1

Cyclic analogues of horseshoe crab peptide tachyplesin I with anticancer and cell

penetrating properties

Felicitas Vernen1, David J. Craik1, Nicole Lawrence1*, Sónia Troeira Henriques1,2*

1Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072,

Australia 2School of Biomedical Sciences, Faculty of Health, Institute of Health & Biomedical Innovation,

Queensland University of Technology, Translational Research Institute, Brisbane, QLD, 4102,

Australia

*Correspondence to:

Dr. Sónia Troeira Henriques Tel: +61 7 3443 7342 E-mail: sonia.henriques@qut.edu.au

Dr. Nicole Lawrence Tel +61 7 3346 2014 E-mail: n.lawrence@uq.edu.au

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ABSTRACT

Tachyplesin-I (TI) is a host defense peptide from the horseshoe crab Tachypleus tridentatus that

has outstanding potential as an anticancer therapeutic lead. Backbone cyclized TI (cTI) has similar

anticancer properties to TI, but has higher stability and lower hemolytic activity. We designed and

synthesized cTI analogues to further improve anticancer potential and investigated structure-

activity relationships based on peptide-membrane interactions, cellular uptake and anticancer

activity. The membrane-binding affinity and cytotoxic activity of cTI were found to be highly

dependent on peptide hydrophobicity and charge. We describe two analogues with increased

selectivity toward melanoma cells and one analogue with ability to enter cells with high efficacy

and low toxicity. Overall, the structure-activity relationship study shows that cTI can be developed

as a membrane-active antimelanoma lead, or be employed as a cell penetrating peptide scaffold

that can target and enter cells without damaging their integrity.

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Despite advances in treatment, cancer remains a leading cause of death worldwide.1 Conventional

chemotherapy is effective against many cancers, but it has adverse effects, such as low specificity

for cancerous cells, and the development of drug-resistances.2, 3 Peptide-based anticancer drugs

have the potential to overcome these drawbacks: they have high target selectivity, low toxicity,

and reduced likelihood to induce resistance.4-8

Host defense peptides (HDPs) are a class of cationic and amphipathic peptides9, 10 that can

selectively target cancerous cells.5, 11-13 Their selectivity toward cancerous cells over host cells is

governed by differences in their cell surface properties;10, 14 the surface of cancerous cells is

negatively charged due to increased exposure of phospholipids with the anionic phosphatidylserine

(PS) headgroup on the cell surface.15-19 In contrast, the surface of healthy eukaryotic cells is rich

in neutral phospholipids14 and phospholipids with anionic PS-headgroups are restricted to the inner

leaflet of the cell membrane.20, 21 Positively-charged HDPs are attracted to the negatively-charged

surface of cancerous cells and insert into their membranes by establishing van-der-Waal’s forces

and hydrophobic interactions with the phospholipid bilayer.10, 22 After insertion into the cell

membrane, HDPs kill cancerous cells via membrane disruption,10 or cross membranes and enter

cells to act on intracellular targets.23, 24

Tachyplesin I (TI) is a cationic HDP isolated from hemocytes of the Japanese horseshoe crab25

with a high affinity for anionic lipid bilayers26 and selective activity against cancer cells.27-38 TI is

composed of 17 amino acid residues with a C-terminal -amidation and is classified as β-hairpin

HDP due to its three-dimensional structure containing an antiparallel β-sheet connected by a β-

turn and stabilized by two disulfide bonds. It has an amphipathic structure in which a large cluster

of hydrophobic side chains protrudes in one side of the β-sheet plane, and side chains from

positively charged amino acid residues locate at the opposite side (Figure 1A-D). 25, 39

TI has been reported to cause cancer cell death by membrane disruption or apoptosis in a dose-

dependent manner34 and has been used as a carrier to deliver a cargo into cancer and plant cells.40

We previously demonstrated that backbone cyclized TI (cTI) has improved stability and hemolytic

properties compared to TI;38 in the current study we were interested in further characterizing cTI

to improve activity and selectivity for cancerous cells. To achieve this goal, we synthesized a set

of cTI analogues (Figure 1C) with different properties (e.g. overall charge and hydrophobicity)

and examined toxicity, ability to bind lipid membranes, selectivity for melanoma cells and ability

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to internalize inside cancerous cells. We describe two cTI analogues with high selectivity for

melanoma cells and one analogue with low cytotoxic and high efficacy to internalize inside

cancerous cells. These results demonstrate the potential of tachyplesin peptides as antimelanoma

leads, and also as a drug delivery scaffold to reach intracellular cancer pathways.

RESULTS AND DISCUSSION

Design, synthesis and characterization of cTI analogues. We designed a set of cTI analogues

to examine the mechanism-of-action and identify peptide features that are important for anticancer

properties. We were especially interested in examining the importance of hydrophobic and charged

residues, and have targeted these residues for substitution with Ser, which has an uncharged polar

side chain and is expected to facilitate peptide solubility. The cTIs were categorized and color-

coded to simplify their distinction: red – peptides with a sequence similar to that of the parent TI;

green – peptides with reduced overall hydrophobicity; blue – peptides with reduced charge and

increased overall hydrophobicity; and purple – peptides with increased charge and/or

hydrophobicity (Figure 1C).

We recently described the characteristics and anticancer activity of TI and cTI,38 and included

them in the current study to allow direct comparison with cTI analogues. [R/K]cTI was produced

to examine the effect of replacing Arg with Lys. Although Arg and Lys are both positively charged,

they differ in their interactions with phospholipid headgroups; the Arg side chain can be involved

in hydrogen bonds with two phospholipid headgroups, whereas the Lys side chain can only form

one hydrogen bond. Compared to Lys, Arg side chains can establish stronger cation-π-interactions

with aromatic residues, preferentially Trp residues, which facilitate peptide self-association within

the membrane and deeper membrane insertion through shielding of positively charged side

chains.41-44 It was expected that comparison between cTI and [R/K]cTI would reveal whether

interactions between Arg side chains and phospholipid headgroups are important for peptide

activity and/or selectivity toward cancerous cells (Figure 1C, group I).

Hydrophobic residues are likely to modulate peptide-lipid binding affinity, insertion into lipid

bilayers and preference for certain lipid compositions.45, 46 These residues facilitate insertion of

positively-charged peptides into negatively-charged membranes, but if too hydrophobic, peptides

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often insert into neutral membranes found in healthy mammalian cells, and induce toxicity.47-49

Thus, reducing the hydrophobicity of peptides can decrease toxicity toward mammalian cells. For

instance, the mutations Y8S or I11S in the β-turn of TI resulted in improved selectivity for bacterial

cells and decreased cytotoxicity against mammalian cells.50 Similarly, TI analogues with the

mutations F4A or I11A had increased selectivity for pathogens and reduced toxicity against red

blood cells.26 The hydrophobic residues Trp2 and Phe4 were shown to be essential for the

interaction of TI with the acyl chains of lipopolysaccharides (LPS) and are important for

antimicrobial activity.51 Also relevant, the two Tyr residues (Tyr8, Tyr13) adjacent to TI disulfide

bonds and part of the β-turn region or antiparallel β-sheet are highly conserved in β-hairpin

peptides.46 Based on these earlier observations, we synthesized three cTI analogues with reduced

hydrophobicity: [W2S]cTI, the Trp residue is known to be involved in membrane partitioning and

it is located in the loop generated by backbone cyclisation;38 [F4S-Y13S]cTI, Phe4 and Tyr13 are

located on opposite strands of the antiparallel β-sheet and also likely to be involved in peptide-

lipid interactions; and [Y8S-I11S]cTI, an analogue with mutations in the β-turn region that could

have high selectivity for negatively-charged membranes and low cytotoxicity against mammalian

cells based on a previous study50 (Figure 1C, group II).

Individual positively-charged residues might impact selectivity of peptides for anionic over

zwitterionic membranes. The charge of the cTI analogues was reduced by substituting Arg residues

9, 14 and 17, with Ser to produce [R9S]cTI, [R14S]cTI, [R17S]cTI (Figure 1C, group III), and

[R9S-R14S-R17S]cTI (Figure 1C, group IV). Arg9 and Arg17 are located at opposite turns and

shield the hydrophobic face from the aqueous solution, whereas Arg14 is located in the β-sheet,

facing away from the hydrophobic patch (see Figure 1B). These substitutions decreased the overall

charge and increased the overall hydrophobicity - the triple mutant [R9S-R14S-R17S]cTI was the

analogue with the highest hydrophobicity in the series (see Figure 1C, peptide charge and RT). It

has previously been proposed that a balance in charge and distribution of hydrophobic residues is

important for membrane penetration and selectivity of peptides.52

The analogue [G18K]cTI was designed to introduce an additional charge in the loop generated

with the backbone cyclization (see Figure 1A,B), whereas the [I11F-G18K]cTI had an additional

charge and a more hydrophobic residue introduced in the β-turn region (Figure1A, group IV).

[I11F-G18K]cTI has sequence similarity to polyphemusin I (RRWCFRVCYRGFCYRKCRx),

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another HDP expressed in horseshoe crab.46 [G18K]cTI, [I11F-G18K]cTI, cTI and [R/K]cTI have

similar overall hydrophobicity.

All peptides described were synthesized using solid-phase peptide synthesis and purified to >95%,

as confirmed by analytical RP-HPLC. Correct folding was confirmed by the expected masses in

ESI-MS (Supplementary Table S1) and by the high similarity of the secondary αH chemical shift

compared to the parent TI (Figure 1D) as determined with two-dimensional NMR spectroscopy.

Retention times (RT) obtained for each analogue in RP-HPLC were compared as an indication of

their overall hydrophobicity (i.e. longer RT indicated higher overall hydrophobicity; see Figure

1C). Stability studies showed that cyclic analogues [F4S-Y13S]cTI and [Y8S-Y13S]cTI were less

resistant to degradation by serum proteases than cTI, but still had improved stability compared to

(non-cyclic) TI38 (supplementary Figure S1).

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Figure 1. Tachyplesin I (TI) and cyclic analogues. (A) Illustration of cTI showing the location of antiparallel beta-

sheet (grey) and disulfide bonds (yellow). (B) Solution structure of cTI (PDB: 6PIN)38 showing the segregation of

hydrophobic and charged amino acids. (C) Primary amino acid sequence; charge at physiological pH; retention time

(RT, min) obtained from a 2% per minute gradient of 90% acetonitrile, 0.1% TFA on RP-HPLC, as a measure of

overall hydrophobicity. Peptides are color coded as follows: group I similar to original sequence (red/orange): group II reduced hydrophobicity (green); group III reduced charge (blue); and group IV increased charge and/or

hydrophobicity (purple/pink). TI has C-terminal amidation (x). Cyclic TI (cTI) analogues are backbone cyclized

between the first and last amino acid. The disulfide bonds between Cys3 and Cys16, and between Cys7 and Cys12 are

in yellow. (D) NMR αH secondary chemical shifts indicate overall structural similarity between the parent TI peptide

and cTI analogues. The location of the β-sheet (i.e. three or more residues with an α-proton secondary chemical shift

larger than 0.1 ppm) are represented by grey arrows.

cTI analogues have high affinity for anionic model membranes. We recently showed that

parent and cyclic tachyplesin I–III peptides exhibit activity against cancer cells and can selectively

bind and insert into lipid membranes that mimic the negatively-charged surface of cancer cells.38

In the current study, we examined the ability of the cTI analogues to bind lipid bilayers using

surface plasmon resonance. Model membranes composed of the zwitterionic phospholipid 1-

palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which forms stable bilayers in a liquid-

disordered phase at 25C, were used to represent the neutral outer membrane of healthy eukaryotic

cells, whereas model membranes with a mixture of POPC and negatively charged 1-palmitoyl-2-

oleoyl-sn-glycero-3-phospho-L-serine (POPS), POPC/POPS (4:1 molar ratio), were used to

represent the negative cell surface charge of cancerous cells (Figure 2). Phospholipids containing

phosphatidylserine (PS)-headgroups or neutral zwitterionic phosphatidylethanolamine (PE)-

headgroups are located in the inner leaflet of healthy eukaryotic cell membranes and are co-

regulated under the same transporters. With the loss of the asymmetric distribution in cancerous

cells, both classes of phospholipids become exposed in the outer membrane leaflet.53, 54 Many

cancer cells, and particularly those in aggressive tumors,55, 56 have also been reported to possess

increased cell membrane rigidity, owing to larger proportions of cholesterol (Chol) and saturated

fatty acids. Chol and sphingomyelin (SM) can segregate and form domains with increased rigidity

in cell membranes, often referred to as raft-like domains with liquid-ordered phase.57-59 Thus,

binding affinities of the peptides to model membranes representing neutral (POPC), neutral with

PE-phospholipids exposed (POPC/POPE (4:1)), negatively charged with PS-phospholipids and/or

with PE-phospholipids exposed (POPC/POPS (4:1), POPC/POPS/POPE (3:1:1)), and model

membranes mimicking raft-like domains (POPC/Chol/SM (2.7:4:3.3))60, 61 were compared.

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Figure 2. Membrane-binding

affinity of TI and its cyclic

analogues. Model membranes

composed of POPC, POPC/POPS (4:1), POPC/POPS/POPE (3:1:1),

POPC/POPE (4:1) and

POPC/Chol/SM (2.7:4:3.3) were

compared. (A) SPR sensorgrams

obtained with 32 µM peptide

injected over lipid bilayers

deposited onto an L1 chip. Peptide

samples were injected for 180 s

(association) and their dissociation

was followed for 600 s. In general,

peptide-membrane association

/dissociation reached equilibrium, as shown by a plateau at the end of

association phase. The response

units (RU) were converted into

peptide-to-lipid ratio (P/L

(mol/mol)) to compare the binding

to the different lipid mixtures by

calculating the amount of peptide

bound to the amount of lipid

deposited onto the L1 chip surface

(1 RU equals 1 pg/mm2 of peptide

or lipid).62, 63 (B) Dose-response curves in which P/L obtained with

peptide-membrane binding at

equilibrium (i.e. at the end of the

association phase and using t = 170

s as a reporting point) was plotted

as a function of the peptide sample

concentration (ranging from 1-64

µM).

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Table 1. Parameters of peptide-membrane binding affinity and kinetics for model membranes composed of

POPC (PC) and POPC/POPS (PC/PS).

Peptide P/Lmax (mol/mol) a P/Loff (mol/mol) b koff (x 10-2 s-1) b KD (µM) a

PC PC/PS (4:1) PC PC/PS (4:1) PC PC/PS (4:1) PC PC/PS (4:1)

TI 0.22 ± 0.03 0.37 ± 0.04 0.05 ± 0.01 0.10 ± 0.01 1.50 ± 0.11 2.75 ± 0.22 16.21 ± 4.61 11.81 ± 2.44

cTI 0.28 ± 0.04 0.42 ± 0.07 0.07 ± 0.01 0.14 ± 0.01 0.91 ± 0.03 0.70 ± 0.03 12.21 ± 3.45 8.16 ± 2.67

[R/K]cTI 0.24 ± 0.03 0.36 ± 0.01 0.08 ± 0.01 0.13 ± 0.01 0.50 ± 0.05 0.28 ± 0.01 14.95 ± 2.87 9.40 ± 0.35

[W2S]cTI 0.11 ± 0.05 0.24 ± 0.02 0.01 ± 0.01 0.03 ± 0.01 1.52 ± 0.08 1.47 ± 0.04 N/A 29.80 ± 3.55

[F4S-Y13S]cTI 0.04 ± 0.01 0.09 ± 0.02 0.01 ± 0.01 0.01 ± 0.01 11.42 ± 0.39 7.05 ± 0.23 N/A 8.89 ± 3.58

[Y8S-I11S]cTI 0.09 ± 0.05 0.08 ± 0.02 0.01 ± 0.01 0.01 ± 0.01 1.59 ± 0.07 2.24 ± 0.05 N/A 41.07 ±16.97

[R14S]cTI 0.45 ± 0.04 0.56 ± 0.04 0.14 ± 0.01 0.26 ± 0.01 0.44 ± 0.01 0.33 ± 0.01 14.04 ± 2.56 7.05 ± 1.13

[R17S]cTI 0.44 ± 0.03 0.56 ± 0.02 0.10 ± 0.01 0.17 ± 0.01 0.67 ± 0.01 0.39 ± 0.01 7.50 ± 1.25 8.03 ± 0.40

[R9S-R14S-R17S] 0.42 ± 0.02 0.73 ± 0.10 0.30 ± 0.01 0.31 ± 0.01 0.42 ± 0.01 0.50 ± 0.09 9.31 ± 0.87 13.59 ± 3.04

[G18K]cTI 0.13 ± 0.01 0.25 ± 0.02 0.05 ± 0.01 0.12 ± 0.01 1.00 ± 0.02 1.35 ± 0.03 7.54 ± 1.11 5.27 ± 0.85

[I11F-G18K]cTI 0.14 ± 0.01 0.35 ± 0.03 0.04 ± 0.01 0.16 ± 0.01 1.18 ± 0.03 1.48 ± 0.03 4.09 ± 0.58 6.44 ± 1.12

a P/Lmax and KD were calculated by fitting the dose-response curves (see Figure 2B) with one-site specific binding

with Hill slope equation (GraphPad Prism). The P/Lmax value represents the maximum binding response of peptide-

to-lipid ratio and was used to compare the overall affinity of analogues for tested lipid systems, KD is the peptide

concentration required to reach half of the maximum binding response, and provides further information on how

analogues compare within a given lipid system.b Peptide remaining bound to the lipid bilayer at the end of the

dissociation phase, P/Loff; and the dissociation rate, koff provide information on how tightly the peptide binds to model

membranes and were determined by fitting sensorgrams obtained with 32 M peptide (see Figure 2A) assuming a

Langmuir kinetic (GraphPad Prism).

All cTI analogues bound with higher affinity for negatively-charged, compared to zwitterionic

membranes (Figure 2A-B, Table 1, and Table S2), as shown with sensorgrams obtained at 32 M,

dose-response curves, and quantified by the binding saturation (i.e. maximum peptide-to-lipid

ratio; P/Lmax).64 The relative membrane-binding affinity of the cTI analogues for tested lipid

compositions in liquid disordered phase was compared with P/Lmax values and followed the order:

POPC/POPS > POPC/POPS/POPE > POPC/POPE ~ POPC. Thus, it is likely that electrostatic

attractions between peptides and anionic PS-phospholipids governed the increased binding affinity

to negatively-charged membranes. cTI analogues showed similar or lower overall membrane-

binding affinity for membranes containing POPE, suggesting no preferential binding to

membranes containing PE-phospholipids. Furthermore, all tested cTI analogues showed a reduced

affinity for membranes composed of POPC/Chol/SM (2.7:4:3.3) compared to POPC membranes

(see Figure 2 and P/Lmax values in Table 1 and Table S2), suggesting that cTI is unlikely to

insert/bind to more rigid domains within cell membranes.14, 57, 65

Comparison of the binding affinity between cTI analogues and model membranes (see P/Lmax

values in Table 1 and Table S2) showed that analogues with high hydrophobicity ([R9S]cTI,

[R14S]cTI, [R17S]cTI, [R9S-R14S-R17S]cTI) had higher overall membrane-binding affinity than

analogues with medium hydrophobicity (TI, cTI, [R/K]cTI, [G18K]cTI and [I11F-G18K]cTI).

Peptides with low hydrophobicity ([F4S-Y13S]cTI, [Y8S-I11S]cTI and [W2S]cTI) had weak

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affinity for all the tested membranes. Thus, the overall hydrophobicity of cTI is important for its

ability to bind lipid membranes of any composition (see Figure 2A,B). The effect of

hydrophobicity on the membrane-binding affinity of cTI analogues on neutral POPC or negatively-

charged POPC/POPS (4:1) bilayers was evident when comparing the binding saturation P/Lmax

and peptide remaining bound to the lipid at the end of the dissociation phase P/Loff. The presence

of anionic PS-headgroups increased the amount of peptide bound (P/Lmax and P/Loff) to

POPC/POPS (4:1) by approximately two, when compared to POPC, with the exception of peptides

of very low hydrophobicity ([F4S-Y13S]cTI and [Y8S-I11S]cTI) and very high hydrophobicity

([R9S-R14S-R17S]cTI). These observations suggest the existence of hydrophobicity thresholds

(as previously reported for other amphipathic peptides) where the first hydrophobicity threshold is

required to enable binding and insertion into negatively-charged membranes, and beyond a second

threshold binding and insertion into neutral membranes occurs.48

Kinetic parameters provided further information and helped distinguish peptide-membrane

interactions between cTI analogues with high affinity for lipid membranes. Membrane dissociation

rates (koff, Table 1) determined by fitting the sensorgrams obtained with 32 M peptide (see Figure

2A) revealed that cTI analogues dissociate slower from POPC/POPS (4:1), than from POPC

membranes. [R/K]cTI, [R14S]cTI and [R9S-R14S-R17S]cTI were the cTI analogues with slowest

dissociation rates from both POPC and POPC/POPS (4:1) bilayers, suggesting that these analogues

established stronger peptide-membrane interactions due to the higher hydrophobicity of

[R14S]cTI, [R17S]cTI and [R9S-R14S-R17S]cTI, or the presence of Lys side chains in [R/K]cTI.

In addition to overall hydrophobicity, the side chains of single residues might also influence the

binding and lipid selectivity of individual analogues. For instance the analogues [R17S]cTI,

[G18K]cTI and [I11F-G18K]cTI reached P/Lmax with POPC membranes at lower peptide

concentrations than TI or other analogues, as quantified with KD, suggesting that these analogues

bind strongly to neutral membranes and are less selective for negatively-charged membranes when

compared at low concentrations.

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Disruption of model membranes induced by cTI analogues. The ability of the peptides to

disrupt lipid bilayers was examined by the release of the fluorescent dye 5-carboxyfluorescein

(CF) from large lamellar vesicles (LUVs) composed with POPC or POPC/POPS (4:1) (Figure 3A).

The analogues were compared according to the maximum vesicle leakage (LCmax) observed and

the concentration required to achieve 50% of maximum leakage (LC50; Figure 3B). As expected,

cTI and analogues disrupted vesicles composed of POPC/POPS (4:1) more efficiently than those

composed of POPC. The membranolytic peptide melittin was included as positive control for

leakage in both lipid systems. Complete release of CF from POPC and POPC/POPS (4:1) vesicles

was achieved by melittin but not by TI or cTI analogues. Leakage from POPC/POPS (4:1) vesicles

induced by TI, cTI and analogues reached a plateau below 100%, which may be explained by

vesicle fusion.66

Figure 3. Lipid vesicle disruption. (A) LUVs with 5 µM lipid composed of POPC or of POPC/POPS (4:1) and loaded

with CF were incubated with up to 10 µM of TI, cTI,38 or analogues. Melittin was included as positive control. Leakage

was determined by following the fluorescence emission signal of CF released from the vesicles (to better distinguish

analogues on their leakage efficacy towards POPC/POPS (4:1) LUVs at low peptide concentrations see supplementary

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Figure S2). (B) Maximum percentage of leakage observed (LCmax) and peptide concentration necessary to achieve

half of the maximum leakage (LC50) fitted with one-site specific binding with Hill slope equation (GraphPad Prism).

A steady-state was not achieved with LUVs of POPC in the concentration range tested, maximum leakage was

assumed to be 100% and used to calculate LC50. Owing to the weak membrane-binding affinity of analogues with

reduced hydrophobicity some of the parameters have large error associated or not possible to fit (see green curves panel A), and are indicated with N/A. Data obtained with [R17S]cTI and POPC/POPS LUVs do not reach saturation

and fitted parameters have a large error associated; nevertheless, values were included for comparison with [R14S]cTI.

In agreement with membrane binding experiments (Figure 2), the cTI analogues with low

hydrophobicity and low membrane binding affinity, [W2S]cTI, [F4S-Y13S]cTI and [Y8S-

I11S]cTI, were the least membrane disruptive of the series (Figure 3). Among these analogues

[W2S]cTI had the highest efficacy for disrupting POPC/POPS (4:1) membranes and reached

higher LCmax than cTI. Furthermore, the highly hydrophobic [R9S-R14S-R17S]cTI disrupted

LUVs composed of POPC and of POPC/POPS (4:1) with similar efficacy as shown by similar

dose-response curves and LCmax around 80% membrane disruption. This loss of selectivity for

negatively-charged membranes is likely due to the higher hydrophobicity, lower overall charge,

high membrane binding of [R9S-R14S-R17S]cTI compared to cTI (see koff and P/Loff in Table 1

and Figure 2).

In contrast, correlation between membrane binding affinity and membrane disruption efficacy was

not observed across all the analogues. For example, cTI had a higher binding affinity than TI to

POPC and POPC/POPS (4:1) bilayers, but TI disrupted vesicles of both lipid compositions more

efficiently than cTI. Additionally, TI and [R/K]cTI with medium hydrophobicity and membrane

binding affinity, disrupted LUVs composed of POPC/POPS (4:1) more efficaciously than the more

hydrophobic [R14S]cTI and [R17S]cTI with higher membrane binding affinity. Although

[R14S]cTI and [R17S]cTI have similar hydrophobicity and binding affinities, their membrane-

disruption against POPC/POPS (4:1) vesicles was considerably different. Both reached high

LCmax, but [R17S]cTI caused a gradual release of dye, whereas [R14S]cTI disrupted the vesicles

at lower concentrations, as seen by comparing the LC50 values.

TI, cTI, [R/K]cTI, [W2S]cTI, [Y8S-I11S]cTI were further compared for their ability to induce

leakage from LUVs composed of POPC/POPE (4:1) and POPC/POPS/POPE (3:1:1)

(supplementary Figure S3, Table S3). TI was the only analogue that showed disruption of LUVs

composed of POPC/POPE (4:1), with an LCmax similar to that obtained with POPC. The ability of

TI, but not cTI and [R/K]cTI, to disrupt LUVs composed of POPC/POPE could suggest an

alteration in the membrane-disruption properties of cyclized peptides due to alterations in peptide

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orientation within the membrane, similar to what has been described for TI and cTI on POPC and

POPC/POPS (4:1) lipid systems.38 All peptides except [Y8S-I11S]cTI induced leakage in vesicles

composed of POPC/POPS/POPE (3:1:1). A lower percentage of vesicles have their membrane

disrupted when incubated with [W2S]cTI than with TI, cTI and [R/K]cTI. The disruption of

POPC/POPS/POPE (3:1:1) vesicles supports the observation that PE-phospholipids do not

facilitate nor obstruct the membrane-binding or disruptive properties of cTI analogues, and

highlights the importance of the electrostatic interactions between the positively-charged peptide

and negatively-charged phospholipids, and the requirement of hydrophobic amino acids for

membrane insertion and/or disruption.

Together these results demonstrate that TI and cTI analogues bind and disrupt negatively-charged

model membranes with higher efficacy than neutral membranes, and in general membrane-binding

affinity correlated with ability to disrupt membranes (see Figure S4). However, disruption of

model membranes and peptide-lipid binding affinity did not correlate directly across all the

analogues, for instance: [R/K]cTI had lower affinity to bind POPC/POPS membranes, but

disrupted these membranes with higher efficacy than cTI; [G18K]cTI and [W2S]cTI bind with

identical affinity to POPC/POPS membranes, but [W2S]cTI disrupted a larger percentage of these

membranes when compared at higher concentrations (see Figure 3 and Figure S4). The leakage

propensity was likely governed by the distribution of the hydrophobic residues, the overall

hydrophobicity and specific location and type of cationic residues. These modifications modulate

the membrane-association/-dissociation rates and in-depth location of the peptides within the lipid

bilayer. For instance, [R14S]cTI and [R17S]cTI, with identical overall charge and hydrophobicity

and differing only on the location of the mutation, had identical overall membrane binding affinity

(i.e. KD and P/Lmax), but more [R14S]cTI remains associated with the membrane (see P/Loff values

in Table 1) which resulted in higher membrane-leakage efficacy (see LC50 values in Figure 3).

Insertion of cTI analogues into lipid membranes. The fluorescence emission properties of Trp

residues vary depending on the local environment. Thus, the Trp fluorescence properties of cTI

analogues were followed in the absence and presence of lipid vesicles to investigate the location

of Trp2 when bound to lipid membranes. A selection of cTI analogues with distinct

hydrophobicity, charge and/or membrane-binding affinities was compared to TI and cTI;38 (i.e.,

[F4S-Y13S]cTI, with weak affinity for membranes; [R14S]cTI, with high hydrophobicity and

14

affinity for membranes; and [I11F-G18K]cTI, more positively-charged and with high affinity and

ability to disrupt lipid membranes). In aqueous solution, the Trp fluorescence emission spectrum

of all the tested analogues possess a maximum at 350 nm (λex = 280 nm) identical to that of L-Trp

amino acid, which indicates Trp2 is exposed and accessible in the solvent (Figure 3A).

To determine whether the Trp2 inserts into lipid bilayers, fluorescence spectra of the selected

analogues was monitored upon titration with vesicles composed of zwitterionic POPC, or of

negatively-charged POPC/POPS (4:1). A shift in the fluorescence emission spectra to a more

energetic wavelength (blue shift) indicates that Trp2 was located in a more hydrophobic

environment and inserted into lipid membranes.67, 68 When titrated with vesicles composed of

POPC (Figure 3A), [R14S]cTI was the only analogue that displayed a blue shift in fluorescence

emission, suggesting that when bound to POPC membranes the analogue [R14S]cTI adopted an

orientation in which the Trp2 inserted into the core of the lipid bilayer, whereas the other analogues

adopted an orientation that exposed the Trp2 residue to the aqueous environment.

The Trp fluorescence emission spectra of all the tested analogues displayed a blue shift upon

titration with vesicles of POPC/POPS (4:1), as shown with the variation in emission maximum

(Figure 4A), and exemplified with spectra obtained with cTI and [R14S]cTI in the absence and

presence of 1 mM POPC or POPC/POPS (4:1) vesicles (Figure 4B). The lowest blue shift was

observed for [F4S-Y13S]cTI, suggesting that the Trp2 in this analogue remained exposed to the

aqueous environment in the presence of 1 mM vesicles of POPC/POPS. This is expected

considering its weak affinity to bind to membranes. cTI, [R14S]cTI and [I11F-G18K]cTI had a

large and similar variation on the fluorescence emission maximum (shift between 27-29 nm) when

titrated with POPC/POPS vesicles and had a slightly larger blue shift than TI (23 nm) (Figure 4A).

These results indicate that changes to charged and hydrophobic residues can alter the orientation

and ability of cTI peptides to insert into lipid bilayers, likely due to changes in amphipathic

character and charge/hydrophobicity distribution within the peptide.

The location of the Trp2 residue when the cTI analogues are bound to lipid bilayers was further

investigated using differential quenching with three fluorescence quenchers: acrylamide, an

aqueous quencher that does not insert into lipid bilayers; and the lipophilic 5- and 16-doxyl stearic

acids (5DS, 16DS), which insert into the lipid bilayer and quench fluorophores located close to the

membrane interface or within the core of the bilayer, respectively.69, 70 The quenching experiments

15

were conducted by incubating 12.5 µM peptide with 1 mM lipid, as the fluorescence emission of

all the peptides had reached a maximum shift under these conditions. Data were fitted with a Stern-

Volmer plot (Figure S5) and the Stern-Volmer constant (KSV; Table 2) was calculated to compare

the quenching efficacy.71, 72

All analogues, except the membrane-inactive [F4S-Y13S]cTI, were quenched with higher efficacy

by acrylamide in aqueous solution (see Figure S5 and fitted KSV values in Table 2) than when

incubated with POPC/POPS membranes, suggesting that Trp2 inserts into these bilayers. In the

presence of POPC all the tested analogues had KSV values identical to those recorded in aqueous

solution. Only the most hydrophobic analogue [R14S]cTI had a lower KSV in the presence of POPC

compared to aqueous solution, confirming that the Trp2 residue of [R14S]cTI inserted into POPC

membranes (see Figure 4B). Overall, the acrylamide quenching results agreed with the Trp

fluorescence emission spectra shifts observed (see Figure 4).

The Trp fluorescence emission of TI and cTI analogues was more efficiently quenched by 16DS

than by 5DS, suggesting that all peptides partition deeply into POPC/POPS (4:1) lipid bilayers.

Comparison between the analogues revealed that cTI, [R14S]cTI and [G18K-I11F]cTI had slightly

higher KSV and fb (i.e. fraction of peptide fluorescence emission accessible to quencher) values for

16DS than TI, indicating deeper insertion and/or a slightly different orientation of the cyclic

analogues within the membrane (Table 2), in agreement with our previous study.38 In regards to

depth of membrane insertion, no major differences could be observed between the individual

cyclic analogues cTI, [R14S]cTI and [I11F-G18K]cTI.

16

Figure 4. Partitioning of TI and cTI analogues into POPC or POPC/POPS (4:1) lipid bilayers, as followed by

Trp fluorescence emission. (A) Trp fluorescence emission spectra maximum (λex = 280 nm) upon titration with

POPC or POPC/POPS (4:1) LUVs. L-Trp amino acid was included as a negative control for membrane partitioning.

(B) Normalized fluorescence emission spectra of 12.5 µM cTI 38 and [R14S]cTI in aqueous solution, with 1 mM

POPC or POPC/POPS (4:1) LUVs.

Table 2. Quenching of Trp fluorescence emission of TI, cTI and analogues by acrylamide, and 5DS and 16DS.

KSV (M-1) a fbb

Peptide Acrylamidea 5DS 16DS 5DS 16DS

Aq. solution POPC POPC/POPS (4:1) POPC/POPS (4:1)

TI 12.8 ± 0.7 12.5 ± 0.5 2.1 ± 0.4 3.7 ± 1.3 19.6 ± 4.3 0.63 ± 0.57 0.74 ± 0.37

cTI 16.4 ± 0.4 17.1 ± 0.3 3.7 ± 0.5 4.9 ± 1.8 26.3 ± 7.1 0.61 ± 0.54 0.83 ± 0.52

[F4S-Y13S]cTI 9.6 ± 0.5 11.1 ± 0.3

[R14S]cTI 21.6 ± 0.6 5.0 ± 0.4 2.8 ± 0.3 4.7 ± 1.5 18.7 ± 2.2 0.63 ± 0.47 0.81 ± 0.22

[I11F-G18K]cTI 12.6 ± 0.7 4.5 ± 0.7 5.0 ± 1.7 24.4 ± 6.9 0.64 ± 0.51 0.80 ± 0.52

aThe quenching efficacy is compared with fitted Stern-Volmer constants (KSV) by following the Trp fluorescence

emission of 12.5 µM peptide in the absence (KSV, aqueous solution) and in the presence of 1 mM LUVs (KSV, POPC

or POPC/POPS (4:1)) followed upon titration with acrylamide (λex = 290 nm), 5- or 16DS (KSV, POPC/POPS (4:1),

λex = 280 nm). KSV was determined by fitting the Stern-Volmer equation, or the Lehrer equation38 when data showed

a downward deviation to linearity. b fb is the fraction of light emitted by the peptide accessible to the quencher and is

obtained from fitting Lehrer equation. Graphs with the fitted data are shown in Figure S5.

17

Anticancer activity and selectivity of cTI analogues. TI and cTI analogues were tested against

a panel of cancerous and non-cancerous cells to investigate their anticancer properties using a

resazurin-based cytotoxicity assay.44, 64 Cancer cell lines included the melanoma cell lines

MM96L, HT144, WM164 and MDA-MB-435S, the cervical cancer cell line HeLa, the breast

cancer cell line MCF-7 and the myelogenous leukemia cell line K562. The immortalized aneuploid

keratinocyte cell line HaCaT and human red blood cells (RBCs) collected from healthy donors

were included as non-cancerous controls. The analogues were compared on their cytotoxicity

against cells by determining the concentration required to achieve 50% cell death (CC50) from

dose-response curves (Table 3). The selectivity of the peptides for cancerous cells was assessed

through the activity/toxicity index (ATI), which was calculated as the quotient of the minimal

hemolytic concentration required for less than 10% hemolysis of RBCs and the median of

cytotoxic concentrations required for 50% cell death.26 Larger ATI values indicate higher

selectivity of the peptide for cancer cells.

TI and cTI have selective activity at low micromolar concentrations against MM96L, HT144 and

WM164, compared to HeLa, HaCaT, and RBCs.38 By expanding the panel of cancerous cell lines

to include MDA-MB-435S (melanoma), K562 (chronic myeloid leukemia) and MCF-7 (breast

cancer) (Table 3), we found that the melanoma cell lines were the most sensitive to treatment with

TI and cTI analogues. However, MDA-MB-435S cells were less susceptible to the peptides than

the other tested melanoma cell lines; K562 cells were as susceptible as MM96L, HT144 and

WM164; and MCF-7 cells were mildly susceptible to cTI and analogues with similar CC50 values

to HeLa and HaCaT. The K562 and melanoma cells were also more susceptible than HeLa and

MCF-7 cells to treatment with gomesin (another -hairpin HDP) and its analogues.73 Furthermore,

the K562 cells were shown to be more susceptible to TI and to other β-hairpin peptides compared

to the robust leukemia cell line KG-1 in another study.27 Similar susceptibility trends, suggest that

cell membranes of K562 and of tested melanoma cells have features, not yet defined, that make

them highly susceptible to cationic membrane-active β-hairpin peptides.

Backbone cyclization reduced the hemolytic activity of cTI compared to TI.38 In the current study

we found that cTI analogues were similarly non-hemolytic at concentrations below 32 µM (Table

3 and supplementary Figure S6), with the exception of [R9S-R14S-R17S]cTI and [I11F-

G18K]cTI, which are more hemolytic than TI and the parent cTI. The increased hemolytic activity

18

of [R9S-R14S-R17S]cTI is likely due to its high hydrophobicity and ability to interact with and

lyse neutral membranes (see Figure 3). [I11F-G18K]cTI did not show increased disruption of

POPC membranes, but it displayed high affinity to bind POPC and POPC/POPS model membranes

at low peptide concentrations, as shown by dose-response curves obtained with SPR (see Figure

2). This analogue has increased local hydrophobicity, due to the substitution of Ile with a Phe, and

an extra positive charge next to the Trp2 residue, due to the substitution of Gly18 with a Lys. These

mutations are likely to increase local concentration and insertion of the peptide into the membrane

of RBCs, followed by membrane disruption and lysis.

Table 3. Cytotoxicity of tachyplesin I and its cyclic analogues against cultured cancer cell lines, an epithelial

control cell line and human RBCs.

CC50 (µM) a Melanoma

ATI c Peptide MM96L HT144 WM164 MDA-MB-435S K562 MCF-7 HeLa HaCaT RBCs b

TI 1.5 ± 0.1 1.7 ± 0.2 2.5 ± 0.1 8.0 ± 0.6 1.7 ± 0.1 16.0 ± 2.0 13.1 ± 1.2 16.0 ± 1.5 34.9 ± 2.8 2.0

cTI 1.3 ± 0.1 1.4 ± 0.1 2.7 ± 0.1 2.7 ± 0.3 2.0 ± 0.1 6.4 ± 0.6 6.7 ± 0.6 7.9 ± 0.5 107 ± 21 1.1

[R/K]cTI 4.3 ± 0.3 3.5 ± 0.4 2.5 ± 0.1 13.4 ± 1.9 31.1 ± 2.1 > 32 2.8

[W2S]cTI 2.0 ± 0.1 3.0 ± 0.2 3.9 ± 0.2 18.9 ± 0.9 2.7 ± 0.2 22.5 ± 2.1 41.4 ± 2.6 > 32 6.8

[F4S-Y13S]cTI > 32 > 64 > 32 > 32 > 32 > 32 > 64 > 64 2.0

[Y8S-I11S]cTI 17.3 ± 1.0 27.8 ± 2.2 28.2 ± 2.0 > 64 13.2 ± 0.9 > 32 > 32 > 64 > 32 0.9

[R9S]cTI 5.3 ± 0.6 > 64 -

[R14S]cTI 1.7 ± 0.1 2.3 ± 0.2 2.7 ± 0.1 3.5 ± 0.3 7.5 ± 0.3 5.3 ± 0.3 > 64 0.7

[R17S]cTI 1.8 ± 0.1 1.2 ± 0.1 1.8 ± 0.1 4.5 ± 0.3 12.2 ± 0.4 10.3 ± 0.4 > 64 0.2

[R9S-R14S-R17S] 11.8 ± 0.9 11.1 ± 0.9 4.8 ± 0.3 15.3 ± 1.4 0.3

[G18K]cTI 1.0 ± 0.1 3.2 ± 0.1 2.7 ± 0.2 1.7 ± 0.1 6.7 ± 1.1 6.9 ± 0.3 > 64 0.3

[I11F-G18K]cTI 1.3 ± 0.1 3.2 ± 0.1 2.9 ± 0.2 1.9 ± 0.1 5.1 ± 0.3 4.1 ± 0.1 8.4 ± 1.2 0.3 a Peptide concentration required to kill 50% of cells (CC50) was determined from dose-response curves

(sigmoidal fit, one site – specific binding with Hill slope, n > 3, + SE). Cell death was determined with a

resazurin-based cytotoxicity assay. The following cancer cell lines were used in this assay: melanoma –

MM96L, HT144, WM164, MDA-MB-435S; chronic myeloid leukemia – K562; breast cancer – MCF-7 and cervical cancer cell line – HeLa. The aneuploid immortal keratinocyte cell line HaCaT and human

RBCs were included as non-cancerous controls. b Hemolytic activity was determined up to 128 µM for TI

and cTI, up to 32 or 64 µM for the other analogues (dose-response curves in Figure S6). c The activity/toxicity indices (ATI) were calculated as the ratio of the minimal hemolytic concentration for 10%

cell death of human red blood cells and the median of CC50 values from all tested melanoma cell lines. A

higher ATI value indicates greater selectivity for cancerous cells.

The cytotoxic activities of the cTI analogues against cancerous cells (Table 3) showed parallels to

the experiments with model membranes (see Figure 2). The reduced cytotoxic activity of the

peptides with lower hydrophobicity, [W2S]cTI, [F4S-Y13S], and [Y8S-I11S], agree with their

reduced membrane binding affinity and membrane disruption ability. Of these peptides, [W2S]cTI

was the most potent. [W2S]cTI showed activities similar to the other cTI analogues against the

susceptible melanoma and leukemia cell lines, but was less active against MDA-MD-435S, HeLa

and HaCaT. Similar tendencies were observed for [Y8S-Y11S]cTI; however, this analogue was

less active than [W2S]cTI. [F4S-Y13S]cTI had no activity against any cell lines at the tested

19

concentrations. cTI and [R14S]cTI were more cytotoxic against the less susceptible cell lines

MDA-BD-435S, HeLa and HaCaT, than TI and [R17S]cTI.

The analogues [W2S]cTI and [R/K]cTI had improved selectivity for melanoma cells, compared to

cTI38 (see Figure S6, ATI values in Table 3). These analogues retained the ability to kill melanoma

cells but were less toxic toward RBCs. These results show that replacement of Trp2 with a Ser

reduced the ability of [W2S]cTI to bind and insert into the more neutral RBC membranes, but it

did not affect the ability to target and disrupt melanoma cell membranes. The higher selectivity of

[R/K]cTI, compared to cTI, might result from the Lys residues interacting weakly with zwitterionic

compared to anionic phospholipids, whereas Arg residues have strong binding affinities for both

zwitterionic and anionic phospholipids.74

Overall, the cytotoxicity and selectivity results agree with previous reports47, 57 and suggest that

positive charge is required for selectivity of negatively charged membranes, whereas the

hydrophobicity of peptides influences the strength of membrane interaction and the activity of the

peptides. However, an optimal balance between charge and hydrophobicity is required for the

desired combination of activity and selectivity.

TI and cTI analogues internalize into cancer cells. TI has been reported to enter cancer cells34

and to act as an efficient carrier for macromolecule delivery into plant and mammalian cells.40 We

were interested in determining whether backbone cyclization and amino acid substitution affected

the cell-penetrating ability of TI into cancerous cells. We labeled TI, cTI, and analogues with

reduced cytotoxic activity [F4S-Y13S]cTI and [Y8S-I11S]cTI, with AlexaFluor 488 through

conjugation to Lys1 (see Figure S7 and Table S4). Labelled and unlabeled peptides had similar

overall hydrophobicity, as suggested by their similar RT in HPLC chromatograms (see Figure S7).

The loss of the Lys1 charge did not affect the ability of previously reported TI analogues to interact

with membranes;26 in addition, we have shown that peptides labeled with AlexaFluor 488 have

similar membrane-binding properties as unlabeled peptides.75, 76 Therefore, conjugation with this

dye is unlikely to modify membrane binding properties of cTI analogues.

We investigated the ability of the labeled peptides to enter into two melanoma cell lines, MM96L

(highly susceptible to treatment with peptide) and MDA-MB-435S (less sensitive to treatment with

peptide), the cervical cancer cell line HeLa, and the epithelial control cell line HaCaT using flow

20

cytometry (Figure 5). Trypan Blue (TB), a non-permeable quencher, was added to distinguish

peptide internalized into intact cells from peptide that was membrane-bound or present inside cells

with compromised membranes. The percentage of fluorescent cells (Figure 5A) informs on the

fraction of cells with internalized peptide, whereas mean fluorescence emission intensity relates to

the amount of peptide internalized into cells (Figure 5B).

Figure 5. Cellular internalization of TI, cTI and cTI analogues. Peptides labelled with Alexa Fluor® 488 (i.e. TI-

A488, cTI-A488, [Y8S-I11S]cTI-A488 and [F4S-Y13S]cTI-A488) were incubated at ≤ 2 µM with the cervical cancer

cell line HeLa, the melanoma cell lines MDA-MB-435S and MM96L, and the keratinocyte cell line HaCaT for 1 h at

21

37C. The internalization was monitored using flow cytometry by following the mean fluorescence emission (λex =

488 nm, λem = 530, 30 nm bandpass) of cells (10 000 cells/sample). Trypan blue (TB) was added to quench the

fluorescence of extracellular and membrane-bound peptide, enabling distinction between internalized and not

internalized peptide. (A) Percentage of fluorescent cells after addition of TB due to internalized labelled peptide (up

to 2 µM). (B) Mean fluorescence emission intensity of cells treated with 2 µM peptide before (-TB, black bar) and

after addition of TB. TAT-A488 (YGRKKRRQRRRPPQG,77 yellow) was included as positive control for

internalization. Data represent mean ± SEM from at least two independent experiments.

We found that TI, cTI, [F4S-Y13S]cTI and [Y8S-I11S]cTI, entered into mammalian cells without

disrupting them, as shown by all the four tested cell lines becoming fluorescently labelled after

incubation with peptide at ≤ 2 M, and after treatment with TB (see percentage of fluorescent cells

in Figure 5A). Interestingly, TI, cTI, and [Y8S-I11S]cTI entered into cells with higher efficacy

than the standard cell penetrating peptide TAT (see for example internalization of HeLa cells in

Figure 5). These results demonstrate the large potential of cTI analogues as cell penetrating

scaffolds.

Comparison of the four cell lines revealed that the tested peptides internalized cells with identical

trend: MDA-MB-435S ≈ MM96L > HeLa > HaCaT, as shown by lower concentrations of peptide

being required to enter 100% of the cells (Figure 5A), and higher mean fluorescence intensities for

MDA-MB-435S and MM96L, than HeLa and HaCaT (Figure 5B). The higher internalization

efficacy into melanoma cells, than HeLa or HaCat, suggests that TI and cTI analogues, can target

and enter into melanoma with higher efficacy than onto other cancer, or non-cancerous cells.

The internalization efficacy of the tested peptides followed the trend: TI-A488 ≈ [Y8S-I11S]cTI-

A488 > cTI-A488 ≈ [F4S-Y13S]cTI-A488 ≈ TAT-A488, as shown by mean fluorescence emission

intensities (Figure 5B). Interestingly, the internalization efficacy of the cTI analogues was not

directly correlated with their cytotoxicity potency (as exemplified with MM96L cells in Figure

S8); for instance, [Y8S-I11S]cTI had almost no cytotoxic activity against all tested cell lines, but

displayed higher internalization efficacy than the more cytotoxic cTI (see Table 3 and Figure S8).

Furthermore, strong membrane binding was not essential for cellular uptake, as [Y8S-I11S]cTI

and [F4S-Y13S]cTI showed only weak binding affinities for all tested model membranes (see

Figure 2).

Overall, our cellular uptake studies showed that cTI analogues, and in particular [Y8S-I11S]cTI,

have great potential for use as a scaffold to target and transport a cargo into melanoma cells,

considering its high internalization efficiency and low cytotoxic activity.

22

CONCLUSION

Our structure-activity studies confirmed the influence of overall and localized hydrophobicity as

well as peptide charge on the membrane-binding properties of cTI analogues. In general, cTI

analogues with reduced hydrophobicity displayed lower ability to bind to lipid membranes, as

shown with analogues [F4S-Y13S]cTI and [Y8S-I11S]cTI, whereas analogues with higher

hydrophobicity, such as [R/K]cTI and [R9S-R14S-R17S]cTI, had higher membrane-binding

affinity and induced more membrane leakage. Nevertheless, individual amino acid mutations can

modulate the balance of charge and hydrophobicity and impact the peptide-membrane interaction

in a non-predictable way, as shown with the analogues [R14S]cTI vs [R17S]cTI, and [I11F-

G18K]cTI vs [G18K]cTI. [R14S]cTI and [R17S]cTI had similar high membrane binding affinities

to negatively-charged POPC/POPS (4:1) bilayers, but disrupted vesicles of the same lipid

composition in a different way. The [I11F-G18K]cTI analogue has medium hydrophobicity, but

only slightly higher membrane binding affinity and membrane disruption ability compared to

[G18K]cTI; however, [I11F-G18K]cTI was highly hemolytic. These results show that the effect

of single amino acid substitutions in a scaffold are not always predictable and highlight the

importance of examining the activity of individual peptides.

In summary, we identified two analogues, [W2S]cTI and [R/K]cTI, with increased selectivity for

melanoma cells, which support the notion that backbone cyclized TI is a promising lead for

development of anti-melanoma therapeutics, e.g. as a topical formulation. cTI analogues can

furthermore enter into cells and have therefore potential as scaffold to target and deliver drugs into

cancerous cells. In particular, the analogue [Y8S-I11S]cTI, with low cytotoxic activity, had high

internalization efficacy into melanoma cells and other tested cells. Together, these findings

showcase the versatility a cTI as a stable scaffold with anticancer properties that is amenable for

use as a system for targeting and delivery of drugs into cancer cells, and in particular melanoma.

METHODS

Peptide synthesis, folding and purification. TI and its backbone cyclized analogues were

synthesized, folded and purified as previously described.38, 78 The correct peptide mass was

confirmed with ESI-MS, the RT were obtained from a 2% per minute gradient of Solvent B (90%

23

(v/v) acetonitrile, 0.1% (v/v) TFA) on RP-HPLC, and the peptide concentrations were determined

from the absorbance at 280 nm using the Beer-Lambert law. The extinction coefficients ɛ280 (Table

S1) were estimated based on the contributions of Tyr and Trp residues, and disulfide bonds.

Peptide stability. The stability of the cTI analogues in 25% (v/v) was carried as previously

described 38, 79. The break-down of the peptides in serum was followed (0, 1 and 24 h) and

quantified using RP-HPLC (10 to 45% of solvent containing 90% acetonitrile and 0.1% TFA at

1%/min gradient). The amount of peptide remaining (%) was calculated by comparing the area of

the peptide peak after 1 h and 24 h to 0 h.

Liposome preparation. Lipid films with mixtures of synthetic lipids (POPC, POPS, POPE),

sphingomyelin (SM) extracted from porcine brain (Avanti Polar Lipids) and synthetic Cholesterol

(Chol; Sigma Aldrich) were resuspended in HEPES buffer (10 mM HEPES, 150 mM NaCl, pH

7.4) and extruded to produce lipid vesicles, as previously described.38, 73, 80 Large unilamellar

vesicles (LUVs, Ø ≤ 100 nm) were used in leakage assays and fluorescent spectroscopy assays,

small unilamellar vesicles (SUVs, Ø ≤ 50 nm) for SPR.

Fluorescence spectroscopy assays. Fluorescence emission spectra (300-400 nm, λex = 280 nm,

3/3 mm slit size) of 12.5 µM peptide in HEPES buffer were determined upon titration with LUVs

composed of POPC (up to 3 mM) or POPC/POPS (4:1) (up to 1 mM lipid concentration) vesicles

in a FluoroMax-4 spectrofluorometer (Horiba) as previously described.38

Trp fluorescence quenching experiments were conducted as before.38 Briefly, the fluorescence

emission spectra of 12.5 µM peptide in HEPES buffer with/without 1 mM LUVs were titrated with

increasing concentrations the aqueous quencher acrylamide (λex = 290 nm) or with the lipidic

quenchers (5DS, 16DS, λex = 280 nm). Quenchers were obtained from Sigma Aldrich. Acrylamide

quenching data were analyzed using the Stern-Volmer representation. Data analysis and

determination of KSV was done as previously detailed.38

Surface plasmon resonance. The affinity and binding kinetics of peptides to membranes of

different compositions were investigated using SPR (BIAcore 3000 instrument GE Healthcare)

and an L1 chip, as previously described.38, 63 The SPR kinetic parameters koff and P/Loff were

determined by fitting the dissociation phase of sensorgrams obtained with peptides at 32 µM in

24

GraphPad Prism 7. The parameters KD and P/Lmax were determined in GraphPad Prism 7 from the

dose-response curves and assuming one-site specific binding with Hill slope.

Vesicle leakage assay. Vesicle leakage assays were carried out as previously described 38, 81.

Briefly, LUVs (Ø ≤ 100 nm) were prepared in HEPES buffer containing fluorescent CF (Sigma

Aldrich) at the self-quenching concentration of 40 mM. Peptides were prepared as two-fold serial

dilutions (starting at 10 μM) with CF-loaded LUVs (5 μM lipid) in 96-well flat bottom black

optiplates (Perkin Elmer). After 20 min incubation, the release of CF was measured in a Tecan

infinite M1000Pro multiplate reader (λex = 490 nm, λem = 513 nm).

Cell culture. Cells were grown in T75 cell culture flasks in a humidified atmosphere (5% CO2,

37°C) and split by dilution upon reaching confluence. HeLa, MCF-7, MDA-MB-435S and HaCaT

were grown in DMEM medium supplemented with 1% (v/v) penicillin/streptomycin and 10%

(v/v) fetal bovine serum (FBS). MM96L, HT144 and WM164 were grown in RPMI medium

supplemented with 1% (v/v) penicillin/streptavidin, 10 % (v/v) FBS, 20 mM L-glutamine, and 10

mM sodium pyruvate. Cell lines identity has been validated using STR profiles.38

Cytotoxicity assay. The resazurin-based cytotoxicity assay was carried out as previously

described.38 Cells were seeded into 96-well flat bottom plates (5 103 cells/well) and incubated

overnight. Peptides were added to each well to follow dose-response with the final concentrations

64, 32, 16, 8, 4,2, and 1 M. PBS was added as blank and 0.1% (v/v) Triton X-100 was used to

establish 100% of cell death. After 2 h incubation, 10 μL of filtered 0.05 % (w/v) resazurin were

added and co-incubated with peptide for 22 h. The following day, the conversion of resazurin to

the fluorescent compound resorufin by viable cells was determined in a Tecan infinite M1000Pro

multiplate reader (ex = 565 nm and em = 584 nm).

Hemolytic assay. The toxicity to human RBCs was determined as previously described.38 Briefly,

RBCs were obtained from blood from healthy donors. RBCs were suspended in PBS by washing

and centrifugation (4-5 times, 1 min, 4000 rpm). The RBCs suspension (0.25% (v/v) in PBS) was

added to a 2-fold peptide dilutions series (0-128 μM) in a 96-well plate. After incubation (1 h,

37˚C), the plates were centrifuged for 5 min at 1000 rpm to precipitate any non-lysed RBCs. 100

μL of the supernatant were transferred to a new 96-well plate82 and the hemoglobin released into

25

the supernatant from lysed cells determined by absorbance measurement at 415 nm in a Tecan

infinite M1000Pro multiplate reader.

Peptide internalization. Peptides labelling with Alexa Fluor 488 5-sulfodichlorophenol ester

(Life Technologies) and the internalization of the labeled peptides into cells was carried out as

previously described.75, 76, 83 Briefly, 0.5-1 mg peptide were dissolved in DMF and Alexa Fluor

488 5-sulfodichlorophenol ester and DIPEA were added to a final ratio of 78/20/2 (v/v/v). The

amide-bond ligation was carried out protected from light for 1-2 hours. Labeled peptide was

separated from unlabeled peptide on an analytical-scale RP-HPLC using a C18 column (0-40%

gradient with solvent B: 90% (v/v) acetonitrile, 0.05% (v/v) trifluoroacetic acid (TFA), at 1%

solvent B/min). The mass of labeled peptides was confirmed with ESI-MS and the peptide

concentrations determined with NanoDrop, measuring the absorbance of Alexa Fluor 488 at 495

nm (ɛ = 73,000 M-1 cm-1).75

ASSOCIATED CONTENT

Supporting Information Available: This material is available free of charge on the ACS

Publications website at DOI:

Supplementary figures and tables (PDF)

Author Contributions. F.V. conducted experimental work, analyzed and interpreted data, and

wrote the manuscript with assistance and input from S.T.H. and N.L. S.T.H., N.L. and D.J.C.

revised and edited the manuscript. S.T.H. and D.J.C. designed the project and acquired the funding.

ACKNOWLEDGMENTS

The National Health Medical Research Council (NHMRC) funded the project (APP1084965). F.V.

was supported by the UQ Research Scholarship, S.T.H. is an Australian Research Council (ARC)

Future Fellow (FT150100398), D.J.C. an ARC Australian Laureate Fellow (FL150100146). The

Translational Research Institute is supported by a grant from the Australian Government. We thank

26

QUEDDI, M. Cooper (IMB, UQ, Australia) and H. Schaider (UQDI, Australia) for providing the

cell lines HaCaT, HT144 and WM164, respectively. We thank O. Cheneval and J. Weidmann

(IMB, UQ, Australia) for the synthesis of peptides used in this study, and Y.-H. Huang (IMB, UQ,

Australia) for assistance with the stability assay.

1-27

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