)
>64 (>38.3)
8 (3.8)
>64 (>38.3)
8 (3.8)
64 (38.3)
4 (1.9)
32 (19.2)
8 (3.8)
32 (19.2)
4 (1.9)
>64
3b
>64 (>41.9)
2 (1.4)
64 (41.9)
8 (5.2)
8 (5.2)
4 (2.6)
33.2
>150 (5%)
(>41.9)
4a
4b
5a
5b
6a
6b
7a
7b
8 (3.3)
8 (4.3)
16 (5.6)
4 (1.8)
2 (0.8)
8 (4.3)
4 (1.4)
2 (0.9)
4 (1.6)
2 (1.1)
8 (2.8)
4 (1.8)
2 (0.8)
2 (1.1)
2 (0.7)
2 (0.9)
4 (1.6)
8 (4.3)
8 (2.8)
2 (0.9)
4 (1.6)
16 (8.6)
2 (0.7)
2 (0.9)
8 (3.3)
8 (4.3)
16 (5.6)
8 (3.7)
4 (1.6)
4 (2.1)
4 (1.4)
4 (1.9)
16 (6.5)
8 (4.3)
8 (2.8)
8 (3.7)
8 (3.2)
8 (4.3)
8 (2.8)
4 (1.9)
8 (3.3)
8 (4.3)
16 (5.6)
8 (3.7)
4 (1.6)
8 (4.3)
4 (1.4)
4 (1.9)
8 (3.3)
16 (8.7)
8 (2.8)
2 (0.9)
4 (1.6)
8 (4.3)
2 (0.7)
2 (0.9)
40.4
38.8
46.9
45.9
36.9
36.1
43.7
41.9
2.6 (21 %)
37.5 (15 %)
<2.3 (100 %)
<2.3 (100 %)
>150 (9 %)
>150 (7 %)
4.7 (68%)
37.5 (22%)
8a
8b
4 (1.7)
4 (2.2)
4 (1.7)
2 (1.1)
2 (0.8)
4 (2.2)
4 (1.7)
2 (1.1)
4 (1.7)
8 (4.4)
4 (1.7)
2 (1.1)
2 (0.8)
2 (1.1)
43.5
41.5
>150 (47%)
>150 (20%)
9a
9b
16 (6.9)
16 (9.1)
16 (6.9)
64 (36.5)
32 (13.7)
>64 (>36.5)
>64
8 (3.4)
8 (4.6)
8 (3.4)
2 (1.1)
4 (1.7)
4 (2.3)
16
64 (>27.4)
32 (18.2)
16 (6.9)
64 (36.5)
32 (13.7)
>64 (>36.5)
>64
16 (6.9)
16 (9.1)
8 (3.4)
8 (4.5)
4 (1.7)
16 (9.1)
0.25
16 (6.9)
16 (9.1)
16 (6.9)
16 (9.1)
4 (1.7)
4 (2.3)
<0.25
16 (6.9)
32 (18.2)
8 (3.4)
64 (36.5)
4 (1.7)
16 (9.1)
0.5
16 (6.9)
32 (18.2)
8 (3.4)
4 (2.3)
4 (1.7)
>64 (>36.5)
<0.25
49.0
45.0
37.3
35.7
35.8
34.6
-
38.5 (88 %)
9.4 (52 %)
>150 (6 %)
>150 (10%)
<2.3 (5 %)
18.8 (9 %)
>150 (2%)
>150 (1%)
10a
10b
11a
11b
Colistin
Gentamicin
<0.25
16
8
1
1
8
1
-
[a] Minimal inhibitory concentrations (i.e. MIC values) for the peptidomimetics and control antibiotics were determined by using 2-fold dilution series in the range 0-
64 μg/mL. The MIC values are given as the median of two independent biological replicates each with three technical replicates in agreement with the CLSI
standards by using visual detection. [b] Hydrophobicity as indicated by the percentage of acetonitrile (%B) at the peak elution time on analytical reversed-phase
HPLC. [c] The hemolytic dose (HD10) is given as the concentration derived from a 2-fold dilution series that resulted in lysis of 10% human erythrocytes. [d]
Staphylococcus aureus [ATCC 29213], [e] Canine clinical methicillin-resistant isolate of Staphylococcus pseudintermedius [29063], [f] vancomycin-resistant
Enterococcus faecium [BM4147], [g] Pseudomonas aeruginosa [ATCC 27853], [h] Escherichia coli [ATCC 25922], [i] Acinetobacter baumannii [ATCC 19606], [j]
Salmonella Typhimurium [U1].
by
the
Clinical
Laboratory
Standards
Institute
Development of a potent and cell-selective antimicrobial
(CLSI)[36].Hemolytic activity was estimated as the peptidomimetic
concentration that gives rise to 10% lysis of human red blood
cells after incubation for 1 hour at 37 °C[37]. Effect on mammalian
cell viability was determined by the MTS/PMS assay[38], and is
reported as the IC50 values in both hepatocytes and fibroblasts.
peptidomimetic is challenging since SAR studies, performed by
us and others[17, 24], on AMPs and antimicrobial peptidomimetics
infer that improved antimicrobial activity often is accompanied by
a similar rise in cytotoxicity. Hence, proper design of stable and
non-toxic lead compounds requires a detailed understanding of
3
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how factors like length, cationicity, amphipathicity and
hydrophobicity define antimicrobial potency and cell selectivity
for a specific class of compounds[39]. The relative hydrophobicity
of peptides and peptidomimetics can be correlated to their
retention in reverse-phase HPLC, and it is usually expressed as
activity against S. aureus and VRE (8- to 16-fold), while potency
toward MRSP and Gram-negative bacteria remained unchanged.
Remarkably, the trisfluorinated 12-mer 7b exhibited high activity
(MIC values of 2-4 µg/mL) against all pathogens included in the
test panel. Likewise, incorporation of cyclohexane-based peptoid
units (to give 8a/b) conferred substantially increased activity
against both Gram-positive and Gram-negative bacteria.
Analogues with an inverse design having lysine-like β-peptoid
residues, but with similar backbone structure (i.e. 11a/b), were
somewhat more hydrophobic than the parent compounds 1a/b,
but still exhibited similar activity profiles to all tested bacteria as
the percentage
of
acetonitrile at
peak
elution[39a]
.
Peptidomimetics were synthesized as 12- and 16-mers (denoted
by suffixes a and b in Figure 1) to examine whether increased
hydrophobicity might compensate for the general observation
that shorter analogues exhibit diminished antimicrobial activity,
whereas cytotoxicity appears to be correlated more distinctly to
oligomer length[20, 22, 40]. Expectedly, a 2- to 16-fold decrease in
antimicrobial activity was generally observed for the shorter
previously found for E. coli[19b]
.
In addition, analogues displaying alternative arrangements of the
cationic and hydrophobic residues, but with identical backbone,
were investigated since all previous work on α-peptide/β-peptoid
oligomers, but for
each bacterium certain shorter
peptidomimetics exhibited equal or even slightly enhanced
potency. Thus, for the Gram-positive S. aureus and VRE only
two short analogues (i.e. 5b and 7b) were equally or slightly
more potent, while this trend was more pronounced for MRSP
as several short analogues (5b-8b) displayed similar or even 4-
fold increased potency. Besides two compounds with slightly
higher activity (5b and 8b), the shorter analogues displayed
retained activity or an up to 16-fold reduction in potency toward
the Gram-negative P. aeruginosa and A. baumannii. For E. coli
and S.Typhimurium the common trend was that the activity of
the shorter analogues ranged from slightly enhanced to
significantly reduced (up to 16-fold) activity (Table 1). Notably,
for most bacteria the highly hydrophobic 12-mers 5b, 7b and 8b
exhibited similar potency (ranging from 2-fold decrease to 2-fold
increase) as the corresponding 16-mers.
hybrids has concerned representatives with alternating cationic
22,
and hydrophobic residues[19b,
40]. Hence, compounds 10a/b
and 9a/b in which all cationic residues were placed pairwise or
completely segregated in each end of the sequence,
respectively, were examined as well. Analogues 10a/b exhibited
similar antimicrobial activity as the parent peptidomimetics 1a/b,
while possessing slightly increased hydrophobicity. By contrast,
compounds 9a/b having a longitudinal amphipathic structure
consistently showed unchanged or decreased antimicrobial
activity albeit being among the most hydrophobic compounds
tested in the present study.
Traditionally, hemolytic activity has been used as a general
indicator of cytotoxicity, and oligomer length and hydrophobicity
appear to be the major determinants of the hemolytic properties
of peptidomimetics[22, 40]. For the pairs 1a/b, 2a/b, and 6a/b no
substantial differences in hemolytic properties were observed
between 16-mers and 12-mers. A slight shortening of the side
chain length of cationic residues (i.e. Lys  Orn) only had a
minor effect on the hemolytic properties (HD10 remained above
150 µM), whereas incorporation of Dab into a 16-mer led to
significantly increased hemolysis (HD10 of 19 µM). Expectedly,
incorporation of more hydrophobic -peptoid residues displaying
methyl (i.e. 4a/b) or trifluoromethyl (i.e. 5a/b) substituents on the
phenyl rings increased the hemolytic activity substantially as
HD10 typically was close to or lower than the MIC value.
Trisfluorinated analogues 7a/b also followed this pattern and
exhibited high hemolysis that may be correlated to high
hydrophobicity ( elution at 46.0% B and 44.1% B) as compared
to 1a/b (35.2% B and 34.7% B). However, compounds 6a/b
containing monofluorinated phenyl rings remained almost non-
hemolytic in accordance with an only slightly increased
hydrophobicity as compared to that of 1a/b.
Interestingly, peptidomimetics with shorter cationic side chains
displayed improved antimicrobial activity against S. aureus and
VRE, whereas the opposite tendency was found for MRSP.
Thus, replacement of Lys with Dab improved the activity against
S. aureus from a MIC of 32 µg/mL to 8 µg/mL (i.e. 1a versus 3a),
while the same modification led to decreased activity against
MRSP (MIC of 2 µg/mL versus 8 µg/mL for 1a and 3a,
respectively). By contrast, no general trend for the potency
against Gram-negative bacteria was observed for this structural
variation. Replacement of Lys (in 1a) with Dab (to give 3a)
confers a slight increase in hydrophobicity as estimated by the
increased retention in reverse phase HPLC (elution at 34.4% B
and 35.3% B, respectively), and these findings are thus in
accordance with earlier observations on the activity of end
group-modified peptidomimetics against S. aureus[24b]
.
To obtain SARs to establish a putative correlation between
increased hydrophobicity and activity, the phenyl rings of the β-
peptoid residues were replaced with substituted aryl moieties
displaying methylation in the 4-position (to give 4a/b) as well as
fluorination in position 4 (i.e. 6a/b) and simultaneously in
positions 3, 4 and 5 (i.e. 7a/b), or trifluoromethylation in position
4 (i.e. 5a/b). Moreover, to extend the hydrophobicity range
further, analogues containing cyclohexane-based peptoid
residues were included. Addition of methyl and trifluoromethyl
groups enhanced the activity against S. aureus and VRE 2- to 8-
fold, e.g., MIC against S. aureus was improved from 32 µg/mL
(1a) to 8 µg/mL (4a), while MIC against VRE was lowered
Besides an influence on the antimicrobial activity, the distribution
of cationic residues within the sequence also had an impact on
the hemolytic properties of the peptidomimetics. Thus, location
of all cationic residues in one end significantly boosted the
hydrophobicity of 9a (51.6% B), which conferred increased
hemolytic properties. Notably, compound 10a with a pairwise
arrangement of cationic residues remained almost non-
hemolytic despite
a
slightly increased hydrophobicity.
Collectively, these results infer that the original design with
significantly for 5b as compared to that of 1b (more than 64-fold). alternating cationic and hydrophobic residues indeed is the most
By contrast, a 2- to 4-fold decrease in activity against Gram-
negative bacteria was typically observed. Fluorination directly on
the aromatic rings (as in 6a and 7a) led to significantly increased
favorable as it allows for potent antimicrobial activity while
keeping hemolysis low. As anticipated from their quite
hydrophobic nature (45.8% B; 43.7% B) compounds 8a/b
4
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Table 2. Effect of selected compounds on mammalian cells given as cell viability (IC50 values given in µg/mL a and selectivity indices (SI)).
Effect on HepG2b
viability
Eukaryote/prokaryote
selectivity index
Effect on NIH 3T3c
viability
Eukaryote/prokaryote
selectivity index
Erythrocyte/prokaryote
selectivity index
Comp´d
IC50/MIC
IC50/MIC
HD10/MIC
IC50 (µg/mL)
IC50 (µg/mL)
G+
G-
G+
G-
G+
G-
318.1 (280.8 – 359.9)
151.2 (133.3 – 173.1)
65.2 (49.3 – 86.6)
44.9 (41.2 – 49.4)
832.3 (723.8 – 959.8)
399.2 (344.6 – 461.6)
527.2 (451-0 – 863.0)
132.4 (113.9 – 154.6)
1a
3a
4a
6a
15
53
38
139
>16
>60
19
12
17
25
7
50
100
49
67
53
27
4.9
1.2
6.6
0.6
9
>140
>75
[a] Values are given as the average (n = 6 in two different cell passages), also stated as 95% confidence interval in brackets. [b] HepG2: human hepatocellular
carcinoma cell line. [c] NIH 3T3: mouse fibroblast cell line.
exhibited intermediate hemolytic activity as compared to that of
1a/b and 5a/b.
selective. Overall, when targeting Gram-positive bacteria it
appears that fluorination (i.e. 6a versus 1a) may be beneficial,
but only to a very limited degree as the absolute IC50 values
were lowered considerably, while the increase in antibacterial
potency was even more pronounced. On the other hand, the
performance of the starting peptidomimetic 1a was superior
against Gram-negative species.
Recent results show that viability testing on different appropriate
cell lines may give a more comprehensive picture of the
selectivity towards bacteria, and thus complement results from
hemolysis
studies[40]
.
Hence,
four
representative
peptidomimetics 1a, 3a, 4a and 6a were selected for testing in
viability assays with hepatocytes (HepG2) and fibroblasts (NIH
3T3) (Table 2). Generally, a correlation between antimicrobial
activity and cytotoxicity was observed. Despite displaying lower
hemolysis than analogues 3a and 4a, peptidomimetic 6a
demonstrated the highest impact on viability of mammalian cells,
indicating a critical effect of fluorination. Surprisingly, compound
4a demonstrated lower cytotoxicity toward fibroblasts than
analogues 3a and 6a, while exhibiting the highest hemolytic
activity. The ranking of compounds in relation to their cytotoxic
effects followed the same trend in both cell lines. Notably,
comparison of the two cell lines infers that HepG2 hepatocytes
appear more sensitive toward all tested compounds than the
NIH 3T3 fibroblasts. In an attempt to reveal a potential direct
correlation between cytotoxicity, hemolytic activity and
antimicrobial activity, selectivity indices (SI) were calculated as
ratios between IC50 or HD10 and an average potency against
Gram-positive or Gram-negative bacteria (Table 2). Interestingly,
all tested analogues displayed similar selectivity indices when
comparing their effect on HepG2 and Gram-positive species,
whereas 1a showed higher selectivity between HepG2 cells and
Gram-negative species. Peptidomimetics 4a and 1a
demonstrated the best cell selectivity between NIH 3T3 cells and
Gram-positive and Gram-negative bacteria, respectively. Also,
6a displayed the highest cell selectivity toward mammalian
erythrocytes and both Gram-positive and Gram-negative species,
while in particular 3a and 4a proved to be considerably less
Conclusions
In the present SAR study, we have explored the effects of
hydrophobicity (via methylation and fluorination), rearrangement
of cationic residues, length of cationic side chains, and oligomer
length on antibacterial activity and cytotoxicity. The results show
that even though hydrophobicity is often considered a key
element in optimization of antibacterial activity, it constitutes a
two-bladed sword as the hydrophobicity must be kept below a
certain threshold level in order to avoid an ensuing loss of
selectivity for bacteria over mammalian erythrocytes and other
relevant cell types. Somewhat surprisingly, modification towards
very high hydrophobicity may in fact even confer diminished
antimicrobial activity, e.g., as seen for the trifluoromethylated
analogues. Nevertheless, limited fluorination at a single position
in the aromatic -peptoid residues proved to be sufficient to
dramatically increase activity against both Gram-positive and
Gram-negative bacteria without affecting the hemolytic
properties (e.g., as seen for 6a), albeit it resulted in acquisition
of some cytotoxicity. By contrast, incorporation of three fluorine
atoms directly on each phenyl ring or as a trifluoromethyl
substituent both led to decreased cell selectivity. Rearrangement
of cationic residues induced increased hydrophobicity without
5
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10.1002/cmdc.201600553
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Several peptidomimetics were prepared manually as previously
described[16]. In brief, H-Rink-Amide ChemMatrix® resin (PCAS BioMatrix
Inc., Quebec, Canada) (loading 0.52 mmol/g, 0.05 mmol) and Teflon
vessels (10 mL; fitted with a polypropylene filter) were used. Coupling
conditions used for peptoid building blocks: 3.0 equiv building block, 3.0
affecting the antimicrobial properties of the compounds, whereas
the hemolytic activity as well as cytotoxicity was increased as
compared to the simple cationic/hydrophobic alternating design.
An interesting trend was seen for MRSP. In this case shorter
peptidomimetics consistently displayed activity comparable to
the corresponding longer analogues despite a clear opposite
trend for other bacteria. Also, the present results further
corroborate our previous observations for this subclass of
peptidomimetics: namely that a slightly increased level of
hydrophobicity is beneficial for both potency and cell selectivity
when Gram-positive bacteria is targeted, whereas such
modifications seem to be of very limited advantage when
combating Gram-negative species.
equiv
benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluoro-
phosphate (PyBOP), and 6.0 equiv DIPEA (>2 h under shaking at room
temperature). Coupling conditions for Fmoc-protected amino acid
building blocks: 5.0 equiv building block, 5.0 equiv PyBOP and 10.0
equiv DIPEA (>2
h under shaking at room temperature). Fmoc
deprotection conditions: 20% piperidine in DMF (2 × 10 min, each time
with 5 mL under shaking at room temperature). Washing conditions: DMF,
MeOH, and DCM (3 × 3 min, each time with 5 mL under shaking at room
temperature). Capping was applied after loading, and after couplings
number 6, 9, and 12: Ac2O–DIPEA–NMP 1:2:3 (5 mL for 10 min under
shaking at room temperature). Cleavage and side chain deprotection
followed by purification were performed as described above for MW-
assisted synthesis of peptidomimetics.
Experimental Section
Determination of antibacterial activity in vitro
General procedure
Six strains were used in MIC determinations: Staphylococcus aureus
Starting materials and solvents were purchased from commercial
suppliers (Iris Biotech, Sigma-Aldrich, and Merck) and used without
further purification. Water used for analytical and preparative HPLC was
[ATCC 29213],
a
canine clinical methicillin-resistant isolate of
Staphylococcus
pseudintermedius [29063], vancomycin-resistant
Enterococcus faecium [BM4147], Pseudomonas aeruginosa [ATCC
27853], Escherichia coli [ATCC 25922], Acinetobacter baumannii [ATCC
19606], Salmonella Typhimurium [U1]. All strains were cryo-preserved in
Brain Heart Infusion (BHI) broth supplemented with glycerol 15% (v/v) at
–80°C, and cultivated aerobically at 37 °C in nutrient agar base (Oxoid,
Basingstoke, UK) supplemented with 5% (v/v) calf blood. Minimal
inhibitory concentrations (MICs) were determined by the broth
microdilution method according to the Clinical Laboratory Standards
Institute (CLSI)[36]. In brief, colonies from fresh overnight cultures were
suspended to a turbidity of 0.5 McFarland, and the resulting suspensions
were further diluted 1:1000 in Mueller−Hinton Broth II. These bacterial
suspensions were added to microtiter plates 96 U-well (Almeco A/S)
containing 2-fold serial dilutions of peptidomimetics in saline followed by
incubation at 37 °C in ambient air for 18 h. The MIC values were
determined as the lowest concentration showing no visible growth as
compared to the control without peptidomimetic added. Experiments
were performed twice as triplicates on two different days.
filtered through
a 0.22 μm Millipore membrane filter. Purity was
determined by analytical HPLC using a Phenomenex Luna C18(2)
column (150 mm × 4.6 mm; 3 µm particle size) on a Shimadzu
Prominence and Shimadzu Nexera system using an aqueous MeCN
gradient with 0.1% TFA added (eluent A: 5:95 MeCN–H2O + 0.1% TFA,
eluent B: 95:5 MeCN–H2O + 0.1% TFA); a flow rate of 0.5 mL/min was
used. For elution of peptidomimetics, a linear gradient of 1060% B
during 10 min was used with UV detection at λ = 220 nm. All tested
compounds had a purity of at least 95%. Preparative HPLC was
performed by using a Phenomenex Luna C18(2) column (250 × 21.2 mm;
5 µm particle size) on a Shimadzu Prominence system by using the
same eluents as for analytical HPLC. Elution was performed with a linear
gradient of 1050% B during 20 min at a flow rate of 40 mL/min with UV
detection at λ = 220 nm. HRMS spectra were obtained by using a Bruker
MicrOTOF-Q II Quadropol MS detector.
General protocol for automated MW-assisted synthesis of
peptidomimetics (1a, 4a, 5a, 5b, 6a, 6b, 10b)
In vitro hemolytic activity
Peptidomimetics were prepared by automated microwave (MW)-assisted
Fmoc-based solid-phase peptide synthesis (SPPS) on a CEMTM Liberty
microwave peptide synthesizer. H-Rink-Amide ChemMatrix® resin
(PCAS BioMatrix Inc., Quebec, Canada) (loading 0.52 mmol/g, 0.1 mmol)
was used. Fmoc (9-fluorenylmethyloxycarbonyl) deprotection conditions:
excess 20% piperidine in DMF, initially 75 °C (MW), 30 s, subsequently
75 °C (MW), 180 s. Coupling conditions: 5.0 equiv of building block, 5.0
equiv N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluoro-
phosphate (HBTU), DIPEA, DMF, 75 °C (MW), 15 min. Resin was
transferred to a Teflon vessel fitted with a polypropylene filter by using
DMF and DCM. Upon draining, the resin was washed with DCM.
Cleavage and side chain deprotection was performed with TFA–H2O
(95:5; 2 × 1 h, each with 5 mL under shaking at room temperature). The
filtrates were collected, and the resin was further eluted with TFA (2 mL),
MeOH (2 mL), and DCM (2 mL). The combined filtrates were
concentrated in vacuo and co-evaporated with MeOH-toluene (3 × 5 mL).
The crude product was purified by preparative HPLC, and the
appropriate fractions were concentrated in vacuo and lyophilized; identity
was verified by HRMS and purity (>95%) was determined by analytical
UHPLC.
The lysis of human red blood cells (hRBCs) was measured as previously
described[37]. In brief, hRBCs from a freshly drawn type 0+ blood sample
were washed with PBS buffer (pH 7.2, 10 mM Tris, 150 mM NaCl), and
then centrifuged once at 700 × g for 8 min and twice at 1000 × g for 8
min each time. Two-fold serial dilutions of the peptidomimetics in PBS
buffer were added to each well in a sterile round-bottom 96-well plate for
a total volume of 20 μL in each well. A 1% v/v hRBC suspension (80 μL
in PBS buffer) was added to reach a total volume of 100 μL in each well.
The plate was incubated (37 °C) for 1 h, and then the cells were pelleted
by centrifugation at 1000 × g for 10 min. The supernatant (60 μL) was
transferred to a new 96-well plate, and the hemoglobin content was
detected by measuring the OD with a Molecular Devices VersaMax
Microplate Reader at 414 nm. OD of cells incubated with melittin (400
μg/mL) defined 100% hemolysis, while OD of cells incubated with PBS
buffer defined 0% hemolysis. Tests were performed twice in triplicates on
two different days.
Cell culturing
Two different cell lines were used: HepG2 and NIH 3T3 (from ATCC,
Manassas, VA, USA), obtained from a similar level of confluence after
21−25 h of culturing under standard conditions (5% CO2/95% O2 at
37 °C); i.e. the seeding densities were the following: HepG2 (6.97 × 104
cells/cm2), NIH 3T3 (6.67 × 104 cells/cm2). HepG2 cells were cultured in
General protocol for manual synthesis of peptidomimetics (1b, 2a,
2b, 3a, 3b, 4b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 11a, 11b)
6
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10.1002/cmdc.201600553
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FULL PAPER
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Acknowledgements
We would like to thank Karina J. Vissing, Birgitte Simonsen,
Anna Mette Hansen and Teresa Piros Dos Santos for practical
help and Maria Pedersen for cell culturing. We thank Drug
Research Academy for partial funding of the POLARstar
equipment. Natalia Molchanova was supported by a PhD grant
from UC-CARE (University of Copenhagen Centre for Control of
Antimicrobial Research) financed by University of Copenhagen.
Keywords: Antimicrobial, peptidomimetics, structure-activity
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8
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Entry for the Table of Contents
From a structure-activity relationship study comprising an array of α-peptide/β-peptoid hybrids with an alternating cationic-
hydrophobic design, displaying variation in length and side chains, it was found that fluorination of the hydrophobic residues led to
increased antibacterial activity. For the longer oligomers a concomitantly increased cytoxicity toward human cells were observed,
while some 12-mers exhibited an acceptable activity profile.
9
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