Potent Antimicrobial Activity of Lipidated Short α,γ-Hybrid Peptides

Article abstract of DOI:10.1002/cmdc.201700370

Herein we report the potent antimicrobial activity of α,γ-hybrid lipopeptides composed of 1:1 alternating α- and γ-amino acids. Along with their potent antimicrobial activity against various Gram-positive and Gram-negative bacteria, these hybrid lipopepti

Full text of DOI:10.1002/cmdc.201700370

Accepted Article
Title: Potent Antimicrobial activity of Lipidated Short alpha, gamma-
hybrid peptides
Authors: Sushil Benke, Hirekodathakallu Thulasiram, and Hosahudya
Gopi
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Link to VoR: http://dx.doi.org/10.1002/cmdc.201700370
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Potent Antimicrobial activity of Lipidated Short , -hybrid peptides
Mr. Sushil N. Benke,[a] Dr. Hirekodathakallu V. Thulasiram,[b]and Dr. Hosahudya N. Gopi*[a]
[a]Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi
Bhabha Road, Pune-41108, [b] CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road,
Pune-411008, INDIA.
*Corresponding author: Dr. Hosahudya N. Gopi, Department of Chemistry, Indian Institute of
Science Education and Research, Dr. Homi Bhabha Road, Pune-41108; E-mail:
hn.gopi@iiserpune.ac.in
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Abstract: We are reporting potent antimicrobial activity of ,-hybrid lipopeptides constituted
with 1:1 alternating - and -amino acids. Along with their potent antimicrobial activity against
various Gram positive and Gram negative bacteria, these hybrid lipopeptides were found to be
less haemolytic. The mechanism of action revealed that these short cationic lipopeptides bind
and disrupt the bacterial cell membrane. Further, the time kill kinetics analysis revealed that the
potent ,-hybrid lipopeptides completely inhibit the bacterial growth in less than 20 minutes.
Overall, the promising antimicrobial activity along with less haemolytic activity displayed by the
,-hybrid lipopeptides can be further explored to design potent lipopeptide antibiotics.
Keywords: Hybrid lipopeptides; Microbial resistance; Antibiotics; Mode of action; Membrane
disruption.
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Introduction
The emergence of drug resistant microorganisms poses a great worldwide threat and created an
immediate need for the development of novel antibiotics.[1] Antimicrobial resistance can affect
any one irrespective of the age and the country, and also jeopardise the progress in medical and
health sciences. In this context, the broad spectrum antimicrobial activity shown by the cationic
host-defence antimicrobial peptides (AMPs) and native lipopeptides have attracted considerable
attention.[2] The AMPs have been serving as defensive agents against various pathogens in plant
and animal kingdoms.[3] However, many natural AMPs have been associated with various
inherent limitations including higher haemolytic activity, poor selectivity and bioavailability.[4]
These intrinsic problems hindered the potential therapeutic applications of natural AMPs. In
contrast to AMPs, lipopeptides are produced only by bacteria, fungi and yeast.[5] The native
lipopeptides are proved to be non-specific and also associated with high toxicity. The
mechanism of action suggested that the cationic AMPs and lipopeptides bind to the bacterial
membrane through charge interactions and disrupt the membrane.[6] Extensive efforts have been
made to improve the specificity and activity of AMPs and lipopeptides.[7] Shai and colleagues[8]
and others[9] have designed a variety of short lipopeptides and examined their potential
antimicrobial properties. In addition to the lipopeptides consisting of completely L-amino acids
as well as alternating L- and D-amino acids,[10] Schweizer and colleagues demonstrated the
antimicrobial activity of short lipo-β-peptides.[11] Besides the utility of β-peptides to mimic
protein secondary structures,[12] they have also been explored to design potent antimicrobial
candidates.[13] In addition to the β-peptides, potential of -peptides built from -amino acids
have been examined to derive protein secondary structure mimetics.[14] In contrast to β-peptides,
the antimicrobial activity of -peptides have not been systematically investigated. The advantage
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of β- and -peptides compared to the -peptides is that they are proteolytically and metabolically
stable.[15] We hypothesize that the conformationally flexible - and hybrid -peptides can be
explored to design potent antimicrobial candidates. Recently, we demonstrated the
supramolecular assembly mediated antimicrobial activity of hybrid lipopeptides containing , β-
unsaturated -amino acids.[16] Herein, we are reporting the design, synthesis and antimicrobial
activity of short ,-hybrid lipopeptides, their mechanism of action and haemolytic activity. In
comparison with -peptide counterparts, these short ,-hybrid lipopeptides showed potent
antimicrobial activity against various bacterial strains and lower hemolytic activity. In addition,
the study of time kill kinetics assay revealed that the ,-hybrid lipopeptides completely
eradicate the bacteria in less than 20 minutes.
Results and Discussion
To examine the antimicrobial activity of short ,-hybrid lipopeptides, we have designed
nine ,-hybrid peptides (P1-P9) and an -peptide P10. The sequences of these lipopeptides
are shown in the Scheme 1. We have utilized both octanoic acid and dodecanoic acid to
understand the importance of lipids in antimicrobial activity. Required -amino acids (-Ala, -
Leu, -Tyr and -Phe) were synthesized starting from commercially available -amino acids as
reported earlier.[17] Using solid phase synthesis we have synthesized all peptides on Knorr
amide resin. All ,-hybrid peptides and the control -peptide were purified using reverse
phase-HPLC on a C18 column. The ,-lipopeptides and the control -peptide were subjected to
antimicrobial and haemolytic activity.
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Scheme1. Sequences of ,-hybrid lipopeptides (P1-P9) and control -lipopeptide (P10)
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Table 1. Minimum inhibitory concentration* of ,-hybrid lipopeptides in µg/mL (µM)
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
E. coli
>250
(418)
163
32
250
>250
13
26
170
6
>250
(249)
(43)
(333)
(>320) (16)
>250 26
(>320) (32)
(31)
(249)
(8.1)
(>366)
E. coli K12
>250
(418)
163
32
250
26
85
12
>250
(249)
(43)
(333)
(31)
(124)
(16.2)
(>366)
K.
>250
(418)
81
16
125
97
6
3.3
85
3
>250
pneumoniae
(124)
(21)
(166)
(124)
(8)
(3.9)
(124)
(4)
(>366)
P.
>250
(418)
163
16
62
97
13
13
42
6
>250
aeruginosa
(249)
(21)
(83)
(124)
(16)
(15.5)
(62)
(8.1)
(>366)
S. aureus
>250
(418)
81
16
125
195
13
13
85
6
>250
(124)
(21)
(166)
(249)
(16)
(15.5)
(124)
(8.1)
(>366)
S.
>250
(418)
81
8
62
195
13
6
42
6
>250
(>366)
typhimurium
(124)
(10.5)
(83)
(249)
(16)
(7.8)
(62)
(8.1)
Hemolysis
HD10
>250
(418)
135
93
>250
>250
136
26
>250
183
>250
(206)
(127)
(>333)
(>320) (168)
(31)
(>367)
(248)
(>366)
*The experiments were performed in triplicates and the least concentration of peptide required
for complete killing of bacteria is reported as minimum inhibitory concentration (MIC).
We examined the antibacterial activity of short lipopeptides, P1-P10 against Gram
negative bacteria Escherichia coli (NCIM 2065), Escherichia coli K12 (NCIM2563), Klebsiella
pneumoniae (NCIM 2957), Pseudomonas aeruginosa (NCIM 5029), Salmonella typhimurium
(NCIM 2501) and Gram positive Staphylococcus aureus (NCIM 5021). The antibacterial activity
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of all peptides were carried out using microbroth dilution method in a 96-well microtiter plate.
The minimum inhibitory concentration (MIC) was measured in triplicate and average values are
given in the Table 1. Among all ,-hybrid lipopeptides, peptides coupled with dodecanoic acid
showed better antimicrobial activity than the peptides coupled with octanoic acid. Among the
dodecanoic acid coupled ,-hybrid peptides (P2, P3, P6, P7 and P9), peptide P9 with -Leu
residues was found to be the best antimicrobial candidate. It inhibits various Gram –ve and Gram
+ve bacteria with low MIC (6 μg/mL) value. The excellent activity of P9 among all the hybrid
peptides motivated us to design -peptide analogue P10. More interestingly, the -peptide
counterpart displayed much higher MIC value under identical conditions. Replacing dodecanoic
acid by octanoic acid (P8) leads to drastic decrease in the antimicrobial activity of the peptide.
Among the C8-fatty acid containing peptides, P8 was found to be the best. The -peptide
counterpart of P9, peptide P10 showed poor antimicrobial activity under identical conditions. It
is worth mentioning that both amino acids side-chains and fatty acids play crucial role in the
antimicrobial activity.
Figure 1. Hemolytic activity of short lipopeptides (The experiments were performed in triplicate
and average value of % hemolysis is reported).
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The encouraging antimicrobial activity of short ,-hybrid lipopeptides motivated us to
investigate their haemolytic activity. We subjected all lipopeptides including control -peptide
P10 for the haemolytic assay. Results of haemolytic activity of all lipopeptides are shown in
Figure 1. The HD10 values are given in the Table 1. Among the active peptides, peptide P7 with
-Tyr residues and P3 with -Ala and -Phe were found to most haemolytic in the series. Highly
active peptide P9 showed comparatively less haemolytic activity. The active peptide P6 with -
Phe was found to be relatively less haemolytic than P7. More importantly, at their MICs these
peptides are not haemolytic. Further, the peptides which displayed weak antimicrobial activity
(P1, P4, P5 and P8) were also found to be less haemolytic.
As ,-hybrid lipopeptides P6 and P9 inhibit the growth of Gram positive and Gram
negative bacteria with comparable MIC values across the panel motivated us to examine their
mode of action. In order to understand whether these peptides act through membrane disruption
similar to other native antimicrobial peptides,[6] we undertook Field-emission scanning electron
microscopy analysis (FE-SEM). Figure 2A depicting the FE-SEM images of the bacteria before
and after the treatment of peptides P6 and P9. The FE-SEM images clearly suggested the
change in morphology of the microorganisms after the treatment of ,-hybrid lipopeptides.
These results indeed indicate that the ,-hybrid lipopeptides disrupt the bacterial membrane. In
order to authenticate whether these peptides really disrupt the bacterial membrane, β-
galactosidase leakage experiment was performed using E. coli containing LacZ reporter gene.[18]
The β-galactosidase is expected to leak out from the E-coli upon disruption of the bacterial cell
wall. The peptides P6 and P9 were incubated with E. coli (TOP 10) and the release of β-
galactosidase was measured using standard fluorogenic substrate, 4-methylumbelliferyl-β-
galactosidase. The results are shown in the Figure 2B. Increased fluorescence intensity
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indicating the release of β-galactosidase upon the treatment of lipopeptides. In contrast, no β-
galactosidase release was observed in the control experiment without lipopeptides. The enzyme
leakage assay suggested that these lipopeptides bind and disrupt the bacterial cell membrane.
Figure 2. A) FE-SEM images of bacteria E. coli K12 (i) and S. aureus (iv) without lipopeptides.
Images of E. coli after the treatment with lipopeptides P6(ii) and P9(iii). Images of S. aureus
after the treatment with lipopeptides P6(v)and P9(vi). B) Peptide mediated β-galactosidase
leakage. The height of the graphs indicates the relative amount of β-galactosidase present in the
medium after the treatment of peptides.
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The potent antibacterial activity of lipopeptide P9 inspired us to further evaluate its
activity through time kill kinetics assay. This experiment gives the information about the rate at
which the lipopeptide is acting on the bacteria. We selected K. pneumoniae for time kill kinetics
experiment as the peptide displayed better MIC values against this microorganism. The peptide
was incubated with the bacterial solution at 2 × MIC (6 μg/mL) and 20 μL of the peptide treated
bacterial solution was drawn with increasing time intervals and diluted to ten times using 0.9%
saline and plated again on Mueller Hinton agar plates. After incubating for about 24 h, the
bacterial colonies were counted. These results were represented in CFU/mL scale. Results of
time kill kinetics assay are shown in Figure 3. These results clearly suggested that the lipopeptide
P9 completely eradicate the bacteria within 20 min.
Figure 3. Time−kill kinetics of peptide P9 (2 × MIC) against K. pneumoniae (asterisks
correspond to <50 CFU/mL). (The experiments were performed in triplicates and the average
CFU values are reported.)
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In conclusion, we have shown the potent antibacterial activity and the mechanism of
action of short ,-hybrid lipopeptides. The hybrid peptides coupled with dodecanoic acids
showed better activity compared to the octanoic acid coupled hybrid peptides. Among the active
lipopeptides, peptides composed of aromatic residues showed higher haemolytic activity
compared to peptides with aliphatic residues. The mechanism of action suggested that these
cationic lipopeptides bind and disrupt the bacterial cell wall. Informatively, lipopeptide P10
with -amino acid showed weaker antimicrobial activity compared to the ,-hybrid
lipopeptides. Moreover, the time kill kinetics assay suggested that the lipopeptide P9 completely
inhibit the growth of bacteria within 20 min. Overall, the potent antibacterial activity, mechanism
of action, and fast killing of bacteria, less haemolytic activity displayed by the short ,-hybrid
peptides, particularly peptide P9, provided an unique opportunity to further design peptide
antibiotics.
Experimental section
General. All reagents, amino acids, solvents were obtained from commercial sources and used
without further purification. Peptides were purified through reverse phase HPLC on a C18
13
column using MeOH/H2O gradient. 1H NMR spectra were recorded on 400 MHz and C NMR
on100 MHz spectrometer using residual solvent as internal standard (DMSO-d6 δH, 2.5 ppm, δc
39.51 ppm) The chemical shifts (δ) were reported in ppm and coupling constant (Ј) in Hz. Mass
spectra of the peptides were obtained from the MALDI-TOF/TOF.
Synthesis of N-Fmoc-protected -amino acids: N-Fmoc-protected -amino acids were
synthesized using reported procedure.[17] Briefly, the N-Boc-α, β-unsaturated γ-amino acid (2
mmol) dissolved in ethanol was subjected to double bond reduction using 20% Pd/C and
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hydrogen. The progress of the reaction was monitored by TLC. After completion (approximately
2 h), Pd/C was filtered and ethanol was evaporated from the reaction mixture to give N-Boc-γ-
amino acid as gummy product in a quantitative yield. Further, the Boc group from N-Boc-γ
gamma amino acid (1 mmol) was deprotected using 5 mL of neat TFA and the free amine was
protected again by Fmoc group using Fmoc-OSu (1 mmol) in basic conditions (10% NaHCO3
and THF). The Fmoc-γ-amino acid was utilized for solid phase peptide synthesis without further
purification.
Synthesis of peptides
All peptides were synthesized on Knorr amide resin at 0.25 mmol scale using standard Fmoc-
chemistry. All coupling reactions were performed using HBTU along with HOBt and monitored
by Kaiser Test. Finally, peptides were cleaved from the resin using the combination of TFA:
water: thioanisole (98:1:1) and filtered. The cleavage solution was evaporated under reduced
pressure to obtain crude product. The crude peptides were purified through reverse phase HPLC
on C18 column using MeOH/H2O gradient.
Antibacterial activity
The bacterial strains Escherichia coli (NCIM 2065), Escherichia coli K12 (NCIM2563),
Klebsiella pneumoniae (NCIM 2957), Pseudomonas aeruginosa (NCIM 5029), Salmonella
typhimurium (NCIM 2501), Staphylococcus aureus (NCIM 5021) used for the antibacterial
activity were obtined from National Collection of Industrial Microorganisms (NCIM).
The antibacterial activity of peptides were carried out in a 96-well microtiter plate using
microbroth dilution method. The cultures of bacteria were incubated over night at 37 °C, and the
solution was diluted with sterile MHB (Muller-Hinton broth) medium to a concentration of 106
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colony forming units/mL. Increasing concentration of peptides P1-P10 with the volume of 50μL
was added to the 50μL bacterial suspension to each well, and incubated for about 18 h at 37 C.
Control experiment was performed without peptides. The inhibition of the growth of bacteria
was monitored through measuring the absorbance at 492 nm. The lowest peptide concentration
required for the complete killing was defined as the MIC value.
Hemolysis assay:
Hemolytic activity was performed on human red blood cells (hRBCs). The fresh hRBCs
collected with EDTA were washed four times with Tris-buffered saline (10 mM Tris, 150mM
NaCl, and pH 7.2). The hRBCs were again diluted to 4% v/v with Tris buffer. To the solution of
hRBCs (50 μL), increasing concentration of the peptides in Tris buffer were added by keeping
the total volume 100 μL. The combined hRBCs and peptides solution was incubated at 37 C for
about 1 h. After 1 h, the solution was centrifuged for about 15 min at 3000 rpm. The supernatant
(50 μL) of the each well was transferred to another 96-well plated and diluted with 50 μL of
water and measure the absorbance of released hemoglobin at 540 nm. Simple Tris buffer
without peptides was used as a negative control and 1% Triton-X was used as a positive control.
All experiments were performed in triplicate.
β-Galactosidase leakage from E.coli cells:
Membrane deformation studies were performed using Escherichia coli (TOP10, Invitrogen) cells
containing LacZ reporter gene as reported earlier. Briefly, 90 μL of Luria Bertani broth
containing the E.coli cells producing β-galactosidase were incubated separately with 10 μL (200
μg/mL) of peptides P6 and P9. After incubating for about 1h, the bacterial solution was
centrifuged for10 min at 4000 rpm. The clear supernatant solution (80 μL) was treated with 20
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μL (400 μg/mL) of 4-methylumbelliferyl-β-galactosidase indicator. The release of the β-
galactosidase was monitored for about 1 h by recording the fluorescence emission at 445 nm
after exciting the solution at 365 nm. The linear plot of fluorescence versus time gave the initial
velocity of the enzyme reaction. The control experiment was performed without peptides.
Time kill kinetics
To understand the rate at which the peptides can completely inhibit bacterial growth we have
performed the time kill kinetics assay. The potent peptide P9 ( 6 μg/mL, 2 × MIC )was added to
the solution of K. peumoniae grown in Mueller Hinton broth (approximately 1.8 × 105 CFU/mL)
and incubated at 37 C. From this solution, 20 μL were drawn at 0, 1, 3, 6, 12, and 24 h
intervals and diluted 10 times with 0.9% saline. The solution extracted at different time intervals
o
were plated again on Mueller Hinton agar plates and incubated at 37 C. Similar experiments
were performed at shorter time intervals 0, 10, 20, 30, 45, and 60 min. After 24 h incubation, the
bacterial colonies were counted.
FE-SEM Analysis
Peptides P6 and P9 (100 μL) at 2 × MIC values were pipetted into a 96-well cell culture plate.
To this peptide solution, 100 μL of either E. coli or S. aureus bacterial solution in Mueller
Hinton broth was added. The bacterial concentration was adjusted to 0.1-0.2 optical density at
600 nm. The peptide treated bacterial solution was incubated for about 2 h at 37 °C. The
bacterial cells were centrifuged and the pellet was washed with PBS. Using 2.5% glutaraldehyde
the cells were fixed and washed with PBS. The bacterial cells were centrifuged and washed with
30%, 50%, 70%, 95%, and 100% graded ethanol solution and finally with water. Finally, the
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dried bacterial cells were mounted on a silicon wafer and imaged under a field emission scanning
electron microscope after the gold coating.
Acknowledgements
This work was supported by Department of Science and Technology, Govt. of India. SNB is
thankful to UGC for research fellowship. We acknowledge Mr. S. Shiv Shankar for his help in
carrying out microbial assays.
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10.1002/cmdc.201700370
ChemMedChem
Table of Contents
We are reporting the design, synthesis and potent antimicrobial activity of short ,-hybrid
lipopeptides composed of 1:1 alternating - and -amino acids. Investigation of the mechanism
18
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700370
ChemMedChem
of action revealed that these short cationic lipopeptides bind and disrupt the bacterial cell
membrane.
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