Arch. Pharm. Res.

(2017) 40:1414–1419 Online ISSN 1976-3786

https://doi.org/10.1007/s12272-017-0963-5 Print ISSN 0253-6269

RESEARCH ARTICLE

Antimicrobial activity and stability of stapled helices

of polybia-MP1
Huy X. Luong1 • Do-Hee Kim2 • Bong-Jin Lee2 • Young-Woo Kim1

Received: 20 August 2017 / Accepted: 27 September 2017 / Published online: 26 October 2017
Ó The Pharmaceutical Society of Korea 2017

Abstract Polybia-MP1 is a well-known natural antimi- Introduction

crobial peptide isolated from the venom of the social wasp
Polybia paulista. A recent study showed that this peptide Antimicrobial resistance is currently one of the biggest
displays a broad antibacterial spectrum as well as low threats to global public health security. Therefore, there is
toxicity to human red blood cells and normal fibroblasts. an urgent need to develop new antibiotics. Antimicrobial
However, its moderate antimicrobial activity and high peptides (AMPs) are a class of naturally occurring antibi-
susceptibility to protease have been a major hurdle for otics exerting their antimicrobial activity via membrane
clinical use. This study examined the possibility of devel- lysis (Tossi et al. 2000; Yeaman and Yount 2003; Jenssen
oping biologically more potent, yet metabolically more et al. 2006; Mahlapuu et al. 2016). This unique mode of
stable, analogues of MP1 using an emerging technology action proves AMPs a promising alternative to conven-
termed ‘‘all-hydrocarbon stapling.’’ The stapled analogues tional small molecule-based antibiotics (Zasloff 2002; Fox
of MP1 showed more than a threefold increase in helicity 2013). However, AMPs also suffer from many drawbacks
as well as an approximately 70-fold enhancement in pro- such as proteolytic instability and hemolytic activity,
teolytic stability. These stapled analogues also exhibited a blocking their practical application in clinic (Marr et al.
significant increase in inhibition against some Gram-posi- 2006).
tive bacteria while displaying a modest enhancement in Polybia-MP1 (MP1), isolated from the venom of the
hemolytic activity. Overall, the current study demonstrated social wasp Polybia paulista, is one of the most widely
that the all-hydrocarbon stapling system is a highly useful studied AMPs due to its broad antimicrobial spectrum and
tool for the development of biologically more potent and relatively low hemolytic activity (Souza et al. 2005).
metabolically more stable analogues of natural antimicro- Clinical use of MP1 is not yet plausible mainly because of
bial peptides. its moderate antimicrobial activity and high susceptibility
to proteolysis. Recently, Zhao and colleagues reported an
Keywords Antimicrobial peptides  a-Helix  Stapled all D-amino acid peptide analogue of MP1 to promote
peptides  Amphipathic peptides  Proteolytic resistance proteolytic stability, but its antimicrobial activity remained
only modest (Zhao et al. 2016).
We have previously reported several cases in which
Verdine’s ‘‘all-hydrocarbon stapling’’ system (Fig. 1) was
successfully applied to improve the pharmacological
properties of AMPs (Pham et al. 2013; Dinh et al.
& Young-Woo Kim 2014, 2015; Luong et al. 2016, 2017). This special cross-
[email protected]
linking system has proved highly effective in stabilizing
1
College of Pharmacy, Dongguk University, Seoul 100-715, helical structures of short peptide stretches (Schafmeister
Korea et al. 2000; Kim et al. 2011). In many previous studies, the
2
College of Pharmacy, Seoul National University, ‘‘stapled’’ helices of various peptides have shown a dra-
Seoul 151-742, Korea matic enhancement in stability against proteolytic

123
Antimicrobial activity and stability of stapled helices of polybia-MP1 1415

Fig. 1 Schematic presentation of the ruthenium-based all-hydrocarbon stapling chemistry that tethers positions 6 and 10 yielding a stapled
peptide. Previous studies indicated that the configuration of the i,i ? 4 staple is exclusively cis (Bhattacharya et al. 2008; Zhang et al. 2008;
Phillips et al. 2011)

degradation (Schafmeister et al. 2000; Verdine and Hilinski

2012). In this study, to develop more potent and yet
metabolically stable analogues of MP1, we designed and
prepared a panel of stapled analogues of MP1 using Ver-
dine’s all-hydrocarbon stapling system and evaluated their
chemical and biological properties.

Materials and methods

General

Commercially available chemicals were used as received.

Fmoc-(S)-a-methyl,a-petenylglycine was purchased from
Fig. 2 Sequences (a) and a wheel diagram (b) of polybia-M1
Okeanos Tech Co. Ltd. (Beijing, China). All other Fmoc-
analogues studied in this report. N-termini of all the peptides are in
protected a-amino acid monomers, Rink Amide MBHA resin, their free form and their C-termini are amidated. Asterisks in position
and 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)- 6 and 10 represent the cross-linked residues. The line connecting
dimethylamino-morpholino)]uranium hexafluorophosphate positions 6 and 10 represents the oct-4-enyl hydrocarbon staple
were purchased from NovaBiochem (San Diego, CA, USA).
Bis(tricyclohexylphosphine)benzylidine ruthenium (IV) MP1 ESIMS m/z for C78H132N20O19 [M?2H]2?/2
dichloride, piperidine, N,N-diisopropylethylamine, N,N- calculated 827.52, found 827.90
dimethylformamide, N-methyl-2-pyrrolidinone, triisopropyl- MP1S ESIMS m/z for C83H138N20O19 [M?2H]2?/2
silane, 1,2-dichloroethane, and trifluoroacetic acid were pur- calculated 860.57, found 861.00
chased from Sigma-Aldrich (St. Louis, MO, USA). MP1S-D8N ESIMS m/z for C83H138N21O18 [M?2H]2?/2
calculated 860.08, found 860.45
Peptide synthesis MP1S-Q12K ESIMS m/z for C49H83N12O7 [M?2H]2?/2
calculated 860.59, found 860.75
MP1 and its stapled analogues (Fig. 2) were prepared by
the standard Fmoc chemistry on Rink Amide MBHA resin
Circular dichroism
with a loading capacity of 0.6 mmol/g using the published
protocol (Kim et al. 2011). The synthesized peptides were
After being dissolved in a 25 mM potassium phosphate
purified to over 95% by reverse phase high-performance
buffer solution (pH 6.5), the concentration of each peptide
liquid chromatography (HPLC) using a Zorbax C18 col-
was determined via absorbance spectroscopy at 280 nm
umn (Agilent, 5 lm, 9.4 9 250 mm). The flow rate of
(extinction coefficient for tryptophan, k280 = 5690 cm-1).
4 mL/min was used with a gradient of 5-100% acetonitrile
The circular dichroism (CD) spectrum of each peptide was
in water containing 0.1% trifluoroacetic acid. Molecular
measured on a Chirascan HP dual polarization CD spec-
weight of each peptide was verified LC/MS (Shimadzu
trometer with a temperature controller. The standard
LCMS-2020).
measurement parameters used are as follows: 1 nm step
resolution, 3 accumulations, 0.5 s response, 1 nm

123
1416 H. X. Luong et al.

bandwidth, and 0.1 cm path length. All spectra were con- Results
verted to a uniform scale of molar ellipticity after back-
ground subtraction. All the curves were smoothed using The secondary structures of the MP1 analogues were
standard parameters. determined by far ultraviolet circular dichroism (CD)
spectroscopy in a phosphate buffer (Fig. 3). Unmodified
Antimicrobial assay analogue MP1 appeared to be partially helical in the given
aqueous solution, showing 25.6% of helicity. All stapled
The minimum inhibitory concentration (MIC) of each analogues of MP1 displayed the characteristic shape of CD
peptide was determined in duplicate by the standard broth spectra for a-helices: two negative minima at around
microdilution method (Jorgensen and Ferraro 1998) with a 208 nm and 222 nm along with a positive maximum near
slight modification. Briefly, bacteria were incubated in 190 nm (Chen et al. 1972). % Helicities of peptides MP1S,
2 ml of Luria–Bertani (LB) broth at 37 °C overnight. The MP1S-D8N, and MP1S-Q12K were 96.1, 89.4, and
peptides were prepared from 0.2 to 200 lg/ml in 96-well 81.8%, respectively.
round-bottom microtiter plates with twofold dilutions in Antimicrobial and hemolytic activities of the peptides
distilled water. The peptides and the bacterial inoculums are summarized in Table 1. In the antimicrobial assay, the
were then diluted using LB broth and treated with bacterial stapled analogue MP1S showed a 7.7-fold increase in
suspension (106–108 colony-forming units (CFU)/ml). antimicrobial activity against Bacillus subtilis (B. subtilis)
After 24 h-incubation at 37 °C, the MIC of each peptide and Staphylococcus aureus (S. aureus) relative to its
was determined based on the visible turbidity in each well. unmodified counterpart MP1 (Table 1, entry 1 and 2).
All bacterial strains tested in this assay were obtained from However, MP1S did not show any improved activity
the Korean Collection for Type Culture (KCTC) at the against Staphylococcus epidermidis (S. epidermidis) and all
Korean Research Institute of Bioscience and Biotechnol-
ogy (KRIBB, Daejeon, Korea).

Hemolysis assay

Mixtures of 10 ll of serially diluted peptides in PBS (0.8–

200 lg/ml final concentration) and 190 ll of 10% sus-
pensions of fresh human red blood cells in PBS were
incubated at 37 °C for 30 min. After centrifugation, the
supernatants were tenfold diluted with PBS, and the
absorbance of each solution was measured at 405 nm. As a
control for 100% hemolysis, a blood suspension treated
with 0.2% Triton X-100 was used. The equation to deter-
mine % hemolysis is as follows:
OD405 nm sample
% Hemolysis ¼ ð1Þ
OD405 nm positive control

Trypsin digestion assay

25 lL of a trypsin solution (0.6 lM, Sigma) in a digestion

buffer (0.1 M NH4HCO3 buffer, pH 8.0) was mixed with
250 lL of a peptide solution (60 lM) containing trypto-
phan (60 lM) in the digestion buffer (enzyme/sub-
strate = 1/1333). The mixture was incubated at room
temperature on a shaker (600 rpm). 50 lL of the mixture
was taken into a PCR tube at the 10-, 20-, 30-, 60-, and Fig. 3 Circular dichroism spectra (a) and % helicity (b) of the
90-min marks and immediately treated with 2 lL of TFA polybia-MP1 and its stapled analogues in a 25 mM potassium
to stop the trypsin digestion. The ratios of the residual phosphate buffer solution at 20 °C. % Helicity was calculated from
substrate and the cleaved products were determined by mean-residue ellipticity at 222 nm ([h]222) using -31,500(1–2.5/n)
and 0 deg cm2 dmol-1 as the values for 100 and 0% helicity,
HPLC with peak detection at 280 nm (40 lL injected). respectively where n is the number of amino acid residues in the
Each experiment was performed in duplicate. peptide (Chen et al. 1972)

123
Antimicrobial activity and stability of stapled helices of polybia-MP1 1417

Table 1 Antimicrobial MICsa

Entry/code Gram (?) Gram (-) Hemolysis (%)b
of peptide analogs against
selected bacteria and their B.s. S.a. S.e. E.c. S.d. S.t. K.p. P.a. 25 lM 12.5 lM
hemolytic activity against
human red blood cells 1 MP1 12.5 18.8 [100 75 75 [100 37.5 [100 3.2 \1
2 MP1S 1.6 2.4 [100 [100 [100 [100 [100 [100 12.3 6.7
3 MP1S-D8N 0.8 0.8 37.5 50 100 [100 37.5 C100 37.8 16.3
4 MP1S-Q12K 0.8 1.2 50 [100 [100 [100 [100 [100 13.9 9.6
c
5 CNT 1.6 1.6 6.3 18.8 508 100 18.8 25 16.6 4.1
The abbreviations used for genus and species names are as follows: B.s., Bacillus subtilis; S.a., Staphy-
lococcus aureus; S.e., Staphylococcus epidermidis; E.c., Escherichia coli; S.d., Shigella dysenteriae; S.t.,
Salmonella typhimurium; K.p., Klebsiella pneumoniae; P.a., Pseudomonas aeruginosa
a
Minimum inhibitory concentration was defined as the lowest peptide concentration (lg/mL) that com-
pletely inhibits the cell growth after 24 h of incubation at 37 °C. The experiment was performed in
duplicate
b
Percent hemolysis is relative to that by 0.1% Triton X-100. The experiment was performed in duplicate
c
Control peptide CNT is an 11-mer peptide reported in a previous study (Won et al. 2011)

Gram-negative strains. The other two stapled analogues,

MP1S-D8N and MPS-Q12K, exhibited even higher
potency against B. subtilis and S. aureus; a 15-fold increase
against B. subtilis and 15–23-fold against S. aureus
(Table 1, entry 3 and 4). Their inhibitory activity against S.
epidermidis was also markedly improved, showing 18.8
and 50 lM of minimum inhibitory concentration (MIC),
respectively, whereas their unmodified counterpart MP1
did not show any inhibition under 100 lM of concentration
in our assay. However, like MP1S, neither analogue dis-
played any improvement in inhibition of Gram-negative
strains. All the stapled analogues showed a higher hemo-
lytic tendency than their unmodified control MP1 at the
given concentration range; compared to MP1, MP1S-D8N
caused the most notable increase in hemolysis (12-fold)
Fig. 4 Tryptic digestion of stapled analogue MP1S-Q12K in com-
whereas the other stapled analogues showed a modest parison with unmodified control MP1. Trypsin and peptides (en-
increase in hemolysis (approximately fourfold). zyme/substrate = 1/1333) were incubated in a digestion buffer
In the trypsin digestion assay, the unmodified control (0.1 M NH4HCO3 buffer, pH 8.0) for the indicated periods
peptide MP1 was rapidly degraded with a half-life of
21 min under the given conditions (trypsin/peptide ration of bacteria whereas the hydrophobic face on the opposite
of 1/1333 at room temperature) (Fig. 4). On the other hand, side plays an important role in membrane perturbation
the cleavage rate of the stapled analogue MP1S-Q12K was through further interaction with the hydrophobic interior of
significantly decreased with a half-life of 1446 min. the membrane (Alvares et al. 2016). We envisioned that
stabilization of polybia-MP1 sequence in the helical con-
formation using Verdine’s all-hydrocarbon stapling system
Discussion would be a promising strategy to the development of more
potent and yet metabolically more stable analogues of its
Polybia-MP1 is a 14-residue peptide (IDWKKLLDAAK own: first, helix stabilization would greatly reduce the
QIL-NH2) and upon binding to membranes is known to entropic penalty of helix formation and thereby increase
adopt an amphipathic a-helix, positioning the hydrophobic potency. Second, helix stabilization would promote prote-
residues on one face and the hydrophilic ones on the other. olytic stability as proteases only recognize their substrate
This helix formation is a prerequisite for polybia-MP1 to sequences in an extended conformation (Tyndall et al.
exert its antimicrobial activity via membrane perturbation; 2005). Verdine’s all-hydrocarbon stapling system has
the cationic face of the amphipathic helix mediates favor- proved the most effective and widely applied helix-stabi-
able ionic interactions with the anionic membrane surface lizing system (Schafmeister et al. 2000; Verdine and

123
1418 H. X. Luong et al.

Hilinski 2012). Furthermore, it is well known that the residue (Lys) at position 12 would reinforce the salt bridge
incorporation of the all-hydrocarbon staple significantly network on the hydrophilic face of the MP1 helix. How-
enhances proteolytic stability of peptides (Schafmeister ever, MP1S-Q12K exhibited an even lower helicity than
et al. 2000; Verdine and Hilinski 2012). Therefore, one can MP1S-D8N. We attribute the helix-destabilizing effect of
anticipate that the stapled analogues of polybia-MP1 would the Gln-to-Lys substitution in the stapled scaffold to the
not only be more potent in antimicrobial activity but also increased electrical field caused by the additional cationic
more stable against proteolytic cleavage. residue (dos Santos Cabrera et al. 2008).
For the sites of staple incorporation in the polybia-MP1 Compared to its unmodified control MP1, the stapled
sequence (MP1), positions 6 and 10 were chosen to gen- analogue MP1S exhibited a significant improvement in
erate a stapled analogue MP1S (Fig. 2a); leucine and ala- inhibitory activity against two Gram-positive strains, B.
nine at the respective positions are located in the middle of subtilis and S. aureus, but failed to show any increased
the hydrophobic face of the amphipathic helix of MP1, and activity against the other Gram-positive S. epidermidis and
replacing these residues with the hydrophobic staple would all Gram-negative strains tested in this study (Table 1,
minimize the disruption of its amphipathicity (Fig. 2b). entries 1 and 2). The incorporation of the helix-stabilizing
Aspartate (Asp) at position 8 is known to play a key role in staple also increased in hemolytic activity, most likely due
contributing to the relatively low hemolytic activity of to the increased hydrophobicity by the hydrocarbon staple.
polybia-MP1 (dos Santos Cabrera et al. 2008). To examine Similar results have been observed in our previous studies,
if Asp residue maintains its role in a stapled scaffold, we and therefore further sequence modifications were required
designed MP1S-D8N, which bears an asparagine (Asn) to optimize the physical and biological properties of the
residue in place of Asp at the position. Polybia-MP1 has six stapled analogues of linear AMPs (Pham et al. 2013; Dinh
charged functional groups in its sequence, three lysine et al. 2014). Compared to MP1S, MP1S-D8N displayed
(Lys) and two Asp residues and a free N-terminal amino more potent inhibitory activity not only against B. subtilis
group. This makes the net charge of MP1 ?2, which is and S. aureus but also against S. epidermidis (Table 1,
relatively small compared to other cationic AMPs. There- entry 3). This improved antimicrobial activity of MP1S-
fore, we designed MP1S-Q12K, which bears an additional D8 N may be attributed to the increased net charge, which
Lys residue in place of glutamine (Gln) at position 12. We would promote a more favorable interaction with the
envisioned that this substitution would expand the cationic anionic bacterial membranes. However, MP1S-D8N also
patch on the hydrophilic face of MP1 helix, which would showed a notably increased hemolytic activity. This is
thereby fortify its interaction with bacterial membrane. possible because, despite the increased net charge, the
Structural analysis of the peptides using CD spec- hydrophobicity of MP1S-D8N is higher than that of MP1S
troscopy confirms the highly effective helix-stabilizing due to the elimination of a charged residue by the Asp-to-
capability of Verdine’s all-hydrocarbon stapling system Asn substitution. This result also confirms the important
(Fig. 3a). Helical contents of all stapled analogues were role of Asp residue at position 8 in lowering hemolytic
increased by more than a three-fold compared to their activity in the polybia-MP1 sequence (dos Santos Cabrera
unmodified counterpart, MP1 (Fig. 3b). MP1S appears to et al. 2008). MP1S-Q12K also displayed improved inhi-
be the most helical among this panel of peptides, showing bitory activity against Gram-positive strains compared to
96% helicity, which is 3.7-fold higher than that of MP1. MP1S (Table 1, entry 4), which may also be explained by
MP1S-D8N and MP1S-Q12K showed slightly lower its increased net charge resulted from the Gln-to-Lys sub-
helical contents (89.4 and 81.8%, respectively). Asp resi- stitution at position 12. Interestingly, unlike MP1S-D8N,
due at position 8 is known to form a salt bridge network the impact of the substitution on hemolytic activity was
with three Lys residues at positions 4, 5, and 11 and a Gln negligible. Although both Asp-to-Asn and Gln-to-Lys
residue at position 12 that makes notable contribution to substitutions result in the same net charge (?3), the latter
helix stabilization in the polybia-MP1 sequence (dos San- would not cause an increase in hydrophobicity since it adds
tos Cabrera et al. 2008). MP1S would still maintain this an ionic residue while the former removes one. Therefore,
salt bridge network as an additional helix-stabilizing ele- the relatively low hemolytic activity of MP1S-Q12K may
ment, which may contribute to the unusually high level of be due to its relatively low hydrophobicity compared to
helical content of MP1S. However, the salt bridge network MP1S-D8N. It is interesting to note that both MP1S-D8N
would be significantly weakened with the Asp-to-Asn and MP1S-Q12K showed relatively low helicity compared
substitution at position 8, to which we attribute the rela- to MP1S but their inhibitory activity was more potent. This
tively low helicity of MP1S-D8N in comparison to MP1S. suggests that, upon stabilization by a staple, biological
With this perspective, we predicted MP1S-Q12K to be activity is determined more by their sequence rather than
more helical than MP1S because we expected that the the subtle difference in helicity.
substitution of a neutral amino acid (Gln) with a cationic

123
Antimicrobial activity and stability of stapled helices of polybia-MP1 1419

Vulnerability to proteolytic cleavage is one of the main Dinh TTT, Kim D-H, Nguyen TQ, Lee B-J, Kim Y-W (2015) N-
drawbacks of many natural AMPs including polybia-MP1. Capping effects of stapled heptapeptides on antimicrobial and
hemolytic activities. Bull Korean Chem Soc 36:2511–2515
To examine the impact of stapling on proteolytic stability dos Santos Cabrera MP, Costa ST, de Souza BM, Palma MS,
of polybia-MP1 analogues, we chose MP1S-Q12K as a Ruggiero JR, Ruggiero Neto J (2008) Selectivity in the
representative stapled analogue and evaluated its prote- mechanism of action of antimicrobial mastoparan peptide
olytic stability against trypsin digestion in comparison with Polybia-MP1. Eur Biophys J 37:879–891
Fox JL (2013) Antimicrobial peptides stage a comeback. Nat
MP1 (Fig. 4). As expected, the unmodified control MP1 Biotechnol 31:379–382
was highly susceptible to trypsin digestion; more than 85% Jenssen H, Hamill P, Hancock REW (2006) Peptide antimicrobial
of the parent sequence was degraded within 60 min (t1/ agents. Clin Microbiol Rev 19:491–511
Jorgensen JH, Ferraro MJ (1998) Antimicrobial susceptibility testing:
2 = 21 min) under the assay conditions used in this study.
general principles and contemporary practices. Clin Infect Dis
On the other hand, more than 97% of the stapled analogue 26:973–980
MP1S-Q12K remained intact after 60 min, showing a Kim Y-W, Grossmann TN, Verdine GL (2011) Synthesis of all-
69-fold increase in stability toward trypsin digestion (t1/ hydrocarbon stapled [alpha]-helical peptides by ring-closing
olefin metathesis. Nat Protoc 26:761–771
2 = 1446 min). This result again confirms the well-estab-
Luong HX, Kim D-H, Lee B-J, Kim Y-W (2016) Antimicrobial and
lished notion that helix-stabilization by a hydrocarbon hemolytic activity of stapled heptapeptide dimers. Bull Korean
staple comes with a significant enhancement in proteolytic Chem Soc 37:1199–1203
stability (Schafmeister et al. 2000). Luong HX, Kim D-H, Mai NT, Lee B-J, Kim Y-W (2017) Mono-
In conclusion, the present study examined the possibility substitution effects on antimicrobial activity of stapled hep-
tapeptides. Arch Pharm Res 40:713–719
of developing biologically more active and yet metaboli- Mahlapuu M, Håkansson J, Ringstad L, Björn C (2016) Antimicrobial
cally more stable analogues of the well-known natural peptides: an emerging category of therapeutic agents. Front Cell
AMP, polybia-MP1, using an emerging technology termed Infect Microbiol 6:194
as the ‘‘all-hydrocarbon stapling system’’. We confirmed Marr AK, Gooderham WJ, Hancock REW (2006) Antibacterial
peptides for therapeutic use: obstacles and realistic outlook. Curr
that the incorporation of a hydrocarbon staple dramatically Opin Pharmacol 6:468–472
stabilizes the helical structure of polybia-MP1 and thereby Pham TK, Kim D-H, Lee B-J, Kim Y-W (2013) Truncated and
significantly promotes proteolytic stability. The current constrained helical analogs of antimicrobial esculentin-2EM.
study also demonstrated that the inhibitory activity of Bioorg Med Chem Lett 23:6717–6720
Phillips C, Roberts LR, Schade M, Bazin R, Bent A, Davies NL,
polybia-MP1 can be greatly improved by the application of Moore R, Pannifer AD, Pickford AR, Prior SH, Read CM, Scott
the stapling system and that the hemolytic activity can be A, Brown DG, Xu B, Irving SL (2011) Design and structure of
modulated via a systemic sequence modification. Further stapled peptides binding to estrogen receptors. J Am Chem Soc
investigation is underway to improve pharmaceutical 133:9696–9699
Schafmeister CE, Po J, Verdine GL (2000) An all-hydrocarbon cross-
properties of the stapled analogues of polybia-MP1. linking system for enhancing the helicity and metabolic stability
of peptides. J Am Chem Soc 122:5891–5892
Acknowledgement This work was supported by the National Souza BM, Mendes MA, Santos LD, Marques MR, César LM,
Research Foundation of Korea (NRF) grant funded by the Korea Almeida RN, Pagnocca FC, Konno K, Palma MS (2005)
government (MSIT) (2015R1D1A1A01060265). Structural and functional characterization of two novel peptide
toxins isolated from the venom of the social wasp Polybia
Compliance with ethical standards paulista. Peptides 26:2157–2164
Tossi A, Sandri L, Giangaspero A (2000) Amphipathic, a-helical
Conflict of interest The authors declare no conflict of interest. antimicrobial peptides. Biopolymers 55:4–530
Tyndall JDA, Nall T, Fairlie DP (2005) Proteases universally recognize
beta strands in their active sites. Chem Rev 105:973–1000
Verdine GL, Hilinski GJ (2012) Stapled peptides for intracellular
References drug targets. Methods Enzymol 503:3–33
Won H-S, Kang S-J, Choi WS, Lee B-J (2011) Activity optimization
Alvares DS, Fanani ML, Ruggiero Neto J, Wilke N (2016) The of an undecapeptide analogue derived from a frog-skin antimi-
interfacial properties of the peptide polybia-MP1 and its crobial peptide. Mol Cells 31:49–54
interaction with DPPC are modulated by lateral electrostatic Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide
attractions. Biochim Biophys Acta 1858:393–402 action and resistance. Pharmacol Res 55:27–55
Bhattacharya S, Zhang H, Debnath AK, Cowburn D (2008) Solution Zasloff M (2002) Antimicrobial peptides of multicellular organisms.
structure of a hydrocarbon stapled peptide inhibitor in complex Nature 415:389–395
with monomeric C-terminal domain of HIV-1 capsid. J Biol Zhang H, Zhao Q, Bhattacharya S, Waheed AA, Tong X, Hong A,
Chem 283:16274–16278 Heck S, Curreli F, Goger M, Cowburn D, Freed EO, Debnath
Chen Y-H, Yang J-T, Martinez HM (1972) Determination of the AK (2008) A cell-penetrating helical peptide as a potential HIV-
secondary structures of proteins by circular dichroism and 1 inhibitor. J Mol Biol 278:565–580
optical rotatory dispersion. Biochemistry 11:4120–4131 Zhao Y, Zhang M, Qiu S, Wang J, Peng J, Zhao P, Zhu R, Wang H, Li
Dinh TTT, Kim D-H, Lee B-J, Kim Y-W (2014) De novo design and Y, Wang K, Yan W, Wang R (2016) Antimicrobial activity and
their antimicrobial activity of stapled amphipathic helices of stability of the D-amino acid substituted derivatives of antimi-
heptapeptides. Bull Korean Chem Soc 35:3632–3636 crobial peptide Polybia-MP1. AMB Express 6:122

123