development of cell‐penetrating peptide‐based drug leads ...390558/uq390558_oa.pdf · disulfide...

Development of cell-penetrating peptide-based drug leads to inhibit MDMX:p53

and MDM2:p53 interactions

Grégoire Philippe, Yen-Hua Huang, Olivier Cheneval, Nicole Lawrence, Zhen Zhang,

David P. Fairlie, David J. Craik, Aline Dantas de Araujo,* Sónia Troeira Henriques*

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

Australia

*corresponding authors

Dr Sónia Troeira Henriques

Email: [email protected]

Tel: +61 7 334 62026

Dr Aline Dantas de Araujo

Email: [email protected]

Tel: +61 7 334 62988

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/bip.22893

This article is protected by copyright. All rights reserved.

2

The transcription factor p53 has a tumor suppressor role in leading damaged cells to

apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2

and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates

p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was

previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53

interactions with nanomolar potency in vitro. With the aim of improving its cellular

inhibitory activity, we produced a series of constrained pDI analogues featuring

lactam staples that stabilize the bioactive helical conformation and fused them with a

cell-penetrating peptide to increase cytosol delivery. We compared pDI and its

analogues on their inhibitory potency, toxicity and ability to enter cancer cells.

Overall, the results show that these analogues keep their nanomolar affinity for

MDM2 and MDMX and are highly active against cancer cells.

Keywords: cell-penetrating peptides, p53 pathway, internalization mechanism,

stapled peptides

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John Wiley & Sons, Inc.

Biopolymers: Peptide Science


3

Introduction

The transcription factor p53 is a tumor suppressor that protects cells from malignant

transformation by inducing cell cycle arrest, apoptosis or senescence in response to

stress, infection or DNA damage.1,2

In healthy cells, p53 levels are kept low and are

downregulated by interactions with MDM2 and MDMX. MDM2 is a ubiquitin E3

ligase that regulates p53 through a negative feedback loop. MDMX is a homolog of

MDM2, inhibiting p53 by masking its transcription domain, and thereby inhibiting

transactivation.3-6

Loss of p53 function is a major contributor to cancer development,

which can result from mutations in the p53 gene or from overexpression or

deregulation of MDM2 and/or MDMX. Therefore, inhibition of MDM2 and/or

MDMX to recover wild-type p53 function is a potential strategy for developing

anticancer therapeutics (Figure 1A).4,7

Validation of MDM2 as a drug target resulted in the development of nutlin-3, a small

molecule that can activate wild-type p53 through hampering its sequestration by

MDM2 in tumor cells.8 This molecule proved that restoring p53 activity by inhibition

of its interaction with MDM2 was possible and could lead to an elegant way of

preventing cancer development.9 However, despite the potent activity of nutlin-3

against MDM2, it cannot bind efficiently to MDMX, and thus fails to activate p53 in

cells overexpressing MDMX.10

Peptides, with their typically larger binding surface areas compared to small

molecules, are attractive binders to inhibit the extended interface of protein-protein

interactions and have therefore been explored as potential inhibitors to efficiently

target both MDM2 and MDMX.10-12

MDMX and MDM2 bind to the N-terminal

region of p53 due to the complementarity between their cleft and the p53 α–helix.

High throughput screening of a 12-mer peptide phage display library was used to

identify an epitope, named peptide dual inhibitor (pDI), which was able to inhibit both

MDMX and MDM2 at nanomolar concentrations. Although this sequence differs

from the original p53 sequence, it contains the three key residues (Phe19, Trp23, and

Leu26) necessary for primary contact with both proteins.13,14

To optimize peptide-

protein interactions, it is necessary to keep the peptide in an α–helical conformation,

with the three key residues displayed at the correct angles (Figure 1B).15

Stabilization of a helical conformation in a peptide can be achieved chemically by

linking adjacent turns of the helix via side-chain covalent linkage (Figure 1B). This

method, known as stapling, can increase protein binding affinity by reducing the

entropic cost of binding and potentially by providing extra contact between the staple

linkers and target proteins. This approach has been efficiently applied to design stable

p53-like peptides.16-20

Various stapling strategies (e.g. lactam bridges, carbon-carbon, triazole, thioether,

disulfide bonds) can be used to constrain peptides into a helical conformation21,22

but

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for the stapled peptides to be active against cancer cells, in addition to inhibiting

MDM2:p53 and/or MDMX:p53 interactions, they must be able to cross the cell

membrane and reach the cytosol where these proteins are located. Promising results

have been achieved and some P53-derived stapled peptides were shown to enter into

cells and activate p53-dependent apoptosis in vivo. For instance, Bernal et al.23

used a

hydrocarbon crosslink incorporated to a native p53 epitope, whereas Chang et al.24

used the same strategy in a pDI-derived peptide. Other helical-inducing tethers

applied to p53 sequences include biphenyl,25

triazole-linked26

and thioether

crosslinkers.27

Although in some cases stapling crosslinkers might assist in binding

affinity and/or the cell penetration of peptides,21

antitumor activity in cancer cells is

not always reported or is only achieved using high concentrations of peptide.

Cell-penetrating peptides (CPPs), which are normally positively-charged, are able to

carry and translocate large proteins or other hydrophilic molecules into cells.28

To

date, several CPPs have been reported.29

The mechanisms they use to penetrate cell

membranes are quite diverse and seem to be cell type, peptide and cargo dependent.29

Additionally, some of these CPPs do not reach the cytosol following penetration, and

instead become entrapped in endosomes, while others enter the nucleus. Recently, a

cytoplasmic transduction peptide (CTP) was designed and shown to have a high

internalization efficiency and the ability to transfer into the cytosol, thus providing a

useful tool for delivery into the cytoplasm.30

In the current study we combined two strategies to enhance α-helicity and cell uptake

of pDI-based peptides to attain increased apoptotic activity against cancer cells.

Specifically, a series of lactam-stapled pDI analogue sequences were fused with CTP.

Their helicity, inhibitory activity, toxicity against cancer cells and intracellular

efficiency rate were examined. The results confirm that the use of side-chain staples

locks the pDI analogues into a α-helical conformation, whereas the CTP sequence

facilitates cellular internalization making the peptides available to inhibit cytosolic

MDMX:p53 and/or MDM2:p53 interactions.

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Material and methods

Peptide synthesis

Peptides (see sequences in Table 1) were synthesized using standard Fmoc-based

solid-phase peptide synthesis on an automated peptide synthesizer (Symphony®

Protein Technologies) with Rink Amide MBHA resin. Amino acid coupling: five

equivalents of Fmoc-protected amino acid, five equivalents of O-(6-

Chlorobenzotriazol-1-yl)-N,N,N ′ ,N ′ -tetramethyluroniumhexafluorophosphate

(HCTU) and five equivalents of N,N-Diisopropylethylamine (DIPEA) were used in

two cycles of 30 min coupling. Fmoc deprotections were achieved by 2 × 3 min

treatments with piperidine: dimethylformamide (DMF) (1:2, v:v). The non-standard

amino acids Fmoc-Lys(Mtt)-OH and Fmoc-Asp(OPip)-OH were employed for

incorporation of orthogonally protected Lys and Asp handles for lactamization at the

respective locations. The on-resin lactamization step was performed mid-term in the

peptide assembly: after the coupling of Fmoc-Lys(Mtt)-OH, ring closing

lactamization was carried out following previously described protocol.31

Briefly, the

Mtt and Pip protecting groups were removed by treatment with 2% (v/v)

trifluoroacetic acid (TFA) in dichloromethane and subsequent cyclization was

performed with (benzotriazol-1-yloxy)tripyrrolidinophosphonium

hexafluorophosphate (PyBOP)/DIPEA. After the lactam bridge was formed, peptide

assembly continued as per the standard protocol. In the case of peptides with two

lactam rings, a second cyclization step was performed after the coupling of the second

Fmoc-Lys(Mtt) residue. When required, the N-terminus was acetylated with acetic

anhydride:DIPEA:DMF (0.87:0.47:15 mL) for 2 x 10 min. After complete assembly,

each peptide was cleaved from the resin by standard TFA acidolysis and purified

using analytical reverse-phase high performance liquid chromatography (RP-HPLC)

as previously described.32

The concentration of unlabeled peptides was determined by

measuring the absorbance at 280 nm (ε280 = 1,530 M-1

cm-1

for CTP, and 8,480 M-

1cm

-1 for the other CTP-peptides) based on the absorbance of Trp and Tyr residues.

Peptide labeling

The pDI used to test the binding to, and inhibition of, MDMX or MDM2, was labeled

with fluorescein. The label was incorporated at the N-terminus by treating the peptide

with fluorescein isothiocyanate (FITC)/DIPEA (2/4 eq) in DMF overnight. The

concentration of the stock solution was determined by NMR spectroscopy using the

PULCON method as described before.31

Peptides used to follow cell internalization by flow cytometry were labeled using

Alexa Fluor®

488 5-sulfodichlorophenol ester (Life Technologies). Dried peptides

were solubilized in 0.1 M sodium bicarbonate (pH 8.3) and incubated with the dye

(dissolved in dimethylsulfoxide (DMSO)) for 2 hours. Labeled and unlabeled peptides

were separated using analytical reverse-phase high performance liquid

chromatography (RP-HPLC) (Agilent) on an analytical C18 column with 1% gradient

of 0−40% of solvent B (90% of acetonitrile (v/v) with 0.045% TFA (v/v)) in solvent

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A (0.05% TFA (v/v)). Peptides with only one label, as confirmed using mass

spectrometry (Shimadzu), were analyzed (≥95% purity on ultra-high performance

liquid chromatography (UHPLC; Shimadzu) or liquid chromatography- mass

spectroscopy (LC-MS; Shimadzu)) and used in this study. Using this labelling method

and CTP analogues we have shown that the label reacts preferentially with the Lys

side chain, instead of reacting with the alfa-amino group at the N-terminus.33,34

To

preferentially achieve labelling at the N-terminus a lower pH (~ 6.5) is normally

required.35

Thus we assume that the single label is incorporated through the formation

of an amide bond with the amine group in the side chain of Lys4, the single free Lys

side chain in the CTP sequence (see Table 1). The concentration of labeled peptide

was determined by measuring the absorbance of Alexa Fluor®

488 at 495 nm (ε495=

71,000 M-1

cm-1

).

Circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy was used to estimate the overall secondary

structure content of the peptides.36

Peptide samples were prepared in sodium

phosphate buffer (10 mM, pH 7.2) at a concentration of 50 µM. CD spectra were

scanned in 1 mm path length cuvettes at wavelengths from 185 to 260 nm with 1 nm

data intervals on a CD spectropolarimeter (Jasco J-810). The percentage of helicity

was determined using the lowest value between 218 and 222 nm and converted as

previously described.37

Briefly, signal was recorded as milli-degrees at 25oC. Zero

was determined based on the recording at 260 nm and the Equation (1) was used to

normalize the milli-degrees into mean-residue ellipticity,

[θ]MRE = θ/(c × l × Nt) (1)

where θ is the data in milli-degrees, c is the peptide concentration in molar, l is the

cuvette path length in mm and Nt is the number of residues. The percentage of

helicity was then calculated using the Luo-Baldwin formula:

Hα(%)=(θ222nm-θC)/(θ∞

222nm – θC) (2)

with θ222nm the lowest value between 218 and 222nm, θC = 2220-53T and θ∞222nm =

(-44000+250T)(1-k/NResidues) with T in oC and k=3.0 as described for

carboxyamidated peptide (peptides with a R-CO-NR’R group given by glutamine (Q)

or asparagine (N)).38

Binding to and competition to MDMX and MDM2 assay

Binding and competition assays were conducted in 96-well plate format using a

fluorimeter microplate reader (PHERAstar FS). Stock solutions of MDM2 (ABCAM

Australia; ab167941) and MDMX (ABCAM Australia; ab167947) were prepared in

Tris buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4) at a maximum

concentration of 500 nM.

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To examine the binding of MDMX and MDM2 to pDI, two-fold dilutions of MDMX

and MDM2 were made to have final concentrations of protein from 125 to 0.06 nM

and a fixed amount of pDI labeled with fluorescein (F-pDI) was added in every well

to obtain a concentration of 10 nM. Fluorescence measurements (excitation at 485 nm

and emission at 520 nm) with polarized light were conducted and fluorescence

polarisation (FP) was calculated experimentally based on the formula:

FP= (I// - I⊥) / (I// + I⊥), (3)

with I// and I⊥ the light emission intensities respectively parallel and perpendicular to

the excitation plane. After subtracting the blank, data were normalized to the

maximum response after the plateau, with 0% being the signal obtained with F-pDI in

buffer. The IC50 and the Hill slope were determined by transformation of the

concentration into log (concentration) and using non-linear sigmoidal 4PL curve on

Prism to obtain the best fit. The IC80 was then determined using Graphpad software.

The ability of CTP-pDI analogues to inhibit and compete with the interaction of

MDMX-pDI or MDM2-pDI was examined by measuring the FP in a competition

assay. Briefly, F-pDI at 10 nM and protein at the IC80 found in the binding assay (i.e.

20 nM for MDMX and 8 nM for MDM2) were mixed and incubated with CTP-pDI

peptides prepared in Tris buffer with two-fold dilution starting from 2 µM. The

percentage of inhibition was determined by considering the FP obtained with 10 nM

of F-pDI without MDMX or MDM2 as 100% of inhibition and F-pDI bound to

MDMX or MDM2 without peptides as 0% of inhibition. The experiment was repeated

three times.

Cell culture

Adherent human cervix epitheloid carcinoma (HeLa) cells were grown in Dulbecco’s

modified Eagle’s medium (DMEM) with 1% (w/v) penicillin/streptomycin and 10%

(v/v) of foetal bovine serum (FBS). Melanoma cells (MM96L) were grown in RPMI

with 1% (w/v) of penicillin/streptomycin and 10% FBS. All cells were maintained at

37°C in a humidified atmosphere containing 5% CO2.

Cytotoxicity assay

Toxicity against HeLa and MM96L cells was tested with two-fold dilutions of non-

labeled peptides starting from 64 µM. Cell death was quantified by a 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were

seeded in 96-well plates at 5×103

cells/well in media and incubated overnight. On the

day of the experiment, 90 µL of fresh serum free media and 10 µL of peptide sample

(solubilized in phosphate buffer saline (PBS) at 10−times the final concentration)

were added to each well and incubated for four hours. MTT was prepared in PBS and

added to the cells for a 2.5 hour incubation. Living cells reduce MTT to water

insoluble crystals of formazan. Media was removed by suction and the crystals were

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8

dissolved using DMSO. Absorbance was then measured at 600 nm and the percentage

of cell death was determined using 0.01% (v/v) Triton X-100 and PBS as controls to

define 100% and 0% of cell death, respectively. Experiments were repeated three

times. The percentage of cytotoxicity was calculated using the Equation (4):

% Cell death = (Asample-APBS)/(ATriton-APBS) x 100 (4)

in which Asample is the absorbance of the sample, APBS is the absorbance of the blank

and ATriton is the absorbance measured with cells incubated with 0.01% (v/v) Triton

X-100.

Hemolysis assay

Hemolytic activity was tested using red blood cells (RBCs) from fresh human blood.

RBCs resuspended in PBS (0.25% (v/v)) were added to peptide samples (two-fold

dilutions, with final maximum concentration of 64 µM) in 96-well round-bottom

plates and incubated for 1 hour at 37ºC. Intact cells were pelleted by centrifugation at

500 g and the supernatant (100 µL) transferred into 96-well flat-bottom plates.

Hemolysis was quantified by measuring the absorbance of hemoglobin in the

supernatant at 405 nm in a UV-visible spectrophotometer plate reader. Triton-X

(0.01% (v/v)) and the hemolytic peptide mellitin were included as positive controls

and PBS was used as blank. The percentage of hemolysis was calculated using the

same equation as for cytotoxicity. Experiments were done in triplicate.

Internalization assay

Internalization assays were conducted as before.33

Briefly, HeLa cells were seeded in

24-well plates at 105 cells/well and grown for 18 to 24 hours. Ten times concentrated

stock solutions of labeled peptides were prepared in PBS/DMSO (1:1, v:v). On the

day of assay, cells were washed with pre-warmed PBS and fresh serum free media

was added to the cells. Alexa Fluor®

488-labeled peptide solutions were added to a

final concentration of 2 µM. After 1 hour incubation at 37ºC, peptide solutions were

removed and cells were washed with cold PBS. Cells were harvested from the plate

by treatment with trypsin (3 min at 37 o

C), transferred into 1.5 mL eppendorf tubes

and centrifuged for 5 min at 500 g at 4oC. Supernatant was removed and cells were

resuspended with cold PBS and kept on ice until measurements on the flow cytometer

(BD FACSCanto II; excitation at 488 nm and emission with a 530/30 nm filter). The

fluorescence was measured before and after addition of trypan blue (TB; 160 µg/mL).

The results were subtracted with the average of the blank and normalized to the mean

fluorescence obtained with TAT (positive control CPP) after TB. Experiments were

repeated three times on independent days.

Preparation of liposomes

Synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-

palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) (Avanti polar lipids) were

solubilized in chloroform and solvent was removed with a nitrogen flow to form lipid

films of POPC or POPC/POPS (80:20, molar ratio) and residual solvent was

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evaporated overnight in a desiccator. HEPES buffer (10 mM HEPES, 150 mM NaCl,

pH7.4) was added to hydrate the lipid films and multilamellar vesicles were formed

by vortexing and freeze-thaw cycles. Small unilamellar vesicles with a 50 nm

diameter were obtained and sized by extrusion.39

Peptide-lipid binding followed by surface plasmon resonance

A Biacore 3000 instrument (GE Healthcare) with a L1 sensor chip were used to

conduct peptide-lipid studies via surface plasmin resonance (SPR). Lipid vesicles

were injected across the chip to deposit a bilayer as described previously.40

Peptides

at various concentrations (starting from 64 µM with two-fold dilutions) were injected

over the deposited membranes as previously described.39

All solutions were freshly

prepared and experiments were conducted at 25ºC; HEPES buffer was used as the

running buffer and to prepare lipid suspensions and peptides samples. Data were

converted in peptide-to-lipid molar ratio (P/L) to normalize the response and compare

the affinity of different peptides as previously described.41

Results were plotted and

fitted with a nonlinear regression equation dose-response binding with variable slope,

P/L = (P/Lmax × [peptide]H)/(KD

H + [peptide]) (5)

where P/Lmax is the maximum binding; KD is the peptide concentration needed to

achieve half-maximum binding at equilibrium and H is the Hill slope.

Results

Constraining α–helix conformation of pDI

To stabilize pDI in its active helical conformation and potentially improve its binding

to MDM2 and MDMX, the peptide was stapled with a side-chain Lys-Asp (KD)

lactam bridge, an α-helical inducing chemical strategy that has been successfully

applied to other bioactive peptides.42

In a comparative study, de Araujo et al

demonstrated that lactam crosslinking of a Lys with an Asp in an i, i + 4 configuration

results in a superior α-helical stabilization compared to other stapling crosslinkings.31

Thus, we prepared a pDI analogue, L03, where a K8-D12 lactam bridge and a binding

enhancer His-to-Glu mutation24

were incorporated at position 5 of the peptide (Table

1). The linkage between positions K8 and D12 should enable the correct display of

the residues important for the binding to MDM2 or MDMX, with the Lys and Asp

anchors displacing residues that are not essential for binding affinity.43

The CD

spectrum of L03 in phosphate buffer (Figure 2) displays two minima at 217 and 208

nm, a typical profile of α-helix confirmation, whereas pDI shows very weak signal at

these wavelengths, confirming that lactam crosslinking can be used to constrain pDI

into a stable α-helix configuration in buffered solution. Nevertheless, neither L03 nor

pDI were cytotoxic against cancer cells, probably due to inability to cross cell

membranes and reach the targets in the cytosol.

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To potentially improve the activity and cellular internalization rate of pDI and of KD-

constrained pDI analogues, a series of pDI analogues featuring one or two KD-lactam

bridges at different positions within the peptide were synthesized with a CTP

sequence fused at the N-terminus (Table 1). In this series, the original pDI sequence

was modified with two additional Ala residues located at the C-terminal and a Leu-to-

cyclobutylalanine mutation known to favor antitumor bioactivity.24

To examine

whether the CTP portion affects the helical content of the pDI constrained region, the

overall helicity of CTP-L03 was compared to that of L03 (Figure 2, Table 2) and

shown to be 30% and 57%, respectively. As CTP and CTP-pDI are not helical in

phosphate buffer (see spectra in Figure 2) and the CTP part of CTP-L03 molecule

accounts for about half of the CTP-L03 sequence, this suggests that the CTP portion

is unstructured and the KD-constrained region retains the same helical stabilization

found in L03.

A comparison of the overall helicity (Table 2) of the KD-constrained analogues

shows that incorporation of a KD-bridge at K19-D23 (CTP-KD1) induces higher

helical stabilization than at K15-D19 (CTP-KD3). Comparison of CTP-KD1 with

CTP-KD2 shows that addition of a second KD bridge at K12-D16 (CTP-KD2),

further improves the helical character of the peptide. This is further confirmed by a

θ222*/θ208 ratio closer to 1, typical of α-helix conformation,44

for CTP-KD2 (0.91)

than for CTP-KD1 (0.75). Although the molar ellipticity of CTP-KD1 and CTP-KD2

did not change at θ222* nm, the CD spectrum for CTP-KD1 shows a significantly

stronger π-π* band at 205 nm, indicative of higher contribution of a random coil

configuration. The presence of a second KD-bridge at the C-terminal position of CTP-

KD3 to form CTP-KD4 also increased the overall helicity from 26% to 41%.

Binding affinity of CTP-pDI analogues for MDM2 and MDMX

The ability of CTP-pDI analogues to bind MDM2 or MDMX was compared using a

competition assay by examining their ability to compete with a fluorescently-labeled

pDI derivatized with fluorescein (F-pDI) and followed by fluorescence polarization.

Prior to the competition assay, the binding affinity of F-pDI with MDM2 or MDMX,

was examined. F-pDI (10 nM) was incubated with various concentrations of each of

the two proteins and the percentage of bound peptide was monitored by an increase in

fluorescence polarization. The protein concentration required to have 80% of F-pDI

bound was determined and found to be 20 nM for MDMX and 8 nM for MDM2.

The ability of CTP-pDI analogues to compete with F-pDI in the binding to MDMX or

MDM2 was quantified by dose-response inhibition curves and the concentration

required to inhibit the binding of 50% of F-pDI to MDMX, or to MDM2 (IC50), was

determined (Table 3) and revealed that all the peptides display affinity in the low

nanomolar range. CTP-pDI analogues with lactam bridges compete with F-pDI to

bind MDM2 or MDMX with 2.5- to 20-fold higher efficiency than the unstructured

CTP-pDI. This experiment showed that fusing the peptides with CTP did not impair

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their ability to bind to MDMX or MDM2 and suggests that constraining the helical

conformation increases the pDI binding affinity.

Toxicity of CTP-pDI analogues

The toxicity of the CTP-pDI analogues against cancer cells was examined by

measuring their activity against melanoma MM96L cells and cervical cancer HeLa

cells. The concentration required to induce 50% of cell death (CC50) was calculated

(Table 4) based on the dose-response curves. CTP, pDI and CTP-pDI are not toxic

against these cancer cells, whereas CTP-pDI analogues with lactam bridges showed

toxicity against both MM96L and HeLa cells, with CTP-KD3 and CTP-KD4 being

the most toxic in the series.

To examine whether CTP-pDI permeabilize cell membranes we investigated the

hemolytic properties of these peptides against fresh human RBCs. The peptides were

non-hemolytic (CC50 > 64 µM), suggesting that they do not permeabilize cell

membranes and thus the activity of the stapled CTP-pDI peptides against cancer cells

is likely to be associated with the activation/potentiation of p53 properties.

Internalization rate into HeLa cells

To examine whether differences in toxic activity of CTP-pDI peptides correlate with

internalization properties, their overall uptake was compared by measuring the mean

fluorescence emission using flow cytometry of HeLa cells incubated for 1 h at 37ºC

with 2 µM of Alexa Fluor®

488-labeled peptides. The percentage of fluorescent cells

gives information on the population of cells that have labeled peptide internalized,

whereas the mean fluorescence emission intensity correlates with the amount of

peptide within cells. Trypan blue (TB) was added to quench the fluorescence of cells

with permeabilized membranes and of membrane-bound peptides with surface

exposed fluorophores.33

TAT (YGRKKRRQRRRPPQG) is a gold standard and well-

studied CPP and was here included as a positive control. The large number of basic

residues gives TAT its cell penetrating properties. A neutral analogue of TAT,

referred to as TAT-G (YGGGKGGQGGGPPQG), was also included as a negative

control. This peptide has all the basic residues of TAT replaced with a glycine, except

Lys5, which was kept for labeling. TAT-G was used to evaluate the non-specific

solute internalization.34

All the tested CTP-pDI peptides were internalized at 2 µM by 90% or more of the cell

population (see controls and CTP-KD3 example in Figure 3A). The internalization

trend, as measured by the mean fluorescence emission signal (Figure 3B), was as

follows: CTP-KD4 > CTP-KD2 > CTP-KD3 > CTP-KD1 ~ CTP-pDI ~ CTP > TAT

> TAT-G. All the CTP-pDI analogues internalized with equal or higher efficiency

than CTP alone, confirming that fusion of pDI analogues with the CPP confers them

with the ability to enter into cells. It is worth noting that upon addition of the

quencher TB the percentage of fluorescent cells remained the same, showing that the

tested peptides do not permeabilize membranes under the conditions of the assay. On

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the other hand, the mean fluorescence emission signal decreases to 50-80% upon

addition of TB, suggesting that a portion of peptide molecules is membrane-bound.

Internalization mode of action

To gain insights into the mode of action used by the peptides to enter cells, we

inhibited endocytic pathways by incubating the cells at 4oC. The percentage of

fluorescent cells (Figure 3C) and the mean fluorescence emission signal (Figure 3D)

was lower at 4ºC than at 37ºC, showing that internalization rate was highly reduced

for the CTP-pDI analogues tested. A drop in the mean fluorescence to 50% or less

after treatment of TB was observed for the tested CTP-pDI analogues, suggesting that

a large portion of the peptide is membrane bound (Figure 3D) and indicating that both

endocytic and membrane-dependent pathways33

are probably involved in the

internalization of the CTP-pDI peptides.

To investigate whether the drop in the percentage of fluorescent cells results from cell

death, we conducted an independent experiment in which the cells were incubated for

1 h at 4ºC, followed by 1 h at 37ºC. The percentage of fluorescent cells and the mean

fluorescence emission was identical to what was observed with cells incubated with

peptides for 1 h at 37 ºC. Although these results are preliminary (involving only one

replicate), they confirm that the cells are viable after 1 hour of incubation at 4ºC.

Interaction of CTP-pDI peptides with lipid bilayers

To evaluate whether CTP-pDI peptides bind to phospholipid bilayers, peptide-

membrane studies were conducted using SPR with model membranes composed of

pure lipid systems. Phospholipids containing PC-headgroups are the most abundant in

mammalian cell membranes and can be used to represent the overall neutral charge of

the cytoplasmic outer layer. The surface of cancer cells is more negatively-charged

than healthy cells, particularly due to the exposure of negatively-charged PS-

phospholipids in the outer leaflet, compared to their location in the inner leaflet of

healthy cells. Thus, a lipid system containing 20% of POPS (i.e., POPC/POPS

(80:20)) was used to mimic the negatively-charged surface of cancer cells.

Sensorgrams and dose-response curves (Figures 4A and 4B) obtained with POPC

show that these peptides have affinity for zwitterionic model membranes. Comparison

of the dissociation phase in the sensorgrams indicates that CTP-KD4 and CTP-KD2

have the slowest membrane-dissociation rate whereas CTP is quickly washed off from

the membrane. The fitted P/L max values (Figure 4 and Table 5) reveal that the

membrane-binding of the tested peptides for POPC follows the trend CTP-KD4 >

CTP-KD2 ~ CTP-KD3 > CTP-pDI > CTP. Overall these results suggest that the

ability of CTP-pDI analogues to bind membranes is not promoted by CTP itself and

the presence of lactam bridges improves affinity for membranes, but is not essential,

as CTP-pDI possesses affinity for membranes. All the peptides had higher affinity for

the more negative POPC/POPS model membranes, probably due to extra electrostatic

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13

attractions between the positively-charged residues in the peptide and the negatively-

charged phospholipid headgroups.

Discussion

In this study we were interested in improving the helical conformation and cellular

uptake of pDI to increase its ability to reactivate p53 activity in cancer cells. The pDI

sequence has been shown to have nanomolar affinity for MDM2 and MDMX, and is

therefore theoretically able to restore p53 activity. Nevertheless, pDI lacks cell

penetration properties, which prevents it from reaching intracellular targets. This led

us to constrain the pDI helical conformation through chemical staples, thus

maximizing its binding affinity to target proteins, and to fuse it with CTP, a CPP able

to reach the cytosol.

CD spectroscopy showed that the CTP-pDI analogues containing lactam bridges have

higher helicity than CTP-pDI without a staple and that the CTP sequence did not

affect the helicity of the pDI analogue portion. All the peptides maintained nanomolar

binding affinity to both MDMX and MDM2. Additionally, the stapled peptides

showed higher affinity for MDMX and MDM2 than non-stapled CTP-pDI. Peptides

with one-linker (CTP-KD1 and CTP-KD3) displayed the highest affinity for MDM2,

but no significant differences in affinity for MDMX were found amongst stapled

peptides.

We demonstrated that by conferring cell-penetration properties the CTP-pDI stapled

analogues were toxic to cancer cells, whereas the unstructured CTP-pDI was not.

None of the tested peptides was toxic to RBCs and thus do not appear to disrupt cell

membranes, allowing it to be concluded that toxicity against cancer cells is probably

correlated to restoration of p53 activity. Although CTP-KD1 has the highest affinity

for both MDM2 and MDMX, CTP-KD3 and CTP-KD4 exhibited higher anti-cancer

activity.

As differences in peptide activity were not directly correlated with affinity for MDM2

or MDMX, we further explored the cell-penetrating properties of the CTP-pDI

analogues. All the CTP-containing peptides internalized into cells, explaining their

toxicity against cancer cells. CTP-KD2, CTP-KD3 and CTP-KD4 have similar

internalization efficiencies, which were higher than CTP-KD1 and CTP-pDI. Despite

a similar internalization rate, CTP-KD2 had lower toxicity against cancer cells than

CTP-KD3 and CTP-KD4. Differences in activity between the peptides are likely to

correlate with translocation efficiency and the amount of peptide that can reach the

cytoplasm. Internalization of peptides into cells can occur via membrane permeation

and endocytic pathways, or via a combination of these two routes. When

internalization is via endocytic pathways, peptides can become entrapped within

endosomes and lysosomes, and are therefore prevented from binding to MDMX or

MDM2 in the cytosol. A drop in the internalization rate when peptides are incubated

at 4ºC might be indicative of internalization via endocytic pathways, but a drop in the

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14

mean fluorescence in the presence of the quencher TB suggests that a portion of the

peptide is membrane bound, indicating that membrane permeation might also be

operative.

Peptides able to translocate through cell membranes possess the ability to bind lipid

bilayers,28

thus the ability of the CTP-pDI analogues to bind to lipid membranes was

further analyzed. Although model membranes composed of synthetic lipid systems

might be regarded as simplistic models, they give useful information on the ability of

peptides to bind lipid bilayers. CTP-pDI demonstrated good binding affinity to lipids,

which suggests that helical constraint or lactam bridges are not required for the CTP-

pDI analogues to bind to membranes. CTP-KD4 has higher binding affinity for both

zwitterionic and negatively charged model membranes, whereas CTP had the lowest

affinity in the series. Overall, this suggests that the binding affinity of a peptide for

the lipids is important for peptide translocation to occur and thus enhanced toxicity

against cancer cells.

It has been reported that two other CPPs, TAT and R9, can internalize via both

endocytic pathway and direct translocation, and that the major pathway is

concentration dependent.45 At low concentration, cellular import was reported to be

due to macropinocytosis and caveolae/lipid raft mediated endocytosis. But after

reaching a threshold concentration, translocation occurred by direct membrane

permeation. Inhibition of the endocytic pathway, when present, tended to increase

overall cellular uptake by promoting accumulation of peptide on the membrane.45

Also, it was noted that the choice of one pathway over another depends on the peptide

sequence, the extracellular peptide concentration and also the cell type.46

Our SPR

studies show that CTP-KD3 and CTP-KD4 have higher affinity for negatively

charged POPC/POPS model membranes, than CTP-KD2, probably due to their extra

positive charge favoring binding with the negatively-charged membrane. Increased

electrostatic attractions between the peptides and the negatively-charged molecules at

the cell surface could lead to an increased local concentration at the cell membrane

and an increased internalization through direct membrane translocation. Overall all

peptides, especially CTP-KD3 and CTP-KD4, showed promising activity with low

nanomolar binding to the target proteins and greatly improved internalization into

cells.

In summary, we have shown that the use of lactam bridges between residues i and i+4

residues is a very effective strategy for stabilizing pDI into a functional helical

conformation. The resulting helical percentage (50 to 80%) is higher or comparable to

other stapling strategies.24,47

In addition, our peptides inhibit both MDMX:p53 and

MDM2:p53 interactions at low nanomolar concentration, which is in the same

concentration range as for the best reported p53-like stapled peptide (IC50 < 10 nM for

both MDM2 and MDMX).14,16,25,48

Fusing pDI to the cell-penetrating CTP sequence

facilitates their delivery into cancer cells. Only few p53-like stapled peptide have

been report to possess both cell penetrating and anti-cancer cell properties. The

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15

concentration required for the CTP-pDI stapled peptide to kill cancer cells is in the

same range as the best reported stapled p53-like peptide.24

Acknowledgements

This work was funded by a National Health and Medical Research Council

(NHMRC) project grant (APP1084965) and an Australian Research Council (ARC)

project grant (DP160104442). D.J.C. is an ARC Australian Laureate Fellow

(LF150100146). D.P.F. is a NHMRC Senior Principal Research Fellow (PP1027369).

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16

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Figure captions

Figure 1. Strategy to restore p53 activity. A: The activity of p53 is inhibited by

MDMX and MDM2. DNA damage, oncogene activation and stress increase the

activity of p53 by inhibiting the production of MDM2 and MDMX, which triggers

apoptosis, senescence and cell cycle arrest. Overexpression of MDM2 and/or MDMX

in some cancer cells downregulates p53 activity. Peptides that mimic the p53 binding

site and have higher binding affinity than the original protein can replace and release

p53 from its inhibitor and restore its activity. B: pDI analogue showing a lactam

bridge (shown in red) between a Lys and an Asp in the last two helical turns at the C-

terminus, and the side chain of the three residues (Phe, Trp and Leu) involved in

binding to MDM2/MDMX.

Figure 2. CD spectra of pDI analogues. A: pDI, L03, CTP, CTP-pDI, CTP-L03 and

B: CTP-KD1, CTP-KD2, CTP-KD3 and CTP-kD4. All peptides were solubilized in

10 mM phosphate buffer (pH 7.2) at 50 µM.

Figure 3. Internalization of CTP-pDI analogues into HeLa cells. HeLa cells (105 per

sample) were incubated with 2 µM of Alexa Fluor® 488-labeled peptide for 1h at

37ºC (A, B) or at 4ºC (C, D) and the internalization was followed by flow cytometry

(excitation at 488 nm and emission at 530/30 nm). A, C: Flow Cytometry dotplots

showing the side scatter (SSC) as a function of the fluorescence emission intensity

obtained after addition of TB. B, D: Mean fluorescence emission (104 per sample)

was monitored before and after addition of trypan blue (TB; 160 µg/mL). The

fluorescence signal was normalized to the signal obtained with cells incubated with

TAT at 37ºC after treatment with TB. Data are mean ± SD of three independent

experiments.

Figure 4. Binding of CTP-pDI analogues to lipid membranes. A: SPR sensorgrams

obtained upon injection of 32 µM of CTP-pDI analogues over POPC bilayers

deposited onto L1 chip. Response units were converted into peptide-to-lipid ratio

(mol/mol) as before.41

B: Dose response binding of CTP-pDI over POPC or

POPC/POPS bilayers. Peptide-to-Lipid ratio obtained at the end of the injection

(t=170s) was plotted as a function of peptide concentration and the curves were fitted

with a dose-response binding with Hill slope (fitted parameters are presented in Table

5).

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Tables

Table 1. Peptide sequences, and calculated and observed masses.a

Peptide Sequence Average mass (Da)

Calculated Observed

pDI Ac-LTFEHYWAQLTS 1537.7 1536.7

L03b Ac-LTFEEYWKQLTD 1596.8 1596.6

CTP YGRKARRRRRR 1529.8 1530.5

CTP-pDI YGRKARRRRRR-LTFEHYWAQLTS 3007.4 3007.2

CTP-L03 YGRKARRRRRR-LTFEEYWKQLTD 3066.5 3066.5

CTP-KD1c YGRKARRRRRR-LTFEEYWKQXTDAA 3220.7 3220.4

CTP-KD2 YGRKARRRRRR-KTFEDYWKQXTDAA 3203.7 3203.1

CTP-KD3 YGRKARRRRRR-LTFKEYWDQXTSAA 3178.7 3178.6

CTP-KD4 YGRKARRRRRR-LTFKEYWDQXKSAAD 3302.8 3302.4 aAll peptides are amidated in C-terminus.

bThe K and D residues in bold denote the site of

the sidechain KD-lactam bridge. cX denotes cyclobutylalanine.

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Table 2. Peptide helicity determined by CD

spectroscopy.

Peptide Helicity (%) θ 222/ θ 208

pDI 10 0.60

L03 57 1.07

CTP-L03 30 0.74

CTP-KD1 33 0.75

CTP-KD2 33 0.91

CTP-KD3 26 0.57

CTP-KD4 41 0.76

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Table 3. Inhibition of MDMX:pDI or

MDM2:pDI interactions by CTP-pDI analogues.a

Peptide MDM2 MDMX

CTP-pDI 37.6 ± 1.2 64.9 ± 1.4

CTP-KD1 3.0 ± 1.3 3.1 ±1.2

CTP-KD2 7.3 ± 1.2 10.1 ± 1.2

CTP-KD3 2.2 ± 1.1 5.3 ± 1.1

CTP-KD4 9.4 ± 1.1 7.7 ± 1.1 a IC50 values and standard deviations are shown in

nM and were determined by fitting dose-response

with sigmoidal curves (three replicates).

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Table 4. Cytotoxicity against RBCs and cultured cancer cells.a

Peptides MM96L HeLa RBC

CTP > 64 > 64 > 64

pDI > 64 > 64 > 64

CTP-pDI > 64 > 64 > 64

CTP-KD1 19.0 ± 0.8 26.9 ± 3.1 > 64

CTP-KD2 16.1 ± 0.6 23.9 ± 2.1 > 64

CTP-KD3 4.9 ± 0.6 5.3 ± 0.8 > 64

CTP-KD4 4.2 ± 0.7 5.1 ± 0.6 > 64 aConcentrations required to induce 50% cell death and

standard deviations are in µM. Peptides were tested in a dose-

response study up to 64 µM in triplicate.

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Table 5. Binding affinity for model membranes.

Peptides POPC POPC/POPS

P/Lmaxa

KD P/Lmax KD

CTP 0.06 ± 0.01 8.59 ± 6.77 0.15 ± 0.03 2.19 ± 1.44

CTP-pDI 0.10 ± 0.01 5.12 ± 0.94 0.25 ± 0.02 6.07 ± 1.71

CTP-KD2 0.13 ± 0.01 6.22 ± 1.05 0.19 ± 0.02 7.53 ± 2.32

CTP-KD3 0.13 ± 0.01 4.79 ± 0.77 0.21 ± 0.01 5.23 ± 0.89

CTP-KD4 0.18 ± 0.01 6.84 ± 1.02 0.24 ± 0.01 5.80 ± 0.57 a Peptide-membrane-binding was studied by SPR and affinity compared with P/Lmax

values (the maximum binding response) and apparent KD (the peptide concentration

required to achieve half-maximum binding) by fitting the dose-response curves

(Figure 4B) with nonlinear regression with Hill slope.

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Figure 1

139x61mm (300 x 300 DPI)

Page 26 of 29




Figure 2

100x82mm (300 x 300 DPI)

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Figure 3

155x136mm (300 x 300 DPI)

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Figure 4

200x48mm (300 x 300 DPI)

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