Cell Membrane Composition Drives Selectivity and Toxicity of

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Cell membrane composition drives selectivity and toxicity of designed cyclic helix-loop-helix peptides with cell penetrating and tumor suppressor properties Grégoire J-B. Philippe, Diana Gaspar, Caibin Sheng, Yen-Hua Huang, Aurélie H. Benfield, Nicholas D. Condon, Joachim Weidmann, Nicole Lawrence, Alexander Löwer, Miguel A. R. B. Castanho, David J Craik, and Sónia Troeira Henriques ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00593 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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Cell membrane composition drives selectivity and toxicity of designed cyclic helix-loop-helix peptides with cell penetrating and tumor suppressor properties Grégoire J-B. Philippe†, Diana Gaspar‡, Caibin Sheng∆, Yen-Hua Huang†, Aurélie H. Benfield†≈, Nicholas D. Condon†, Joachim Weidmann†, Nicole Lawrence†, Alexander Löwer∆, Miguel A. R. B. Castanho‡, David J Craik†, Sónia Troeira Henriques†≈*

†Institute for Molecular Bioscience, the University of Queensland, QLD 4072, Australia ‡ Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal ∆Technische Universität Darmstadt, 64287 Darmstadt, Germany ≈

School of Biomedical Sciences, Institute of Health & Biomedical

Innovation, Queensland University of Technology, Translational Research Institute, Brisbane, QLD 4102, Australia.

*To whom correspondence should be addressed: Sónia T. Henriques, Email: [email protected]

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ABSTRACT: The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance towards proteases, cell-penetrating properties, and toxicity towards cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.



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Introduction The tumor suppressor protein p53, ‘the guardian of the genome’, blocks cancer progression, but is inactive in more than 50% of human cancers. p53 is a transcription factor that regulates the cell cycle and is activated by many cellular stresses, such as DNA-damage.1 In healthy cells, p53 is located in the cytosol and its activity is inhibited by binding to MDM2 or to MDMX.2, the

transactivation

3

In normal conditions, both proteins block

domain

of

p53

preventing

its

upregulation.

Following the detection of damaged DNA, the complex between p53 and its inhibitors

is

disrupted.

The

degradation

of

MDM2

together

with

upregulation and accumulation of p53 causes either DNA repair, cell cycle arrest, senescence or apoptosis.4 Replication of cancer cells and tumor growth can occur when this pathway is disrupted, either by mutations in p53, or by overexpression of its inhibitors MDM2 and/or MDMX.5 Re-activation of the p53 pathway is a promising targeted strategy to treat

established

development

of

tumors.6,

nutlin-3,

7

a

This

strategy

was

validated

small

molecule

able

to

by

the

prevent

the

sequestration of p53 by MDM2 and to reactivate its function as a tumor suppressor (Figure 1A).8 Although effective against MDM2, nutlin-3 failed to reactivate p53 in cells that overexpress MDMX.9 Investigation of

the

structure

showed

that

the

hydrophobic

cleft

of

MDMX

is

differently shaped and smaller than that of MDM2, preventing effective targeting of both interactions at the same time by small molecules.10


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Figure 1: Targeting p53 pathway. (A) Reactivation of p53 pathway using peptides is a strategy that can be applied to treat tumors with wild-type p53 and with overexpression of MDM2 and/or MDMX. Upon reaching the cytosol, the peptides compete with p53 for binding to the inhibitors MDM2 and MDMX. The binding affinity is dependent on the correct display of the residues Phe, Trp and Leu of TAp53 in a helical conformation. (B) Small molecules (e.g., nutlin-3a) have been compared with p53 mimetics; different strategies have been applied to ameliorate p53 mimetics (e.g. MCO-PMI11, cHLH-p53-R12, SAH-p5313 and ATSP-704114) these approaches aimed at increasing the stability of the helical structure, improve the affinity, enhance cell-penetrating properties and/or resistance to proteases. Reactivating the p53 pathway in cancer cells will trigger specific apoptotic cell death.

Peptides and peptidomimetics have emerged as good alternatives to small

molecule

drugs

for

inhibiting

intracellular

protein-protein

interactions.15 They have some advantages over small molecules, as they possess larger binding site favoring specific binding to proteins. Two promising peptides; pDI (peptide dual inhibitor, LTFEHYWAQLTS) and PMI (TSFAEYWNLLSP);

identified

from

peptide

phage

display

libraries,

possess high binding affinity for both MDM2 and MDMX and are able to disrupt their interactions with p53.16,

17

Both peptides acquire helical

conformation when bound to the inhibitors MDM2/X, and possess the same three key residues (Phe, Trp and Leu) that mimic the Phe19, Trp23, and Leu26 in the native structure of TAp53 (transactivation domain of p53).18 Li et al. showed that Phe3 and Trp7 within PMI are essential for binding to the inhibitors MDM2/X, whereas Leu10 and Tyr6 further 4 ACS Paragon Plus Environment


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improve the binding affinity of the peptide. The other amino acid residues in the peptide sequence do not directly contact MDM2 and MDMX, but instead contribute to the helical conformation, to facilitate primary contact then deep insertion into the inhibitors.19 Owing to the importance of a helical conformation for the activity of these peptides, several strategies have been developed to constrain the conformation to optimize presentation of the essential residues (Figure 1B). One strategy is to use a chemical staple, in which a constraining bond between the side chains of amino-acids from adjacent turns of a helix is formed. This strategy has proved to be very efficacious at increasing the affinity of p53-mimetic peptides for MDM2 and MDMX and, in some cases, to increase the stability and cellpenetrating properties of the peptide.14,

20, 21

A recent study suggested

that placing a chemical staple at the amphipathic boundary of the peptide extends its hydrophobic surface and consequently its ability to enter inside cells.22 Another strategy to constrain these peptides is to graft them into a stable scaffold. For example, the cyclotide Momordica cochinchinensis trypsin inhibitor I (MCoTI-I) has been successfully used as a scaffold to stabilize PMI.11 MCoTI-I possesses a cyclic backbone and three disulfide

bonds

organized

in

a

knot,

which

confers

outstanding

stability.23 Furthermore, MCoTI-I is nontoxic and able to internalize inside human cells.24,

25

PMI was grafted in loop 6 of MCoTI-I and the

peptide retained the toxicity and internalization properties of the scaffold.11 Cyclized helix-loop-helix peptide (cHLH) is another scaffold that has been used to improve stability and cell penetrating properties of a p53 mimetic, as shown with the peptide cHLH-p53-R.12 This peptide possesses two helices – a cell penetrating and a p53 mimetic – linked together by a Gly loop and cyclized with a thioether bond (Figure 1B) to improve structural stability and resistance to proteases. cHLH-p53-R was shown to have high affinity for MDM2, to internalize into cancer cell lines and to possess toxicity towards cancer cell lines with wild type p53. 5 ACS Paragon Plus Environment

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In this study, we were interested in further investigating the internalization properties, mode-of-action and the importance of the cell membrane and peptide-lipid interactions for the activity of cHLHp53-R. Thus, we designed a set of cHLH-p53-R analogues and compared their affinity for MDM2 and MDMX, internalization properties, ability to target cancer over non-cancerous cells, toxicity towards cancer cells, and whether their mode-of-action involves re-activating the p53 pathway. We demonstrate that cHLH-p53-R does not reactivate the p53 pathway and does not lead cancer cells to apoptosis. Instead, it enters and kills cancer cells by a mechanism dependent on interaction with cell membranes. RESULTS AND DISCUSSION

Design and synthesis of cHLH analogues Sequence and structure of designed cHLH-p53-R analogues are shown in Figures 2A&B. All peptides were successfully synthesized and cyclized through formation of a thioether bond as confirmed by observed masses identical to those calculated (Table S1). All peptides were purified to > 95% as confirmed with HPLC-MS.

Figure 2: Structure, sequence and activity of cHLH analogues. (A) Diagram of the three-dimensional structure and sequence of peptides included in this study. The helix in blue is the cell-penetrating sequence and the other helix is a p53-mimetic sequence (native p53 sequence in grey; pDI in green; p53 with F30A mutation in orange). The residues essential for the inhibitory activity are highlighted in red. The helices are connected by a Gly loop and the cyclization is done with a thioether



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linker (chloroacetic acid and side chain of cysteine). Putative salt bridges between Glu and Lys are shown in blue and red for positive and negative charge, respectively, and the hydrophobic interactions between Leu-Leu residues are represented with dashed lines. Cyclization through thioether bond is shown in yellow. (B) Peptide sequences: the color scheme is the same as in panel (A), only p53 mimetic helices were represented in black and mutated residues were underlined. (C) Binding affinity for MDMX and MDM2 as calculated using a competitive assay followed by fluorescence polarization. The values are in nM and are the fitted parameters +/- SD. aThe affinity of cHLH-KD3-R was determined with a different stock of MDM2 and MDMX (IC50 determined were 239.1 ± 10.7 nM for MDM2 and 96.9 ± 10.1 nM for MDMX) and values shown in the table were normalized using IC50 obtained with cHLH-pDI1-R using the same stock of proteins.

The analogue [F30A]cHLH-p53-R was designed as a putative inactive control; Phe30, which mimics the Phe in the p53 pocket and is essential for binding to MDM2 and MDMX, was replaced with an Ala residue, and therefore, the peptide was expected to be unable to bind to MDM2 and MDMX. With the intention of improving affinity for MDM2 and MDMX, while maintaining the stability and cell-penetrating ability of the parent peptide, we designed the analogues cHLH-pDI1-R and cHLH-KD3-R. In these peptides, the p53 helix was replaced with pDI, or with KD3,21 a stapled version of pDI stabilized with a lactam bridge (i; i+4 between the lysine and aspartic acid) previously shown to possess a 10-fold increase in the affinity for MDM2 and MDMX, compared to the unstapled pDI. Both peptides have the three key residues (Phe30, Trp34 and Leu37) necessary to mimic the pocket of the p53 protein and the Tyr5 that enhances the affinity for both MDM2 and MDMX. In the analogue cHLH-pDI2-R, the residues His32, Ala35 and Gln36 were replaced with Leu32, Leu35 and Lys36, and Thr38 was removed to match the length of the parent p53 domain. In addition, Glu9, located in the helix with cell penetrating properties, was replaced with a Lys. These modifications were included to increase structural stability, compared to cHLH-pDI1-R, by facilitating intramolecular interactions between the two helices through Leu-Leu hydrophobic bonds and Lys-Glu salt

bridges.

As,

in

the

future,

we

would

like

to

consider

extension/modification of the peptide in the Gly loop, we have also designed [G17K]cHLH-p53-R in which a Lys residue was inserted within the Gly loop.


7

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Inhibition of MDM2/X-p53 interactions with cHLH-p53-R analogues To examine the binding affinity of cHLH-p53-R, and of its analogues, for

MDM2

and

interactions,

MDMX we

and

their

conducted

a

ability

to

competition

disrupt assay

the

using

MDM2/X:p53

fluorescein-

labeled pDI (F-pDI) as a mimetic of p53 protein and monitored its fluorescence polarization as previously described and optimized with unlabeled analogues of pDI.21 Briefly, we incubated F-pDI with MDM2, or with MDMX, at fixed concentrations (i.e., 10 nM F-pDI with 8 nM MDM2, or with 45 nM MDMX) to achieve at least 80% of bound F-pDI (i.e., MDM2/X:F-pDI)

and

measured

the

variation

of

F-pDI

fluorescence

polarization with increasing concentrations of cHLH-p53-R, or of its analogues. Binding of cHLH-p53-R to MDM2, or to MDMX, displaces F-pDI from the complexes MDM2/X:F-pDI and results in a decrease in its fluorescence polarization.

The

calculated

binding

affinities

(IC50;

peptide

concentrations required to displace 50% of F-pDI from the complexes MDM2/X:F-pDI) are shown in Figure 2C. cHLH-p53-R and its analogues bind to MDM2 and to MDMX with nanomolar affinity. The inactive control [F30A]cHLH-p53-R does not bind to MDM2, nor to MDMX, for concentrations up to 3 µM. We have also included nutlin-3, and as expected it inhibits MDM2 in the conditions of the assay, but does not bind to MDMX. The binding affinity of cHLH-pDI1-R and of cHLH-pDI2-R to MDM2 and MDMX are identical to that of cHLH-p53-R. Although the linear pDI has higher affinity for MDM2 and MDMX compared to linear versions of sequences that mimic the p53 binding domain,16 no differences were detected when pDI and the p53-like peptide were constrained in the cHLH scaffold. The analogue [G17K]cHLH-p53-R also had a binding affinity identical to that of cHLH-p53-R, suggesting that replacing Gly17 with a Lys does not affect the formation of a helix-loop-helix structure, or the correct display of the cHLH-p53-R active sequence. Overall, we have confirmed that cHLH-p53-R inhibits the activity of MDM2 with high efficacy,12 and we found for the first time its ability to inhibit MDMX. Thus, cHLH-p53-R and analogues with dual inhibitor activity have the potential to reactivate the p53 pathway in cells that ACS Paragon Plus Environment

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overexpress MDM2 and/or MDMX,14 in contrast to nutlin-3a able to only inhibit MDM2.

Stability, resistance and alpha-helical conformation of cHLH-p53-R analogues The contribution of alpha-helical conformation to the overall structure (Figure 3A), and the thermal stability of the cHLH-p53-R analogues (Figure 3B) was examined by CD spectroscopy. The resistance of cHLHp53-R analogues to enzymatic degradation was examined by following the percentage of peptide remaining in solution upon incubation with human serum over time (Figure 3C): the analogues [G17K]cHLH-p53-R, cHLH-pDI1 and

cHLH-pDI2

were

chosen

to

elucidate

the

importance

of

the

intramolecular interactions for resistance to serum proteases.

Figure 3: Overall structure and stability of cHLH-p53-R analogues. (A) CD spectra acquired with 50 µM peptide in water at 25°C. The CD spectra show minima at 205 and 222 nm, suggesting that portions of the peptide are in helical conformation. (B) Stability of cHLH-p53-R and analogues to high temperatures as followed by variations in the CD spectra. The mean residue ellipticity obtained at 222 nm was followed upon increase in temperature (1°C/min). The percentage of helicity was calculated from the mean residue ellipticity obtained at 222 nm using the Luo-Baldwin formula and plotted as a function of temperature. (C) Percentage of remaining peptides upon incubation with 25% of human serum at 37°C for up to 24 h. The presence of intact peptide was identified using quantitative LC/MS and the percentage was calculated by comparing the area of the chromatographic peak at a given time with that obtained at time 0.

CD spectra of all the analogues showed the typical profile of alpha-helical contribution

conformation, of

the

with

helical

minima

at

conformation

205 to

and

the

222

overall

nm.

The

three-

dimensional structure was estimated using the Leo-Baldwin formula as ACS Paragon Plus Environment

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previously described,21 and cHLH-p53-R was found to have 65% of its residues in a helical conformation, in agreement with published results (63% in helical conformation), and with the cell-penetrating and p53mimetic portions forming two helices.12 The structure of cHLH-p53-R remained substantially folded at high temperature (55.3% at 75°C). Furthermore, the peptide recovered the original structure when cooled from 75°C to 25°C. Overall, these results support the hypothesis that the

formation

hydrophobic

of

salt

bridges

interactions

between

between

Lys

and

Leu-Leu

Glu

residues,

residues

favor

and the

stabilization and formation of the two helices.26 The analogue cHLH-pDI1-R has fewer amino acid residues in a helical conformation (41% at 25°C), compared to cHLH-p53-R. With increasing temperature, the peptide becomes even less structured, with only 16.6% of its residues in helical conformation at 75°C. This peptide is also less resistant to degradation in serum, with a half-life time of 6.1 hours. Loss of structure and stability suggest that at least some of the

amino-acid

residues

replaced

in

cHLH-pDI1-R

are

involved

in

intramolecular bonds between the two helices. The analogue cHLH-KD3-R, with a lactam bridge staple to maintain the pDI helix, has a low synthesis yield, and low helical content (20% at 25°C). We hypothesize that stapling the p53 helix disturbs the intramolecular

interactions

between

the

two

helices

within

the

scaffold. Due to its low synthesis yield, lack of improvement of the binding affinity for MDM2/X or on the helical conformation, we did not test this analogue further. The single mutations F30A and G17K had slightly enhanced helicity and thermal stability (Figures 3A&B), as shown by comparison of the analogues [F30A]cHLH-p53-R and [G17K]cHLH-p53-R with the parent cHLHp53-R. Although [G17K]cHLH-p53-R possess an extra Lys residue, this analogue has high resistance to serum proteases, as 75% of the peptide remained intact after 24 h incubation with 25% serum. This high degree of protease-resistance is similar to the previously reported resistance of cHLH-p53-R in similar conditions.12


10


Page 12 of 44

The overall structure of the analogue cHLH-pDI2-R, in which the

1 pDI portion was redesigned to improve intramolecular interactions, had 2 3 a low percentage of residues with helical conformation (41% at 25°C) 4 5 and low stability in serum, similarly to cHLH-pDI1-R. 6 7 Overall, we confirmed that cHLH-p53-R has a robust helix-loop8 9 helix conformation, and showed that alterations in the intramolecular 10 interactions, such as the salt bridge and hydrophobic interactions, 11 12 impact the helical conformation and subsequently decrease its stability 13 14 in human serum. Despite the changes in the overall structure, the 15 binding affinity of all the cHLH analogues to both MDM2 and MDMX are 16 17 near-identical and in the nanomolar range. The exception is the inactive 18 19 [F30A]cHLH-p53-R, confirming the importance of the Phe for the activity. 20 cHLH analogues bind to MDM2 with affinity similar to nutlin-3, but 21 22 lower than previously reported with other constrained helices (e.g., 23 24 MCO-PMI,11 stapled peptides (KD3,21 ATSP704114)), suggesting that the 25 26 display of the p53 active sequence might be hindered within the cHLH 27 scaffold. 28 29 30 31 Toxicity of cHLH-p53-R towards non-cancerous and cancerous cell lines 32 33 Table 1: Toxicity against cancerous, non-cancerous and primary cells induced by cHLH-p53-R and analogues.a 34 Skin melanocytes/fibroblasts (M) Breast epithelium cells (M) Blood cells (M) 35 MM96L MDA-MB-435S HFF-1 MCF-7 MDA-MB-231 MCF 10A K562 PBMCs RBCsc Peptide Assayb (h) Cancer Cancer Non-cancer Cancer Cancer Non-cancer Cancer Non-cancer Non-cancer 36 WT mutant WT WT mutant WT mutant WT WT 37 (26) 5.4 ± 0.3 5.7 ± 0.4 11.9 ± 1.0 16.0 ± 0.5 15.9 ± 2.3 8.0 ± 0.8 8.2 ± 0.4 48.9 ± 5.3 > 64 R 38 cHLH-p53R (48) 3.1 ± 0.2 10.7 ± 1.5 9.6 ± 2.0 5.2 ± 0.3 39 L (2) 14.6 ± 0.3 14.3 ± 0.8 24.5 ± 2.2 > 32 (26) 5.3 ± 0.5 7.1 ± 0.6 8.8 ± 1.2 25.7 ± 3.2 42.8 ± 7.0 > 64 40 [G17K]cHLH-p53R R (48) 1.8 ± 0.1 6.8 ± 1.0 5.4 ± 0.3 41 (26) > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 R 42 (48) 11.4 ± 1.4 42.9 ± 8.7 > 64 41.8 ± 3.9 cHLH-pDI1-R 43 L (2) > 64 44 (26) > 64 > 32 > 64 > 64 > 64 > 64 36.7 ± 1.9 > 64 > 64 R (48) > 64 > 64 > 64 28.0 ± 1.1 [F30A]cHLH-p53-R 45 L (2) > 64 > 32 > 32 > 32 46 a Values are concentrations required to induce 50% of cell death, or 50% of membrane disruption, and respective standard deviation in µM. Values were obtained from fitting dose-response 47 curves from three independent experiments. Peptides were tested up to 64 µM. Cancerous cells are identified with ‘cancer’, non-cancerous cells are identified with ‘non-cancer’, WT refers to cells with wild-type p53 and ‘mutant’ refers to cells with mutant p53. b Toxicity was quantified using a resazurin assay (R). Peptides were either incubated with cells for 26 h in total (2h 48 pre-incubation with peptide followed by 24h co-incubation with resazurin), or for 48 h (24h pre-incubation with peptide followed by 24h co-incubation with resazurin). Membrane disruption 49 was quantified by measuring the release of LDH (L) into the supernatant following 2 h peptide incubation. c RBC hemolysis was assessed by measuring the absorbance of released hemoglobin 50 (at 405 nm) following 1 h peptide incubation. 51 52 53 To examine whether cHLH-p53-R and analogues selectively kill cancer 54 cells with wild-type p53 we screened their toxicity against a panel of 55 56 cells (Table 1). We have tested cHLH-p53-R and compared it with three 57 58 59 11 ACS Paragon Plus Environment 60

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analogues: [G17K]cHLH-p53-R, which possess similar MDM2/X inhibitory activity and -helical structure; cHLH-pDI1-R, with similar MDM2/X inhibitory

activity,

cell

penetrating

helix,

but

lower

helical

conformation; and [F30A]cHLH-p53-R, unable to inhibit MDM2/X activity but with similar -helical structure. We included model cells of three tissues: skin, breast epithelium and blood; and compared toxicity against immortalized cancerous cells with wild-type p53 (i.e. melanoma cells MM96L, and epithelial breast cancer cells MCF-7), with mutant p53 (i.e., metastatic melanoma cells MDA-MB-435S, epithelial breast cancer cells MDA-MB-231, and leukemia cells K562), immortalized non-cancerous cells (i.e., breast epithelial cells MCF 10A, foreskin HFF-1), and primary blood cells (RBCs and PBMCs). Immortalized cells and PBMCs were treated for 26 h with two-fold dilutions of peptides. Resazurin was added to the cells and co-incubated with peptide in the last 24 h. Resazurin is converted into fluorescent resofurin

by

viable

cells

and

the

percentage

of

cell

death

was

calculated by comparing the fluorescence emission signal of resofurin obtained with treated and untreated cells (used to establish 0% of cell death and discount potential effects of resazurin in cell proliferation rate).27

The

hemoglobin

in

toxicity the

towards

supernatant

peptide.28

dilutions

of

sigmoidal

curve

RBCs

for

Dose

each

was

after

1

response

peptide-cell

quantified h

by

treatment

curves pair

with

were

and

release

two-fold

fitted

the

of

with

a

concentration

required to induce cytotoxicity against 50% of the cells (CC50) ± SD determined from fitted curves (see values on Table 1). Our results demonstrate that cHLH-p53-R is toxic to cancer cells with either wild-type or mutant p53, and also non-cancerous cells, as clearly demonstrated with the three skin cell lines included (MM96L, MDA-MB-435S and HFF-1, see Table 1). The peptide was toxic to all tested cultured cell lines (CC50 64 µM against RBCs and CC50 = 48.9 ± 5.3 µM against PBMCs).

Overall,

these

results

disagree


with

an

earlier

report 12


Page 14 of 44

suggesting that cHLH-p53-R was only toxic towards cancer cells with wild-type p53.12 The toxicity profile obtained with tested cHLH-p53-R analogues also shows lack of selectivity for cancer cells with wild-type p53. The analogue [G17K]cHLH-p53-R, with identical ability to disrupt MDM2/X:p53 interactions as cHLH-p53-R, had similar toxicity profile as the parent peptide; whereas the cHLH-pDI1-R, also able to disrupt MDM2/X:p53 interactions,

was

not

toxic

against

any

of

the

tested

cells.

Interestingly, [F30A]cHLH-p53-R, which lacks affinity for MDM2/X, was non-toxic against cells with wild-type p53 but was mildly toxic against K562 with mutated p53. If the mechanism by which cHLH peptides induce cell death was exclusively dependent on reactivation of the p53 pathway, active cHLH-p53-R analogues should only be toxic against cancer cells containing wild-type p53, whereas [F30A]cHLH-p53-R should be non-toxic against all types of cells. Thus, the toxicity profile obtained with cHLH analogues suggests that the activity of these peptides is not directly related to their ability to inhibit MDM2/X:p53 interactions and are likely to act by mechanism(s) not exclusively dependent on reactivation of p53 pathway. Reactivation of the p53 pathway can induce a delayed apoptosis (after repeated pulses of p53 accumulation and the cell not being able to repair damage).29 To investigate whether longer incubation times resulted in cell death via reactivation of the p53 pathway, we treated a smaller panel of cells with peptides for 48 h and co-incubated with rezasurin in the last 24 h. We tested cHLH-p53-R and the analogues against cancer cells with wild-type (MM96L), or with mutant p53 (K562) and non-cancerous cells (HFF-1, MCF 10A). Although a lower concentration of peptide was required to induce cell toxicity upon treatment for 48 h (seec in Table 1), the toxicity profile did not change, i.e. cHLHp53-R is toxic against non-cancerous cell lines, and also against cancerous cells with mutant p53. Thus, the increased toxicity is unlikely to be directly related to reactivation of p53 and supports an alternative toxicity mechanism.


13

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Comparison of the toxicity of cHLH analogues (see Table 1) revealed the

trend:

cHLH-p53-R

[F30A]cHLH-p53-R, properties,

such

and

suggesting as

[G17K]cHLH-p53-R that

alterations

in

a

>

cHLH-pDI1-R

combination

the

stability

of of

and

peptide’s

the

helical

conformation (see Figure 3), and local impact from single mutations, are important for their potency. The lower overall charge and/or increased overall hydrophobicity (Supplementary information, Table S1) of cHLH-pDI1-R might contribute to its lower activity when compared to cHLH-p53-R. cHLH-peptides possess features typical of peptides with membranedisruptive properties, including an amphipathic overall structure, with a high positive charge and high proportion of hydrophobic residues.30 We investigated whether cHLH-p53-R analogues compromises cell membrane integrity by following the enzymatic activity of cytosolic lactate dehydrogenase (LDH) leaked into the supernatant. Our results show that cHLH-pDI1-R and [F30A]cHLH-p53-R did not permeabilize cell membranes of MM96L within 2 h, in agreement with their lack of toxicity (see Table 1). cHLH-p53-R permeabilized MM96L, MDA-MB-435S and MCF-7 cell membranes, and although the concentrations required to induce membrane leakage were slightly higher than the determined CC50, this permeability data suggests that toxicity induced by cHLH-p53-R is at least partially dependent on a mechanism that disrupts cell membranes. To further examine whether cell death involved necrosis via cell membrane disruption, we have conducted live-cell imaging studies with MCF-7 cells and SYTOX® Green, a non-permeable dye with a similar reporter function as propidium iodide (i.e. it only enters cells with compromised membranes and becomes fluorescent upon binding to nucleic acids). After 2 h of incubation with 32 M cHLH-p53-R, the nuclei of MCF-7 cells became fluorescent (Figure S1A, Video S1), confirming the ability of this peptide to induce cell-membrane permeabilization with subsequent uptake of SYTOX® Green. These studies show that toxic concentrations

of

cHLH-p53-R

induce

necrosis

via

cell

membrane

permeabilization.


14


Page 16 of 44

We also conducted an xCELLigence real time cell assay with MCF-7 cells to monitor cell proliferation upon treatment with cHLH-p53-R, or [F30A]cHLH-p53-R, over 48 h (Figure S1B). The cell index variation obtained with various concentrations of cHLH-p53-R showed that at nontoxic concentration (4 M) the proliferation rate was identical to that of

untreated

cells

(blank),

whereas

upon

addition

of

toxic

concentrations (64 M) there was a sudden drop in cell index, suggesting rapid cell death. When treated with 64 M of the inactive [F30A]cHLHp53-R, cell proliferation started to decrease after 5 h and cell viability was close to zero after 30 h. As [F30A]cHLH-p53-R is unable to reactivate p53, these results support the notion that cell death induced by cHLH analogues occurs by a mechanism that is independent of the p53 pathway. The trend obtained in the susceptibility of the cells to cHLH analogs is irrespective of their p53 status (e.g. MM96L > K562 > HFF-1> MCF-7). This trend has previously been observed with other peptides that are membrane active,30 thus we hyptothesise that variation in toxicity might depend on cell membrane properties.

Internalization of cHLH analogues into cells To examine if toxicity is linked to the amount of peptide reaching the inside of cells, the internalization efficiency of cHLH-p53-R and its analogues was compared. Fluorescently-labeled peptides were incubated with MCF 10A, MCF-7 or with MM96L cells (1 h at 37°C), and the mean fluorescence emission intensity and the percentage of fluorescent cells was monitored using flow cytometry.27 Peptides were labeled with Alexa Fluor® 488 through amide-bond ligation on the side chain of Lys as previously described.31 cHLH-pDI1-R was labeled on the introduced Lys in the glycine loop. cHLH-p53-R has five Lys residues and we obtained three isomers with different retention times containing a single label, as confirmed by mass using LC-MS (Table S1). We isolated the three isomers and compared their internalization rate into MCF 10A cells; no differences were detected between the three isomers (Figure 4A), suggesting that individual lysines and the position of the label do not impact the internalization efficacy of the peptide. 15 ACS Paragon Plus Environment

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cHLH-pDI1-R internalized cells with an efficiency rate identical to that of TAT (HIV-derived CPP), assuming the quantum yield of the probe

is

not

affected

by

the

peptides;

noteworthy,

cHLH-p53-R

internalized into MCF 10A with 20-fold higher efficiency than TAT (Fig 4A). A similar internalization degree was observed with MM96L and MCF-7 cells (Figure 3B), and the trend in the internalization efficacy was as follows: cHLH-p53-R > [G17K]cHLH-p53-R > [F30A]cHLH-p53-R > cHLHpDI1-R ~ TAT.

Figure 4: Internalization of cHLH-p53 analogues into immortalized cell lines. Cells were incubated (1 h at 37°C) with Alexa Fluor® 488-labeled peptide and internalization monitored using flow cytometry (excitation (EX) at 488 nm and emission (EM) at 530/30 nm). (A,B,C) Mean fluorescence emission intensity of cells (5,00010.000 cells/sample) treated with peptides, before and after addition of trypan blue (TB; final concentration of 0.4 mg/mL; dashed columns). Values were normalized to fluorescence emission signal obtained with cells treated with 2 µM (MM96L) or 4 µM (MCF-7; MCF 10A) TAT for 1 h, and after addition of TB. (A) MCF 10A incubated with peptides at 4 µM. p1, p2 and p3 indicate location of the label at three different positions on [G17K]cHLH-p53-R. (B) MM96L incubated with peptides at 2 µM. (C) MCF-7 incubated with peptides at 4 µM. (D) Flow cytometry dot plots of the side scatter (SSC) as a function of the intensity of fluorescence obtained with MCF-7 incubated with peptides at 4 µM and after quenching with TB. At least 90% of the cells had ACS Paragon Plus Environment

16


Page 18 of 44

peptide internalized for all the peptides tested. (E) Spinning disc confocal microscopy of MCF-7 cells incubated with 4 µM of cHLH-p53-R labelled with Alexa Fluor® 488. The white circle indicates the shape of the expanded cell at the final timepoint, arrows indicate fluorescent endosomes, the scale bar represents 10 µm. Images have been pseudocoloured with a red-yellow gradient look-up-table (LUT; “Muscle and Bones” Arivis Vision 4D). Frames displayed are individual time-points from Video S2 (See also Video S3 for 2D observations).

Addition

of

trypan

blue

(TB,

a

quencher

of

extracellular

fluorescence) did not change the fluorescence emission signal of tested peptides for any of the cell lines tested, nor the percentage of fluorescent cells (Figure 3D; for example, more than 90% of the cells are fluorescent upon treatment with 1 µM of cHLH-p53-R). This suggests that at the concentration tested these peptides are located inside cells, not bound to the cell surface, and internalize without disrupting cell membranes.32 Interestingly, among the analogues compared, cHLH-p53-R and/or [G17K]cHLH-p53-R, shown to be toxic (see Table 1), internalized into the

tested

cell

lines

with

higher

efficiency

than

the

non-toxic

analogues [F30A]cHLH-p53-R and cHLH-pDI1-R. We hypothesize that the amount of peptide inside cells may have a role in the toxicity and/or that uptake and toxicity mechanisms are governed by similar peptide features. This correlation might not be true for other cell lines not tested. To confirm internalization and gain insight on the localization of the peptide, we conducted live-cell imaging on the MCF-7 cell line using 4 µM of labeled cHLH-p53-R. At first, cHLH-p53-R appeared to accumulate in endosomes (Figure 4E, arrows) and as the intensity of peptide increased, the size of the vesicles increased too. One to two hours after addition of the peptide, the cells were permeabilized and we observed a rapid uptake of peptide into cells (starting from 1 h 20 min 21 s in Video S1 and shown in micrographs in Figure 4E). The peptide localized mainly in the nucleus, and the volume of the cell increased (potential indicator of oncosis - an early event of necrosis; Video S1 and S2).

Time-lapse microscopy with p53 reporter cells ACS Paragon Plus Environment

17

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Owing to their dual inhibitor activity, cHLH analogues should lead to upregulation of p53, its accumulation within the cell, and apoptosis of cancer cells that over-express MDM2 and/or MDMX.14 To determine whether the p53 pathway is activated by cHLH peptides, we quantified the dynamics of accumulation of p53 in MCF-7 (overexpressing MDM2 and MDMX) and in A549 cells (overexpressing MDM2) using a fluorescent reporter (mVenus-p53). The cells were incubated with peptide, nutlin3a

or

with

the

radiomimetic

drug

neocarcinostatin,

and

their

fluorescence emission measured every 15 min. A549 cells possess a fluorescent nuclear marker, which allows computer-aided identification and quantification of p53 in hundreds of individual cells. In MCF-7 reporter cells, which do not possess such a nuclear marker, reactivation of p53 was examined by comparing the overall fluorescence signal obtained with untreated and treated cells. A549 cells that overexpress MDM2 treated with neocarninostatin (500 ng/mL) and nutlin-3 (20 µM) show an increase in the fluorescence emission signal of the mVenus-p53 reporter cells over time, suggesting reactivation and accumulation of p53. In contrast, we did not detect a significant increase of the fluorescence emission signal in cells treated with any of the cHLH-p53-R analogues (Figure 5, with replicate in Figure S2), even at toxic concentrations. It was previously reported, that proliferation of A549 cells decreases when incubated with 25 µM cHLH-p53-R,12 here we demonstrate that toxicity is unlikely to be linked with elevated p53 levels as the cell structure seems to be affected (Video S4) MCF-7 cells incubated with toxic concentrations of cHLH-p53-R (20 µM) also did not demonstrate increased levels of mVenus-p53 (Video S5). Instead, at these toxic concentrations the peptide induced localized bursts in the cell membrane and nucleus condensation (Video S6). These results corroborate a mode-of-action not related to reactivation of the p53 network.


18


Page 20 of 44

Figure 5: p53 response after treatment with peptide was monitored over time in A549 lung cancer cells (wildy type p53) using fluorescence microscopy. (A) Varying concentrations of cHLH-p53-R ranging from 1 to 32 µM were tested and showed no significant effect on p53 activation. (B) 32 µM of [F30A]cHLH-p53-R was tested and also showed no significant effect on p53 activation. (C) A negative control (blank or vehicle) and two positive controls (neocarcinostatin (NCS; 500 ng/mL) induces DNA damage and thereby activate p53; nutlin-3a (20 µM) inhibits interaction between p53 and MDM2 and thereby stabilize p53). p53 levels in single cells were quantified and grouped together. The number of analysed cells are indicated. Bold lines represent median p53 level, shaded areas 25th to 75th percentiles.

Ability of cHLH-P53-R analogues to bind, disrupt and aggregate model membranes The involvement of peptide-lipid interactions in cellular uptake and toxicity of cHLH analogues was investigated by following their binding affinity for model membranes using surface plasmon resonance (SPR). In healthy cells, phospholipids are asymmetrically distributed across the two layers within the cell membranes: the outer layer is composed mainly of

phospholipids

containing

zwitterionic

phosphatidylcholine

(PC)-

headgroups, whereas the inner leaflet has a proportion of phospholipids containing PC- headgroups, zwitterionic phosphatidylethanolamine (PE)headgroups and anionic phosphatidylserine (PS)-headgroups. In cancer cells,

the

outer

leaflet

is

more

negatively

charged

due

to

the

inactivation of transmembrane proteins and cell surface exposure of phospholipids composed

of

containing

PS-headgroup.33,

34

Thus,

model

membranes

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ACS Paragon Plus Environment

(POPC) 19

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were used to mimic properties of outer leaflet in healthy cells, and a model composed with a mixture of POPC/POPS (1-palmitoyl-2oleyol-snglycero-3-phospho-L-serine; with 4:1 molar ratio) was used to examine the effect of having negatively-charged phospholipids exposed, as in cancer cells. All the tested peptides bound to negatively-charged POPC/POPS (4:1) with higher affinity than to zwitterionic POPC model membranes, as shown with sensorgrams, dose-response curves (Figure 6A, 6B) and calculated membrane partition constant (Kp, i.e. the ratio of peptide molecules distributed between the lipid phase and aqueous phase, Figure 6C and Figure S3).35 Sensorgrams obtained with the four tested peptides show a fast off-rate (see dissociation phase (2), Figure 6A, 6B), suggesting a shallow location at the lipid bilayer. A small fraction of cHLH-p53-R and [G17K]cHLH-p53-R molecules remained bound to the bilayer within the time of the experiment (Figure 6B, dissociation phase). Comparison

of

[G17K]cHLH-p53-R

the

have

four

similar

peptides membrane

showed binding

that

cHLH-p53-R

properties

to

and each

other, and higher peptide-lipid binding affinity than [F30A]cHLH-p53-R and cHLH-pDI1-R, as shown by comparison of the KP values (see Figure 6C and Figure S3). This trend correlates with the similarly high toxicity of cHLH-p53-R and [G17K]cHLH-p53-R, and low toxicity of [F30A]cHLH-p53R and cHLH-pDI1-R, against the tested cells lines (see Table 1). Interestingly, this is also the trend observed for internalization efficiency, suggesting that internalization is correlated to membrane binding. To examine whether cHLH-p53-R analogues disrupt lipid bilayers we measured leakage from large unilamelar vesicles (LUVs) composed of POPC, or of POPC/POPS (4:1), upon incubation with peptides (Figure 6D). None of the peptides induced leakage from POPC vesicles, but cHLH-p53-R and [G17K]cHLH-p53-R disrupted vesicles composed of POPC/POPS (4:1). A plateau was reached at about 50% of vesicles being disrupted, which was distinct

from

the

effect

observed

with

other

membrane-disruptive

peptides (see for instance mellitin in Figure 6D). cHLH-p53-R did not ACS Paragon Plus Environment

20


Page 22 of 44

aggregate in solution but upon addition of peptide to LUVs composed of POPC/POPS (4:1) we observed an increase in light scattering, which suggested fusion and/or aggregation of the vesicles (Figure 6E).36,

37

Overall, these results show that toxicity of cHLH analogues is independent of their ability to inhibit MDM2/X:p53 interactions and correlates with their ability to bind and disrupt lipid membranes. This is clearly evident with the analogue cHLH-pDI1-R, despite its ability to disrupt MDM2/X:p53 interactions with potency as high as cHLH-p53-R and [G17K]cHLH-p53-R, it displays low toxicity and low membrane-binding properties similar to [F30A]cHLH-p53-R.

Figure 6: Interaction of cHLH-p53-R analogues with model membranes monitored by SPR. (A,B) Sensorgrams (left panel) and dose response curves (right panel) obtained with cHLH-p53-R analogues injected over (A) POPC or (B) POPC/POPS(4:1) bilayers. Peptide samples (64 µM) were injected for 180 s (1; association phase), and dissociations was followed for 600 s (2; dissociation phase). Dose-response curves show peptide-tolipid ratio (P/L; mol/mol) obtained at the end of the injection (t = 170 s) as a function of concentration of peptide and fitted with a non-linear dose-response binding with Hill slope on graphpad prism (version 8.0.1). (C) Maximum binding response (P/Lmax) fitted from the dose-response curves shown in A&B; KP is the membrane ACS Paragon Plus Environment

21

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partition constant and correlates to the peptide-lipid binding affinity; KP was determined by fitting dose response curves using a partition formalism equation previously published (see fitted curves and equation in Figure S3),35 (D) POPC and POPC/POPS (4:1) vesicles (lipid concentration 10 µM) containing a self-quenched concentration of carboxyfluorescein were incubated with increasing concentrations of peptide. The percentage of leakage was determined by following release of fluorescein into solution via fluorescence emission intensity (EX = 485 nm, EM = 520 nm). (E) Variation of optical density of POPC or POPC/POPS LUVs (100 µM lipid concentration) upon addition of 25 µM of cHLH-p53-R was measured at 436 nm. An increase in light scattering suggests increase of vesicles size due to fusion.

Binding of cHLH-p53-R analogues to model membranes followed by Trp fluorescence spectroscopy To examine whether cHLH-p53-R inserts in the membrane, we measured the Trp fluorescence emission spectra of cHLH-p53-R in the absence and presence of LUVs and compared it with [F30A]cHLH-p53-R. The fluorescence emission spectra of cHLH-p53-R and [F30A]cHLH-p53-R in buffer had a maximum emission around 355 nm (Figure 7A), similar to that obtained with L-Trp amino acid (data not shown), suggesting that in both peptides the Trp residue was fully exposed to the aqueous environment. The fluorescence emission spectra of both peptides did not change upon addition

of

POPC

vesicles

(Figure

S2).

In

contrast,

addition

of

POPC/POPS (4:1) LUVs induced a blue shift in the fluorescence emission spectra of cHLH-p53-R (maximum shifted from 357.5 to 333 nm, Figure 7A). This large shift indicates that the Trp residue became less exposed to water and located in a more hydrophobic environment, likely by inserting into the lipid bilayer. A concomitant increase in the quantum yield of the fluorescence is normally observed, but a decrease of quantum yield upon addition of vesicles was observed here for both peptides

(Figure

S4).

This

effect

suggests

quenching

of

the

Trp

fluorescence by intermolecular interactions when the peptide is bound to lipid membranes. Insertion of cHLH-p53-R into POPC/POPS (4:1) bilayers was further examined using acrylamide to quench the fluorescence of Trp residues exposed to the aqueous environment. The quenching efficacy of the peptides by acrylamide was compared in the absence and presence of 2 mM POPC or POPC/POPS (4:1) LUVs (Figure 7B, 7C) and by fitting the data with Stern-Volmer plot, in which the Stern-Volmer constant (KSV) is 22 ACS Paragon Plus Environment


Page 24 of 44

directly proportional to the quenching efficiency. KSV obtained with cHLH-p53-R and [F30A]cHLH-p53-R in aqueous solution is identical to that obtained with L-Trp amino acid, confirming that the Trp residue is exposed to the aqueous environment. In the presence of 2 mM POPC LUVs the quenching efficiency of both peptides was identical to that in aqueous solution, but there was a significant drop in quenching efficiency in the presence of 2 mM POPC/POPS (4:1) vesicles. A lower quenching efficiency was particularly evident for cHLH-p53-R in the presence of POPC/POPS (4:1) vesicles (Figure 7B). Overall, the fluorescence emission studies confirmed that none of cHLH-p53-R analogues had strong binding to zwitterionic POPC membranes, and cHLH-p53-R binds to POPC/POPS (4:1) vesicles with higher affinity than [F30A]cHLH-p53-R (Figure 6B). In addition, it revealed that when cHLH-p53-R is bound to POPC/POPS (4:1) the Trp residue is not exposed to the aqueous environment and is likely to be inserted into the hydrophobic region of the bilayer. Studies with model membranes suggest that the mechanism of cellular uptake and toxicity of the cHLH-p53-R analogues involves the ability to

target

and

bind

to

lipids

bilayers

within

cell

membranes.

In

particular, a stronger binding affinity and ability to disrupt model membranes composed with negatively-charged membranes, might explain the stronger susceptibility of cancer cells, compared to non-cancerous primary

cells,

and

corroborates

a

cell

death

mechanism

involving

disruption of cell membranes. The overall lipid binding affinity and leakage efficiency of these peptides is not as high as observed for other cationic peptides tested under the same conditions (e.g. gomesin, as shown with SPR using similar conditions) known to exert their activity by disrupting cell membranes.30 This suggests that peptidelipid binding through electrostatic attractions improves targeting and selectivity for cancer cells, but other mechanisms, rather than direct cell membrane disruption, might be operative.


23

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Figure 7: Interaction of cHLH-p53-R analogues with model membranes monitored with Trp fluorescence (A) Trp fluorescence emission spectra of cHLH-p53-R and [F30A]cHLH-p53-R upon increasing concentrations of LUVs composed of POPC/POPS (4:1). Fluorescence emission spectra were adjusted to the dilution factor and light scattering, and normalised to the maximum to put in evidence the blue shift when the tryptophan changes from aqueous to hydrophobic environment. (B) Stern-Volmer plot of the quenching of Trp fluorescence emission of cHLH-p53-R and [F30A]cHLH-p53-R induced by acrylamide showing the ratio of the fluorescence emission intensity in absence of quencher (I0) and in presence of increasing concentration of acrylamide (I). (C) The Stern-volmer constant (KSV) was obtained from the slope in (B) and is directly proportional to the quenching efficacy of acrylamide: a reduced slope compared to buffer suggests the Trp is less accessible to the aqueous environment.

Effect of cHLH-p53-R on the cell membrane surface charge and morphology Some cationic peptides with high affinity for lipid bilayers have been shown

to

neutralize

the

surface

charge

of

cell

membranes.38

To

investigate whether cHLH-p53-R neutralizes the surface of cancer cells, we performed zeta potential measurement and monitored the surface charge of MCF-7 and of MDA-MB-231 cells in the presence of cHLH-p53-R at nontoxic concentrations and compared with the least active analogue, [F30A]cHLH-p53-R (Figure S5). Our results showed that none of peptides neutralize the charge of the cell membrane at the concentrations tested. The lack of surface neutralization supports the notion that cHLH-p53R, and analogues, do not stay bound to the outer membrane, but cross the cell membrane and locate intracellularly, as shown above (see Figure 4). To gain insights into the mechanism of disruption of the membrane, we used

atomic

force

microscopy

(AFM)

to

visualize

changes

on

the

morphology of the model cell line MCF-7 (Figure 8A) after treatment ACS Paragon Plus Environment

24


Page 26 of 44

with cHLH-p53-R or with [F30A]cHLH-p53-R. We repeated the experiment with MDA-MB-231 (Figure 8B), a cell-line previously reported to have low percentage of PS-phospholipids exposed on its outer membrane33 and, unlike MCF-7, is not permeabilized by the peptide after incubation for 2 h as suggested by the LDH assay (see Table 1).

Figure 8: The effect of cHLH-p53-R and [F30A]cHLH-p53-R on the morphology of cancer cells monitored by AFM. (A,B) Micrographs of (A) MCF-7 and (B) MDA-MB-231 cells after 24 h treatment with peptide; graphical representation of the height and length one selected cell (black bar). (C,D) Maximum height of (C) MCF-7 and (D) MDA-MB-231 cells after treatment with peptide. The results are represented as means ± SD of five cells for MCF-7, nine cells for MDA-MB-231. **p value < 0.005, *p value < 0.05, ns represents non-significant change between conditions (One-way ANOVA followed by Tukey’s multiple comparisons test).

cHLH-p53-R

induced

morphological

changes

on

MCF-7

cells

at

concentrations as low as 2 µM. In particular, cells expanded and covered a larger area, probably due to loss of membrane rigidity, and the height of the nucleus was lower compared to the controls (Figure 8A, 8C). At 4 µM, the peptide induced various alterations: cells became more compact around the nucleus; cell membranes lost cohesion; and vesicle fusion 25 ACS Paragon Plus Environment

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events occurred, similar to what was seen previously in time lapse microscopy

(see

Video

S4).

[F30A]cHLH-p53-R

did

not

impact

significantly on the morphology of the cells, even at a concentration as high as 32 µM, which is in agreement with its low toxicity, low binding affinity for lipid membranes and inability to induce leakage on model vesicles for up to 64 µM. We did not detect changes in the morphology of MDA-MB-231 cells treated with either cHLH-p53-R or [F30A]cHLH-p53-R. This finding agrees with the lack of cell membrane disruption even when MDA-MB-231 cells were incubated with peptides at high concentration (32 µM). Necrosis events are often characterized by cytosolic swelling (decrease in roughness) and loss of plasma-membrane integrity, whereas apoptosis leads to cell shrinkage (increase in roughness), membrane blebbing, and nuclear fragmentation.39 We determined the roughness of plasma and nuclear membranes for both cell lines (Figure S6).40 The peptides did not impact the membrane roughness of MCF-7 cells, nor induced formation of pores. MDA-MB-231 cells treated with cHLH-p53-R (4 µM) show an increase in nuclear membrane roughness, but no changes in the cellular volume or height of the nucleus. From these results, we cannot conclude whether cHLH-p53-R induces apoptosis or necrosis at low concentration, but AFM results corroborate a mechanism independent on p53 pathway and involving modifications in intracellular organelles.

Conclusion Peptides and peptidomimetics able to enter inside cells and mimic natural sequences that mediate specific intracellular PPIs are perfect candidates to develop targeted cancer therapeutics15 and have inspired our studies with cHLH scaffold. We have confirmed that this scaffold is well-suited for the grafting of helical therapeutic peptides due to its helical structure, high stability, and exceptional internalization properties.

The

salt

bridge

and

hydrophobic

interactions

between

opposite helices contribute to the scaffolds’ conformation. Impairing these interactions by mutations of Leu and Lys residues leads to loss of helicity, stability and internalization properties. Thus, the cHLH ACS Paragon Plus Environment

26


Page 28 of 44

scaffold can form stable helices by introducing a grafted sequence that follows the pattern ELXXLEXELXXLEX or KLXXLEXKLXXLEX. We

also

demonstrated

that

cHLH-p53-R

toxicity

to

cells

is

independent to the reactivation of p53, as shown by similar levels of toxicity against mutant p53 cells (e.g. MDA-MB-435S and K562), the lack of fluorescence response in MCF-7 and A549 reporter cells expressing mVenus-p53, and supported by AFM studies showing no evidence of typical apoptotic behaviors. Based on our findings, we hypothesized that at concentrations close to CC50 cHLH-p53-R induces cell death by a mechanism that involves four steps, as schematically represented in Figure 9 (1) the positivelycharged peptide targets the negative surface of cancer cells via electrostatic attractions, as supported by SPR studies (see Figure 6AB); (2) the peptide inserts into cell membranes and enters inside cells very efficaciously, as supported by flow cytometry, zeta potential and tryptophan fluorescence studies (see Figures 4, S4 and 7); (3) once inside

cells,

disruption

of

the

peptide

intracellular

induces

toxicity

organelles;

through

(4)

the

fusion

cell

and/or

membrane

is

disrupted and the cells die. Evidence for these steps is supported by cell toxicity, leakage and fusion of lipid membranes, and microscopy with live cells (see Table 1 and Figures 4, 5, 6D-E). When cells are incubated

with

highly

toxic

concentrations,

a

faster

mechanism

involving direct disrupting of cell membrane is likely to occur (Figure 9), as supported by cell membrane leakage studies (see Table 1), livecell microscopy and real-time proliferation studies (see Figure S1). Given the high cellular uptake and stability, cHLH arrangement has potential application as scaffold to internalize and stabilize helical peptides involved in intracellular PPI, but needs further improvements. For instance, this scaffold could be improved to enhance the selectivity toward cancer cells by adding tumor- or metastatic-targeting domain.41 In addition, its vesicle fusion properties could be utilized to escape intracellular compartments and reach the cytosol, without damaging the intracellular

organelles

by

using

already

reported

mutation

strategies.42 ACS Paragon Plus Environment

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Figure


9:

Schematic

representation

of

the

mode-of-action

of

cHLH-p53-R.

At

concentrations close to the CC50 (1) the positively-charged peptide targets the negative surface of cancer cells via electrostatic attractions; (2) the peptide inserts into cell membranes and efficiently enters inside cells; (3) once inside cells, the peptide induces toxicity through fusion of intracellular organelles. (4) the cell membrane is disrupted and the cells die, no signs of apoptosis were observed. At high concentrations (>CC50) the peptide induces direct cell membrane disruption.


28


Page 30 of 44

Material and Methods Peptide synthesis. cHLH-p53-R and analogues were synthesized using Bocchemistry

as

before.31

After

last

deprotection,

the

resin

was

neutralized with N,N-Diisopropylethylamine and chloroacetic acid was coupled to the glycine at the N-terminus. Peptides were cleaved using hydrogen fluoride acid with the p-cresol scavenger for two hours at 0°C,

and

precipitated

in

diethyl

ether

before

filtration.

The

precipitate was solubilized in a mixture 1:1 of solvent A (0.05% (v/v) trifluoroacetic

acid

(TFA)

in

water)

and

solvent

B

(90%

(v/v)

acetonitrile, 0.045% (v/v) TFA in water). Crude peptides were cyclized in 4 mL dimethylsulfoxyde (DMSO) and 20 µL of triethanolamine, forming a thioether bond between the side chain of the cysteine at the Cterminal and the chloroacetic acid. Formyl group (-CHO) was removed from the side chain of the tryptophan using 2% (v/v) ethanolamine. Between each step, the peptides were purified using reverse phase highperformance liquid chromatography (RP-HPLC (Shimadzu)) using gradient of solvent B in solvent A.

Peptide labeling. F-pDI used in the binding and competition assay was labeled on the N-terminus during solid phase synthesis using fluorescein isothiocyanate and the concentration of the stock was determined as previously described.43 Peptides used to follow cell internalization by flow

cytometry

were

labeled

using

Alexa

Fluor

sulfodichlorophenol ester (Life Technologies) as before.21, and

unlabeled

peptides

were

separated

using

488 27

analytical

5-

Labeled RP-HPLC

(Agilent) on an analytical C18 column with 1% gradient of solvent B in solvent A from 0 to 40% of solvent B or 0.5% gradient from 20 to 40% of solvent B. Peptides with only one label, as confirmed using liquid chromatography-mass spectrometry (Shimadzu) (95% purity), were used to conduct the experiments. The three isomers of [G17K]cHLH-p53-R with one label were digested with trypsin and the position of the label was determined using tandem mass spectrometry MS/MS. The concentration of labeled peptide was determined measuring the absorbance of Alexa Fluor® 488 at 495 nm (495= 71,000 M-1cm-1)44 ACS Paragon Plus Environment

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Circular dichroism spectroscopy. Samples were prepared and measured as before.21

To

examine

the

temperature

effect

in

the

structure

and

stability of the peptides, the samples were heated from 25°C to 75°C with increments of 1°C/min and the signal was recorded at 222 nm. In addition, full spectra were acquired at 25, 40, 50, 60 and 75°C. Samples spectra were corrected by subtracting the blank, and by applying Savitzky-Golay deconvolution smoothing. Milli-degrees were converted to

mean

residue

ellipticity,

and

the

percentage

of

helicity

was

determined using Luo-Baldwin formula with mean residue ellipticity at 222 nm, as before.21

Peptide-protein

affinity

assay.

Binding

and

competition

assays

to

proteins MDM2 and MDMX were conducted as before.21 Each peptide was incubated (two-fold dilution starting from 3 µM) with F-pDI at 10 nM and MDM2 at 8 nM, or with MDMX at 45 nM. The concentrations of MDMX and MDM2 chosen are the corresponding IC80 from the binding assay with FpDI. The maximum of inhibition was determined using F-pDI free in buffer as a control, and the 0% inhibition as the signal obtained with F-pDI bound to the proteins in buffer. The experiment was made in triplicate.

Isolation of RBCs and PBMCs. Fresh human blood was collected on the day of the experiment from healthy donors following protocols approved by the Human Research Ethics Committees at the University of Queensland. RBCs were isolated and washed by centrifugation (1500g, 1 min) and resuspended in Dulbecco’s Phosphate buffer saline (DPBS; 2.7 mM KCl, 1.8 mM KH2PO4, 137.9 mM NaCl, 8 mM Na2HPO4). PBMCs were isolated by density

gradient

centrifugation

using

Ficoll-Paque

PREMIUM

(GE

Healthcare) as previously reported.45 PBMCs were resuspended in RPMI supplemented with 1% (v/v) penicillin/streptomycin and 10% (v/v) fetal bovine serum (FBS) and counted before plating.

Hemolytic assay. Disruption of RBCs induced by cHLH peptides was followed by measuring the release of hemoglobin in the medium as 30 ACS Paragon Plus Environment


Page 32 of 44

previously reported.29 Briefly, peptide samples with two-fold dilutions starting from 64 µM were prepared in PBS and incubated for 1 h at 37ºC with RBCs (0.25% (v/v)).

Cell culture. Adherent human breast epithelial cells (MCF 10A) were grown in Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (DMEM/F12) with 10% (v/v) horse serum, 20 ng/mL of epidermal growth factor (EGF), 0.5 µg/mL of hydrocortisone, 100 ng/mL of cholera toxin and 10 µg/mL of insulin. Melanoma cells (MM96L) were grown in RPMI with 10% (v/v) FBS. Epithelial metastatic breast cancer cell lines (MCF-7) and metastatic melanoma cell lines (MDA-MB-435 and MDA-MB-231) were grown in DMEM with 10% (v/v) FBS. Human foreskin fibroblast (HFF-1) were grown in DMEM with 15% FBS. All media were complemented with 1% (v/v) Penicillin-Streptomycin. Cells were grown in incubator at 37°C with 5% CO2.

Transgenic cell line. Fluorescent p53 reporters in A549 and MCF-7 cells have been previously described.46-48 In brief, cells express a fusion of p53 and the yellow fluorescent protein mVenus under control of the human metalothionin promoter. A549 reporter cells additionally express a fusion of the histone H2B with the cyan fluorescent protein mCerulean under control of the human ubiquitin C promoter. Cells were grown in RPMI plus 10% FBS supplemented with selective antibiotics (400 µg/ml neomycin and 50 µg/ml hygromycin) as appropriate.

Cytotoxicity Assay and membrane disruption followed with LDH enzymatic activity. Toxicity against non-cancerous and cancer cells was tested by a rezasurin assay as previously reported.27,

45

Cells were seeded in

96-well plates at 5103 cells/well in media and incubated overnight. LDH assay was performed following Promega kit instructions. Briefly, after 2 h treatment with peptide and before adding resazurin, 10 µL of supernatant was collected into a new plate and incubated with LDH substrate for 40 minutes at room temperature in the dark. The reaction ACS Paragon Plus Environment

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was stopped using Stop solution and the absorbance read at 490 nm in a plate reader.

Cell proliferation followed with xCELLigence. Real-time cell viability was monitored using the xCELLigence cell assay (ACEA Bioscience). Medium background was evaluated before addition of the cells to the plate. Then, cells were seeded on a 16 well-plate equipped with biosensor at 104 cells/well and incubated at 37°C with 5% CO2 for 24 hours. The medium was then replaced with medium without FBS before addition of peptides. The impedance of the cells was monitored every 10 minutes for 2 hours after addition of the peptides and every 15 minutes otherwise.

Cell membrane permeabilization and peptide intracellular localization followed by microscopy imaging. MCF-7 cells were seeded in 8-well Borosilicate plates (Thermo Fisher Scientific) at 3104 cells/well in media and incubated overnight. Before the assay, cells were washed and imaged in serum- and phenol red-free DMEM complemented with 1% (v/v) Penicillin-Streptomycin. During all live cell imaging experiments, samples were incubated at 37°C with 5% CO2. Membrane

permeabilization

of

MCF-7

cells

incubated

with

different

concentration of cHLH-p53-R, was monitored using 1 µM of SYTOX® Green dye. Images were captured every 79 seconds for 2 hours and 40 minutes using a custom built inverted widefield microscope with a Nikon TiE inverted stand with Perfect Focus System, and OKO labs incubation. Images were acquired using a 20x 0.75NA Plan Apochromat objective, SYTOX® Green signal was detected using the 485 nm line of a Lumencor LED array and filtered with a 525/30 nm emission filter, and captured using a Hamamatsu Orca Flash 4.0 sCMOS camera running NIS Elements AR. A custom ImageJ macro script was used to identify individual nuclei and record the mean nuclear green intensity fluorescence value at each time point. Measured green intensity values were normalised against the initial timepoint (t = 0) and the average of multiple cells from multiple fields of view was used for each treatment condition.


32


Page 34 of 44

Localisation within the cells was monitored using Alexa-Fluor® 488 labelled cHLH-p53-R. Images were acquired using a Zeiss Spinning Disc confocal microscope fitted with a LCI Plan-Neofluar 63x 1.3NA water immersion objective and Yokogawa CSU-X1 scanhead fitted with twin Photometrics Evolve EMCCD cameras, running Zen Blue software.

Serum stability assay. Human serum (Sigma-Aldrich, human male AB plasma) was centrifuged at 17,000 g (2  10 min) to remove lipid, diluted to 25% in PBS (v/v), pre-warmed at 37°C, and incubated with individual peptides at 20 µM for 0, 1, 4, 10, and 24 h. At each time point, samples were

removed

from

the

incubator

and

the

serum

proteases

were

precipitated with acetonitrile supplemented with 3% TFA (v/v). Samples were vortexed and centrifuged at 17,000 g for 10 min. The supernatant of each sample was collected and spiked with equal amount of the internal standard (N-methylated cyclic penta-alanine peptide). The amount of peptide in the supernatant was determined using quantitative LC/MS analysis (SCIEX X500R QTOF System), where the area of the mass chromatogram at each time point was normalized to the peak area of the internal standard. The percentage of remaining peptide was calculated by comparing the normalized peaks to that obtained at time 0 to assess analytes loss.

Internalization

assay.

Internalization

assays

were

conducted

as

before.27 The mean fluorescence intensity obtained with the samples was subtracted

with

that

of

the

blanks

and

normalized

to

the

mean

fluorescence obtained with TAT at 4 µM after treatment with TB (Sigma, stock of 4 mg/mL) as before,32,

49, 50

at a final concentration of 0.4

mg/mL. This assay has been previously optimised with Alexa Fluor ® 488labelled peptide and 0.1-0.2 mg/mL of TB was enough to distinguish peptide internalized versus peptide bound to the cell membrane and/or located inside cells with their membrane disrupted.27,

32

We have also

compared the fluorescence of cells incubated after treatment with 0.2 mg/mL and with 2 mg/mL TB, and no differences were detected, confirming


33

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cHLH peptides do not remain attached to the cell surface. Experiments were conducted in triplicate on independent days.

Lipid vesicle preparation. Synthetic lipids POPC and POPS were purchased from Avanti Polar Lipids. The lipids were solubilized in chloroform and solvent was evaporated with a nitrogen flow to form lipid films of POPC or POPC/POPS (4:1, molar ratio) and kept in a desiccator overnight to evaporate residual solvent. The lipids were resuspended in HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.4) and vortexed. Multilamellar vesicles were formed by cycles of freeze and thaw. LUVs (diameter of 100 nm) and small unilamellar vesicles (SUVs; diameter of 50 nm) were prepared by extrusion, as previously described.51

Leakage

of

POPC/POPS

lipid (4:1)

vesicles

induced

containing

a

by

peptides.

self-quenched

LUVs

of

POPC

and

concentration

of

carboxyfluorescein (CF; 50 mM) were prepared as before,52 diluted to a final concentration of 10 µM of lipid and incubated with two-fold dilution of peptides up to 10 µM in a 96 well plate (PerkinElmer, flat bottom black polystyrene). The signal from vesicles with dye at selfquenching concentration is discounted, as previously described. The amount of dye that is released into solution is proportional to the percentage of vesicles that are disrupted, and its concentration is in a range in which fluorescence signal is directly proportional to the concentration of dye in aqueous solution.

52

Peptides and LUVs were

prepared in fresh HEPES buffer and the fluorescence was measured in a fluorimeter microplate reader (Tecan M1000 Pro Plate Reader).

Aggregation/fusion of vesicles induced by peptides. The optical density of LUVs composed of POPC, or POPC/POPS (4:1), was monitored over time via absorbance at 436 nm as before.36 The optical density of LUVs suspensions containing 100 µM of lipid prepared in HEPES buffer was followed before and after addition of 10 µM of cHLH-p53-R for 10 min in a UV-Vis spectrophotometer (Varian Cary 50 Bio).


34


Page 36 of 44

Peptide-membrane interactions studied by surface plasmon resonance. Peptide-lipid interactions were studied using SPR on a Biacore 3000 instrument (GE healthcare) at 25°C. SUVs of POPC and POPC/POPS (4:1) were prepared in HEPES and deposited onto a L1 biosensor chip to form a

lipid

bilayer.

Peptides

were

prepared

in

HEPES

at

various

concentration (two-fold dilutions starting from 64 µM) and injected over the lipid bilayers in HEPES as the running buffer. Response units were converted into moles of peptide and lipid and normalized as peptide-to-lipid ratio (P/L) as previously described.53

Peptide insertion into lipid bilayers, as followed by Trp fluorescence spectroscopy. The peptides from this study have one Trp residue and their fluorescence emission spectra were monitored upon titration of 12.5

M

peptide

with

LUVs

suspensions

as

previously

described.51

Acrylamide is an aqueous quencher of Trp fluorescence and was here used to estimate the exposure of Trp residues to the aqueous environment. Briefly, samples of 12.5 M cHLH-p53-R or [F30A]cHLH-p53-R in buffer, or with 2 mM LUVs composed of POPC or of POPC/POPS (4;1) were titrated with

a

stock

solution

of

acrylamide

as

before.45

Its

quenching

efficiency was quantified by calculating the Stern-Volmer constant (KSV). Fluorescence measurements were carried on a LS50B PerkinElmer fluorescence spectrophotometer using a quartz cuvette with l = 0.5 cm.

Zeta potential measurements to follow changes in cellular surface charge. Cells were grown to confluency, trypsinized on the day of the experiment and resuspended in medium, centrifuged, resuspended in PBS and stored at 4°C. Disposable zeta cuvettes (Malvern, Folded capillary zeta cell) were activated with 96% ethanol and rinsed. Samples were prepared by adding 90 µL of buffer, or peptide, to 810 µL of cell suspension (2.5 × 105 cells/mL) and added to activated cuvettes, placed in the zeta sizer (Malvern, Zetasizer Nano ZS) and equilibrated for 30 min at 37°C; 15 measurements were acquired at an applied voltage of 40 V, with 40 sub runs with 90 second pause between each of them. The


35

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experiments were conducted at least two times using independent cellular suspensions on different days.

Examination of p53 activation by time-lapse microscopy. One day before experiments, 6104 cells were plated in 24-well µ-plates (ibidi) in normal growth medium. Right before experiments, medium was replaced with

fluorobrite®

DMEM

supplemented

with

1%

(v/v)

Penicillin-

Streptomycin, 2% HEPES, 1% BSA and 1% glutamate for live-cell imaging. Cells were placed in a humidified incubation chamber at 37 °C containing 5% CO2, and imaged every 15 min on a Nikon Ti inverted fluorescence microscope with a Nikon DS-Qi2 camera and a 20x plan apo objective (NA 0.75) controlled by Nikon Elements software. Appropriate filter sets were used (mCerulean: EX = 438/24 nm, dichroic beam splitter (BS) = 458 nm, EM = 483/32 nm emission; mVenus: EX = 500/24 nm, BS = 520 nm, EM 542/27 nm). Peptide samples (10x concentrated) and control reagents were added to cells 1 h after the start of the acquisition.

Image analysis. As previously described,47 cells were identified and tracked from time-series images using custom-written Matlab scripts based on codes developed by the Alon lab54 and the CellProfiler project.55 In

brief,

we

first

applied

flat

field

correction

and

background

subtraction to raw images. We next segmented individual nuclei from nuclear marker images (H2B-CFP) using thresholding and seeded watershed algorithms. Segmented cells were then tracked in time series images using a greedy match algorithm. Only cells trackable through the full period of experiments were considered.

Atomic force microscopy imaging. Cells (800 µL of 5104 cells/mL) were added to ibiTreat 35 mm µ-dishes (Ibidi) and incubated for 24h (MDAMB-231) or 48h (MCF-7) in complete medium. Peptide was added to cells in fresh medium without serum and incubated for 24h; the medium was removed and the plate washed with warm PBS and with sterile water. Cells were fixed using 1% (v/v) glutaraldehyde for 10 minutes, washed ACS Paragon Plus Environment

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Page 38 of 44

with sterile water and left to air-dry. The acquisition of the images was performed as before,38 with a JPK Nano Wizard II (Berlin, Germany) mounted on a Zeiss Axiovert 200 inverted microscope (Carl Zeiss, Germany) and an uncoated silicon ACL cantilever from AppNano (resonance frequencies between 145 – 230 KHz and average spring constant 45 Nm-1). The cell height corresponds to the difference between the bottom and highest point of each cell. The highest point was acquired after drawing the height profile for each cell using JPK SPM Data processing version 5.1.8. The roughness of the cell membrane was determined as before on Gwyddion software 2.24 and was defined as the mean of the deviation in heights

from

the

value.38,

mean

56

Cells

were

observed

and

images

collected from samples prepared on two to three different days, and on two independent areas within each plate.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. File 1: Supplementary figures and tables (pdf) File 2: Video S1: Permeabilization of MCF-7 cells when treated with 32 µM cHLH-p53-R peptide monitored by binding of SYTOX® Green with nucleic acids using time lapse microscopy. Cells with green fluorescence have been permeabilized. Scale bar is 50 m. File 3: Video S2: Spinning disc confocal microscopy of MCF-7 cells incubated

with

4

µM

of

Alexa

Fluor®

488

labelled

cHLH-p53-R.

4D

rendering was obtained from the Arivis Vision4D software pseudocoloured with a red-yellow gradient LUT (“Muscle and Bones”) and cropping the full field of view to a single cell. Scale bar is 10 m. File 4: Video S3: Spinning disc confocal microscopy of MCF-7 cells incubated with 4 µM of Alexa Fluor® 488 labelled cHLH-p53-R. Images were acquired every 30 seconds for 120 min. Scale bar is 10 m.


37

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File 5: Video S4: Side-by-side observation on bright-field microscopy of MCF-7 cells treated or non-treated with 32 µM cHLH-p53-R. The treated cells are strongly affected by the peptide. Scale bar is 10 m. File 6: Video S5: Fluorescence microscopy of mVenus-p53 MCF-7 reporter cells with 20 µM cHLH-p53-R. Images were recorded with EX = 500/24 nm and EM = 542/27 nm every 15 min for 24 h, peptide was added after 1 h. Scale bar is 50 m. File 7: Video S6: Bright-field microscopy of mVenus-p53 MCF-7 reporter cells treated with 20 µM cHLH-p53-R. Images were recorded every 15 min for 24h; peptide was added after 1 h. Acknowledgement The project was supported by a National Health Medical Research Council (NHMRC) project grant (APP1084965) by a Fundação para a Ciência e a Tecnologia (FCT I.P., Portugal) project grant (PTDC/BBB-BQB/1693/2014) and by a Marie Skłodowska-Curie Research and Innovation Staff Exchange grant (RISE; call: H2020-MSCA-RISE-2014, grant agreement 644167). Live cell microscopy (Spinning disc confocal microscopy and SYTOX® green experiment) was performed at the Australian Cancer Research Foundation (ACRF)/Institute

for

Molecular

Bioscience

Cancer

Biology

Imaging

Facility, which was established with the support of the ACRF. S.T. Henriques

is

(FT150100398),

an

Australian

D.J.

Craik

Research is

an

Council

ARC

(ARC)

Australian

Future

Fellow

Laureate

Fellow

(FL150100146). G.J-B. Philippe has a UQ scholarship. The Translational Research

Institute

is

supported

by

a

grant

from

the

Australian

Government. The authors thank A. Dantas (IMB, UQ) and N. Fletcher (CAI, UQ) for valuable suggestions, J. Koehbach (IMB, UQ, Australia) for peptide synthesis, F. Oliveira (FMU, Portugal) for advice on data analysis, O. Cheneval (IMB, UQ, Australia) and P. Snyder (TU, Germany) for assistance in the lab.

Abbreviations AFM, atomic force microscopy; KSV, Stern-Volmer constant, Rms, Root-mean-square roughness; MDM2, Murine double minute 2 protein; MDMX, Murine double minute X protein; POPC, 1-palmitoylACS Paragon Plus Environment

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2-oleoyl-sn-glycero-3-phosphocholine; POPS, 1-palmitoyl-2oleyol-sn-glycero-3-phospho-L-serine; TB trypan blue


39

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