The Outcomes of Decorated Prolines in the Discovery of Antimicrobial Peptides from Temporin-L

Article abstract of DOI:10.1002/cmdc.201900221

Previously, we identified a potent antimicrobial analogue of temporin L (TL), [Pro³]TL, in which glutamine at position 3 was substituted with proline. In this study, a series of analogues in which position 3 is substituted with non-natural proline derivatives, was investigated for correlations between the conformational properties of the compounds and their antibacterial, cytotoxic, and hemolytic activities. Non-natural proline analogues with substituents at position 4 of the pyrrolidine ring were considered. Structure–activity relationship (SAR) studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with circular dichroism (CD) and NMR spectroscopic analyses for selected compounds. The most promising peptides were additionally evaluated for their activity against some representative veterinary microbial strains to compare with those from human strains. We identified novel analogues with interesting properties that make them attractive lead compounds.

Full text of DOI:10.1002/cmdc.201900221

Accepted Article
Title: The Outcomes of Decorated-Prolines in Discovering Novel
Antimicrobial Peptides from Temporin-L
Authors: Paolo grieco, Elisabetta Buommino, Alfonso Carotenuto,
Ignazio Antignano, Rosa Bellavita, Bruno Casciaro, Maria
Rosa Loffredo, Francesco Merlino, Ettore Novellino, Maria
Luisa Mangoni, Francesca Paola Nocera, Diego Brancaccio,
Pasqualina Punzi, Daniela Roversi, Raffaele Ingenito, and
Elisabetta Bianchi
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To be cited as: ChemMedChem 10.1002/cmdc.201900221
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10.1002/cmdc.201900221
ChemMedChem
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The Outcomes of Decorated-Prolines in Discovering Novel
Antimicrobial Peptides from Temporin-L
E. Buommino,[a],† A. Carotenuto,[a],† I. Antignano,[a] R. Bellavita,[a] B. Casciaro,[b] M. R. Loffredo,[b] F.
Merlino,[a] E. Novellino,[a] M. L. Mangoni,[b] F. P. Nocera,[e] D. Brancaccio,[a] P. Punzi,[c] D. Roversi,[c] R.
Ingenito,[c] E. Bianchi,[c],* and P. Grieco [a],[d],
*
[a]
[b]
[c]
Prof. P. Grieco, Prof. A. Carotenuto, Prof. E. Novellino, Dr E. Buommino, Dr. D. Brancaccio, Dr. F. Merlino, Dr. R. Bellavita, Dr. I. Antignano
Department of Pharmacy, University of Naples “Federico II”, Naples, 80131, Italy
paolo.grieco@unina.it
Prof. M.LK. Mangoni, Dr. B. Casciaro, Dr. M.R. Loffredo,
Department of Biochemical Sciences, Laboratory affiliated to Pasteur Institute Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome,
00185, Italy
Dr. E. Bianchi, Dr. R. Ingenito, Dr. P. Punzi, Dr. D. Roversi
Peptide Chemistry Unit, IRBM S.p.A., via Pontina km 30,600, Pomezia, 00071, Italy
E.Bianchi@irbm.com
[d]
[e]
Prof. P. Grieco
Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPEB) University of Naples “Federico II”, Naples, 80134, Italy.
Dr. F.P. Nocera
Department of Veterinary Medicine and Animal Production, University of Naples Federico II, 80137 Naples. Italy
Supporting information for this article is given via a link at the end of the document.
1
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Abstract: Previously, we identified
a
potent antimicrobial
of the physical integrity of the microbial membrane and/or by
translocating across the membrane into the cytoplasm of bacteria
to act on intracellular targets.[21] Only a few AMPs are currently in
clinical trials because of their low metabolic stability, which
generally represents the limit of therapeutic peptides.[22,23] The
amphibian peptides temporins represent one of the largest
families (around 100 members) and are among the smallest-sized
AMPs (10-16 amino acid residues) found in nature to date. In the
sequence, they are C-terminally amidated and only one or two
basic residues are present.[24] Temporins are known to be
particularly active against Gram-positive bacteria, with minimal
inhibitory concentrations (MIC) ranging from 2.5 to 20 μM.[25,26]
The exception is given by the isoform L (temporin L, TL), Phe-Val-
Gln-Trp-Phe-Ser-Lys-Phe-Leu-Gly-Arg-Ile-Leu-NH2 (1), as it is
also active against Gram-negative bacteria and yeast strains.[24]
Most of the temporins are practically non-hemolytic, but the highly
potent TL kills human erythrocytes at microbicidal concentrations
as well.[27] Therefore, for further development as a broad spectrum
antibiotic, it is necessary to increase its therapeutic index, referred
to as the ratio between the concentration of hemolytic activity
(minimum hemolytic concentration, MHC) and antimicrobial
activity (MIC), MHC/MIC.[28] Studies focused on the structure-
activity relationships of both native TL and some synthetic
analogues revealed the existence of a tight correlation between
their hemolytic activity and α-helix content,[29] while less
correlation was found between α-helix content and antimicrobial
activity.[30] Previously, we identified an interesting TL analogue,
[Pro3]TL (2), through the replacement of glutamine in position 3
with a proline. [Pro3]TL (2) showed a reduced cytolytic effect in
vitro from 2- to 5-fold lower than that of the natural TL (1), while
preserved antimicrobial effectiveness against both bacteria and
yeasts.[29,30]
analogue of temporin L (TL), [Pro3]TL, in which glutamine at
position 3 was substituted with proline. In this study, a series of
analogues, where the position 3 was substituted with non-natural
proline derivatives, was investigated for the correlation between
the conformational properties of the compounds and their
antibacterial, cytotoxic and hemolytic activities. Non-natural
proline analogues with substituents at position 4 of the pyrrolidine
ring were considered. Structure-activity relationship studies of
these analogues were performed by means of antimicrobial and
cytotoxicity assays along with circular dichroism (CD) and NMR
spectroscopy analysis for selected compounds. The most
promising peptides were additionally evaluated for their activity
against some representative veterinary microbial strains to
compare with those from human strains. We identified novel
analogues with interesting properties that make them attractive
lead compounds.
Introduction
The growing development of antibiotic resistance poses a major
human health concern.[1,2] Although antimicrobial agents have
saved lives of millions of people, their inappropriate and intensive
use in both veterinary and human medicine as well as in food
animals, has promoted the emergence of antimicrobial resistance
(AMR) and the higher risk of untreatable infections.[3,4] Diseases
caused by drug-resistant bacteria may transfer their resistance to
drug susceptible bacteria that ultimately may act as reservoir of
resistance to pathogenic organisms.[5] During the last decades,
free movement of people and goods around the world as well as
the intensive international transport of livestock, have contributed
even more to AMR. Moreover, the AMR emergence is associated
with a decline in the rate of development of new antimicrobial
agents.[6] In fact, only a few novel antibiotics against Gram-
positive bacteria have become available in recent years,[7] such
as the new cephalosporins Ceftobiprole and Ceftaroline, active
against methicillin-resistant Staphylococcus aureus (MRSA), and
the oxazolidinone Tedizolid that has activity against clinically
relevant pathogens, e.g. MRSA, methicillin-susceptible S. aureus
(MSSA) and various Streptococcus species.[8,9] Instead, treating
Gram-negative bacterial infections is more challenging because
of the rapid antimicrobial resistance that they develop. Indeed, no
new class of drugs has been introduced during the last four
decades.[10,11] Besides, it has been estimated that most of the
antibiotics currently used for common human and animal
infections will be useless within five to ten years, moving back to
a pre-antibiotic era when infections were used to cause high
morbidity and mortality.[12] In this context, antimicrobial peptides
(AMPs), existing in all life forms,[13] exert killing activity on a large
number of microorganisms.[14] Belonging to the host’s innate
defense system, they represent the most ancient and fast-acting
elements against microbial pathogens.[15] To date, these AMPs
are promising candidates for a new generation of anti-infective
agents, in both human and veterinary medicine. AMPs are short
and generally positively charged peptides,[16,17] many of which
have the ability to kill microbial pathogens directly, whereas others
act indirectly by modulating the host defense systems.[18-20] Many
AMPs display a rapid antimicrobial activity by causing disruption
Table 1. Peptide sequences of synthesized [Pro3]TL analogues
Cmpd
Sequencea
TL, 1b
F-V-Q-W-F-S-K-F-L-G-R-I-L-NH2
F-V-Pro-W-F-S-K-F-L-G-R-I-L-NH2
[Pro3]TL, 2b
3
4
F-V-(cis-4F-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
F-V-(4,4diF-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
F-V-(cis-4CF3-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
F-V-(cis-4MeS-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
F-V-(cis-4NH2-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
F-V-(trans-4Chx-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
F-V-(trans-4Ph-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
F-V-(trans-4Bn-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
F-V-(cis-4PhO-Pro)-W-F-S-K-F-L-G-R-I-L-NH2
5
6
7
8
9
10
11
[a] Residue variation compared to 1 are highlighted in bold. [b] Already
described in literature [29].
Considering this prominent result, we designed a focused library
of derivatives in which the pyrrolidine group was decorated with
various chemical functionalities at position 4 of the ring (Table 1
and Figure 1) to accomplish an extensive structure- and
2
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10.1002/cmdc.201900221
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conformation-activity relationship study. Such compounds were Results and Discussion
analyzed by means of antimicrobial and cytotoxicity assays on
both human bacterial strains and some veterinary bacterial strains
in order to compare both activities. Conformational analysis by CD
spectroscopy was performed for all compounds, while the NMR
spectroscopy was carried out only for selected analogues.
Synthesis
Peptide synthesis of 1-11 was manually performed by microwave-
assisted solid-phase peptide synthesis (SPPS) and using Fmoc/t-
Bu chemistry. After cleavage from the solid support, crude
peptides were purified and characterized by reverse-phase high
pressure liquid chromatography (RP-HPLC) and ESI-MS (Table
S1 and Figure S1, see Supporting Information).
Biological activity
Antimicrobial activity. The primary structures of the designed
temporin L analogues are reported in Table 1, where position 3
was replaced with several proline residues (Figure 1) decorated
with different chemical groups at position 4 of the pyrrolidine ring.
In particular, polar electron-withdrawing (fluoro, trifluoromethyl),
apolar (methylthio), positively charged (amino), aliphatic
(cyclohexyl) or aromatic (phenyl and benzyl) groups were
employed since these groups can interact with the microbial
membrane envisaged as the biological target of TL and its
derivatives.[30]
Figure 1. Proline derivatives used in this study.
Table 2. Antimicrobial activity of designed compounds 3-11.
Microorganisms
MIC values (μM)a
1
2
3
4
5
6
7
8
9
10
11
Gram-negative bacteria
E. coli ATCC 25922
12.5
12.5
12.5
25.0
12.5
12.5
12.5
25.0
12.5
25.0
12.5
25.0
3.12
12.5
50.0
100.
12.5
50.0
25.0
50.0
25.0
50.0
P. aeruginosa ATCC
27853
Gram-positive bacteria
B. megaterium Bm11
3.12
3.12
3.12
6.25
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
3.12
S. epidermidis ATCC
12228
Yeasts
C. albicans ATCC 24433
6.25
12.5
12.5
12.5
12.5
6.25
12.5
25.0
12.5
25.0
12.5
Aliquots (50 μl) of bacteria in mid-log phase at a concentration of 2 × 106 colony-forming units (CFU)/ml (5 x 104 CFU/ml for Candida) in culture medium (Müller-
Hinton broth for bacteria, Winge broth for C. albicans) were added to 50 μl of MH broth (Winge broth for C.albicans) containing the peptide in serial twofold dilutions.
The minimal inhibitory concentration (MIC) is the concentration (μM) of peptide at which 100 % inhibition of microbial growth is observed, after an incubation of 16-
18 h at 37°C (at 30 °C for C, albicans).
a Each MIC value is the average of at least three independent experiments.
3
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The antimicrobial activity against some reference bacterial and
Candida strains was evaluated in the broth microdilution assay to
determine the MIC (Table 2). All synthesized compounds
displayed a good antimicrobial activity towards the Gram-positive
bacterial strains considered in this study, with MICs values
comparable to those of 1 and 2. Notably, almost all compounds
displayed a similar efficacy against Candida albicans ATCC
24433, as compound 2. Peptide 3, in which the residue of Pro3
compared to both reference peptides TL (1) and [Pro3]TL (2).
However, the anticandidal activity remained almost unchanged
with respect to 2, except for compound 10, showing a MIC of 25.0
µM.
Subsequently, the peptides were evaluated for their activity
against some veterinary bacterial strains, namely E. coli and P.
mirabilis (Gram-negative), S. pseudintermedius (Gram-positive),
M. pachydermatis (yeast).
was replaced with cis-4-fluoro-proline, showed
a
similar
The veterinary clinical isolates were first analyzed by disc
diffusion test for the occurrence of antibiotic resistance,
demonstrating a strong resistance to most common antibiotics
used in the therapy of pet animals (data not shown). Regarding
the activity on Gram-negative strains, only compounds 6 and 7
showed a decent activity on E. coli. In particular, compound 7
resulted in the most potent one with a MIC of 3.12 µM. On the
contrary, all other synthesized compounds were inactive against
P. mirabilis (MIC >100 µM). It is important to underline that this
strain was resistant to almost all classes of antibiotics tested by
disc diffusion test (data not shown). Further interesting results
were obtained on S. pseudintermedius. With the sole exception of
compounds 4 and 5 all peptides showed a good activity.
Specifically, compounds 3 and 11 displayed the best activity
against both MSSP and MRSP with a MIC of 1.56 µM. Additionally,
the compounds were tested on the yeast M. pachydermatis. Good
activity was shown for all compounds, especially for 6, 9 and 11
with a MIC of 12.5 µM. Overall, compound 11 showed the best
activity on both M. pachydermatis and S. pseudintermedius.
Since both strains can be etiological agents in canine piodermite
and otitis, compound 11 can be considered a potential lead
compound for combined therapy in the care of pet animals.
Taking into account the antimicrobial activity against reference
strains and clinical isolates, the compounds that showed a better
activity than compound 2 were found to be 6, 7 and 11 and for this
reason they were selected for further characterization in the
hemolysis assay.
antibacterial activity as the parent peptide 2, on both Gram-
negative and Gram-positive bacterial strains. In addition, the
activity towards C. albicans resulted to be 2-fold lower respect to
TL. Peptides 5 and 6, featuring cis-trifluoromethyl-Pro and cis-
methylthio-Pro, respectively, had a similar behavior. In fact, they
resulted to be 2-fold less active than TL toward P. aeruginosa
ATCC 27853, while the activity on E. coli ATCC 25922 was
unchanged. The addition of another fluorine atom on the
pyrrolidine in peptide 4 (4,4-difluoro-Pro) had little effect on the
antimicrobial activity, either compared to 5 (cis-4-trifluoromethyl-
Pro TL) or to the parent [Pro3]TL (2). Differently, peptide 7,
characterized by
a cis-4-amino-Pro residue, displayed a
significant lower MIC (3.12 µM) on E. coli ATCC 25922, while it
maintained similar activity against P. aeruginosa ATCC 27853.
Furthermore, the anticandidal activity was slightly decreased with
respect to 1. Surprisingly, by replacing the Pro3 with its trans-4-
cyclohexyl-Pro derivative, the resulting peptide 8 showed a
dramatic reduction in activity on E. coli ATCC 25922 and lost
effect on P. aeruginosa ATCC 27853. In addition, the activity
towards C. albicans resulted to be 2-fold lower than the parent
peptide 2. Finally, analogues 9-11, all characterized by an
aromatic-containing functionality in position 4 of the pyrrolidine
ring, trans-4-phenyl-Pro (9), trans-4-benzyl-Pro (10), cis-4-
phenoxy-Pro (11), respectively, resulted to be poorly active on P.
aeruginosa ATCC 27853 (MIC 50 µM) while the activity on E. coli
ATCC 25922 was 2-fold lower for compounds 10 and 11,
Table 3. Antimicrobial activity of designed compounds 3-11 on veterinary strains.
Microorganisms
MIC values (μM)a
1
2
3
4
5
6
7
8
9
10
11
Gram-negative bacteria
E. coli
12.5
12.5
>100
>100
>100
>100
>100
>100
12.5
3.125
>100
>100
>100
>100
>100
>100
>100
>100
>100
Proteus mirabilis
>100
>100
>100
Gram-positive bacteria
MSSP
MRSP
3.125
3.125
3.125
3.125
1.56
1.56
>100
>100
>100
>100
3.125
3.125
6.25
6.25
3.125
3.125
3.125
6.25
3.125
3.125
1.56
1.56
Yeasts
M. pachydermatis
25
25
25
25
25
12.5
25
25
12.5
25
12.5
Aliquots (50 μl) of bacteria in mid-log phase at a concentration of 2 × 106 colony-forming units (CFU)/ml (5 x 104 CFU/ml for yeasts) in culture medium (Müller-Hinton
broth for bacteria, Sabouraud broth for M.pachydermatis) were added to 50 μl of MH broth (Sabouraud broth for M.pachydermatis) containing the peptide in serial
twofold dilutions. The minimal inhibitory concentration (MIC) is the concentration (μM) of peptide at which 100 % inhibition of microbial growth is observed, after an
incubation of 16-18 h at 37°C (at 30°C for yeasts) aEach MIC value is the average of at least three independent experiments.
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Hemolytic activity. The compounds with the best antimicrobial
activity (6, 7 and 11) were tested for their effect on circulating
blood cells (i.e. erythrocytes) and the percentages of hemolysis
are reported in Figure 2 and compared to those of the parent
peptide (2). Compound 11 showed a potent hemolytic effect even
at low concentrations (>40% at 3.12 or 6.25 µM). In comparison,
compound 6 was found to be less hemolytic than compounds 11
and 2 at a concentration range from 3.12 to 25.0 µM, but more
active than the parent peptide 2 at the highest concentration (i.e.
50 µM). Interestingly, compound 7 was less toxic than compound
2 (p<0.01) and all the other tested analogs at all concentrations
tested. This makes peptide 7 the compound with the best
biological profile.
epidermitis (Table 2). This is in accordance with our previous
finding that helical percentage of TL derivatives has minor effect
on their antibacterial activity.[35] The relationships between the
conformations of the synthesized peptides and the antibacterial
activity on Gram-negative bacteria is more intriguing. Three
peptides (8, 10, and 11) showed a reduced activity in comparison
to the parent 2 against both the human Gram-negative bacteria
tested (Table 2). Those peptides had different degrees of helical
content in SDS (from 21 to 48%) thus, again, there is not a clear
correlation between antibacterial activity and helical content.
Interestingly enough, peptides 8, 10, and 11 showed a relatively
high propensity to the β-sheet or turn formation in PB solution
which could indicate their tendency to aggregate in amyloid-like
structures. It can be hypothesized that this aggregation tendency
increases in contact with the external membrane of the Gram-
negative bacteria, in particular, in the presence of
lipopolysaccharides (LPS). This possibility was tested by
acquiring the CD spectrum of peptide 8, which was almost
inactive against Gram-negative bacteria, in the presence of LPS
micelles (Figure S2, Supporting Information).
Figure 2. Hemolytic activity of compounds 6, 7 and 11 compared to the parent
peptide 2. Percentages of hemolysis ± SEM were calculated with respect to the
complete lysis of erythrocytes in distilled water.
Structural Studies
Figure 3. CD spectra of peptide 1 and its analogues 2-11 in PB a), SDS b), DPC
c), and DPC/SDS 9:1 micelle solutions d). (1, black solid line; 2, blue solid line;
3, magenta solid line; 4, red solid line; 5, green solid line; 6, orange solid line; 7,
black dashed line; 8, blue dashed line; 9, magenta dashed line; 10, red dashed
line; 11, green dashed line).
CD analysis. To explore the conformational changes of TL
analogues synthesized in this study, we performed CD
spectroscopy studies in phosphate buffer (PB, pH 7.4), sodium
dodecylsulphate (SDS), dodecylphosphocholine (DPC), and
DPC/SDS 9:1 micelle solution (Figure 3). These micelles were
used as rough mimetics of bacterial, mammalian, and yeast
membranes, respectively. [31-33]
The spectrum displayed a minimum at 220 nm and a maximum at
196 nm and its deconvolution by Bestsel algorithm gave a β-sheet
content higher than 40%, a situation often associated to amyloid-
like peptide oligomers.[35] Henceforth, peptide 8 inactivity against
Gram-negative bacteria could be due to its oligomeric state
induced by the contact with LPS. The oligomers large size makes
it more difficult for the peptide to diffuse through the cell wall into
the target cytoplasmic membrane.[36] It is worth noting that TL,
differently from peptide 8, has a high helical propensity in the
same experimental conditions.[35,37]
Compound 7 showed a very high activity against E. coli, being 4-
fold higher than both its parent 2 and the native TL (1). Notably,
peptide 7 activity against E. coli was also confirmed on a
veterinary strain (Table 3). Therefore, this peptide conformation
was deeply investigated by solution NMR (see below).
Secondary structure content (Table 4) was evaluated from CD
spectra by the Bestsel method.[34] CD spectra in PB revealed the
prevalence of disordered conformers for all compounds
characterized by a minimum close to 200 nm (Figure 3a and Table
4). For some analogues (8, 10, and 11) a relatively intense
minimum at 228 and maximum at 200 nm were observed. These
spectral features were associated by Bestsel prediction to an
increase of the β-sheet and/or β-turn structures content (Table 4).
In SDS, DPC, and DPC/SDS 9:1 membrane mimetic solutions,
CD spectra indicated more structured peptides (Figures 3b-d). In
all cases, two minima close to 208 and 222 nm were obtained,
indicating helical propensity. Indeed, helical percentage of the
peptides varied from 21 to 52% in SDS and from 24 to 60% in
DPC/SDS according to Bestsel prediction (Table 4). Nonetheless,
antibacterial activity on Gram-positive strains and antifungal
activity were almost identical regardless the helical content. For
example, peptides 7 (42.7% helix in SDS) and 8 (21.3% helix in
SDS) had the same activity on both B. megaterium and S.
Considering the effect of the conformational propensities of the
peptides on the hemolytic effect, peptide 11 showed the highest
hemolytic effect (Figure 2) and also the highest helical content
(Table 4) in the eukaryotic mimetic DPC solution. This result is in
5
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10.1002/cmdc.201900221
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accordance with our previous result correlating peptide helicity in
DPC with its hemolytic effect.[35,38] Peptides 2, 6, 7 displayed
similar helical contents (Table 4). The lower hemolytic potency of
peptide 7 could be tentatively explained considering our previous
result on the mechanism of the hemolytic action of TL (1) and its
derivatives. In fact, studying the interaction between 1 and
zwitterionic DPC micelles by NMR[29] and MD simulations, [30] we
found that 1 inserts into the micelle, perpendicularly to the
membrane, with the N-terminus deeply buried in the micelle
hydrophobic core, thus unveiling the barrel-stave mechanism. [30]
The presence of a hydrophilic group on Pro3 within the N-terminus
region, such as the amino group of 7, probably prevents the
peptide to exert the above described mechanism, thus explaining
its low toxicity.
Table 4. Secondary structure* percentages of TL analogues.
Peptide
PB solution
DPC solution
SDS solution
DPC/SDS Solution
h
t
r
h
t
r
h
t
r
h
t
r
b
b
b
b
1
2
12.7
31.5
14.2
41.5
62.2
2.0
9.5
26.4
47.7
8.7
11.0
32.6
60.5
3.7
11.1
24.7
9.6
11.9
12.3
15.3
0.0
19.1
25.1
26.9
17.9
51.7
14.5
36.7
31.0
35.8
22.1
18.7
16.3
16.2
14.8
13.1
16.1
16.1
16.1
18.0
27.6
52.7
46.7
44.5
51.9
35.2
54.5
41.0
39.7
41.3
44.8
42.8
42.2
40.2
39.5
42.6
44.3
25.0
28.8
39.4
51.8
16.2
9.9
11.2
12.8
11.0
11.3
12.4
12.0
13.5
13.4
12.9
10.8
29.8
35.2
31.7
35.9
36.9
27.5
48.2
39.9
41.6
30.6
45.6
42.0
43.5
51.7
44.1
42.7
21.3
32.3
30.9
48.4
3.4
10.9
5.9
2.8
2.3
9.4
15.8
6.7
4.2
4.2
15.0
14.3
12.0
12.4
16.1
12.8
13.6
14.7
14.3
12.7
36.0
32.8
38.6
33.1
37.5
35.2
49.3
46.3
50.6
34.8
43.2
43.5
41.0
40.9
45.3
45.1
24.4
26.5
35.8
49.9
14.5
8.4
12.7
13.8
11.2
11.9
13.0
11.5
12.3
16.6
12.4
13.9
29.5
34.3
31.3
32.7
36.7
28.2
42.1
41.7
39.3
28.2
3
4
17.1
13.4
8.1
16.5
14.5
5.1
5
6
7
14.9
6.1
16.1
13.4
17.9
6.1
15.2
21.2
15.2
12.5
8.0
8
9
13.3
4.8
10
11
5.5
6.8
*h helix, β beta-sheet, t turn, r random coil. Peptide 8 in LPS: h 1.6, β 41.3, t 13.9, r 43.2.
NMR analysis of peptides 7 and 8. Considering the very
interesting antimicrobial activity of compound 7, in particular
against Gram-negative bacteria, and its low hemolytic effect, it
was also investigated by NMR spectroscopy. Moreover, peptide
8, which was almost inactive against Gram-negative, was also
investigated by NMR for comparison. NMR spectroscopy was
performed in SDS micellar solution. Complete 1H NMR chemical
shift assignments were effectively achieved for both peptides
according to the Wüthrich procedure (see Supporting Information,
Tables S2–S3).[39]
Peptide 7 in SDS solution exhibited NMR spectral features
pointing to helical propensity. Upfield shift of the Hα NMR signals
and temperature coefficients of many amide protons (Table S2),
and diagnostic NOEs (Table S4) indicated that many residues in
the central and C-terminal region are in a helical conformation.
Structure calculation gave an ensemble of 20 structures (Figure
4a) satisfying the NMR-derived constraints (violations smaller
than 0.10 Å). Backbone is well defined, with a rmsd = 0.34 Å. An
inverse γ-turn centered on the amino-Pro3 can be observed at the
N-terminus followed by an α-helix from Trp4 to Leu13 (Figure 4a).
Also, the side chains are well defined apart those of Phe1 which
is very flexible and Phe8 which shows both trans and gauche+
orientations. Interestingly, the peptide assumes an amphipathic
structure with a positive side (upper side in Figure 4a) and a
hydrophobic side (lower side).
In particular, the cis-4-amino group on Pro3 points towards the
other positively charged groups (Lys7 amino and Arg11
Figure 4. Superposition of the 20 lowest energy conformers of 7 (a, PDB ID
code: 6QXB) and 8 (b, PDB ID code: 6QXC) in SDS. Structures were
superimposed using the backbone heavy atoms. Only heavy backbone atoms
are shown for clarity. Backbone atoms were evidenced as a ribbon. Heavy
atoms are shown with different colors (carbon, green; nitrogen, blue; oxygen,
red). *Pro: cis-4-amino proline. **Pro: trans-4-cyclohexyl proline.
guanidinium groups) enlarging the charged peptide surface.
Diagnostic NMR parameters observed for 8 also indicated
conformational propensity toward helical or turn structures but
with higher flexibility (Tables S3 and S5). In fact, the up-field shifts
of the Hα NMR signals were less intense than those observed for
6
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10.1002/cmdc.201900221
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Materials and general methods. Nα-Fmoc-protected amino acids, Fmoc-
Phe, Fmoc-Val, Fmoc-Gln(Trt), Fmoc-Pro, Fmoc-Trp(Boc), Fmoc-Ser(tBu),
Fmoc-Lys(Boc), Fmoc-Leu, Fmoc-Gly, Fmoc-Arg(Pbf), Fmoc-Ile, all were
purchased from NovaBiochem. Other non-natural Nα-Fmoc-amino acids,
namely Fmoc-L-Pro(4,4-diF2), Fmoc-L-Pro(cis-4-F), Fmoc-L-Pro(cis-4-
2 and 7. Calculated structures, shown in Figure 4b, were less
defined than those obtained for 7 (rmsd backbone heavy atoms =
0.72 Å) which is in accordance with the CD result indicating a
minor content of helix structure compared to 1, 2, and 7 (Table 4).
A helix structure is observable from residue 5 to 12 in many
structures of the ensemble (18/20), while the N-terminal region is
in extended conformation. Notably, the cyclohexyl group is
positioned near the phenyl rings of Phe1 and Phe5 forming a
hydrophobic cluster. The overall structure is amphipathic with the
cyclohexyl cluster oriented on the same side of Phe8, Leu9, and
Ile12 (lower side in Figure 4b). Differently from peptides 7 and 8,
parent peptide 1 showed a tendency to the amphipathic α-helix
along the entire sequence in SDS solution (Figure S3, Supporting
Information) except some flexibility at the N-and C-termina.[29]
However, the loss of the N-terminal helical segment, observed in
7 and 8, doesn’t affect the antimicrobial activity of the TL
derivatives, as already demonstrated for peptide 2.[29,30]
Henceforth, both peptides 7 and 8 keep the amphipathic character
of their parent peptides 1 and 2 irrespective of the chemical
modification on Pro3. This is due to the high flexibility of the N-
terminal region of these peptides, which allows to orient the
additional group on the proline in the most suitable way for
membrane interactions. Extending this result to all the developed
peptides (Table 1), this could explain the high activity against
Gram-positive bacteria and good activity against fungi observed
for all of them (Table 2). In the same way, a properly orientation
of the positively charged amino group of 7 can explain its
increased activity against many Gram-negative strains.
CF3),
Fmoc-L-Pro(cis-4-OPh),
Fmoc-L-Pro(trans-4-Bzl),
Fmoc-L-
Pro(trans-4-Ph), Fmoc-L-Pro(trans-4-Chx) and Fmoc-L-Pro(cis-4-SMe),
were purchased from Polypeptide Laboratories. Fmoc-L-Pro(cis-4-NHBoc)
was purchased from 3B Scientific Corp. The Rink amide resin was
purchased from Chem-Impex. Coupling reagents were purchased as
follows: N,N′-Diisopropylcarbodiimide (DIC) from Sigma-Aldrich, Ethyl
cyano(hydroxyimino)acetate (Oxyma Pure®) from Chem-Impex, 1-
Hydroxy-7-azabenzotriazole
(HOAt)
from
CreoSalus.
N,N-
diisopropylethylamine (DIEA), piperidine and trifluoroacetic acid (TFA)
were purchased from Sigma-Aldrich. Peptide synthesis solvents and
reagents were purchased as follows: N,N-dimethylformamide (DMF),
dichloromethane (DCM), tert-Butyl methyl ether (MTBE), methanol,
acetonitrile (CH3CN) HPLC grade were all from Sigma-Aldrich; diethyl
ether (Et2O) from ITW Reagents; N-Methyl-2-pyrrolidone (NMP) from
Merck Millipore. All were reagent grade acquired from commercial sources
and used without further purification.
Peptide synthesis. The synthesis of peptides 1-11 was performed by solid-
phase peptide strategy (SPPS) using Fmoc/t-Bu chemistry. All peptides
were assembled on a Rink amide PS resin 100-200 mesh, (0.2 mmol scale
with a 0.47 mmol/g of loading). The synthesis of peptides 1 and 2 was
performed manually using a Microwave CEM Accent instrument. Amino
acids (3 equiv) were acylated using equimolar amount of DIC and Oxyma
as activators. Double coupling conditions: 75°C-170W-15’’, then 90°C-
30W-110’’, were used for all amino acids. The synthesis of all the other
peptides was performed on a Liberty Blue microwave synthesizer (CEM
corporation) using a 5-fold excess of amino acids and equimolar amounts
of DIC and Oxyma. The synthesis proceeded on the synthesizer up to Trp4,
followed by acylation with the different pyrrolidine analogues for each
sequence by manual synthesis using 3 equivalents of each amino acid and
equimolar amounts of activators DIC and HOAt in DMF. The last two
residues Val2 and Phe1 were acylated by double acylation with DIC and
HOAt as activators. The final peptide-resins were washed and dried with
DCM, MeOH and Et2O. Peptide 1 and its analogues 2-11 were cleaved
from the resin using a solution of TFA/TIS/Phenol/H2O, 87/3/5/5 for 2 h.
The resins were filtered, and peptide solution were precipitated with MTBE.
The peptide precipitates were centrifuged and washed with fresh MTBE.
The peptide pellets were dried dissolved in 50:50 water/acetonitrile 0.1%
TFA and lyophilized.
On the other hand, reduction or loss of activity against Gram-
negative bacteria observed in some of the novel peptides is likely
due to peptide aggregation as stated above. In fact, the
hydrophobic cluster found in peptide 8 structure (Figure 4b), but
also predictable in other peptide like 10 and 11, can work as an
aggregation point leading to the oligomerization.
Conclusions
Peptide 1 has unique anti-yeast and anti-Gram-negative activity
among the temporins. These activities were maintained by its
analogue 2, which also showed a reduced cytotoxicity against red
blood cells. By decorating the Pro residue in 2, we have now
obtained interesting candidates for antimicrobial clinical studies.
In particular, peptide 7 characterized by a cis-4-amino-Pro
residue has shown an improved activity than the parent peptide 2
against human and veterinary Gram-negative bacterial strains
and, at the same time, it has conserved the activity of 2 on Gram-
positives and yeasts and the low toxicity on red blood cells.
Peptide 11 is also interesting due to its activity against veterinary
strains but, because of its toxicity, it can be considered as a
starting point for further chemical alterations.
Purification of peptides was performed by preparative reverse phase
chromatography on a Waters 2545 HPLC system equipped with a 2489
UV detector on a Waters XBridge ™ C18 preparative column (130 Å, 5 µm,
150x50mm) using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in
acetonitrile. All fractions with the highest purity were collected and
lyophilized obtaining peptides with a purity >95% (20-65% overall yield).
Purified peptides were characterized on an Acquity UPLC Waters
Chromatograph, using a Waters BEH130 C18 Acquity column (2.1x100
mm, 1.7µm, at 45°C) for compounds 1-6 and an Aeris PEPTIDE XB-C18
100Å column (2.1x100 mm, 2.6µm, at 45°C) for compounds 7-11, using
as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and by
ESI on an Acquity SQ2 Mass Detector (Table S1 and Figure S1,
Supporting Information).
More in general, we demonstrated that the decoration of Pro3 on
peptide 2 is a successful strategy to obtain active antimicrobial
peptides, since the flexibility of the N-terminal peptide region
allows the additional group to orient adequately for membrane
interaction.
Biology
Microorganisms. The strains used for the antimicrobial assays were the
following: the Gram-negative bacteria, Escherichia coli ATCC 25922 and
Pseudomonas aeruginosa ATCC 27853; the Gram-positive bacteria
Bacillus megaterium Bm11 and Staphylococcus epidermidis ATCC 12228;
and the yeasts Candida albicans ATCC 24433.
Experimental Section
Chemistry
7
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10.1002/cmdc.201900221
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FULL PAPER
Veterinary clinical strains of E. coli, Proteus mirabilis, S. pseudintermedius,
and M. pachydermatis were isolated from pet animals and processed at
the Microbiology Laboratory of the Department of Veterinary Medicine and
Animal Production, University of Naples Federico II (Italy). The strains
were identified by matrix-assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF MS) (Bruker Daltonik). For this study
were selected multidrug-resistant bacterial strains. Furthermore, two S.
pseudintermedius strains were chosen, one methicillin-resistant (MRSP)
and the other was methicillin-susceptible (MSSP).[40]
experiments performed at different temperatures in the range 298-320 K
by means of linear regression.
Structural Determinations. The NOE-based distance restraints were
obtained from NOESY spectra collected with a mixing time of 100 ms. The
NOE cross peaks were integrated with the XEASY program and were
converted into upper distance bounds using the CALIBA program
incorporated into the program package CYANA.[49] Only NOE derived
constraints were considered in the annealing procedures. For each
examined peptide, an ensemble of 200 structures was generated with the
simulated annealing of the program CYANA. Nonstandard cis-4-amino-
Pro and trans-4-cyclohexyl-Pro residues were added to CYANA residue
library using the Biopolymer module of InsightII (Accelrys, San Diego, CA).
Then, 20 structures were chosen, whose interproton distances best fitted
NOE derived distances, and refined through successive steps of
restrained and unrestrained energy minimization calculations using the
Discover algorithm (Accelrys, San Diego, CA) and the consistent valence
force field.[50] The minimization lowered the total energy of the structures;
no residue was found in the disallowed region of the Ramachandran plot.
The final structures were analyzed using the InsightII program (Accelrys,
San Diego, CA). Molecular graphics images were realized using the UCSF
Chimera package.[51]
Antimicrobial assay. The MIC values were obtained by adapting the
microbroth dilution method outlined by the Clinical and Laboratory
Standards Institute, using sterile 96-well plates (Falcon NJ, USA). Aliquots
(50 μl) of bacteria in mid-log phase at a concentration of 2×106 colony-
forming units (CFU)/mL (5 x 104 CFU/ml for C. albicans) in culture medium
(Mueller-Hinton, MH or Winge broth, WB, for C. albicans, and Sabouraud
broth for M. pachydermatis) were added to 50 μl of MH/WB containing the
peptide in serial 2-fold dilutions in water. Peptide dilutions ranged from 0.1
to 100 μM. MIC were considered as the peptide concentration at which
100% inhibition of microbial growth is visually observed after 18-20 h of
incubation at 37 °C (30 °C for yeasts). Each MIC value is the average of
at least three independent experiments.[38]
Hemolytic assay. The hemolytic activity was measured on sheep red
blood cells (OXOID, SR0051D). Briefly, aliquots of erythrocyte suspension
(1x108 cells/mL) in 0.9% (w/v) NaCl were incubated with serial two-fold
dilutions of the peptides in water for 40 min at 37 °C with gentle shaking.
The samples were then centrifuged 5 min at 900 x g and the absorbance
of the supernatant was measured at 415 nm using the microplate reader
(Infinite M200; Tecan, Salzburg, Austria). The complete lysis of red blood
cells was measured by suspending erythrocytes in distilled water.[31]
Statistical analyses. Quantitative data were expressed as the mean ±
standard error of the mean (SEM). The statistical analyses were performed
using Student’s t test with the PRISM software (GraphPad, San Diego, CA).
Differences were considered to be statistically significant for p< 0.05.
Acknowledgements
This work was supported by grants from Regione Campania,
Progetti Retrospettivi Coerenti con la Misura 3.5 del FET
Campania 2007-2013 (art.41 del Reg. (CE) 1198/2006); and
Finanziamento della ricerca di Ateneo - Università degli Studi di
Napoli Federico II - Annualità 2016, prot. N. 16503 and Sapienza
University of Rome (project RM11816436113D8A)
Conformational analyses
Circular dichroism spectroscopy. Circular dichroism spectra were recorded
at 25°C using quartz cells of 0.1 cm path length with a JASCO J-710 CD
spectropolarimeter (JASCO International Co. Ltd.). Measurements were
run in the 260-185 nm spectral range, 1 nm bandwidth, 16 s response time
and 2 nm/min scanning speed. The concentration of peptides was 100μ
M in 10 mM sodium phosphate buffer (pH 7.4), SDS (20 mM), DPC (20
mM), or DPC/SDS (18/2 mM) micellar solutions. The Bestsel algorithm
was used to extract secondary structure contents.[34] A CD spectrum of
peptide 8 was also acquired in LPS 100 µM.
Conflict of interest
The authors declare no conflict of interest.
NMR spectroscopy. The samples for NMR spectroscopy were prepared by
dissolving the appropriate amount of peptides 7 and 8 in 0.55 mL of 1H2O,
0.05 mL of 2H2O to obtain a concentration of 1-2 mM peptides and 200 mM
SDS-d25. The NMR experiments were performed at pH 5.0. NMR spectra
were recorded on a Varian Unity INOVA 700 MHz spectrometer equipped
with a z-gradient 5 mm triple-resonance probe head. All the spectra were
recorded at a temperature of 25 °C. The spectra were calibrated relative
to TSP (0.00 ppm) as internal standard. One-dimensional (1D) NMR
spectra were recorded in the Fourier mode with quadrature detection. Two-
dimensional (2D) DQF-COSY,[41,42] TOCSY,[43] and NOESY spectra,[44]
were recorded in the phase-sensitive mode using the method from
States.[45] Data block sizes were 2048 addresses in t2 and 512 equidistant
t1 values. A mixing time of 80 ms was used for the TOCSY experiments.
NOESY experiments were run with mixing times in the range of 100-200
ms. The water signal was suppressed by gradient echo.[46] The 2D NMR
spectra were processed using the NMRPipe package.[47] Before Fourier
transformation, the time domain data matrices were multiplied by shifted
sin2 functions in both dimensions, and the free induction decay size was
doubled in F1 and F2 by zero filling. The qualitative and quantitative
analysis of DQF-COSY, TOCSY, and NOESY spectra were obtained using
Keywords: Antimicrobial peptides • Temporin L analogues•
Synthesis • Biological activity • Conformational studies
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9
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
In this study, using some non-canonical proline derivatives, we investigated the correlation between the conformational properties of
the synthesized compounds and their antibacterial, cytotoxic and hemolytic activities. A SAR study was performed with CD and NMR
spectroscopy analysis. We have identified novel analogues with interesting properties that make them promising leads for further
chemical optimization.
10
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