In e na ional Jou nal o
Molecula Sciences
A icle
Cha ac e iza ion o Tachyplesin Pep ides and Thei
Cyclized Analogues o Imp o e An imic obial
and An icance P ope ies
Felici as Ve nen 1, Pe a J. Ha ey 1, Susana A. Dias 2, Ana SaloméVeiga 2,
Yen-Hua Huang 1, Da id J. C aik 1, Nicole Law ence 1and Sónia T oei a Hen iques 1,3,*
1Ins i u e o Molecula Bioscience, The Uni e si y o Queensland, B isbane, Queensland 4072, Aus alia
2Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa,
1649-028 Lisboa, Po ugal
3
School o Biomedical Sciences, Facul y o Heal h, Ins i u e o Heal h & Biomedical Inno a ion, Queensland
Uni e si y o Technology, T ansla ional Resea ch Ins i u e, B isbane, Queensland 4102, Aus alia
*Co espondence: [email p o ec ed]; Tel.: +61-7-34437342
Recei ed: 26 July 2019; Accep ed: 21 Augus 2019; Published: 26 Augus 2019


Abs ac :
Tachyplesin I, II and III a e hos de ense pep ides om ho seshoe c ab species wi h
an imic obial and an icance ac i i ies. They ha e an amphipa hic
β
-hai pin s uc u e, a e highly
posi i ely-cha ged and di e by only one o wo amino acid esidues. In his s udy, we compa ed
he s uc u e and ac i i y o he h ee achyplesin pep ides alongside hei backbone cyclized
analogues. We assessed he pep ide s uc u es using nuclea magne ic esonance (NMR) spec oscopy,
hen compa ed he ac i i y agains bac e ia (bo h in he plank onic and bio ilm o ms) and a panel o
cance ous cells. The impo ance o pep ide-lipid in e ac ions was examined using su ace plasmon
esonance and luo escence spec oscopy me hodologies. Ou s udies showed ha achyplesin
pep ides and hei cyclic analogues we e mos po en agains G am-nega i e bac e ia and melanoma
cell lines, and showed a p e e ence o binding o nega i ely-cha ged lipid memb anes. Backbone
cycliza ion did no imp o e po ency, bu imp o ed pep ide s abili y in human se um and educed
oxici y owa dhuman edbloodcells. Pep ide-lipidbindinga ini y, o ien a ion wi hin hememb ane,
and abili y o dis up lipid bilaye s di e ed be ween he cyclized pep ide and he pa en coun e pa .
We show ha achyplesin pep ides and cyclized analogues ha e simila ly po en an imic obial and
an icance p ope ies, bu ha backbone cycliza ion imp o es hei s abili y and he apeu ic po en ial.
Keywo ds:
achyplesin; hos de ense pep ide; an icance ; an imic obial; an ibio ilm; pep ide-memb ane
in e ac ion; s uc u e-ac i i y; model memb anes; nuclea magne ic esonance solu ion s uc u e
1. In oduc ion
The hos de ense pep ides (HDPs) achyplesin I, II and III (TI, TII and TIII) a e ac i e agains a
b oad ange o G am-nega i e and G am-posi i e bac e ia and ungi [
1
–
4
] and possess an icance
p ope ies [
5
–
15
]. Each analogue was isola ed om a di e en species o ho seshoe c ab, bu hey sha e
high sequence homology (Table 1). TI, TII and TIII possess 17 amino acid esidues, wo disul ide bonds,
a C- e minal
α
-amida ion, and hei s uc u e is o ganized in a
β
-hai pin [
2
,
3
,
16
]. Like o he HDPs [
17
],
TI, TII and TIII possess an amphipa hic seconda y s uc u e (i.e., posi i ely cha ged and hyd ophobic
amino acids seg ega e in o dis inc clus e s), hough o be essen ial o hei an imic obial ac i i y.
Ca ionic amphipa hic HDPs selec i ely a ge he anionic su aces o mic obes, a he han
he neu al su ace o hos cells, and kill hem by a mechanism ha in ol es binding o and
inse ion in o cell memb anes. The ini ial binding is media ed by elec os a ic a ac ions be ween he
posi i ely-cha ged esidues o HDPs and he anionic mic obial su ace [
18
], and is ollowed by he
In . J. Mol. Sci. 2019,20, 4184; doi:10.3390/ijms20174184 www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2019,20, 4184 2 o 25
inse ion o hyd ophobic esidues in o lipid memb anes in a p ocess ha in ol es an-de -Waal’s
in e ac ions wi h he phospholipids [18,19].
Simila o bac e ial cells, he su ace o cance cells is nega i ely-cha ged due o he
inc eased exp ession and exposu e o phospholipids con aining he anionic phospha idylse ine
(PS) headg oup [
20
–
23
]. In con as , cell memb anes o heal hy mammalian cells a e asymme ic
and phospholipids con aining PS-headg oups a e ound exclusi ely in he inne lea le . Va ia ions
in he o e all cell su ace cha ge [
24
] in phospholipids wi h exposed [
25
,
26
] memb ane luidi y and
cu a u e [
18
,
27
], ha e been shown o modula e he selec i e oxici y o HDPs owa ds cance cells.
These di e ences egula e he a ini y o pep ides o cell memb anes, he e ec i e pep ide- o-lipid
a io and he abili y o HDP o kill cance ous cells o pa hogens a he han heal hy cells [28–30].
Se e al s udies ha e in es iga ed he ac i i y, s uc u e and mechanism o he ac ion o TI, bu ew
ha e examined he ac i i y o TII and TIII. Ea ly s udies sugges ed ha TI kills bac e ial cells by a
mechanismin ol inginne memb anepe meabiliza ionand he apide luxo K
+
[
31
–
33
]. La e , TI was
shown o ansloca e ac oss lipid bilaye s, cause a phospholipid lip- lop and o m o oidal po es [
33
,
34
].
In cance ous cells, TI was epo ed o induce cell dis up ion and la e apop osis/nec osis [
12
,
35
].
Pa edes-Game o e al. p oposed ha he cance cell dea h mechanism was dependen on he pep ide
dose: a high concen a ions, he pep ide-induced di ec cell memb ane dis up ion, and a lowe
concen a ions, i ac i a ed in acellula cell dea h mechanisms [12].
Because o he exp ession o TI, TII and TIII in dis inc species o ho seshoe c ab, we we e
speci ically in e es ed in compa ing hei s uc u e, ac i i y and mode-o -ac ion. As hese pep ides ha e
a po en ial applica ion as an icance and/o an imic obial agen s, we in es iga ed whe he backbone
cycliza ion would inc ease he s abili y and main ain ac i i y. Ou s udies show ha TI, TII and TIII,
and hei cyclic analogues cTI, cTII and cTIII, ha e simila s uc u es and ac i i ies agains bac e ia and
cance ous cells. Backbone cycliza ion educed he hemoly ic ac i i y and inc eased pep ide s abili y
while main aining po en an icance and an imic obial ac i i ies. cTI and cTIII especially showed
po en ial o be conside ed o he de elopmen o an icance pep ide-based d ugs.
2. Resul s
2.1. P ope ies o Tachyplesin I–III and Thei Cyclic Analogues
The amino acid sequences o TI, TII and TIII di e in posi ions 1 o 15, which can be a lysine o an
a ginine esidue (Table 1). So a , TI is he mos s udied and he only analogue wi h epo ed s uc u e
calcula ions [
36
–
38
]. We we e in e es ed in compa ing TI, TII and TIII and hei backbone-cyclized
analogues (cTI–III) o iden i y simila i ies and di e ences in hei h ee-dimensional s uc u e and
s abili y; and o de e mine whe he hese cha ac e is ics a ec he memb ane in e ac ions and biological
ac i i y o he pep ides.
All pep ides we e syn hesized using solid-phase pep ide syn hesis, oxidized and co ec ly olded,
as sugges ed by he obse ed masses using elec osp ay ioniza ion mass spec oscopy (ESI-MS;
Supplemen a y Figu e S1 and Table 1) and con i med h ough clea ly dispe sed peaks in he amide
egion o hei espec i e One-dimensional (1D)
1
H NMR spec a [
39
]. The pep ides we e pu i ied o
>95%, as con i med by analy ical e e se-phase high-pe o mance liquid ch oma og aphy (RP-HPLC;
see ch oma og ams o pu e pep ides in Supplemen a y Figu e S1a).
Despi e mino di e ences, hei o e all hyd ophobici y ollows he end cTI >cTII >cTIII >TI >
TII >TIII (Table 1), as indica ed by hei e en ion ime (RT) on analy ical RP-HPLC (Supplemen a y
Figu e S1). The cyclic analogues appea o be o e all mo e hyd ophobic (less pola ) han he pa en
pep ides, which is consis en wi h he loss o he N- e minal cha ge and he C- e minal amida ion
esul ing om cycliza ion.
In . J. Mol. Sci. 2019,20, 4184 3 o 25
Table 1.
The sequence and physicochemical p ope ies o achyplesin I–III (TI-TIII) and hei cyclic
analogues (cTI-cTIII).
Pep ide Sequence 1Mass (Da) 2RT (min) 3Cha ge 4
Calc. Obs.
TI KWCFRVCYRGICYRRCR * 2263.8 2263.5 18.04 +7
TII RWCFRVCYRGICYRKCR * 2263.8 2263.5 17.79 +7
TIII KWCFRVCYRGICYRKCR * 2235.8 2235.6 17.68 +7
cTI KWCFRVCYRGICYRRCRG2303.8 2303.7 18.69 +6
cTII RWCFRVCYRGICYRKCRG 2303.8 2303.7 18.53 +6
cTIII KWCFRVCYRGICYRKCRG 2275.8 2275.5 18.44 +6
1
Pep ide sequences and amino acid esidues di e ing om TI a e in bold. * deno es C- e minal amida ion.
2
A e age
mass calcula ed (Calc.) om he amino acid sequence and expe imen ally obse ed (Obs.) using syn he ic pep ide
and de e mined om m/z 3+in ESI-MS.
3
Re en ion ime (RT) o pep ides on an analy ical RP-HPLC; ch oma og ams
shown in Supplemen a y Figu e S1a we e ob ained wi h a 2%/min g adien o 0–40% sol en B (90% ace oni ile;
0.05% i luo oace ic acid (TFA) ( / )) in sol en A (H
2
O, 0.05% TFA ( / )) a a low a e o 0.3 mL/min.
4
Cha ge o
he pep ides a pH 7.4.
2.2. S uc u e o Tachyplesins and Backbone-Cyclised Analogues
The h ee-dimensional (3D) s uc u es o TII and TIII, and o he backbone-cyclized analogues
cTI–cTIII we e de e mined wi h solu ion NMR spec oscopy. All backbone esonances we e ully
assigned apa om he N- e minal amides o he wo linea pep ides, and he R1/G18 amides o cTII,
e lec i e o some deg ee o lexibili y in hese egions. The seconda y
α
H chemical shi s o he na i e
pep ides and he cyclic analogues we e highly simila , indica ing a negligible change in he backbone
s uc u e and he β-s ands (W2-Y8; I11-R17) (Figu e 1a,b).
The 3D solu ion s uc u es o each pep ide we e calcula ed om dis ance es ain s, anging om
154 o he linea pep ides, o 172–284 o he cyclic analogues, along wi h dihed al angle es ain s
o aling 34 o 36. The inal amily o s uc u es o each o he pep ides has good s uc u al and ene gy
s a is ics, as indica ed by an o e all MolP obi y sco e o less han 1.6, shown in Table S1. Analysis
o he s uc u es by PROMOTIF [
40
] de ines an ipa allel
β
-s ands being o med by esidues W2-Y8
and I11-R17 in all bu one o he pep ides. The excep ion is TIII which has sligh ly sho e s ands
o med be ween esidues C3-Y8 and I11-C16. All disul ide bonds a e de ined by PROMOTIF as
adop ing he sho igh -hand hook con igu a ion. S uc u es o he achyplesin pep ides and he
cyclized analogues di e ed p ima ily in he lexibili y o he N- and C- e mini o he pa en pep ides
(Figu e 1b–d). The educ ion o he amino acid side-chain lexibili y in he e minal egions due o
backbone cycliza ion is emphasized in Figu e 1d wi h he side chain o he esidues K/R1 and K/R15,
which di e be ween TI/cTI, TII/cTII and TIII/cTIII; he side chain o W2 is also shown, as his esidue is
used o moni o he pep ide pa i ioning in o lipid bilaye s (see Sec ion 2.5.2). No signi ican ca ion-
π
in e ac ions be ween he R/K1 and W2 we e no ed o any o he achyplesin pep ides. Such an
in e ac ion migh be e ealed by NMR in he o m o subs an ial de ia ions o he chemical shi s o
a ginine/lysine esidues bu none we e obse ed (see Figu e 1a). The lack o ca ion-
π
in e ac ions is
also suppo ed by he NMR solu ion s uc u es ha e eal a high deg ee o lexibili y in he e mini o
he linea pep ides, and by he di e en o ien a ions ha hese esidues acqui e in he cyclic pep ides,
as shown in Figu e 1d.
The pep ide s uc u es we e deposi ed wi h he P o ein Da a Bank (PDB) and he Biological
Magne ic ResonanceDa a Bank (BMRB):TII—PDB ID:6PI2, BMRB ID:30617; TIII—PDB ID:6PI3, BMRB
ID: 30618; cTI—PDB ID: 6PIN; BMRB ID: 30619; cTII—PDB ID: 6PIO, BMRB ID: 30620; cTIII—PDB ID:
6PIP, BMRB ID: 30621.
In . J. Mol. Sci. 2019,20, 4184 4 o 25
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 o 25
Figu e 1. The NMR s uc u es o TI and cTI ( ed), TII and cTII (cyan), TIII and cTIII (magen a). (a)
Seconda y αH chemical shi a 298 K de e mined om
1
H NMR spec a. Shi s we e calcula ed by
sub ac ion o andom coil
1
H NMR shi s
[41]
om he expe imen al alues. Fo achyplesin I (TI),
he shi s o he pep ide syn hesized in ou lab we e compa ed o shi s ob ained om da a banks:
TI—PDB ID 2RTV
[37]
, and TI—BMRB ID 1135
[38]
. Posi i e shi s g ea e han 0.1 ppm sugges β-
s ands, indica ed by g ey a ows. (b) O e lay o he cyclic analogues (colo ed backbone) wi h hei
espec i e pa en pep ide (g ey backbone). Hyd ogen bonds be ween β-s ands a e indica ed in blue.
The s uc u e o TI was ob ained om he PDB (ID 1WO0). (c) O e lay o TI–TIII and cTI–cTIII. (d)
Mobili y o he esidues (K o R) a posi ion 1 and 15, which di e be ween he pep ides, and o W2
o he na i e sequences and cyclic analogues, espec i ely.
2.3. Imp o ed S abili y and Reduced Hemoly ic Ac i i y o Cyclized Tachyplesin Pep ides
Cycliza ion has been shown o inc ease s abili y [42,43] and educe he hemoly ic ac i i y o
some pep ides [44,45]. Compa ison o esis ance o human p o eases showed ha he cTI analogue
did no deg ade a e ea men o 24 h in 25% ( / ) human se um, whe eas only 25% o TI emained
in he solu ion (see analy ical RP-HPLC ch oma og ams in supplemen a y Figu e S2). A pep ide
(linea and wi hou disul ide bonds) used as a con ol was ully deg aded unde he same condi ions
Figu e 1.
The NMR s uc u es o TI and cTI ( ed), TII and cTII (cyan), TIII and cTIII (magen a).
(
a
) Seconda y
α
H chemical shi a 298 K de e mined om
1
H NMR spec a. Shi s we e calcula ed by
sub ac ion o andom coil
1
H NMR shi s [
41
] om he expe imen al alues. Fo achyplesin I (TI),
he shi s o he pep ide syn hesized in ou lab we e compa ed o shi s ob ained om da a banks:
TI—PDB ID 2RTV [
37
], and TI—BMRB ID 1135 [
38
]. Posi i e shi s g ea e han 0.1 ppm sugges
β
-s ands, indica ed by g ey a ows. (
b
) O e lay o he cyclic analogues (colo ed backbone) wi h
hei espec i e pa en pep ide (g ey backbone). Hyd ogen bonds be ween β-s ands a e indica ed in
blue. The s uc u e o TI was ob ained om he PDB (ID 1WO0). (
c
) O e lay o TI–TIII and cTI–cTIII.
(
d
) Mobili y o he esidues (K o R) a posi ion 1 and 15, which di e be ween he pep ides, and o W2
o he na i e sequences and cyclic analogues, espec i ely.
2.3. Imp o ed S abili y and Reduced Hemoly ic Ac i i y o Cyclized Tachyplesin Pep ides
Cycliza ion has been shown o inc ease s abili y [
42
,
43
] and educe he hemoly ic ac i i y o some
pep ides [
44
,
45
]. Compa ison o esis ance o human p o eases showed ha he cTI analogue did no
deg ade a e ea men o 24 h in 25% ( / ) human se um, whe eas only 25% o TI emained in he
solu ion (see analy ical RP-HPLC ch oma og ams in Supplemen a y Figu e S2). A pep ide (linea and
In . J. Mol. Sci. 2019,20, 4184 5 o 25
wi hou disul ide bonds) used as a con ol was ully deg aded unde he same condi ions (Figu e 2a).
TI has p e iously been shown o be comple ely s able o 2 h in mouse o human se um [46].
The pe cen age o hemolysis o human ed blood cells (RBCs) ollowed he end TI >TII >
TIII >cTII >cTI >cTIII when compa ed a 128
µ
M, he highes concen a ion o pep ide es ed.
TI was he mos hemoly ic o he na i e sequences. Backbone cycliza ion educed he hemoly ic
ac i i y o he cTI and cTIII compa ed o hei pa en coun e pa s, bu led o no clea imp o emen
o cTII. The C-amida ed pep ides TI–TIII lysed 66%, 56% and 41% o RBCs a 64
µ
M espec i ely
(Figu e 2b and Table 2). A simila end bu lowe hemoly ic ac i i y had been epo ed o TI, TII,
and TIII lacking C- e minal amida ion a highe pep ide concen a ions [
47
], which is known o impac
pep ide ac i i y [48,49].
The posi i e con ol meli in, a hemoly ic pep ide om honeybee enom, induces 100% hemolysis
in human RBCs a concen a ions abo e 2
µ
M. By compa ison, a his concen a ion, all achyplesin
pep ides ha e a low hemoly ic ac i i y o a ound 10%.
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 o 25
(Figu e 2a). TI has p e iously been shown o be comple ely s able o 2 h in mouse o human se um
[46].
The pe cen age o hemolysis o human ed blood cells (RBCs) ollowed he end
TI > TII > TIII > cTII > cTI > cTIII when compa ed a 128 µM, he highes concen a ion o pep ide
es ed. TI was he mos hemoly ic o he na i e sequences. Backbone cycliza ion educed he
hemoly ic ac i i y o he cTI and cTIII compa ed o hei pa en coun e pa s, bu led o no clea
imp o emen o cTII. The C-amida ed pep ides TI–TIII lysed 66%, 56% and 41% o RBCs a 64 µM
espec i ely (Figu e 2b and Table 2). A simila end bu lowe hemoly ic ac i i y had been epo ed
o TI, TII, and TIII lacking C- e minal amida ion a highe pep ide concen a ions [47], which is
known o impac pep ide ac i i y [48,49].
The posi i e con ol meli in, a hemoly ic pep ide om honeybee enom, induces 100%
hemolysis in human RBCs a concen a ions abo e 2 µM. By compa ison, a his concen a ion, all
achyplesin pep ides ha e a low hemoly ic ac i i y o a ound 10%.
Figu e 2. The pep ide s abili y in human se um and ac i i y agains human ed blood cells (RBCs). (a)
S abili y o TI and cyclic analogue cTI in 25% ( / ) human se um o 24 h. The samples we e analyzed
by analy ical RP-HPLC and he pe cen age o pep ide emaining was de e mined om he a ea unde
he pep ide peak in he ch oma og am in compa ison o peak a ea a ime ze o. A linea 18 amino acid
pep ide (KGGGGSGQLIDSMANSFV) was included as he posi i e con ol. (b) Hemoly ic ac i i y o
achyplesins and hei cyclic analogues we e es ed up o 128 µM agains human RBCs (0.25% ( / ),
1 h incuba ion a 37 °C). Meli in, a highly hemoly ic pep ide, was included as he posi i e con ol.
Table 2. Concen a ion o achyplesin I–III (TI-TIII) and o hei cyclic analogues (cTI-cTIII) equi ed
o induce 50% lysis in RBCs (HC50).
a.
Pep ide HC50 (µM)
TI 34.9 ± 2.8
TII 55.4 ± 6.6
TIII 86.4 ± 12.2
cTI 106.9 ± 21.0
cTII 64.1 ± 9.4
cTIII > 128
a
Values we e de e mined om a minimum o h ee independen eplica es and depic ed as mean ± SEM.
2.4. Biological Ac i i y o Tachyplesin I–III and o Thei Cyclic Analogues
2.4.1. Ac i i y agains Bac e ia
The G am-nega i e Esche ichia coli s ains ATCC 25922 and DC2 CGSC 7139 and he G am-
posi i e S aphylococcus au eus s ains ATCC 25923 and ATCC 6538 we e used o de e mine he
an imic obial ac i i y o he pa en achyplesin pep ides and hei cyclic analogues agains bac e ia
wi h di e ences in he physical and chemical p ope ies o hei cell wall. E. coli ATCC 25922
Figu e 2.
The pep ide s abili y in human se um and ac i i y agains human ed blood cells (RBCs).
(
a
) S abili y o TI and cyclic analogue cTI in 25% ( / ) human se um o 24 h. The samples we e analyzed
by analy ical RP-HPLC and he pe cen age o pep ide emaining was de e mined om he a ea unde
he pep ide peak in he ch oma og am in compa ison o peak a ea a ime ze o. A linea 18 amino acid
pep ide (KGGGGSGQLIDSMANSFV) was included as he posi i e con ol. (
b
) Hemoly ic ac i i y o
achyplesins and hei cyclic analogues we e es ed up o 128
µ
M agains human RBCs (0.25% ( / ), 1 h
incuba ion a 37 ◦C). Meli in, a highly hemoly ic pep ide, was included as he posi i e con ol.
Table 2.
Concen a ion o achyplesin I–III (TI-TIII) and o hei cyclic analogues (cTI-cTIII) equi ed o
induce 50% lysis in RBCs (HC50). a.
Pep ide HC50 (µM)
TI 34.9 ±2.8
TII 55.4 ±6.6
TIII 86.4 ±12.2
cTI 106.9 ±21.0
cTII 64.1 ±9.4
cTIII >128
aValues we e de e mined om a minimum o h ee independen eplica es and depic ed as mean ±SEM.
2.4. Biological Ac i i y o Tachyplesin I–III and o Thei Cyclic Analogues
2.4.1. Ac i i y agains Bac e ia
The G am-nega i e Esche ichia coli s ains ATCC 25922 and DC2 CGSC 7139 and he G am-posi i e
S aphylococcus au eus s ains ATCC 25923 and ATCC 6538 we e used o de e mine he an imic obial

In . J. Mol. Sci. 2019,20, 4184 6 o 25
ac i i y o he pa en achyplesin pep ides and hei cyclic analogues agains bac e ia wi h di e ences in
he physical and chemical p ope ies o hei cell wall. E. coli ATCC 25922 (“smoo h” LPS) and S. au eus
ATCC 25923 a e common con ol s ains o an imic obial suscep ibili y es ing [
50
,
51
]. E. coli DC2
CGSC 7139 is hype sensi i e o an ibac e ial agen s and mo e pe meable o dyes [
52
],
while S. au eus
ATCC 6538 is known o o m bio ilms [
53
]. Tachyplesin pep ides and hei cyclic analogues we e es ed
agains plank onic cul u es o all s ains and agains S. au eus ATCC 6538 in he bio ilm o m o di ec
compa ison o hei an imic obial ac i i y.
Tachyplesin I–III we e wo- o- ou imes mo e po en han hei cyclic analogues agains all
bac e ial s ains in hei plank onic g ow h o m (Table 3). Gene ally, he G am-nega i e s ains we e
mo e suscep ible han he G am-posi i e s ains. Simila MICs ha e been epo ed p e iously o
TI agains E. coli ATCC 25922, [
1
] and TI and TII agains S. au eus ATCC 25923 [
2
,
3
]. Tachyplesin
pep ides we e mos ac i e agains E. coli DC2 CGSC 7139 and E. coli ATCC 25922. The ac i i y agains
hese bac e ial s ains was educed when he pep ides we e cyclized. The pep ides we e leas ac i e
agains S. au eus ATCC 6538 in he plank onic o m, and no di e ence in ac i i y was obse ed be ween
achyplesin pep ides and cyclic analogues (Table 3).
Table 3.
The minimal inhibi o y concen a ions (MICs) o achyplesin I–III (TI-TIII) and o hei cyclic
analogues (cTI-cTIII) agains plank onic bac e ia.
MIC (µM)
Pep ide E. coli DC2
CGSC 7139
E. coli
ATCC 25922
S. au eus
ATCC 25923
S. au eus
ATCC 6538
TI 0.5–1 0.0625–0.5 1–4 4–8
TII 0.5–1 0.125–0.5 1–4 4–8
TIII 0.25–0.5 0.0625–0.5 1–4 4–8
cTI 4 1 2–8 8
cTII 4–8 1–2 2–8 8
cTIII 4–8 1–2 2–8 8
To de e mine whe he he achyplesin pep ides could ac agains bac e ial bio ilms, he cells
me abolic ac i i y was measu ed o bio ilms o med by S. au eus ATCC 6538 (Figu e 3and Table 4).
All pep ides exhibi ed simila ac i i y agains he bac e ia in he bio ilm o m, wi h a 50% loss o cell
me abolic ac i i y obse ed a ~20
µ
M. Howe e , app oxima ely 40% o he bio ilm was me abolically
ac i e a 32
µ
M, he highes concen a ion es ed, sugges ing a educed ac i i y agains bio ilm
compa ed o he plank onic S. au eus ATCC 6538 bac e ia.
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 o 25
(“smoo h” LPS) and S. au eus ATCC 25923 a e common con ol s ains o an imic obial suscep ibili y
es ing [50,51]. E. coli DC2 CGSC 7139 is hype sensi i e o an ibac e ial agen s and mo e pe meable
o dyes [52], while S. au eus ATCC 6538 is known o o m bio ilms [53]. Tachyplesin pep ides and
hei cyclic analogues we e es ed agains plank onic cul u es o all s ains and agains S. au eus
ATCC 6538 in he bio ilm o m o di ec compa ison o hei an imic obial ac i i y.
Tachyplesin I–III we e wo- o- ou imes mo e po en han hei cyclic analogues agains all
bac e ial s ains in hei plank onic g ow h o m (Table 3). Gene ally, he G am-nega i e s ains we e
mo e suscep ible han he G am-posi i e s ains. Simila MICs ha e been epo ed p e iously o TI
agains E. coli ATCC 25922, [1] and TI and TII agains S. au eus ATCC 25923 [2,3]. Tachyplesin
pep ides we e mos ac i e agains E. coli DC2 CGSC 7139 and E. coli ATCC 25922. The ac i i y agains
hese bac e ial s ains was educed when he pep ides we e cyclized. The pep ides we e leas ac i e
agains S. au eus ATCC 6538 in he plank onic o m, and no di e ence in ac i i y was obse ed
be ween achyplesin pep ides and cyclic analogues (Table 3).
Table 3. The minimal inhibi o y concen a ions (MICs) o achyplesin I–III (TI-TIII) and o hei cyclic
analogues (cTI-cTIII) agains plank onic bac e ia.
MIC (µM)
Pep ide E. coli DC2
CGSC 7139
E. coli
ATCC 25922
S. au eus
ATCC 25923
S. au eus
ATCC 6538
TI 0.5–1 0.0625–0.5 1–4 4–8
TII 0.5–1 0.125–0.5 1–4 4–8
TIII 0.25–0.5 0.0625–0.5 1–4 4–8
cTI 4 1 2–8 8
cTII 4–8 1–2 2–8 8
cTIII 4–8 1–2 2–8 8
To de e mine whe he he achyplesin pep ides could ac agains bac e ial bio ilms, he cells
me abolic ac i i y was measu ed o bio ilms o med by S. au eus ATCC 6538 (Figu e 3 and Table 4).
All pep ides exhibi ed simila ac i i y agains he bac e ia in he bio ilm o m, wi h a 50% loss o cell
me abolic ac i i y obse ed a ~20 µM. Howe e , app oxima ely 40% o he bio ilm was me abolically
ac i e a 32 µM, he highes concen a ion es ed, sugges ing a educed ac i i y agains bio ilm
compa ed o he plank onic S. au eus ATCC 6538 bac e ia.
Figu e 3. The ac i i y o achyplesin pep ides and hei cyclic analogues agains an S. au eus ATCC
6538 bio ilm. The esponse cu e o bio ilm ea ed wi h inc easing he concen a ions o pep ide (n =
4, ± SEM).
Figu e 3.
The ac i i y o achyplesin pep ides and hei cyclic analogues agains an S. au eus ATCC
6538 bio ilm. The esponse cu e o bio ilm ea ed wi h inc easing he concen a ions o pep ide (n=4,
±SEM).
In . J. Mol. Sci. 2019,20, 4184 7 o 25
Table 4.
Ac i i y o achyplesin I–III (TI–TIII) and cyclic analogues (cTI–cTIII) agains S. au eus ATCC
6538 in he bio ilm g ow h o m.
Pep ide CC50 (µM) 1Me abolically Ac i e Cells (%) 2
TI 21.5 ±2.2 37.7 ±4.3
TII 23.1 ±2.9 39.9 ±2.9
TIII 24.2 ±4.1 43.3 ±4.1
cTI 17.8 ±3.6 31.9 ±6.0
cTII 19.4 ±1.5 34.6 ±7.1
cTIII 18.0 ±1.4 37.5 ±7.2
1
Pep ide concen a ion equi ed o induce he educ ion o 50% o he me abolically ac i e cell popula ion (CC50)
de e mined using one-si e speci ic binding wi h he Hill slope.
2
Pe cen age o cells emaining me abolically ac i e
a 32 µM, he highes pep ide concen a ion es ed.
2.4.2. Ac i i y agains Cance ous Cells
TI, TII, TIII, and hei cyclic analogues we e es ed agains h ee melanoma (MM96L, HT144
and WM164) and one ce ical cance (HeLa) cell line (Supplemen a y Tables S2 and S3) o de e mine
whe he he pep ides exhibi di e en cy o oxic ac i i ies. The aneuploid immo al ke a inocy e
cell line HaCaT was included as a non-cance ous con ol. Cy o oxici y owa d cance cell lines was
compa ed by de e mining pep ide concen a ions equi ed o achie e 50% o cell dea h (CC50) om
dose- esponse cu es (Table 5).
Table 5.
The cy o oxici y o achyplesin I–III (TI-TIII) and cyclic analogues (cTI-cTIII) agains
cul u ed cells.
CC50 (µM) 1Melanoma
Selec i i y 5
Pep ide MM96L 2HT144 2WM164 2HeLa 3HaCaT 4
TI 1.5 ±0.1 1.7 ±0.2 2.5 ±0.1 13.1 ±1.2 11.6 ±1.6 2–21
TII 1.6 ±0.1 2.0 ±0.1 1.6 ±0.1 18.0 ±3.9 3.7 ±0.2 3–35
TIII 1.8 ±0.1 2.0 ±0.1 1.7 ±0.1 21.7 ±1.1 7.3 ±0.5 5–48
cTI 1.3 ±0.1 1.4 ±0.1 2.7 ±0.1 6.7 ±0.6 7.9 ±0.5 2–76
cTII 1.1 ±0.1 0.8 ±0.04 2.4 ±0.3 7.2 ±0.4 2.4 ±0.3 3–58
cTIII 1.7 ±0.1 0.9 ±0.03 1.3 ±0.1 9.3 ±0.4 7.5 ±0.3 3–98
1
The concen a ion necessa y o kill 50% o cells was calcula ed om dose- esponse cu es (n
≥
3,
±
SEM).
2
Melanoma cell lines: MM96L, HT144, WM164,
3
ce ical cance cell line: HeLa,
4
heal hy epi helial con ol
cell line: HaCaT (aneuploid immo al ke a inocy e). Desc ip ion and e i ica ion o each cell line a e de ailed in
Supplemen a y Tables S2 and S3).
5
selec i i y o melanoma cell lines was es ima ed h ough he ac i i y- oxici y
index (ATI). ATI =MHC/MCC50 (modi ied om Re e ence [
46
]), wi h MHC being he minimal concen a ion
necessa y o induce 10% (lowe alue) o 50% (highe alue) cell dea h in human RBCs and MCC50 being he
median o CC50 alues o all melanoma cell lines. Values abo e 1 indica e a highe selec i i y o he cance ous cells
o e RBCs.
The cy o oxic ac i i ies o he achyplesin pep ides and hei cyclic analogues a e dependen
on he cell lines (Table 5). All pep ides we e mos e ec i e agains he melanoma cell lines MM96L,
HT144 and WM164 (cy o oxic ac i i ies anged be ween CC50 0.8–2.7
µ
M). Compa ed o he con ol
cell line HaCaT, he cy o oxic ac i i ies o he pep ides agains melanoma we e signi ican ly di e en
(p<0.05) wi h he excep ion o cTII agains WM164. The ce ical cance cell line HeLa was mo e
esis an owa ds he achyplesin pep ides compa ed o he melanoma cell lines and highe pep ide
concen a ions we e necessa y o each 50% cell dea h. Di e ences in cy o oxic ac i i y agains HeLa,
compa ed o he con ol cell line HaCaT, can be obse ed o all pep ides excep TI and cTI. The cyclic
analogues cTI–cTIII we e app oxima ely 2x mo e po en agains HeLa han he pa en pep ides TI–TIII
(p<0.05).
The pep ides ha e a simila selec i i y o he melanoma cell lines a lowe pep ide concen a ions
which would induce ≤10% hemolysis in RBCs. A concen a ions inducing ≤50% hemolysis in RBCs,
In . J. Mol. Sci. 2019,20, 4184 8 o 25
he cyclic analogues cTI–cTIII we e mo e selec i e. O e all, he mos p omising he apeu ic ange was
obse ed o TIII, cTI and cTIII (Table 5).
2.5. Mechanis ic S udies
Pep ides wi h an amphipa hic a angemen o cha ged and hyd ophobic esidues a e known
o ac agains bac e ial and cance cells ia selec i e memb ane a ge ing, pene a ion and/o lysis.
To cha ac e ize how pep ide-memb ane in e ac ions a ec biological ac i i y, we unde ook de ailed
pep ide-memb ane binding s udies ha compa e pa en and cyclic achyplesin pep ides.
2.5.1. Pep ide Binding o Model Memb anes
The abili y o TI o bind o model memb anes has been p e iously shown and a p e e ence
o nega i ely-cha ged memb anes was ound [
31
,
46
]. We we e in e es ed in compa ing he
memb ane-binding p ope ies o he pa en achyplesins e sus he cyclic analogues o de e mine
whe he di e ences in memb ane binding could explain he ela i e biological ac i i ies (see Table 5).
Gi en he simila po encies among he h ee achyplesins, and among he h ee cyclic analogues,
we compa ed he in e ac ion o TI and cTI, as ep esen a i e o pa en and cyclic achyplesin
pep ides, espec i ely, wi h model memb anes using su ace plasmon esonance (SPR). Phospholipids
con aining PC-headg oups a e he mos common in he ou e lea le o he mammalian plasma
memb ane [
54
,
55
]; hus, we p epa ed model memb anes composed o he zwi e ionic POPC
(1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine), which o ms luid bilaye s a 25
◦
C and mimics
he o e all luidi y and neu al su ace o heal hy euka yo ic cells [
56
]. Phospholipids con aining
he nega i ely-cha ged PS-headg oups a e no mally es ic ed o he inne lea le in euka yo ic cell
memb anes bu a e exposed a he cell su ace o cance ous cells [
20
,
22
,
23
]. The e o e, we used
model memb anes wi h 20% o POPS (1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine), POPC/POPS
(4:1 mola a io), o ep esen he nega i ely-cha ged su ace o cance cells.
Bo h TI and cTI bound wi h highe a ini y o nega i ely-cha ged POPC/POPS (4:1) han o
zwi e ionic POPC lipid bilaye s. cTI had s onge a ini y o bo h lipid sys ems han he pa en
pep ide, as shown by a highe pep ide- o-lipid (P/L) a io du ing associa ion, a slowe dissocia ion
a e (k
o
) om he lipids, and a highe amoun o pep ide emaining associa ed o he memb ane a
he end o dissocia ion (P/L
o
) (Figu e 4a,b and Table 6). Addi ionally, TI and cTI dis up ed la ge
unilamella esicles (LUVs) o POPC/POPS (4:1) wi h highe e icacy han LUVs o POPC, which ag ees
wi h hei p e e ence o nega i ely-cha ged o e neu al memb anes. Howe e , TI dis up ed LUVs
o POPC and o POPC/POPS (4:1) mo e e icien ly han he cyclic analogue cTI (Figu e 4c, Table 6).
Thus, he highe binding sa u a ion and a ini y o cTI (see P/L
max
and kine ic pa ame e s in Table 6)
o he memb anes did no co ela e wi h i s abili y o dis up memb anes. The highe e icacy o TI
in dis up ing memb anes, compa ed o he cyclic analogue, migh explain he highe ac i i y o he
pa en TI–TIII o plank onic bac e ial cells (see Table 3), compa ed o hei cyclic analogues.
In . J. Mol. Sci. 2019,20, 4184 9 o 25
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 o 25
hemolysis in RBCs, he cyclic analogues cTI–cTIII we e mo e selec i e. O e all, he mos p omising
he apeu ic ange was obse ed o TIII, cTI and cTIII (Table 5).
2.5. Mechanis ic S udies
Pep ides wi h an amphipa hic a angemen o cha ged and hyd ophobic esidues a e known o
ac agains bac e ial and cance cells ia selec i e memb ane a ge ing, pene a ion and/o lysis. To
cha ac e ize how pep ide-memb ane in e ac ions a ec biological ac i i y, we unde ook de ailed
pep ide-memb ane binding s udies ha compa e pa en and cyclic achyplesin pep ides.
2.5.1. Pep ide Binding o Model Memb anes
The abili y o TI o bind o model memb anes has been p e iously shown and a p e e ence o
nega i ely-cha ged memb anes was ound [31,46]. We we e in e es ed in compa ing he memb ane-
binding p ope ies o he pa en achyplesins e sus he cyclic analogues o de e mine whe he
di e ences in memb ane binding could explain he ela i e biological ac i i ies (see Table 5). Gi en
he simila po encies among he h ee achyplesins, and among he h ee cyclic analogues, we
compa ed he in e ac ion o TI and cTI, as ep esen a i e o pa en and cyclic achyplesin pep ides,
espec i ely, wi h model memb anes using su ace plasmon esonance (SPR). Phospholipids
con aining PC-headg oups a e he mos common in he ou e lea le o he mammalian plasma
memb ane [54,55]; hus, we p epa ed model memb anes composed o he zwi e ionic POPC (1-
palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine), which o ms luid bilaye s a 25 °C and mimics he
o e all luidi y and neu al su ace o heal hy euka yo ic cells [56]. Phospholipids con aining he
nega i ely-cha ged PS-headg oups a e no mally es ic ed o he inne lea le in euka yo ic cell
memb anes bu a e exposed a he cell su ace o cance ous cells [20,22,23]. The e o e, we used model
memb anes wi h 20% o POPS (1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine), POPC/POPS (4:1
mola a io), o ep esen he nega i ely-cha ged su ace o cance cells.
Bo h TI and cTI bound wi h highe a ini y o nega i ely-cha ged POPC/POPS (4:1) han o
zwi e ionic POPC lipid bilaye s. cTI had s onge a ini y o bo h lipid sys ems han he pa en
pep ide, as shown by a highe pep ide- o-lipid (P/L) a io du ing associa ion, a slowe dissocia ion
a e (k
o
) om he lipids, and a highe amoun o pep ide emaining associa ed o he memb ane a
he end o dissocia ion (P/L
o
) (Figu e 4a,b and Table 6). Addi ionally, TI and cTI dis up ed la ge
unilamella esicles (LUVs) o POPC/POPS (4:1) wi h highe e icacy han LUVs o POPC, which
ag ees wi h hei p e e ence o nega i ely-cha ged o e neu al memb anes. Howe e , TI dis up ed
LUVs o POPC and o POPC/POPS (4:1) mo e e icien ly han he cyclic analogue cTI (Figu e 4c, Table
6). Thus, he highe binding sa u a ion and a ini y o cTI (see P/L
max
and kine ic pa ame e s in Table
6) o he memb anes did no co ela e wi h i s abili y o dis up memb anes. The highe e icacy o TI
in dis up ing memb anes, compa ed o he cyclic analogue, migh explain he highe ac i i y o he
pa en TI–TIII o plank onic bac e ial cells (see Table 3), compa ed o hei cyclic analogues.
Figu e 4. Memb ane binding and dis up ion induced by achyplesin I (TI) and cyclic achyplesin I
(cTI). Model memb anes composed o 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine (POPC) and
POPC/1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine (POPS) (4:1) we e compa ed. (a) Su ace
plasmon esonance senso g ams ob ained wi h 32 µM pep ide injec ed o e lipid bilaye s deposi ed
Figu e 4.
Memb ane binding and dis up ion induced by achyplesin I (TI) and cyclic achyplesin
I (cTI). Model memb anes composed o 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine (POPC)
and POPC/1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine (POPS) (4:1) we e compa ed. (
a
) Su ace
plasmon esonance senso g ams ob ained wi h 32
µ
M pep ide injec ed o e lipid bilaye s deposi ed on
an L1 chip su ace o 180 s (associa ion); dissocia ion was moni o ed o 600 s. Response uni s (RU) we e
con e ed in o a pep ide- o-lipid a io (P/L (mol/mol)) o ake in o conside a ion he di e ences in lipid
packing esul ing in di e en amoun s being deposi ed o co e he chip su ace.
(b) The dose- esponse
cu es show P/L ob ained a he end o he associa ion phase ( =170 s) and plo ed as a unc ion o
pep ide concen a ion injec ed. (
c
) Pe cen age o esicle leakage de e mined by luo escence emission
in ensi y o 5-ca boxy luo escein (
λex
=490 nm,
λem
=513 nm) leaking om LUVs a inc easing
concen a ions o pep ide. Lipid concen a ion used was 5
µ
M, and he pep ide was es ed up o
10
µ
M. Dose- esponse cu es we e i ed wi h one-si e speci ic binding wi h Hill slope equa ion in
G aphPad P ism.
Table 6.
The kine ic and a ini y pa ame e s om su ace plasmon esonance analysis o he in e ac ion
o 32
µ
M achyplesin I (TI) and cyclic achyplesin I (cTI) wi h neu al (POPC) and nega i ely-cha ged
model memb anes (POPC/POPS (4:1)) and leakage induced by he same pep ides and lipid sys ems.
Pep ide Lipid
Sys em
P/Lmax
(mol/mol) 1KD
(µM) 1ko
(x 10−2s−1)2P/Lo
(mol/mol) 2LCmax
(%) 3
TI POPC 0.26 ±0.06 22.4 ±10.9 1.50 ±0.11 0.046 ±0.001 39.5 ±4.6
cTI 0.33 ±0.07 16.7 ±8.4 0.91 ±0.03 0.065 ±0.001 32.9 ±9.4
TI POPC/POPS
(4:1)
0.37 ±0.04 11.8 ±2.4 2.75 ±0.22 0.096 ±0.001 76.0 ±2.8
cTI 0.48 ±0.08 9.2 ±3.4 0.70 ±0.03 0.137 ±0.002 58.5 ±3.6
1
P/L
max
and K
D
we e calcula ed om he dose- esponse cu es (one-si e speci ic binding wi h Hill slope equa ion,
G aphPad P ism) in Figu e 4b. The P/L
max
alue ep esen s he pep ide- o-lipid a io (mol/mol) when pep ide-lipid
binding eaches sa u a ion, K
D
is he pep ide concen a ion necessa y o each he hal -maximal binding esponse.
2
k
o
is he dissocia ion cons an and P/L
o
is he pep ide-lipid a io a he end o associa ion phase calcula ed om
he senso g ams ob ained wi h 32
µ
M pep ide in Figu e 4a. k
o
and P/L
o
we e i ed in G aphPad P ism, assuming
a Langmui kine ic.
3
Pe cen age o leakage achie ed when incuba ing 10
µ
M pep ide wi h 5
µ
M LUVs. POPC is
1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine; POPS is 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine.
To examine he abili y o cTI o bind model memb anes ha mimic bac e ial
cell memb anes, we p epa ed esicles wi h an E. coli pola lipid ex ac composed o
zwi e ionic phospha idyle hanolamine (PE)-phospholipids, nega i ely-cha ged phospha idylglyce ol
(PG)-phospholipids, and ca diolipin (CA) in he p opo ion 67:23.2:9.8 (w /w %). The SPR senso g am
and dose- esponse cu es (Supplemen a y Figu e S3) show ha cTI has a high a ini y o E. coli lipids.
Compa ison o he dose- esponse cu es and i ed pa ame e s show ha he maximum amoun o
cTI bound o E. coli lipids (P/L
max
, Supplemen a y Table S4) is no as high as o he o he es ed
nega i ely-cha ged memb anes o o he zwi e ionic POPC, bu he P/L
o
is highe han om he o he
es ed memb anes (see Supplemen a y Table S4), sugges ing ha a la ge amoun o pep ide emains
bound o he bilaye s ha mimic bac e ial memb anes. To in es iga e whe he cTI dis inguishes
he nega i ely-cha ged headg oups p esen in bac e ia (i.e., PG) om hose in cance cells (i.e., PS),
we compa ed he binding o cTI o POPC/POPG (1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphoglyce ol;
In . J. Mol. Sci. 2019,20, 4184 16 o 25
0.05% ( / ) TFA) un il he desi ed pu i y o >95%. The co ec pep ide mass was con i med wi h
ESI-MS, while na i e disul ide connec i i y was in e ed om he dispe sion o peaks in he 1D NMR
spec a using a B uke A ance 600 MHz spec ome e (Bille ica, USA). The pep ide concen a ion was
de e mined om he abso bance a 280 nm (
E280
=8730 M
−1
.cm
−1
as es ima ed ex inc ion coe icien
based on he con ibu ion o Ty and T p esidues, and disul ide bonds).
4.2. NMR Spec oscopy
Fo he s uc u al analysis o TI–III and cTI–III, pep ide (1 mg/mL) was dissol ed in H
2
O/D
2
O
(10:1, / ) and he pH adjus ed o pH 4–5. 1D
1
H spec a, wo-dimensional o al co ela ed spec oscopy
(TOCSY) and nuclea O e hause e ec spec oscopy (NOESY) we e acqui ed wi h a B uke A ance
600 MHz NMR spec ome e (Bille ica, USA) a a empe a u e o 298 K. Addi ional spec a o
1
H-
13
C
HSQC and
1
H-
15
N HSQC in H
2
O/D
2
O (10:1, / ) and exclusi e co ela ion spec a (E.COSY) in D
2
O
we e acqui ed. Spec a we e e e enced o an in e nal s anda d 2,2-dime hyl-2-silapen one-5-sul ona e
(DSS) a 0 ppm. CYANA 3.97 was used o au oma ically calcula e and e ine s uc u es based on
dis ance es ain s de i ed om he NOESY spec a [
72
], and o sion angles (
φ
and
ϕ
) gene a ed
using TALOS-N and H
α
, C
α
, C
β
, HN chemical shi s de i ed om NOESY,
1
H-
13
C HSQC and
1
H-
15
N
HSQC spec a [
73
]. Se e al
χ
1 side-chain angle es ain s we e added based on E.COSY and NOESY
da a. A inal se o s uc u es was gene a ed wi h CNS [
74
] using o sion angle dynamics, e inemen
and ene gy minimiza ion in explici sol en . Final s uc u es we e assessed o s e eochemical quali y
using MolP obi y [75].
4.3. Se um S abili y
The se um s abili y assay was ca ied ou as p e iously desc ibed [
42
] wi h some modi ica ions.
B ie ly, achyplesin a ian s we e incuba ed in 25% ( / ) human se um dilu ed in phospha e-bu e ed
saline (PBS) a a inal concen a ion o 50
µ
M a 37
◦
C. T iplica es we e collec ed a ime 0 h and 24 h and
he se um p o eins we e p ecipi a ed wi h ace oni ile (1:3 a io) supplemen ed wi h 3% TFA. Samples
we e kep on ice o 10 min be o e cen i uga ion a 17,000
×
g o 10 min a 4
◦
C. The pep ide con aining
supe na an o each sample was ha es ed and quan i ied using RP-HPLC (10 o 45% sol en B, 1%/min
g adien ). The pe cen age o pep ide s abili y in human se um was calcula ed by compa ing he a ea o
he pep ide peak ob ained a 24 h o ha a ime 0 h. A linea pep ide con aining 18 amino acid esidues
(KGGGGSGQLIDSMANSFV) was included as a con ol suscep ible o p o eoly ic deg ada ion.
4.4. Hemoly ic S udies
A small amoun o blood was collec ed om h ee heal hy human dono s. The blood was
immedia ely dilu ed in PBS and cen i uged 4–5 imes o 1 min a 4000 pm o wash and sepa a e he
human ed blood cells (RBCs). RBCs suspension (0.25% ( / ) in PBS) was incuba ed wi h pep ides wi h
wo- old se ial dilu ions o he pep ide (highes concen a ion es ed was 128
µ
M, and he lowes was
0.25
µ
M) in a 96-well pla e. Meli in, a memb ane dis up i e pep ide, was used as con ol. The pla es
we e incuba ed o 1 h a 37
◦
C. A e incuba ion, he pla es we e cen i uged o 5 min a 1000 pm o
pelle any non-lysed RBCs. A o al o 100
µ
L o he supe na an we e ans e ed o a new 96-well
pla e [
76
]. The hemoglobin eleased in o he supe na an om lysed cells was measu ed by abso bance
a 415 nm using he Tecan in ini e M1000P o mul ipla e eade (Männedo , Swi ze land).
4.5. Cell Cul u e
Cells we e g own in cell cul u e lasks and incuba ed in a humidi ied a mosphe e (5% CO
2
,
37
◦
C). The cance cell lines HeLa and he con ol cell line HaCaT we e g own in a DMEM medium
supplemen ed wi h 1% ( / ) penicillin/s ep omycin and 10% ( / ) e al bo ine se um (FBS). The cance
cell lines MM96L, HT144 and WM164 we e g own in a RPMI medium supplemen ed wi h 1% ( / )
penicillin/s ep a idin, 10% ( / ) FBS, 20 mM l-glu amine, and 10 mM sodium py u a e. Cell cul u es

In . J. Mol. Sci. 2019,20, 4184 17 o 25
we e main ained by dilu ion upon eaching con luence, each 48–72 h. Mo e in o ma ion abou he cell
lines [77] is a ailable in Tables S2 and S3.
4.6. Cy o oxici y Assays
Cells we e seeded in o 96-well la -bo om pla es a 5
×
10
3
cells/well and incuba ed o e nigh .
The medium was emo ed and eplaced wi h 90
µ
L se um- ee medium, 10
µ
L o 10x concen a ed
pep ide solu ions in PBS we e added. PBS was added as blank and 0.1% ( / ) T i on X-100 was used o
es ablish 100% o cell dea h. A e 2 h incuba ion a 37
◦
C, 10
µ
L o il e ed 0.05% (w/ ) esazu in solu ion
was added o each well [
70
]. Resazu in is con e ed o he pink and luo escen compound eso u in by
iable cells [
78
]. A e incuba ion o e nigh , he luo escence in ensi y (
λex
=565 nm and
λem =584 nm
)
was measu ed wi h he Tecan in ini e M1000P o mul ipla e eade (Männedo , Swi ze land).
The selec i i y was calcula ed h ough he ac i i y- oxici y index (ATI) [
46
]. ATI =MHC/MCC50,
wi h MHC being he minimal concen a ion necessa y o induce 10% o 50% cell dea h in human ed
blood cells and MCC50 he median o cy o oxic concen a ions (CC50) o all es ed melanoma cell lines.
4.7. An imic obial S udies
S. au eus ATCC 25923, S. au eus ATCC 6538, E. coli ATCC 25922, and E. coli DC2 CGSC 7139 we e
g own in Muelle Hin on B o h (Sigma Ald ich, S . Luis, USA). Bac e ial cul u es in he exponen ial
g ow h phase we e dilu ed o an OD600nm o 0.001 and seeded in o 96-well pla es. Pep ides a di e en
concen a ions, s a ing a 64
µ
M and wi h wo- old se ial dilu ions (i.e., 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25,
0.125 and 0.0625
µ
M), we e incuba ed wi h cells [
43
]; 0.05% ( / ) esazu in was added o he cul u es
he nex day and he con e sion o eso u in was measu ed a e 1 h o incuba ion a 37
◦
C using a
565 nm exci a ion and 584 nm emission wa eleng h, as abo e.
4.8. Bio ilm S udies
S. au eus ATCC 6538 (1
×
10
6
c u/mL) was cul u ed in T yp ic Soy B o h, con aining 0.25% (w/ )
glucoseand incuba ed in96-well mic o i e la -bo omed polys y enepla es o 24ha 37
◦
C.P e o med
bio ilms we e hen washed wi h Muelle Hin on B o h o emo e non-adhe en cells. Two- old se ial
dilu ions o each pep ide (highes concen a ion es ed was 32
µ
M and lowes was 0.25
µ
M) we e
added o he bio ilms o 4 h. Un ea ed 24 h p e o med bio ilms we e used as a con ol. The me abolic
ac i i y o bio ilm-embedded cells was de e mined using a esazu in educ ion luo ome ic assay as
p e iously desc ibed [53].
4.9. Lipid Vesicle P epa a ion
Mix u es wi h syn he ic lipids (POPC, POPS, POPG, A an i Pola Lipids) o E. coli lipid ex ac
(A an i Pola Lipids, Alabas e . USA) we e ex uded in HEPES bu e (10 mM HEPES, 150 mM NaCl,
pH 7.4) o p oduce lipid esicles, as p e iously desc ibed [
43
,
79
]. LUVs (Ø
≤
100 nm) we e used in
luo escence spec oscopy assays and small unilamella esicles (SUVs, Ø ≤50 nm) o SPR.
4.10. Fluo escence Spec oscopy Assays
Fluo escence emission spec a (300–400 nm, exci a ion a 280 nm, sli s 3/3 mm) o 12.5
µ
M pep ide
and l-T p in HEPES bu e (in qua z cu e es, pa h leng h o 0.5 cm) we e scanned upon i a ion wi h
LUVs composed o a ious lipid composi ions (up o 3 mM POPC, and up o 1.5 mM POPC/POPS (4:1)
o POPC/POPG (4:1)) [
80
] using a Fluo oMax-4 spec o luo ome e (Ho iba, Kyo o, Japan). In eg a ed
a eas o he luo escence emission spec a we e co ec ed o luo opho e dilu ion and ligh dispe sion
due o i a ion wi h LUVs suspension; he blank was discoun ed.
In . J. Mol. Sci. 2019,20, 4184 18 o 25
4.11. Quenching o T yp ophan Fluo escence
The memb ane in-dep h loca ion o he T p esidue wi hin he pep ides was ollowed using
Ac ylamide (Sigma Ald ich, S . Luis, USA) and 5- and 16-DS (Sigma Ald ich, S . Luis, USA).
Quenching induced by ac ylamide was moni o ed wi h 12.5
µ
M pep ide in a HEPES bu e , in he
p esence o 1 mM o POPC, 0.1 mM POPC/POPS (4:1), 1 mM POPC/POPS (4:1), o 0.1 mM POPC/POPG
(4:1) i a ed wi h inc easing concen a ion o ac ylamide [
81
]. The luo escence emission spec a
we e de e mined wi h an exci a ion wa eleng h o 290 nm o educe he quenche / luo opho e ligh
abso p ion a io. The luo escence emission spec a a ea was co ec ed o he inne il e e ec [
82
]
due o inc eased abso bance o ac ylamide. Da a poin s we e analyzed using he S e n-Volme
ep esen a ion (Equa ion (1)), he KSV is de e mined om he slope.
I0
I=1+KSV[Q](1)
A o al o 12.5
µ
M o TI, o o cTI, in HEPES bu e and 1 mM POPC/POPS (4:1) we e i a ed wi h
inc easing concen a ion o 5DS, o o 16DS. The luo escence emission spec a a ea was co ec ed o
he inne il e e ec [
82
] due o inc eased abso bance o 5-,16DS. The e ec i e concen a ion o 5DS and
16DS in he lipid bilaye was calcula ed: pa i ion coe icien s o 5DS and 16DS in o luid memb anes
a e 89,000 and 9730, espec i ely [
59
,
81
]. The da a poin s had a nega i e de ia ion o linea i y and he
KSV was de e mined by i ing he da a wi h he Leh e equa ion (Equa ion (2)) [59,81].
I0
I=1+KSV[Q]
(1+KSV[Q])(1− B)+ B
(2)
I0= luo escen in ensi y in bu e
I= luo escen in ensi y in p esence o quenche
KSV =S e n-Volme cons an
[Q]=quenche concen a ion
B= ac ion o ligh accessible o he quenche =I0,B
I0
I0,B = luo escen in ensi y o he accessible popula ion o he quenche when [Q] =0
The a e age dis ance o he T p esidue om he bilaye cen e (Å) was de e mined using he
pa allax me hod [62] ollowing he equa ion,
z1F=1
−πCln F1
F2−L2
21
2L21 (3)
zcF =z1F+Lc1(4)
in which he z
1F
is he dis ance be ween he T p esidue and he quenche moie y in 5DS, L
21
is he
dis ance be ween he quenche g oups in 5 and 16DS (5 Å), F
1
is he luo escence o 12.5
µ
M o TI,
o o cTI, in he p esence o 1 mM POPC/POPS (4:1) and 0.4 mM 5DS, F
2
is he luo escence o 12.5
µ
M
o TI, o o cTI, in he p esence o 1 mM POPC/POPS (4:1) and 0.4 mM 16DS. C is he mola ac ion
o quenche wi hin he o al lipid concen a ion pe uni a ea (assuming he su ace o lipid o be
70 Å2) [62].
z
cF
is he dis ance be ween he T p esidue and he cen e o he bilaye , L
c1
is he dis ance
be ween he cen e o he bilaye s and he quenche g oup o 5DS (15 Å).
4.12. Su ace Plasmon Resonance (SPR)
SPR was used o in es iga e he a ini y and binding kine ics o pep ides o memb anes o di e en
composi ions. The expe imen s we e conduc ed wi h an L1 biosenso chip (GE Heal hca e) a 25
◦
C
using a BIAco e 3000 ins umen (GE Heal hca e, Chicago, USA) [
83
,
84
]. The HEPES bu e was used
o sample p epa a ion and as a unning bu e . Lipid bilaye s we e immobilized on o L1 chip by
In . J. Mol. Sci. 2019,20, 4184 19 o 25
injec ion o SUVs a a low a e o 2
µ
L/min. Pep ide samples wi h wo- old se ial dilu ions ( he highes
concen a ion es ed was 64
µ
M and he lowes was 1
µ
M) we e injec ed o e he lipid bilaye a a
low a e o 5
µ
L/min. Associa ion o he pep ides on o he lipid bilaye was ollowed o 180 s and
he dissocia ion om he lipid o 600 s. The BIAe al so wa e was used o analyze he senso g ams.
The esponse uni s (RU) we e no malized o he pep ide- o-lipid a io (P/L); he P/L ob ained a a ixed
ime poin a he end o he associa ion cu e (a 170 s), a which he esponse has eached a pla eau
and he binding is close o equilib ium, was used o compa e he a ini y o he pep ides o he di e en
lipid sys ems [84].
4.13. Vesicle Leakage Assay
LUVs (Ø
≤
100 nm) we e p epa ed wi h he HEPES bu e con aining 40 mM o he luo escen
5-ca boxy luo escein (CF, Sigma Ald ich, S . Luis, USA). LUVs illed wi h CF a sel -quenching
concen a ions we e sepa a ed om he non-encapsula ed dye on a Sephadex G-50 column equilib a ed
wi h HEPES bu e [
85
]. The concen a ion o CF-LUVs was de e mined h ough a calib a ion cu e
p epa ed om he o iginal lipid mix u e using S ewa ’s assay (abso bance a 485 nm) [
86
]. Pep ides
we e incuba ed (25
◦
C, 20 min) a wo- old dilu ions (s a ing a 10
µ
M) wi h LUVs (5
µ
M) in he
HEPES bu e in a 96-well la -bo om black op ipla es (Pe kin Elme , Wal ham, USA). Vesicles and
pep ides we e incuba ed o 20 min in da k and he elease o CF was measu ed in a Tecan in ini e
M1000P o mul ipla e eade using 490 nm as he exci a ion and 513 nm as emission wa eleng hs.
4.14. S a is ical Analysis
Values (mean o i
±
SEM) we e analyzed in G aphPad P ism 7 o es o signi ican di e ences
in he cy o oxic ac i i y o he pep ides. The mul iple - es unc ion wi h he Holm–Sidak me hod was
applied when indica ed. P alues below 0.05 we e conside ed o be signi ican .
Supplemen a y Ma e ials:
Supplemen a y ma e ials can be ound a h p://www.mdpi.com/1422-0067/20/17/
4184/s1.
Au ho Con ibu ions:
F.V. concep ualized his s udy wi h supe ision and inpu om S.T.H. and N.L. F.V., S.A.D.,
and P.J.H. conduc ed expe imen al wo k: F.V. olded and pu i ied he pep ides, conduc ed oxici y assays, model
memb anes, su ace plasmon esonance and luo escence spec oscopy assays; S.A.D. conduc ed he bio ilm assay
and helped F.V. wi h he an imic obial assays; P.J.H. pe o med NMR spec oscopy and s uc u al analysis o he
pep ides; Y.-H.H. es ablished he se um s abili y assay. F.V. analyzed and in e p e ed da a wi h assis ance om
S.T.H. and expe con ibu ions om all he co-au ho s. S.T.H., N.L., P.J.H., D.J.C., Y.-H.H. and A.S.V. e ised and
edi ed he manusc ip . S.T.H. and D.J.C. p o ided he esou ces and acqui ed he unding.
Funding:
This p ojec was unded by a Na ional Heal h Medical Resea ch Council (NHMRC) p ojec g an
(APP1084965). F.V. was suppo ed by he UQ Resea ch Schola ship, S.T.H. is an Aus alian Resea ch Council (ARC)
Fu u eFellow(FT150100398), D.J.C. isanARC Aus alianLau ea e Fellow(FL150100146). Ma ie Skłodowska-Cu ie
Resea ch and Inno a ion S a Exchange g an (RISE; call: H2020-MSCA-RISE-2014, g an ag eemen 644167)
unded secondmen s o S.A.D. and o A.S.V. o he Uni e si y o Queensland. The T ansla ional Resea ch Ins i u e
is suppo ed by a g an om he Aus alian Go e nmen .
Acknowledgmen s:
Many hanks o QUEDDI, he Ma Coope lab and he Helmu Schaide lab o he cell lines
HaCaT, HT144 and WM164 espec i ely. The au ho s hank Oli ie Chene al and Joachim Weidmann (IMB, UQ)
o hei assis ance wi h pep ide syn hesis.
Con lic s o In e es :
The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he
s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , o in he decision o
publish he esul s.
Abb e ia ions
1D One-dimensional
2-CTC 2-chlo o i yl esin
3D Th ee-dimensional
ACN Ace oni ile
ATI Ac i i y/ oxici y index
ATTC Ame ican Type Cul u e Collec ion
In . J. Mol. Sci. 2019,20, 4184 20 o 25
BMRB Biological magne ic esonance da a bank
CC Cy o oxic concen a ion
CF 5-ca boxy luo escein
cTI Cyclic Tachyplesin I
cTII Cyclic Tachyplesin II
cTIII Cyclic Tachyplesin III
cTI–cTIII Cyclic Tachyplesin I, II and III
DS Doxyl-s ea ic acid
E.COSY Exclusi e co ela ion spec a
ESI-MS Elec osp ay ioniza ion mass spec oscopy
FBS Fe al bo ine se um
Fmoc 9- luo enylme hoxyca bonyl
HC50 Hemoly ic concen a ion necessa y o 50% lysis o RBCs
HDP Hos de ense pep ide
KSV S e n – Volme cons an
LPS Lipopolysaccha ide
LUV La ge unilamella esicle
MCC Minimal cy o oxic concen a ion
MHC Minimal hemoly ic concen a ion
MIC Minimal inhibi o y concen a ion
NMR Nuclea magne ic esonance
NOESY Nuclea O e hause e ec spec oscopy
PBS Phospha e bu e ed saline
PDB P o ein da a bank
PC Phospha idylcholine
PS Phospha idylse ine
PG Phospha idylglyce ol
P/L Pep ide- o-lipid a io
POPC 1-palmi oyl-2-oleoyl-glyce o-3-phosphocholine
POPS 1-palmi oyl-2-oleoyl-sn-glyce o-3-phospho-L-se ine
POPG 1-palmi oyl-2-oleoyl-sn-glyce o-3-phospho-(10- ac-glyce ol)
RBCs Red blood cells
RP-HPLC Re e se-phase high-pe o mance liquid ch oma og aphy
RT Re en ion ime
RU Response uni
SEM S anda d e o o mean
SPPS Solid phase pep ide syn hesis
SPR Su ace plasmon esonance
SUV Small unilamella esicle
TI Tachyplesin I
TII Tachyplesin II
TIII Tachyplesin III
TI–TIII Tachyplesin I, II and III
TFA T i luo oace ic acid
TOCSY To al co ela ed spec oscopy
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