Bioportide: an emergent concept of bioactive cell-penetrating peptides

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Cellular and Molecular Life Sciences ISSN 1420-682X Cell. Mol. Life Sci.DOI 10.1007/s00018-012-0979-4

Bioportide: an emergent concept ofbioactive cell-penetrating peptides

John Howl, Sabine Matou-Nasri, DavidC. West, Michelle Farquhar, JiřinaSlaninová, Claes-Göran Östenson,Matjaz Zorko, Pernilla Östlund, et al.

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RESEARCH ARTICLE

Bioportide: an emergent concept of bioactive cell-penetratingpeptides

John Howl • Sabine Matou-Nasri • David C. West • Michelle Farquhar •

Jirina Slaninova • Claes-Goran Ostenson • Matjaz Zorko • Pernilla Ostlund •

Shant Kumar • Ulo Langel • Jane McKeating • Sarah Jones

Received: 18 October 2011 / Revised: 20 March 2012 / Accepted: 22 March 2012

� Springer Basel AG 2012

Abstract Cell-penetrating peptides (CPPs) have proven

utility for the highly efficient intracellular delivery of

bioactive cargoes that include peptides, proteins, and oli-

gonucleotides. The many strategies developed to utilize

CPPs solely as pharmacokinetic modifiers necessarily

requires them to be relatively inert. Moreover, it is feasible

to combine one or multiple CPPs with bioactive cargoes

either by direct chemical conjugation or, more rarely, as

non-covalent complexes. In terms of the message-address

hypothesis, this combination of cargo (message) linked to

a CPP (address) as a tandem construct conforms to the

sychnological organization. More recently, we have

introduced the term bioportide to describe monomeric

CPPs that are intrinsically bioactive. Herein, we describe

the design and biochemical properties of two rhegnylogi-

cally organized monometic CPPs that collectively

modulate a variety of biological and pathophysiological

phenomena. Thus, camptide, a cell-penetrant sequence

located within the first intracellular loop of a human cal-

citonin receptor, regulates cAMP-dependent processes to

modulate insulin secretion and viral infectivity. Nosangi-

otide, a bioportide derived from endothelial nitric oxide

synthase, potently inhibits many aspects of the endothelial

cell morphology and movement and displays potent anti-

angiogenic activity in vivo. We conclude that, due to their

capacity to translocate and target intracellular signaling

events, bioportides represent an innovative generic class of

bioactive agents.

J. Howl (&) � S. Jones

Research Institute in Healthcare Science, School of Applied

Sciences, University of Wolverhampton, Wolverhampton

WV1 1LY, UK

e-mail: [email protected]

URL: http://www.wlv.ac.uk/default.aspx?page=16517

S. Matou-Nasri

Institute for Biomedical Research into Human Movement

and Health, Manchester Metropolitan University,

Manchester M1 5GD, UK

D. C. West

School of Biological Sciences, University of Liverpool,

Biosciences Building, Crown Street, Liverpool L69 7ZB, UK

M. Farquhar � J. McKeating

School of Immunity and Infection, University of Birmingham,

Edgbaston, Birmingham B15 2TT, UK

J. Slaninova

Department of Antimicrobial Peptides, Institute of Organic

Chemistry and Biochemistry, Academy of Sciences of the Czech

Republic, 166 10 Prague 6, Czech Republic

C.-G. Ostenson

Department of Molecular Medicine and Surgery,

Karolinska Institute, 171 77 Stockholm, Sweden

M. Zorko

Medical Faculty, Institute of Biochemistry,

University of Ljubljana, 1105 Ljubljana, Slovenia

P. Ostlund � U. Langel

Department of Neurochemistry, Stockholm University,

10691 Stockholm, Sweden

S. Kumar

Faculty of Medical and Human Sciences, School of Medicine,

University of Manchester, Manchester M13 9PT, UK

Cell. Mol. Life Sci.

DOI 10.1007/s00018-012-0979-4 Cellular and Molecular Life Sciences

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Author's personal copy

Keywords Angiogenesis � Bioportide � Camptide �Cell-penetrating peptide � Nosangiotide � Second

messenger � Insulin secretion � Viral infectivity

Introduction

Cell-penetrating peptides (CPPs), alternatively named

protein transduction domains, have proven utility for the

highly efficient intracellular delivery of bioactive moieties

that include peptides, proteins, oligonucleotides, and other

bio-conjugates [1]. The majority of CPPs identified to date,

including more common sequences such as Penetratin [2],

HIV-encoded Tat [3], and transportan [4], are polycationic

in nature. Moreover, differential positive charge distribu-

tion provides some CPPs with an additional amphipathic

character, at least when modeled as a 3.613 helix, and these

properties are major determinants of effective intracellular

transduction [5, 6]. However, the mechanisms by which

CPPs gain entry to the intracellular milieu are not fully

resolved. Moreover, it is certain that some CPPs, including

Penetratin [2], mitoparan [7], and cytochrome c-derived

sequences [8], have a propensity to accumulate at specific

intracellular sites.

The many strategies developed to utilize CPPs solely as

transport vectors or pharmacokinetic modifiers necessarily

requires them to be relatively inert at concentrations

required (usually in the low micromolar range) for effec-

tive delivery kinetics [1]. To achieve a desired biological

action, it is feasible to combine one or multiple CPPs with

bioactive cargoes either by direct chemical conjugation or,

more rarely, as non-covalent complexes. In terms of the

message-address hypothesis this combination of cargo

(message) linked to a CPP (address) as a tandem construct

conforms to the sychnological organization and represents

a common strategy employed to exploit the effective

translocation properties of numerous CPPs [9, 10].

Based initially upon studies with predicted CPPs derived

from the primary sequence of cytochrome c, we more

recently introduced the term bioportide to describe a class

of CPP that is intrinsically bioactive [10]. CPPs such as Cyt

c77–101 are able to promote apoptosis in brain tumor cell

lines following their highly efficient uptake. Moreover, the

intracellular distribution of Cyt c77–101 indicates that the

peptide interacts with multiple protein targets to induce

caspase 3-dependent cell death [8]. These developments

reveal that CPPs may contain additional pharmacophores

that facilitate the discrete modulation of the activity of

intracellular proteins. These rhegnylogically organized

bioportides differ from the more usual synchnologic com-

bination of an inert CPP with another bioactive cargo [e.g.

11]. Thus, prediction algorithms primarily designed to

discover CPP sequences within the primary sequences of

proteins [12, 13] are also capable of identifying bioportides

in which the pharmacophores for effective translocation

and biological activity are discontinuously located within a

single peptide [8, 10].

Previous studies have indicated that G protein-coupled

receptors are a viable source of bioportides [14]. In an

effort to identify additional bioportides, we employed a

QSAR-based algorithm [12] to identify a 20 AA fragment

within the first intracellular loop of the human type 1 cal-

citonin receptor (hCTR-1) that was predicted to be a highly

probable CPP. This sequence, hCTR-1174–193, includes a

splice variant 16 AA insert that modulates the pharma-

cology of hCTRs by inhibiting receptor-stimulated inositol

phosphate metabolism, but facilitating the synthesis of

cAMP [15]. Additionally, a similar strategy was employed

to identify nosangiotide, a cell permeable sequence from

within a calmodulin binding domain of endothelial nitric

oxide synthase [16]. The studies reported herein, and

additional data recently emerging from other laboratories,

clearly indicate that cell permeable bioportides can be

exploited to selectively modulate a range of biological

events and so extend the utility of CPPs beyond their more

common employment as pharmacokinetic modifiers.

Materials and methods

Peptide design and synthesis

To identify putative bioportides we employed a QSAR-

based algorithm [12] to predict CPPs within the primary

sequences of the human type 1 calcitonin receptor [15] and

a calmodulin binding domain of endothelial nitric oxide

synthase [16]. As a strategy towards the identification of

highly efficient translocation-competent peptides, we chose

to focus our efforts entirely on highly probable CPP

sequences (Fig. 2).

Peptides prepared for the studies described herein were

manually synthesized (0.1–0.2 mmol scale) on Rink amide

methylbenzhydrylamine (MBHA) resin (Novabiochem,

Beeston, UK) employing an N-a-Fmoc protection strategy

with O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-te-

tramethyluronium hexafluorophosphate (HCTU; AGTC

Bioproducts, Hessle, UK) activation. Fluorescent peptides,

to be used in confocal live cell imaging, were synthesized

by amino-terminal acylation of CPP sequences with

6-Carboxy-tetramethylrhodamine (rho) or 5-Carboxyfluo-

rescein (fluo; Novabiochem) as previously reported [7, 8].

Crude peptides were purified to apparent homogeneity by

semi-preparative scale high performance liquid chroma-

tography [7, 8]. The predicted masses of all peptides used

(average M ? H?) were confirmed to an accuracy of ±1

J. Howl et al.

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by matrix-assisted laser desorption ionization (MALDI)

time of flight MS operated in positive ion mode using

a-cyano-4-hydroxycinnamic acid (Sigma) as a matrix.

Cell culture

U373MG human astrocytoma cells [17] and ECV304

human bladder cancer cells [18] were routinely maintained

in a humidified atmosphere of 5 % CO2 at 37 �C in Dul-

becco’s modified Eagle’s Medium (DMEM) (U373MG) or

M199 medium (ECV304) supplemented with L-glutamine

(0.1 mg/ml) 10 % (wt/vol) fetal bovine serum (FBS),

penicillin (100 U/ml) and streptomycin (100 lg/ml). The

human hepatoma cell line Huh-7.5 [19] (provided by

Charles Rice, The Rockefeller University, NY, USA) was

propagated in DMEM supplemented with 10 % FBS, 1 %

non-essential amino acids (Invitrogen, CA) and grown in a

humidified atmosphere of 5 % CO2 at 37 �C.

Bovine aortic endothelial cells (BAECs) were grown in

complete medium composed of DMEM supplemented with

15 % FBS, 2 mM glutamine, 100 U/ml penicillin, and

100 lg/ml streptomycin. BAECs were seeded into T75

flasks pre-coated with 0.1 % gelatine and incubated in a

humidified atmosphere of 5 % CO2 at 37 �C. Every 3 days,

cells underwent a passage at a split ratio of 1:2 or 1:3 by

treatment with PBS without Ca2? and Mg2?, then cell

detachment by enzymatic digestion with 0.05 % trypsin/

0.02 % EDTA. Cells were used throughout the study

between passages 15 and 17.

Human dermal microvascular endothelial cells (Pro-

moCell) were grown in endothelial medium plus growth

supplement (with final supplement concentrations of 5 %

(vol/vol) FBS, 0.4 % (vol/vol) endothelial growth supple-

ment, 10 ng/ml EGF, 22.5 lg/ml heparin and 1 lg/ml

hydrocortisone, (PromoCell) and further supplemented

with penicillin (100 U/ml), streptomycin (100 lg/ml) and

amphotericin B (250 lg/ml). Cells were incubated in a

humidified atmosphere of 5 % CO2 at 37 �C and every

3 days, cells underwent a passage at a split ratio of 1:2 or

1:3. Cells were used throughout the study between pas-

sages 3 and 10.

Cell viability assays

To fully evaluate the potential cytoxicity of bioportides,

cellular viability was measured using the 3-(4,5-dim-

ethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)

conversion assay [7, 8] in a broad range of different cell

types. ECV304, human dermal microvascular endothelial,

U373MG astrocytic tumor, H630WT colorectal cancer and

HCT113 colon cancer cells were cultured in 96-well plates

and treated with peptides (0.1–30 lM) for 24–72 h at

37 �C. Following the removal of stimulation medium, cells

were incubated with MTT (0.5 mg/ml) for 3 h at 37 �C.

Medium was aspirated and the insoluble formazan product

solubilized with DMSO. MTT conversion was determined

by colorimetric analysis at 540 nm.

Live cell-imaging analyses

Cells, maintained as above, were transferred to 35-mm

sterile glass base dishes (PAA Laboratories) and grown to

75 % confluence. Immediately prior to the addition of

fluorescent peptides, cells were washed with and trans-

ferred into medium lacking phenol red. During the period

of exposure to peptides, cell layers were maintained at

37 �C in a humidified atmosphere of 5 % CO2. Immedi-

ately prior to observation, cells were washed gently (89)

and analyzed with a Carl Zeiss LSM510Meta confocal

microscope equipped with a live cell-imaging chamber.

Hepatitis C viral infectivity and release

Cell culture derived hepatitis C virus (HCV) strains, J6/

JFH and JFH-1, were generated as previously described

[20]. Using the Megascript T7 kit (Ambion, TX) RNA was

transcribed in vitro from full-length genomes and electro-

porated into Huh-7.5 cells. High titer stocks were generated

by serial passage through naive Huh-7.5 cells [21].

Supernatants were collected at 72 and 96 h post-infection

pooled and stored at -80 �C.

To determine the influence of camptide upon HCV

infection, Huh-7.5 cells were seeded at 1.5 9 104/cm2 and

the following day subjected to a 3-h serum starvation prior

to incubating with increasing concentrations of peptide

diluted in serum free DMEM for 1 h. HCVcc-containing

medium was added to target cells and incubated for 1 h.

Unbound virus/peptide was removed by washing and the

medium replaced with 3 % FBS/DMEM. After 48-h

infection, infected cells were detected by methanol-fixation

and staining for NS5A using the anti-NS5A 9E10 antibody

(C. Rice, Rockefeller University, USA). Bound antibody

was detected with an Alexa-488 conjugated anti-mouse

IgG (Invitrogen) and antigen positive cells enumerated on a

Nikon TE2000. Infectivity is defined as the number of

infected cells and expressed relative to control untreated

cells.

To evaluate the level of infectious virus released from

J6/JFH and JFH-1-infected cultures, cells were seeded at

6 9 104/cm2 in 48-well plates and the following day serum

starved for 3 h prior to incubation with increasing con-

centrations of peptide for 1 h. Cells were washed

extensively and cell-free medium collected for quantifica-

tion of infectious virus. To quantify extracellular virus

infectivity, the collected medium was allowed to infect

naive Huh-7.5 target cells at various dilutions for 1 h at

Bioportide: an emergent concept

123


37 �C. Viral infection was detected after 48 h by staining

for NS5A as described above.

Insulin secretion from isolated rat pancreatic islets

Pancreatic islets were isolated from four male diabetic

Goto-Kakizaki (GK) rats, a model of type 2 diabetes [22],

and four male control Wistar rats, all 2–3 months old.

p glucose levels (non-fasting) at the time of killing were

10.4 ± 1.4 and 6.8 ± 0.5 mM, respectively, (p \ 0.01).

Islets were isolated by collagenase digestion and main-

tained overnight in tissue culture (RPMI1640 with

11.1 mM glucose, 10 % FBS and antibiotics) prior to

incubations. After a pre-incubation for 45 min in Kreb-

Ringer bicarbonate buffer (KRb) with 2 mg/ml bovine

albumin and 3.3 mM glucose, batches of three islets were

incubated for 1 h in 300 ll of KRb with albumin, either 3.3

or 16.7 mM glucose and with addition of 100 nM, 1 lM or

10 lM of the camptide bioportide. After incubations, ali-

quots were taken for radioimmunoassay of insulin. Results

are expressed as lU insulin released per islet/h, calculated

as mean ± SEM of four experiments, where the mean of

each experiment is based on quadruplicates (four batches).

Measurement of cAMP content

As previously described [18, 23], cAMP assays utilized

confluent monolayers of ECV304 cells in 24-well plates.

Cells were washed twice with M199 medium and incu-

bated for 15 min at 37 �C in balanced salt solution (BSS)

containing 0.5 mM isobutylmethylxanthine. Peptides were

added for further periods as indicated and reactions ter-

minated by washing cell monolayers with ice-cold 70 %

(v/v) ethanol. Cytoplasmic contents were extracted by

scraping cells in ice cold 70 % ethanol (0.5 ml/well) and

stored on ice for a further 1 h. Supernatants were separated

from cell debris by centrifugation and the pelleted material

re-extracted with 70 % ethanol. Supernatants were com-

bined and evaporated to dryness under vacuum. Samples

were dissolved in buffer (50 mM Tris, 4 mM EDTA; pH

7.5) and cAMP measured using Biotrak assay kits

(Amersham) according to the manufacturer’s instructions.

Chemotaxis assays

BAECs were seeded at 7.3 9 104 cells/ml under 100 ll of

serum-poor medium (SPM, complete medium with 2.5 %

v/v FBS) onto the porous membrane (Costar; 8-lm pore

filter) of transwell inserts plated into a 24-well plate. The

wells with inserts contained basal medium supplemented

with 0.1 % FBS with or without nosangiotide (0.1–10 lM)

in the presence or absence of 25 ng/ml recombinant bovine

basic fibroblast growth factor (FGF-2; R&D systems,

Minneapolis, MN). After 20 h incubation, the cells which

did not migrate on the upper surface of the membrane were

removed with a cotton swab soaked with PBS then wiped

with dried cotton swab. Cells which migrated were fixed

with 4 % paraformaldehyde, left to air dry for cell staining

with Giemsa, then counted with an optical microscope.

Reported data were derived from three independent

experiments performed in duplicate.

Cell proliferation assays

BAECs were seeded in complete medium at a concentra-

tion of 4 9 104 cells/ml at 500 ll per well of a 24-well

plate. After 4 h incubation for cell attachment, the com-

plete medium was renewed with SPM, in which the cells

grew at a significantly reduced rate for BAEC, with addi-

tion or not of nosangiotide (0.01–10 lM) in the presence or

absence of 25 ng/ml FGF-2. For each experimental con-

dition, endothelial cells were treated in duplicate for

BAEC. After 72 h incubation, cells were washed with PBS

without Ca2? and Mg2?, detached with trypsin then

counted using a Coulter counter (Coulter Electronics,

Hialeah, FL). Reported data were derived from three

independent experiments performed in triplicate.

Tube formation assays

BAECs at a concentration of 2 9 106 cells/ml were mixed

in equal volume with growth factor-reduced Matrigel

(10 mg/ml; BD Biosciences, San Jose, CA) with or without

nosangiotide at concentrations varying between 0.01 and

100 lM in the presence or absence of 25 ng/ml FGF-2.

Half the volume of the mixture was put under a spot shape

into 48-well plates and each experimental condition was

performed in duplicate. The gel mixed with cells was

allowed to polymerize for 1 h at 37 �C. After polymeri-

zation, each spot of Matrigel with cells was bathed in

500 ll of complete medium for 24 h. The cells were then

fixed with 4 % paraformaldehyde for 10 min. To quantify

responses, closed areas per field were recorded as indicated

in Fig. 7b.

Chorioallantoic membrane (CAM) assays

The influence of nosangiotide on angiogenesis in vivo was

determined in the chick chorioallantoic membrane (CAM)

assay as previously described [29]. Fertilized hens eggs

were washed and incubated at 37 �C, without turning. To

expose the CAM, a window was created in the shells of

4 day-old fertilized eggs. On day 8, a 2-mm methylcellu-

lose pellet (5 ll of 1 % sterile methylcellulose; 4,000

centipoises, Sigma) containing either no additions (con-

trol), 200 ng FGF-2 (R&D Systems), 0.5 pmol of

J. Howl et al.

123


nosangiotide (compound control) or both FGF-2 and

0.005–0.5 nmoles nosangiotide, was applied to the cho-

rioallantoic membrane. The degree of resultant

angiogenesis was determined on day 10. The angiogenic

reaction was scored as: 0-negative; 0.5-change in vessel

architecture but not directed to the point of sample appli-

cation; 1-partial spokewheel (1/3 of circumference exhibits

directional angiogenesis); 2-partial spokewheel (2/3

spokewheel), 3-full spokewheel and 4-strong and extensive

full spokewheel. This approach enabled calculation of

accumulated responses for the test samples in each group.

Statistical analysis was performed using the Mann–Whit-

ney U test.

For photography, 2 ml of a 50 % (v/v) aqueous emulsion

of paraffin oil, containing 2 % (v/v) Tween 80, was injected

under the membrane at the site of sample application and

the angiogenic reaction photographed under a Leitz bin-

ocular dissecting microscope and indirect fiber-optic

illumination.

Analyses of the rate of GTPcS binding

As previously described [22], we determined the influence

of camptide on the initial rate of binding of [35S] GTPcS to

rat brain cortical membranes. Membranes (0.5 mg/ml

protein) were incubated with 5 mM MgCl2, 1 mM dithio-

threitol, 150 mM NaCl, 1 lM GDP and 0.5 nM [35S]

GTPcS at 13 �C in TE buffer (10 mM Tris–

HCl ? 0.1 mM EDTA) for 3 min in the presence or

absence of camptide. The unbound [35S] GTPcS was

washed out by rapid filtration of the reaction mixture

through Millipore GF/C glass-fiber filters under vacuum

three times with 5 ml of TE buffer. After extraction of the

radioactive material overnight in 20 ml of Emulsifier-Safe

(Packard, USA) scintillation liquid, radioactivity was

determined using an LKB 1214 Rackbeta liquid scintilla-

tion counter. Blank values were determined by the same

procedure in samples in which the membranes were

replaced with buffer.

Uterotonic test in vitro

The uterotonic test was performed in vitro using strips of

Wistar rat uterus in the absence or presence (1 mM) of

Mg2? ions [24–26]. Estrus was induced in rats by the

injection of estrogen 48 h before the experiments were

performed. After decapitation, the uterine horns were

excised, longitudinally cut, placed into a bathing chamber

and hooked up to a mechanical transducer to monitor

contractions. Doses of standard oxytocin (in the presence

or absence of nosangiotide) or the peptide alone were

added to the uterus in the organ bath. The height of the

single isometric contraction of a uterine strip was

measured. Experiments were performed using uteri from a

total of four different animals.

Pressor assay in situ

The effect of peptides on blood pressure was analyzed

using phenoxybenzamine-treated male Wistar rats [26, 27]

under urethane anesthesia. Arginine vasopressin (AVP)

was used as a standard pressor agent. Changes of the

arterial blood pressure were registered. Concentration–

response curves (single administration into vena femoralis)

were constructed for AVP and nosangiotide alone and for

AVP in the presence of nosangiotide. When co-adminis-

tered, nosangiotide was delivered 1 min prior to the

injection of vasopressin. Both peptides were tested in 3–5

independent experiments.

Handling of the experimental animals was performed

under supervision of the Ethics Committee of the Academy

of Sciences according to §23 of the law of the Czech

Republic no. 246/1992.

Results

Prediction of cell-penetrating peptides using QSAR

analyses

As illustrated herein (Fig. 1), a consideration of the

sequences of biologically active cell-penetrating peptides

indicates that two conceptually different organizations are

possible. Based upon the message-address hypothesis [9], a

majority of studies that employ synchnologically organized

CPPs combine an active cargo (message) with an inert CPP

(address) as a tandem linkage (Fig. 1). However, as con-

firmed by the studies reported herein, rhegnylogically

organized bioactive peptides are also possible (Fig. 1). We

have introduced the term biportide [10] to distinguish this

type of CPP from the more usual inert CPPs employed

solely as pharmacokinetic modulators. Moreover, and as

indicated in Table 1, bioportides are capable of regulating

a diverse plethora of biological processes.

The QSAR prediction algorithm developed by Hallbrink

and coworkers [12], previously utilized to identify cryptic

CPPs and bioportides within the human cytochrome c pro-

tein [8], was employed to identify putative CPPs within the

primary sequence of the human calcitonin type 1 receptor

(hCTR-1) and endothelial nitric oxide synthase (eNOS;

Fig. 2). In contrast to our previous studies with cytochrome

c [8] and G protein-coupled receptors [14], these QSAR

analyses (Fig. 2) were restricted to previously identified

functional domains within target proteins [15, 16]. Thus,

we identified two peptides, hCTR-1174-193 (hereafter

referred to as camptide) and eNOS492-507 (hereafter


123


referred to as nosangiotide) that were predicted to be highly

probable CPPs and, therefore, candidate bioportides.

Translocation and intracellular distribution

of bioportides

Confocal microscopy employing fluorophore-conjugated

CPPs and bioportides is a convenient methodology to

determine the intracellular distribution of peptides fol-

lowing translocation [1–4, 7, 8]. As indicated (Fig. 3),

there was an obvious time-dependent distribution to the

intracellular distribution of nosangiotide. Thus, at earlier

time periods (45 min; Fig. 3a) a punctate distribution of

rhodamine fluorescence indicated that nosangiotide was

mostly located within a discrete lyso-endosomal compart-

ment. Moreover, nosangiotide is obviously capable of

accessing other cellular compartments since, at later time

periods (80 min; Fig. 3b), fluorescence was distributed

within both cytoplasmic compartments and the nucleus.

This change in distribution may represent the release of

either intact peptide or degradation products from endo-

somal compartments. However, it is noteworthy that

biological responses to exogenously applied nosangiotide

are observed at peptide concentrations much lower than

those used in these experiments (c.f. Fig. 7a).

In contrast, fluorescein-conjugated camptide assumed a

vesicular cytoplasmic distribution and minor plasma

membrane association following translocation, and was

entirely excluded from the nucleus (Fig. 3c).

Bioportides are without influence upon cellular

viabilities

Since membrane perturbations leading to cellular necrosis

can occur at high concentrations of some CPPs, it was

necessary to establish that the observed intrinsic activities

of bioportides were not compromised by cellular cytoxic-

ity. MTT conversion assays were therefore employed to

determine whether bioportides adversely influenced cellu-

lar viabilities [7]. These investigations determined that, at

concentrations between 0.1 and 30 lM and over a period of

24 h and up to 72 h, nosangiotide exposure was without

influence upon the viability of ECV304 bladder cancer

cells, human dermal microvascular endothelial cells,

U373MG astrocytic tumor cells, H630WT colorectal cancer

Fig. 1 Theoretical molecular structures of bioportides conforming to

the sychnologic or rhegnylogic pattern of organization. In the most

extreme case a sychnologically organized bioportide could include a

polyarginine CPP such as R8 (address) joined to a bioactive peptide in

which every amino acid side-chain is a message pharmacophore. The

linker joining these two distinct peptides as a tandem structure can be

a conventional peptide bond, a disulphide or other covalent structure.

Moreover, non-covalent complexes between CPPs and larger macro-

molecules could also be developed as bioportides. In the rhegnylogic

organization the pharmacophores for penetration (address) and

bioactivity (message) are discontinuously distributed within a single

peptide. It is most probable that a majority of rhegnylogically

organized bioportides contain amino acid side chains that play either a

dual role in both penetration and bioactivity and others that contribute

little if anything to either of these distinct processes. This type of

organization is illustrated here as theoretical bioportide

Table 1 The functional diversity of bioportides

Sequence Target Activity

Rhegnylogically organized

Nosangiotide ND Anti-angiogenic

Camptide G proteins cAMP modulation

Cyt c77–101 [8] ER Apoptogenic

BIP [30] BAX Anti-apoptotic

Mouse PrP1–28 [31] Prion proteins Anti-prion infection

AT1AR304–318 [14] G proteins Blood vessel

contraction

Mitoparan [7] VDAC Apoptogenic

Sychnologically organized

Tat-acp-TrkA666–676 [32] TrkA TrkA antagonism

Hph-1-ctCTLA-4 [33] TcR signaling Negative regulator

of TcR signaling

STAT-6-IP [34] STAT-6 Inhibitor of Th2

cytokine

production

Tat48-60-P10 [11] PCNA Apoptogenic

TAT-MAK19 [35] ND Apoptogenic

Antp-MEK1 [36] ERK Inhibitor of ERK2

activation

TP10-Gi3a346–355 [37] ND MAP kinase

activation

Nup153-Cyt c [8] Nuclear pore

complex

Apoptogenic

AT1AR angiotensin type 1A receptor, BAX Bcl-2-associated X pro-

tein, BIP Bax-inhibiting peptide, CTLA-4 cytotoxic T-lymphocyte

antigen 4, ER endoplasmic reticulum, ERK extracellular signal-reg-

ulated kinase, MEK mitogen activated protein kinase, ND not

determined, Nup153 nucleoporin 153, PCNA proliferating cell nuclear

antigen, PrP prion protein, STAT-6 signal transducer and activator of

transcription 6, Tat Trans-activator of transcription, TcR T cell

receptor, TP10 transportan 10, TrkA neurotrophic tyrosine kinase

receptor type A, VDAC voltage-dependent anion channel

J. Howl et al.

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cells and HCT113 colon cancer cells. Similarly, and over a

24 h period, camptide (0.1–30 lM) had no influence upon

the viability of ECV304 cells. Furthermore, no evidence of

cytotoxicity was observed when employing either biopor-

tide in the variety of cellular assay systems described

herein.

Biological activities of camptide

Camptide modulates intracellular cAMP concentration

For these assays, we employed ECV304 bladder cancer

cells that express the type 1 hCTR functionally coupled to

Gs [23]. As previously reported [23], stimulation of these

cells with salmon calcitonin (sCT) induced a robust

increase in [cAMP]i. As indicated (Fig. 4a), exogenous

application of camptide (1 lM) stimulated cAMP synthesis

when added alone, and also augmented hCTR-stimulated

cAMP synthesis.

Camptide directly activates G proteins in rat brain cortical

membranes

As reported in Fig. 4b, camptide markedly increased the

initial rate of GTPcS binding to G proteins in rat cortical

membranes. This activation of G proteins was character-

ized by the following parameters: maximal activation =

151.4 ± 4.9 %; Hill coefficient (nH) = 1.3 ± 0.4; EC50 =

2.5 ± 1.4 lM. These data and studies of insulin secretion

(see below), indicate that the most likely intracellular

target of camptide is the Gs subtype of heterotrimeric G

protein.

Camptide modulates viral infectivity

Our previous studies have revealed an essential role for

cAMP activated protein kinase A in HCV infection where

forskolin stimulation of cAMP enhanced HCV entry and

promoted the levels of extracellular infectious virus [28].

Given our earlier observation that camptide stimulates

cAMP synthesis (Fig. 4), we investigated the effect of this

peptide on HCV infection. Treatment of Huh-7.5 cells with

increasing concentrations of camptide enhanced the

infectivity of two different HCV strains J6/JFH and JFH-1

(Fig. 5a). Consistent with the enhanced infectivity of both

viral strains for Huh-7.5 cells, we observed a significant

increase in the levels of extracellular infectious virus

(Fig. 5b). These data clearly demonstrate a biological

effect that is dependent upon camptide stimulated cAMP

synthesis.

KLTTIFPLNWKYRKAL ( insert)

RKLTTIFPLNWKYRKALSLG Camptide

1 2 3 4 5 6 7

N

C

Sequence CPP IndexRKKTFKEVANAVKISASLMG 1RKKTFKEVANA 3RKKTFKEVANAVK 3RKKTFKEVANAVKI 3RKKTFKEVANAVKISA 3 Nosangiotide

a b

Fig. 2 Identification and sources of bioportides described in this

study. As illustrated in a, the bioportide camptide consists of a 16 AA

insert expressed by hCTR-1 (KLTTIFPLNWKYRKAL) flanked by

additional residues from the native sequence predicted to be a highly

probable (score 3) CPP using the QSAR algorithm. b (modified from

[16]) Indicates the loop of eNOS that is bound by calmodulin. The

full-length 20 AA peptide described by Aoyagi and coworkers [16]

was predicted to be only a possible (score 1) CPP. Thus, nosangiotide

represents the longest homologue (16 AA) of this structural loop

predicted to be highly probable CPP. Both camptide and nosangiotide

were synthesized as C-terminal amidated peptides


123


Camptide modulates insulin secretion

As indicated in Fig. 6, in both GK and control Wistar rat

islets, insulin secretion at 3.3 mM glucose was significantly

increased about twofold by 0.1 and 1 lM camptide, while

10 lM did not stimulate secretion. As expected, high

glucose concentration (16.7 mM) augmented insulin

release more in Wistar than in GK rat islets. At 16.7 mM

glucose camptide did not influence insulin secretion except

in GK islets where there was an inhibition at 10 lM.

Fig. 3 Intracellular distribution of bioportides nosangiotide and

camptide. Human dermal microvascular endothelial cells were

incubated with rho-nosangiotide (5 lM) for 45 min (a) and 80 min

(b) then visualized localization by live confocal cell imaging.

a Earlier time points indicate that rho-nosangiotide assumes a

predominant vesicular distribution throughout the cytoplasm. b Fol-

lowing 80-min incubation, however, rho-nosangiotide becomes

evenly dispersed throughout the cytoplasm and nucleus. Both

temporal observations are represented as fluorescent images alone

[a(i) and b(i)] and superimposed onto images taken using differential

interference contrast [a(ii) and [b(ii)] so as to facilitate subcellular

localization of the peptide. c Live confocal cell-imaging analysis

further demonstrated that fluo-camptide translocates the plasma

membrane of ECV304 cells to assume a minor degree of plasma

membrane staining but predominantly a cytoplasmic vesicular

distribution. ECV304 cells were treated with fluo-camptide (5 lM)

for 60 min [c(i)] and CellMaskTM deep red plasma membrane stain

(Invitrogen) at 5 lg/ml for the final 5 min of incubation [c(ii)]. c(iii)represents merged images

J. Howl et al.

123


In similar studies using 30 mM KCl and 0.25 mM

diazoxide to depolarize beta cells and maintain the open

state of ATP-dependent K? channels, camptide had no

influence upon insulin release from GK islets at 3.3 mM

glucose. Under these conditions insulin release in the

presence of camptide (1 lM) was 39.2 ± 5.8 lU/islet/h

(n = 4) compared with normal levels of 35.5 ± 6.2 lU/

islet/h (n = 4) in the absence of peptide. These data

indicate that camptide does not exert any modulatory

action upon the distal component of the exocytotic

pathway.

Biological activities of nosangiotide

Lack of influence of nosangiotide on NO-mediated

physiological processes

Uterotonic assays indicated that nosangiotide possessed no

endogenous activity at maximal concentrations of 15 lM

either in the absence and presence of magnesium ions.

Moreover, a 1 min or 10 min pre-incubation with nosan-

giotide did not inhibit contractions evoked by standard

doses of oxytocin.

A B C D E

0

1

2

3

4

5

6

7

8

9

10

11

A sCT (100 nM)B sCT (100 nM) + camptide (1μM)C sCT (1μM)D sCT (1μM) + camptide (1μM)E camptide (1 μM)

*

cAM

P f

orm

atio

n (

fold

/bas

al)

80

90

100

110

120

130

140

150

160

-8 -7 -6 -5 -4

log { [camptide (M) }

Init

ial r

ate

of

[35S

]GT

Pγ S

bin

din

g (

% b

asal

)

a b

Fig. 4 Camptide modulates intracellular cAMP concentration.

a Camptide (1 lM) independently stimulates cAMP formation in

ECV304 cells (column E) and significantly (p \ 0.05, Mann–

Whitney U test, one-tailed) augments hCTR1-stimulated cAMP

formation by the application of 100 nM salmon calcitonin (sCT)

(c.f. columns A and B). Augmentation of sCT (1 lM)-induced cAMP

formation by camptide (1 lM) (c.f. columns C and D) was not

calculated to be of statistical significance. Asterisk indicates a

significant difference between A and B. b Camptide directly activates

heterotrimeric G proteins as measured by the initial rate of binding of

[35S] GTPcS to rat brain cortical membranes. Basal values (100 %)

represent the initial rate of [35S] GTPcS binding of 93 fmol/min/mg

protein. Each point is the mean value of three independent determi-

nations, bars are SEM. Curves were obtained by fitting the sigmoidal

dose–response equation with variable slope (Hill coefficient) to the

experimental points using nonlinear regression algorithm (PRISM4

computer program)


123


Co

ntr

ol Mµ

1

Mµ5

Mµ10

Co

ntr

ol Mµ

1

Mµ5

Mµ10

0

50

100

150

200

250J6/JFH

JFH-1

camptide camptide

Rel

ativ

e H

CV

cc In

fect

ivit

y

a b

Co

ntr

ol Mµ

1

Mµ5

Mµ10

Co

ntr

ol Mµ

1

Mµ5

Mµ10

0

100

200

300

400

500

600

J6/JFH

JFH-1

camptide camptide

Rel

ativ

e E

xtra

cellu

lar

Vir

us

Infe

ctiv

ity

Fig. 5 Camptide modulates hepatitis C virus infectivity. a Huh-7.5

cells were incubated with camptide (1, 5, 10 lM) for 1 h prior to

incubation with HCV J6/JFH (black bars) or JFH-1 (grey bars) for

1 h in the continuous presence of camptide. HCV-infected cells were

fixed after 48 h and infectivity quantified as described, b J6/JFH

(black bars) or JFH-1 (grey bars) infected Huh-7.5 cells were

incubated with increasing concentrations of camptide (1, 5, 10 lM)

for 1 h. Cells were extensively washed, supernatant was collected

after 1 h, and virus infectivity in the harvested supernatant determined

by infection of naive Huh-7.5 cells. Infectivity is expressed relative to

untreated control cells and represents the mean ± SEM for three

replicate infections

Control Mµ0.1

Mµ

1 Mµ

10

Control Mµ0.1

Mµ

1 Mµ

10

0

10

20

30

40

50

60

70

80

90

100

110

120GK islets

Wistar islets

**

U in

sulin

rel

ease

d p

er is

let/

h

a b

Control Mµ0.1

Mµ

1 Mµ

10

Control Mµ0.1

Mµ

1 Mµ

10

0

5

10

15

20

25

30

35

40

45GK islets

Wistar islets

*

* *

*

camptide camptidecamptide camptide

µ

µ

U in

sulin

rel

ease

d p

er is

let/

h

Fig. 6 Camptide modulates

insulin secretion from isolated

rat pancreatic islets. a Islets

were incubated with 3.3 mM

glucose. At low glucose

concentrations, camptide

significantly enhances insulin

secretion compared to 3 mM

glucose alone (*p \ 0.05,

Student’s paired t test). b Islets

were incubated with 16.7 mM

glucose. At high glucose

concentrations, camptide

significantly suppresses insulin

release compared to 16.7 mM

glucose alone (**p \ 0.05,

Student’s paired t test)

J. Howl et al.

123


In vivo assays indicted that nosangiotide possessed no

intrinsic pressor activity at maximal concentrations of

0.3 mg/kg. Furthermore, nosangiotide displayed no antag-

onistic activity against standard doses of vasopressin

(6.6 9 10-5 mg/kg and 1.5 9 10-5 mg/kg).

Nosangiotide exhibits potent anti-angiogenic activities in

vitro

As indicated in Fig. 7a, exogenous application of nosan-

giotide abrogated many biological features of angiogenesis,

Thus, in a concentration-dependent manner, nosangiotide

inhibited the proliferation (Fig. 7a, A), chemotaxis

(Fig. 7a, B) and tube-forming capacity (Fig. 7a, C) of

primary BAECs induced by FGF-2 (25 ng/ml). These data

suggest a common locus of action of nosangiotide that can

be negatively modulated at concentrations of peptide

within the nanomolar range. Tri-dimensional Matrigel

studies further demonstrated the ability of nosangiotide

to inhibit FGF-2-induced tube formation of primary

BAECs (Fig. 7b). While FGF-2-stimulated BAECs gave a

mean ± SEM of 194.2 ± 38.5 closed areas per field,

there was statistically no significant difference (p [ 0.5)

between control cells (non-FGF-2-treated, mean ±

SEM = 79.5 ± 22.6) and FGF-2-treated cells in the pres-

ence of 10 lM nosangiotide (mean ± SEM = 99.2 ±

27.1). Statistical analysis was performed using the non-

parametric Mann–Whitney Test and from three indepen-

dent experiments performed in triplicate.

Nosangiotide inhibits angiogenesis in vivo

As indicated in Fig. 8, exogenous application of nosangi-

otide completely abrogated the ability of FGF-2 to induce

revascularization in the CAM assay. Furthermore, similar

effects were observed when nosangiotide was added in

aliquots of 50–5,000 ng mixed with FGF-2 (200 lg). A

summary of these data is provided in Table 2.

Discussion

The results presented herein corroborate previously pub-

lished reports [7, 8, 11, 30–37], including examples in

Table 1, to indicate that the intrinsic bioactivities of CPPs

can be variously exploited to achieve a potent and selective

influence on cell biology. As a majority of studies seek to

employ CPPs as inert carrier vectors, we have introduced

the term bioportide [10] to distinguish this novel class of

bioactive CPP. We anticipate that the further study of

bioportides will identify agents capable of modulating the

activity of other generic drug targets. Thus, in addition to

the many recent advances in CPP vector technologies in

medicine and biology (reviewed in [38]), it is certain that

bioportides will also be utilized as innovative research

tools and further developed as diagnostic and therapeutic

agents. Indeed, as exemplified by our studies with nosan-

giotide, some bioportides display potent and

therapeutically beneficial activities that could be further

improved by a variety of strategies alluded to below. Other

biportides such as camptide that modulate common second

messenger systems display a wider spectrum of biological

activities. Thus, by virtue of an interaction with G proteins

and promotion of cAMP synthesis, camptide is able to

modulate both insulin secretion from pancreatic islets and

hepatitis C infectivity in hepatoma cells. Clearly, there is

enormous potential to develop other bioportides that can

readily translocate into eukaryotic cells to influence dis-

crete protein targets.

A variety of strategies can and have been employed to

identify polycationic CPP sequences. For example, studies

by Futaki and coworkers [39] revealed that a majority (nine

of ten) of arginine-rich basic sequences from human RNA-

binding proteins were efficient CPPs. Moreover, these

peptides were identified simply by analogy to HIV-1 Tat

(48–60) a widely employed CPP vector derived from a

viral transcription factor [3]. Moreover, other cryptic CPPs

within the primary sequences of target proteins can be

predicted using a QSAR algorithm [8, 12–14]. In our hands

this algorithm has proven particularly useful and has

enabled the identification of CPPs within GPCRs [14] and

cytochrome c [8]. Similar computational analyses also

supported the identification of both nosangiotide and

camptide as described herein. Moreover, such approaches

are currently restricted only to the prediction of cellular

penetration rather than bioactivities. Perhaps as we learn

more of the molecular mode of action of bioportides, and

begin to understand the peptide-protein interactions that

facilitate their bioactivities, additional computational

methodologies might also be gainfully employed (reviewed

in [40]).

As recently summarized [41, 42], there is increasing

recognition that CPP vectors can often display unwanted

activities by virtue of their abilities to interfere with a

variety of cellular process. Particularly at higher concen-

trations ([10 lM), some CPPs can adversely modify

membrane integrity to compromise cellular viability [43].

Other studies suggest that the activity of a bioactive cargo

can be profoundly influenced by the CPP to which it is

sychnologically conjugated [8, 44]. Though there remains

much debate as to the precise mechanisms of cellular

penetration (c.f. [45] and [46]), it is obvious that larger

macromolecular complexes delivered by CPP technologies

are mostly trapped within intracellular endosomes of var-

ious types [47]. Thus, there would appear to be a significant

advantage to the employment of relatively smaller,


123


rhegnylogically organized bioportides, including mitoparan

[7], camptide and nosangiotide, which escape endosomal

entrapment to specifically target a variety of intracellular

organelles and compartments. Of course, conjugation of

bioportides to other bioactive agents, including small drugs

such as methotrexate and temozolomide, is also feasible

100 µm

600

800

1000

1200

1400

1600

-8 -7 -6 -5

log { [nosangiotide] (M) }

nu

mb

er o

f m

igra

ted

cel

ls

60,000

70,000

80,000

90,000

-9 -8 -7 -6 -5


cell

nu

mb

er

60

80

100

120

140

160

180

200

220

240

-9 -8 -7 -6 -5 -4


mea

n o

f cl

ose

d a

reas

per

fie

ld

(A) (B) (C)

Examples of closed areas

(A)

(B) (C)

(a)

(b)

J. Howl et al.

123


and could expand the CPP-based therapeutic arsenal for

serious human disease including cancer [38, 48–50].

The mechanisms of action of bioportides are highly

variable and, in some cases, presently unknown. Moreover,

our contention that both rhengnylogic and sychnologic

organizations (Fig. 1) are compatible with the general

bioportide concept is also supported by many data sum-

marized in Table 1. Thus, in addition to camptide and

nosangiotide reported herein, a growing number of publi-

cations have described monomeric CPPs that are

intrinsically bioactive (Table 1 and references therein).

Further investigations to more precisely determine the

influence of individual pharmacophores within these bio-

portides are clearly warranted and necessary to support our

current theoretical exposition of bioportide topologies

illustrated in Fig. 1.

The bioportide camptide includes a 16 AA insert from

the first intracellular loop of type 1 human calcitonin

receptor (hCTR-1). This structural motif completely

inhibits the inositol phosphate (IP) signal transduction

pathway when hCTR-1 is stimulated by calcitonin. In

contrast, agonist activation of hCTR-2, a receptor lacking

the 16 AA splice insert, stimulates both cAMP and IP

signaling pathways [51, 52]. Thus, our data suggest that the

actual biological task of this short sequence is to selectively

bind and activate Gs enabling the specific elevation of

cAMP in preference to IP metabolism. As previously

suggested [51, 52], such a mechanism, the result of alter-

native gene splicing, may serve to differentially regulate

CT signaling via competing cAMP and IP signaling

pathways.

Our studies confirm that, by a receptor-independent

mechanism, camptide is able to modulate diverse biologi-

cal processes that are specifically regulated by

heterotrimeric G proteins. Thus, by virtue of an interaction

with G proteins and promotion of cAMP synthesis,

camptide can modulate both insulin secretion and viral

infectivity. It is most likely that the stimulation of insulin

secretion in both healthy and diabetic rats is due to

enhancement of cAMP content. Indeed, although GK rat

islets exhibit a defective insulin response to high glucose

concentrations, the responses to cAMP increasing agents

such as forskolin and glucose-like-polypetide-1 are not

decreased [22]. Moreover, it is likely that, in common

with other bioactive peptides including mastoparan ana-

logues, camptide is able to interact with different

intracellular loci, including multiple G proteins and other

targets, in a concentration-dependent manner. Such a

mechanism is a possible explanation for the reduced

secretory potency of camptide at higher concentrations as

reported herein.

The mechanism of action of nosangiotide is, to date, less

clearly defined though clues are emerging. As reported

herein, this bioportide has no impact upon NO-mediated

phenomena in vivo. Thus, nosangiotide is without influence

upon resting blood pressure in rodents, neither does it

modulate the pressor activity of vasopressin. Similarly,

nosangiotide has no influence upon oxytocin-induced

myometrial contraction. However, nosangiotide is quite

clearly a very potent anti-angiogenic agent. Our pre-

liminary investigations (Jones and Howl, unpublished)

have identified at least eight genes that are modulated at the

transcriptional level by exogenous nosangiotide treatment.

It is also interesting to note that, following longer incu-

bation times with nosangiotide, we have observed minor

changes in endothelial cell morphology. We are therefore

currently employing a proteomics approach to identify

cognate protein binding partners for nosangiotide in cel-

lular extracts. Theoretically at least, bioportides including

nosangiotide could bind a spectrum of intracellular protein

targets and it is highly likely that some bioactivities are the

result of peptides being able to modulate protein–protein

interactions. Such activities could result from peptides

directly occupying functional domains or from an indirect

allosteric modulation [40].

There are numerous approaches by which the pharma-

cokinetic and/or pharmacodynamic properties of

bioportides can be further improved. CPPs in general show

little cell-type selectivity. Thus, it may be desirable to add

a homing peptide sequence, usually as a tandem extension,

to direct chimeric peptides to appropriate cell or tissues.

Indeed, numerous homing sequences have been described

[53, 54], and a major goal of such studies is to target

therapeutic agents to human disease sites, particularly

cancer. Another approach is to engineer an enzymatically

labile site into a non-permeable but ‘activatable’ peptide

that is locally cleaved to generate a functional CPP [55].

A variety of other structural modifications, particularly

main-chain peptidomimetics, (reviewed in [56]), are also

feasible in the design of CPPs and bioportides that maintain

the capacity to recognize appropriate molecular topology

but are intrinsically much more resistant to proteolysis. It

should be noted that although a variety of retro and retro-

inverso analogues of the tat CPP have proven highly effi-

cient delivery vectors both in vitro and in vivo (reviewed in

Fig. 7 Nosangiotide inhibits the biological features of angiogenesis

in vitro. (a) FGF-2-induced tube formation is inhibited by nosangi-

otide. Representative photomicrographs show the organization of

primary cultured bovine aortic endothelial cells into tube-like

structures in tri-dimensional Matrigel culture from control cells

(A) and cells treated with 25 ng/ml FGF-2 (B) after 24 h of incubation.

The addition of 10 lM nosangiotide (C) inhibited FGF-2-induced

tube formation. The inset presents two examples of closed areas as

parameters of quantification. (b) Exogenous application of nosangi-

otide inhibits FGF-2 (25 ng/ml)-induced proliferation (IC50 = 83.7

nM, A), chemotaxis (IC50 = 38.2 nM, B) and tube formation (IC50 =

509 nM, C) of primary bovine aortic endothelial cells

b


123


Fig. 8 Nosangiotide attenuates FGF-2-induced angiogenesis of the

chorioallantoic membrane. a Carrier control; b FGF-2 (200 ng);

c nosangiotide (0.5 nmole); d FGF-2 (200 ng) with nosangiotide

(0.5 nmole); e FGF-2 (200 ng) with nosangiotide (0.05 nmole);

f FGF-2 (200 ng) with nosangiotide (0.005 nmole). Arrow indicates

sample membrane. All photographs are at 925 magnification

Table 2 The influence of

nosangiotide on FGF-2

stimulated angiogenesis in the

CAM assay

Compound Angiogenic response of compound alone:

median response (m) and significance

(p) respect to the control (n = 5)

Inhibition of FGF-induced angiogenic

response. Median response (m) and

significance (p) versus FGF-2 alone

(n = 5)

Control m = 0 ND

FGF-2 (200 ng) m = 3; p = 0.0079 ND

Nosangiotide

(0.5 nmole)

m = 0; p = 1 m = 0; p = 0.0079

Nosangiotide

(0.05 nmole)

ND m = 0; p = 0.0079

Nosangiotide

(0.005 nmole)

ND m = 0.5; p = 0.0079

J. Howl et al.

123


[57]), similar modifications of other CPPs may be com-

promised by increased cellular cytotoxicity [58].

Conclusions

The present study identifies and characterizes in detail

some of the biological properties of two novel peptides,

camptide, and nosangiotide. Clearly, these bioportides are

able to modulate the functions of intracellular signaling

pathways following their efficient translocation. We con-

tend that, as a class of bioactive CPP, bioportides offer

enormous potential for the study and manipulation of both

normal and pathophysiological processes mediated by

intracellular proteins that are inaccessible to cell imper-

meable agents. Thus, bioportides could be utilized as

diagnostic and therapeutic agents, targeting intracellular

proteins that are intractable to moieties incapable of

intracellular translocation.

References

1. Langel U (ed) (2011) Cell-penetrating peptides (Methods in

molecular biology 683) Humana Press, New York

2. Derossi D, Joliot AH, Chassaing G, Prochiantz A (1994) The

third helix of the Antennapedia homeodomain translocates

through biological membranes. J Biol Chem 269:10444–10450

3. Vives E, Brodin P, Lebleau B (1997) A truncated HIV-1 TAT

protein basic domain rapidly translocates through the plasma

membrane and accumulates in the cell nucleus. J Biol Chem

272:16010–16017

4. Pooga M, Hallbrink M, Zorko M, Langel U (1998) Cell pene-

tration by transportan. FASEB J 12:67–77

5. Deshayes S, Konate K, Aldrian G, Heitz F, Divita G (2011)

Interactions of amphipathic CPPs with model membranes. In:

Langel U (ed) Cell-penetrating peptides (Methods in Molecular

Biology 683). Humana Press, New York, pp 41–56

6. Verdurmen WPR, Brock R (2011) Biological responses towards

cationic peptides and drug carriers. Trends Pharamcol Sci

32:116–124

7. Jones S, Martel C, Belzacq-Casagrande A, Brenner C, Howl J

(2008) Mitoparan and target-selective chimeric analogues:

membrane translocation and intracellular redistribution induces

mitochondrial apoptosis. Biochim Biophys Acta 1783:849–863

8. Jones S, Holm T, Mager I, Langel U, Howl J (2010) Character-

isation of bioactive cell-penetrating peptides from cytochrome c:

protein mimicry and the development of a novel apoptogenic

agent. Chem Biol 17:735–744

9. Portoghese P (1989) Bivalent ligands and the message-address

concept in the design of selective opioid receptor antagonists.

Trends Pharmacol Sci 10:230–235

10. Howl J, Jones S (2008) Proteomimetic cell-penetrating peptides.

Int J Pept Res Ther 14:359–366

11. Baker RD, Howl J, Nicholl ID (2007) A sychnological cell-

penetrating peptide mimic of p21WAF1/CIP1 is pro-apoptogenic.

Peptides 28:731–740

12. Hallbrink M, Kilk K, Elmquist A, Lundberg P, Lindgren M, Jiang

Y, Pooga M, Soomets U, Langel U (2005) Prediction of cell-

penetrating peptides. Int J Pept Res Ther 11:249–259

13. Hansen M, Kilk K, Langel U (2007) Predicting cell-penetrating

peptides. Adv Drug Deliver Rev 60:572–579

14. Ostlund P, Kilk K, Lindgren M, Hallbrink M, Jiang Y, Budhina

M, Cerne K, Bavec A, Ostenson C-G, Zorko M, Langel U (2005)

Cell-penetrating mimics of agonist-activated G-protein coupled

receptors. Int J Pept Res Ther 11:237–247

15. Moore EE, Kuestener RE, Stroop SD, Grant FJ, Matthewes SL,

Brady CL, Sexton PM, Findlay DM (1995) Functionally different

isoforms of the human calcitonin receptor result from alternative

splicing of the gene transcript. Mol Endocrinol 9:959–968

16. Aoyagi M, Arvai AS, Tainer JA, Getzoff ED (2003) Structural

basis for endothelial nitric oxide synthase binding to calmodulin.

EMBO J 22:766–775

17. Ponten P, Macintyre EH (1968) Long term culture of normal and

neoplastic human glia. Acta Pathol Microbiol Scand 74:465–486

18. Brown J, Reading SJ, Jones S, Fitchett CJ, Howl J, Martin A,

Longland C, Michelangeli F, Dubrova YE, Brown C (2000)

Critical evaluation of ECV304 as a human endothelial cell model

determined by genetic analysis and functional responses: a

comparison with the human bladder cancer derived epithelial cell

line T24/83. Lab Invest 80:37–45

19. Blight KJ, McKeating JA, Rice CM (2002) Highly permissive

cell lines for subgenomic and genomic hepatitis C virus RNA

replication. J Virol 76:13001–13014

20. Lindenbach BD, Evans MJ, Syder AJ, Wolk B, Tellinghuisen TL,

Liu CC, Maruyama T, Hynes RO, Burton DR, McKeating JA,

Rice CM (2005) Complete replication of hepatitis C virus in cell

culture. Science 309:623–626

21. Evans MJ, von Hahn T, Tscherne DM, Syder AJ, Panis M, Wolk

B, Hatziioannou T, McKeating JA, Bieniasz PD, Rice CM (2007)

Claudin-1 is a hepatitis C virus co-receptor required for a late step

in entry. Nature 446:801–805

22. Ostenson C-G, Efendic S (2007) Islet gene expression and

function in type 2 diabetes; studies in the Goto-Kakizaki rat and

humans. Diabetes Obes Metab 9:180–186

23. Howl J, Mondszein RM, Wheatley M (1998) Characterization of

G protein-coupled receptors expressed by ECV304 human

endothelial cells. Endothelium 6:23–32

24. Holton P (1948) A modification of the method of Dale and

Lindlow for standardization of posterior pituitary extract. Br J

Pharmacol 3:328–334

25. Munsick R (1960) Effect of magnesium ion on the response of the

rat uterus to neurohypophysial hormones and analogues. Endo-

crinology 66:451–457

26. Slaninova J (1987) Fundamental biological evaluation. In: Lebl

M, Jost K, Brtnik F (eds) Handbook of neurohypophyseal hor-

mone analogs, vol I. CRC Press, Boca Raton, pp 83–107

27. Dekanski J (1952) The quantitative assay of vasopressin. Br J

Pharmacol 7:567–572

28. Farquhar MJ, Harris HJ, Jones S, Nielsen SU, Brimacombe CL,

Molina S, Toms GL, Maurel P, Howl J, van Ijzendoorn SVD,

Balfe P, McKeating A (2008) Protein kinase A dependent

step(s) in hepatitis c virus entry and infectivity. J Virol

82:8797–8811

29. West DC, Thompson WD, Sells PG, Burbridge MF (2001)

Angiogenesis assays using the chick chorioallantoic membrane.

In: Murray JC (ed) Methods in molecular medicine—angiogen-

esis: reviews and protocols. Humana Press, New York,

pp 107–130

30. Li Y, Gama V, Yoshida T, Gomez JA, Ishikawa K, Sasaguri H,

Cohen HY, Sinclair DA, Mizusawa H, Matsuyama S (2007) Bax-

inhibiting peptide protects cells from polyglutamine toxicity

caused by Ku70 acetylation. Cell Death Differ 14:2058–2067

31. Lofgren K, Wahlstrom A, Lundberg P, Langel U, Graslund A,

Bedecs K (2008) Antiprion properties of prion protein-derived

cell-penetrating peptides. FASEB J 22:2177–2184


123


32. Hirose M, Takatori M, Kuroda Y, Abe M, Murata E, Isada T,

Ueda K, Shigemi K, Shibazaki M, Shimizu F, Hirata M,

Fukazawa K, Sakaguchi M, Kegeyama K, Tanaka Y (2008)

Effect of synthetic cell-penetrating peptides on TrkA activity in

PC12 cells. J Pharamcol Sci 106:107–113

33. Choi J-M, Ahn M-H, Chae W-J, Jung Y-G, Park J-C, Park J-W,

Park T-K, Lee J-H, Seo B-F, Kim K-D, Kim E-S, Lee D-H, Lee

S-K, Lee S-K (2006) Intranasal delivery of the cytoplasmic

domain of CTLA-4 using a novel protein transduction domain

prevents allergic inflammation. Nat Med 12:574–579

34. McCusker CT, Wang Y, Shan J, Kinyanjui MW, Villeneuve A,

Michael H, Fixman ED (2007) Inhibition of experimental allergic

airways disease by local application of a cell-penetrating domi-

nant-negative STAT-6 peptide. J Immunol 179:2556–2564

35. Makino E, Sakaguchi N, Iwatsuki K, Huh N-H (2004) Intro-

duction of an N-terminal peptide of S100C/A11 into human cells

induces apoptotic cell death. J Mol Med 82:612–620

36. Keleman BR, Hsiao K, Goueli SA (2002) Selective in vivo

inhibition of mitogen-activated protein kinase activation using

cell-permeable peptides. J Biol Chem 277:8741–8748

37. Jones S, Farquhar M, Martin A, Howl J (2005) Intracellular

translocation of the decapeptide carboxyl terminal of Gi3ainduces the dual phosphorylation of p42/p44 MAP kinases.

Biochim Biophys Acta 1745:207–214

38. Fonseca SB, Pereira MP, Kelley SO (2009) Recent advances in

the use of cell-penetrating peptides for medical and biological

applications. Adv Drug Deliv Rev 61:953–964

39. Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K,

Sugiura Y (2001) Arginine-rich peptides. An abundant source of

membrane-permeable peptides having potential as carriers for

intracellular protein delivery. J Biol Chem 276:5836–5840

40. Vanhee P, van der Sloot AM, Verschueren E, Serrano L, Rous-

seau F, Schymkowitz J (2011) Computational design of peptide

ligands. Trends Biotechnol 29:231–239

41. Verdurmen WPR, Brock R (2011) Biological responses towards

cationic peptides and drug carriers. Trends Pharmacol Sci

32:116–124

42. Brugnano J, Ward BC, Panitch A (2010) Cell-penetrating pep-

tides can exert biological activity: a review. BioMol Concepts

1:109–116

43. Saar K, Lindgren M, Hansen M, Eirıksdottir E, Jiang Y, Rosen-

thal-Aizman K, Sassian M, Langel U (2005) Cell-penetrating

peptides: a comparative membrane toxicity study. Anal Biochem

345:55–65

44. Ward B, Seal BL, Brophy CM, Panitch A (2009) Design of a

bioactive cell-penetrating peptide: when a transduction domain

does more than transducer. J Pept Sci 15:668–674

45. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ,

Chernomordik LV, Lebleu B (2003) Cell-penetrating peptides. A

reevaluation of the mechanism of cellular uptake. J Biol Chem

278:585–590

46. Ter-Avetisyan G, Tunnemann G, Nowak D, Nitschke M, Herrmann

A, Drab M, Cardoso MC (2009) Cell entry of arginine-rich peptides

is independent of endocytosis. J Biol Chem 284:3370–3378

47. Raagel H, Saalik P, Hansen M, Langel U, Pooga M (2009) CPP–

protein constructs induce a population of non-acidic vesicles

during trafficking through endo-lysosomal pathway. J Control

Release 139:10–117

48. Fry DC, Vasilev LT (2005) Targeting protein–protein interac-

tions for cancer therapy. J Mol Med 83:955–963

49. D’Andrea LD, Del Gatto A, Pedone C, Benedetti E (2006)

Peptide-based molecules in angiogenesis. Chem Biol Drug Des

67:115–126

50. Low W, Mortlock A, Petrovska L, Dottorini T, Dougan G, Cri-

santi A (2007) Functional cell permeable motifs within medically

relevant proteins. J Biotechnol 129:55–564

51. Moore EE, Kuestener RE, Stroop SD, Grant FJ, Matthewes SL,

Brady CL, Sexton PM, Findlay DM (1995) Functionally different

isoforms of the human calcitonin receptor result from alternative

splicing of the gene transcript. Mol Endocrinol 9:959–968

52. Nussenzveig DR, Thaw CN, Gershengorn MC (1994) Inhibition

of inositol phosphate second messenger formation by intracellular

loop one of a human calcitonin receptor. J Biol Chem

269:28123–28129

53. Rivinoja A, Laakkonen P (2011) Identification of homing pep-

tides using the in vivo phage display technology. In: Langel U

(ed) Cell-penetrating peptides. Methods and Protocols. Humana

Press, New York, pp 401–415

54. Aina OH, Liu R, Sutcliffe JL, Marik J, Pan C-X, Lam KS (2007)

From combinatorial chemistry to cancer-targeting peptides. Mol

Pharm 5:631–651

55. Jiang T, Olson ES, Nguyen QT, Roy M, Jennings PA, Tsien RY

(2004) Tumor imaging by means of proteolytic activation of cell-

penetrating peptides. Proc Natl Acad Sci USA 101:17867–17872

56. Chorev M, Goodman M (1995) Recent developments in retro

peptides and proteins—an ongoing topochemical exploration.

Trends Biotechnol 13:438–445

57. Howl J, Jones S (2009) Transport molecules using reverse

sequence HIV-Tat polypeptides: not just any old Tat? Expert

Opin Ther Pat 19:1329–1333

58. Holm T, Raagel H, El Andaloussi S, Hein M, Mae M, Pooga M,

Langel U (2011) Retro-inversion of certain cell-penetrating

peptides causes severe cellular toxicity. Biochim Biophys Acta

1808:1544–1551

J. Howl et al.

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