Synthetic constrained peptide selectively binds and antagonizes death receptor 5

Synthetic constrained peptide selectively binds andantagonizes death receptor 5Johanna Vrielink1,*, Mariette S. Heins1,*, Rita Setroikromo1, Eva Szegezdi2, Margaret M. Mullally1,Afshin Samali2 and Wim J. Quax1

1 Department of Pharmaceutical Biology, University of Groningen, the Netherlands

2 Department of Biochemistry, Cell Stress and Apoptosis Research Group, National Centre for Biomedical Engineering Science,

National University of Ireland, Galway, Ireland

Introduction

Tumour necrosis factor-related apoptosis-inducing

ligand (TRAIL ⁄Apo2L), a member of the tumour

necrosis factor (TNF) ligand family, is well known for

its ability to induce apoptosis in many cancer cells but

not in most untransformed cells [1]. This quality makes

TRAIL an interesting and promising target for cancer

therapy and the main focus of TRAIL research has

therefore been on its role in cancer. Less attention has

been paid to the role of TRAIL in neurodegenerative

diseases. Normally, mature neurones will last the lifespan

Keywords

apoptosis; DR5; phage display; R2C16;

TRAIL

Correspondence

W. J. Quax, Department of Pharmaceutical

Biology, University of Groningen, Antonius

Deusinglaan 1, 9713 AV, Groningen, the

Netherlands

Fax: +31 50 363 3000

Tel: +31 50 363 2558

E-mail: [email protected]

*These authors contributed equally to this

work

(Received 12 October 2009, revised 17

December 2009, accepted 25 January

2010)

doi:10.1111/j.1742-4658.2010.07590.x

Apoptosis or programmed cell death is an inherent part of the development

and homeostasis of multicellular organisms. Dysregulation of apoptosis is

implicated in the pathogenesis of diseases such as cancer, neurodegenera-

tive diseases and autoimmune disorders. Tumour necrosis factor-related

apoptosis-inducing ligand (TRAIL) is able to induce apoptosis by binding

death receptor (DR)4 (TRAIL-R1) and DR5 (TRAIL-R2), which makes

TRAIL an interesting and promising therapeutic target. To identify

peptides that specifically interact with DR5, a disulfide-constrained phage

display peptide library was screened for binders towards this receptor.

Phage-displayed peptides were identified that bind specifically to DR5 and

not to DR4, nor any of the decoy receptors. We show that the synthesized

peptide, YCKVILTHRCY, in both monomeric and dimeric forms, binds

specifically to DR5 in such a way that TRAIL binding to DR5 is inhibited.

Surface plasmon resonance studies showed higher affinity towards DR5 for

the dimeric form then the monomeric form of the peptide, with apparent

Kd values of 40 nm versus 272 nm, respectively. Binding studied on cell

lines by flow cytometry analyses showed concentration-dependent binding.

Upon co-incubation with increasing concentrations of TRAIL, the peptide

binding was reduced. Moreover, both the monomeric and dimeric forms of

the peptide reduced TRAIL-induced cell death in Colo205 colon carcinoma

cells. The peptide, YCKVILTHRCY, or its derivates, may be a useful

investigative tool for dissecting signalling via DR5 relative to DR4 or could

act as a lead peptide for the development of therapeutic agents in diseases

with dysregulated TRAIL-signalling.

Abbreviations

DcR, decoy receptor; DR, death receptor; EAE, experimental autoimmune encephalomyelitis; FACS, fluorescence-activated cell sorting;

HRP, horseradish peroxidase; OPG, osteoprotegerin; PE, phycoerythrin; pfu, plaque forming unit; RU, response unit; sTRAIL, soluble TRAIL;

TMB, tetramethylbenzidine; TNF, tumour necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL-receptor.

FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS 1653

of the organism. However, in neurodegenerative

diseases, the balance is shifted towards death and the

major cause of neuronal loss is apoptosis. TRAIL is

present in the central nervous system and can induce

apoptosis in brain cells. Studies have shown the

involvement of TRAIL in neurodegenerative diseases

such as HIV-1-associated dementia, Alzheimer’s

disease and multiple sclerosis [2–4]. Molecules that

counteract the dysregulation of apoptosis could com-

prise effective therapeutic agents in these degenerative

disorders.

TRAIL induces apoptosis by binding to the cyste-

ine-rich extracellular domain of death receptors DR4

(TRAIL-R1) [5] and DR5 (TRAIL-R2) [6–8]. Binding

of the trimeric ligand leads to clustering of the

cytoplasmic death domains of the receptors and

recruitment of signalling molecules to form the death-

inducing signalling complex, which activates the

caspase cascade and thus initiates apoptosis [9,10]. In

addition to DR4 and DR5, TRAIL is capable of bind-

ing to three decoy receptors (DcR): DcR1 ⁄TRAIL-R3

[6,7], DcR2 ⁄TRAIL-R4 [11] and osteoprotegerin

(OPG) [12]. DcR1 lacks a functional death domain,

DcR2 contains a truncated death domain and OPG is

a soluble receptor; therefore, they cannot trigger a pro-

apoptotic signal.

Although the crystal structure of TRAIL in complex

with DR5 is known, the exact mechanism of binding

and signal initiation is still not completely understood

[13–15]. The first step in signalling by members of the

TNF family is considered to comprise ligand-induced

trimerization of the receptor. However, the identifica-

tion of a pre-ligand assembly domain suggested that

receptors may already be pre-assembled as trimers

before ligand binding [16–18]. Another intriguing fea-

ture of DR5 is that it appears to be able to mediate

distinctly different cell signals depending on the inter-

action with different receptor agonists [19,20]. Further-

more, depending on the cell type, TRAIL can signal

apoptosis either via DR4 [21–23] or DR5, or both

[24–26]. The reasons for the differences between DR4

versus DR5 signalling are not yet fully understood. To

study the differences in mechanism of ligand binding

and subsequent intracellular signalling through DR4

versus DR5, receptor-selective agonists and antagonists

are necessary. Recently, we have described DR5 [25]

and DR4 [27] selective agonistic variants of TRAIL.

To identify an antagonist to address the differences in

DR4 versus DR5 signalling, we now select for a pep-

tide that binds specifically to DR5. Peptides and small

proteins were demonstrated to bind their targets with

high affinity and specificity and to have advantage

over antibodies [28]. Phage display, a sophisticated

technique that links genotype and phenotype, was used

to select for such ligand-mimicking peptides. Earlier

studies have shown that it is a practical method for

identifying peptides with either agonistic [29–32] or

antagonistic properties for various receptors [33–35].

In the present study, by screening a disulfide-con-

strained phage display peptide library, we report the

identification of a peptide that specifically interacts

with DR5 and blocks binding of TRAIL to DR5. The

identified DR5-binding phage-displayed peptides shows

a strong consensus sequence and the monomeric and

dimeric forms of one of these peptides, YCK-

VILTHRCY, were further characterized. Both the

monomeric and dimeric peptide show selective binding

to DR5 in vitro. To confirm the binding specificity of

the monomeric and dimeric peptides on the membrane

of intact cells, we show binding towards Jurkat cells

that can be competed with soluble TRAIL (sTRAIL).

Finally, we demonstrate that the peptides can reduce

TRAIL-induced apoptosis on Colo205 cells. Compared

to the monomeric form, the dimeric form has higher

affinity for DR5 and increased antagonistic activity.

The identified peptide, or its derivatives, can be a use-

ful tool for elucidating the mechanism of TRAIL

signalling or the mechanism of controlling differential

signalling through DR4 or DR5. In addition, this pep-

tide may act as a lead peptide for the development of

therapeutic agents in diseases with dysregulated

TRAIL-signalling.

Results

Identification of DR5-binding phages

To select for peptides able to bind to DR5 with high

affinity, we used a cystein-constrained heptamer pep-

tide phage library. After three rounds of selection

(as described in the Experimental procedures), 25 indi-

vidual clones were picked and sequenced (Table 1).

The binding ability of the phages displaying these

peptides to DR5 was analysed using ELISA. The wells

were coated with DR5-Fc, the extracellular domain of

DR5 fused to the Fc-portion of human IgG1, and the

bound phages were detected with an horseradish per-

oxidase (HRP)-antibody against the phage coat protein

g8p. The background signal measured for a well with

no receptor coated was subtracted. Fourteen out of 25

phages showed binding to DR5 (Table 1). These

peptides share a highly homologous consensus C(K ⁄I ⁄L)V(Y ⁄ I ⁄A)LT(Q ⁄H ⁄L)(K ⁄R)C. Phage 77-R2C16

(CKVILTHRC) showed the highest affinity for DR5

and was selected for further investigation. Purified

phage 77-R2C16 showed a dose-dependent binding to

Inhibition of DR5 signalling J. Vrielink et al.

1654 FEBS Journal 277 (2010) 1653–1665 ª 2010 The Authors Journal compilation ª 2010 FEBS

DR5. Even when using increased amounts, no binding

to DR5 of control phage (i.e. a phage that displays

g3p without a C7C peptide) was observed (Fig. 1A).

Phage 77-R2C16 was also used to determine selectivity

towards DR5 using ELISA. Wells were coated with

different TNF family receptors (i.e. DR4-Fc, DR5-Fc,

DcR1-Fc, DcR2-Fc, OPG-Fc, mouse OPG-Fc, mouse

receptor activator of nuclear factor-jB-Fc and TNF-

receptor 1-Fc) and binding of 77-R2C16 or control

phage was measured. Phage 77-R2C16 exclusively

binds to DR5 and not to any of the other receptors

tested. The control phage demonstrated no binding to

any of the receptors, confirming that binding of

77-R2C16 to DR5 was via the displayed peptide, and

not via other regions of the phage (Fig. 1B). To assess

where the peptide binds to the receptor ⁄ ligand inter-

face of DR5, we tested whether sTRAIL competes for

DR5-binding with phage 77-R2C16. Phage 77-R2C16

was added to the wells at a concentration of 1 · 1010

plaque forming units (pfu)ÆmL)1 and the sTRAIL

concentration was increased. With an increasing

concentration of sTRAIL, the binding of the phage to

the receptor DR5 decreased, suggesting that phage

77-R2C16 attaches to a binding patch on DR5 over-

lapping with sTRAIL (Fig. 1C).

Competition studies with synthetic constrained

peptides

Because the phage-displayed peptide may have differ-

ent binding characteristics compared to the constrained

peptide alone, the corresponding constrained peptide

YCKVILTHRCY (peptide R2C16) was synthesized.

Tyrosine residues were added to the ends of this

hydrophobic peptide to increase its solubility. During

the synthesis, next to the monomer, a dimeric peptide

was also formed. This dimeric peptide was separated

from the monomeric peptide by HPLC. The mass of

the dimeric peptide and measurements using MALDI-

TOF indicated that all cysteines in the dimeric peptide

Table 1. Sequences of the 25 clones picked after three rounds of biopanning against DR5. The sequences are denoted by the single-letter

amino acid code. Of these 25 clones, 14 clones showed binding to DR5 with ELISA (indicated by an asteriak). These 14 clones show a

strong consensus sequence with valine at position 2, leucine at position 4, threonine at position 5 and a basic amino acid (arginine or lysine)

at position 7. The random residues are shown in bold; fixed cysteines and the preceding alanine in are shown in normal text; and consensus

residues are shaded grey.

Clone Sequence

77-R2C16 A C K V I L T H R C *

89-R2C2 A C K V I L T H R C *

77-R2C5 A C K V A L T L R C *





77-R2C8 A C L V Y L T Q R C *



77-R2C2 A C I V Y L T Q K C *



77-R2C1 A C I L Y L T Q K C *

77-R2C4 A C K L A M T M K C



77-R2C6 A C F L V M S Q R C

77-R2C10 A C L W F P R E Q C

77-R2C14 A C L W F P R E Q C

89-R2C3 A C M L P L Y F P C

77-R2C11 A C E L P R S P S C

77-R2C7 A C T V P A F P A C

89-R2C1 A C T N S A M A D C

77-R2C17 A C K H E P T P N C

Consensus A C K V Y L T Q R C

L A H K

I I L

J. Vrielink et al. Inhibition of DR5 signalling


were oxidized and that covalent bonds were formed

between the two monomers. This suggests that the two

monomers are linked to each other via disulfide

bridges. To determine the orientation of the two

monomeric peptides in relation to each other (i.e. par-

allel or symmetrical), the dimeric peptide was digested

with trypsin and measured using MALDI-TOF. The

analysis showed that the dimeric peptide sample con-

tained both parallel and antiparallel orientated mono-

mers (data not shown).

The monomeric and dimeric peptides were used in

competitive studies with ELISA. By adding increasing

concentrations of the peptide R2C16, a competition

with the phage 77-R2C16 (1 · 1010 pfuÆmL)1) for

DR5-binding could be seen with ELISA. A known

TNFa antagonist peptidomimetic, WP9QY [36], was

used as a control peptide. When used at the same con-

centrations, it did not compete with phage 77-R2C16

for binding to DR5 (Fig. 2A). This indicates that the

peptide R2C16, in both monomeric and dimeric forms,

is capable of binding to DR5. Competitive ELISA was

also used to analyse the effect of an increasing concen-

tration of the monomeric and dimeric forms of R2C16

on TRAIL DR5-binding. In this competition ELISA,

the amount of bound sTRAIL was measured. The

results obtained show that both forms of the R2C16

peptide could compete with sTRAIL for binding to

DR5, not only confirming that the R2C16 peptide is

indeed a DR5-binding peptide, but also suggesting that

R2C16 and TRAIL bind to an overlapping area on

DR5 (Fig. 2B).

Binding studies with surface plasmon resonance

Binding of the monomeric and dimeric form of peptide

R2C16 to immobilized DR4-Fc and DR5-Fc receptor

was assessed in real time by using surface plasmon

resonance. Both forms of R2C16 bound in a dose-depen-

dent manner to DR5 (Fig. 3A, B). It was observed

that, after saturation was reached, injection of higher

concentrations of peptides resulted in increasing

response units (RUs), indicating the accumulation of

the peptide. Furthermore, at higher concentrations

(> 2000 nm monomer or > 120 nm dimer), some

binding to DR4 was observed (Fig. 3C, D). Because of

the hydrophobic nature of the peptide, we consider

that the accumulation on DR5 and binding to DR4 is

caused by aggregation of the peptides. Thus, for Kd

determination, we decided to use the lane coated with

DR4-Fc as a control lane instead of an empty lane.

The signal obtained at equilibrium (176 s after injec-

tion) was plotted against the concentration of the pep-

tide and apparent Kd values were calculated from these

A

B

C

Fig. 1. ELISAs with phage 77-R2C16 and control phage. (A) Wells

are coated with DR5-Ig. Phage 77-R2C16 (•) bound to DR5 in a dose-

dependent manner; control phage ( ) did not show any binding. This

indicates that binding to DR5 is not mediated by nonspecific binding

of the phage particle. (B) Wells are coated with different receptors of

the TNF-family. Phage 77-R2C16 showed specific binding to DR5,

and control phage showed no binding to any of the receptors. This

confirms that binding of the phage 77-R2C16 to DR5 is via the dis-

played peptide, and not via other parts of the phage particle. (C) Com-

petition ELISA of sTRAIL with phage 77-R2C16 (1 · 1010 pfuÆmL)1)

for binding to DR5. Increasing amounts of sTRAIL decreased the

binding of phage 77-R2C16 to DR5, suggesting that phage 77-R2C16

and sTRAIL bind to an overlapping region on DR5.



plots. The monomer has an apparent Kd value of

272 nm (range 251–294 nm) and the dimer has a value

of 40 nm (37–44 nm) (Fig. 4A, B).

Binding studies towards Jurkat cells with

fluorescence-activated cell sorting (FACS) analysis

To further evaluate the binding of the monomeric and

dimeric peptides, we characterized their binding

towards Jurkat cells by flow cytometry. Jurkat cells are

a widely used model of DR5 only cells. The con-

strained peptide R2C16 was synthesized with a biotin

label at the C-terminus (YCKVILTHRCY-K[biotin]).

Again, both a monomeric and a dimeric form of the

peptide were formed and they were separated from

each other by HPLC. Both forms of biotin-R2C16

bound in a dose-dependent manner to DR5 (Fig. 5A1,

B2). Compared to the control, the increasing amounts

of biotinylated monomeric and dimeric peptide showed

an increased fluorescence signal. At higher concentra-

tions of peptide (> 23 nm biotinylated monomer or

> 12 nm biotinylated dimer), the fluorescence signal

suddenly and drastically dropped to almost control

levels (data not shown). Again, this suggests the for-

mation of aggregates as a result of the hydrophobic

nature of the peptides, which would only be increased

by the addition of the biotin label.

To determine the specificity of this binding interac-

tion, the Jurkat cells were co-incubated with the bioti-

nylated peptides (5.71 nm of monomer and 1.43 nm

of dimer) and increasing concentrations of sTRAIL

(0.21 pm to 2.1 nm). Compared to the monomeric or

dimeric biotinylated-R2C16 alone, analyses showed

that the fluorescence signal can be gradually reduced

by increasing the concentrations of sTRAIL

(Fig. 5A2, A3, B2, B3). This indicates that the bioti-

nylated peptides and sTRAIL bind at a similar posi-

tion on the cells, most likely at an overlapping patch

of DR5.

Constrained R2C16 peptide inhibits

TRAIL-induced apoptosis

To assess the effect of R2C16 on DR5 apoptosis

induction, Colo205 colon carcinoma cells were used.

We have previously reported that Colo205 cells were

sensitive to TRAIL-induced apoptosis and the

TRAIL-death signal was primarily transmitted by

DR5 in these cells [25]. Treatment of the cells with

increasing concentrations of the monomeric R2C16

peptide caused no cell death, as measured by annexin

V labelling of the dying cells (Fig. 6A). Treatment with

the dimeric form of R2C16 lead to similar results,

where only the highest concentration (3.6 lm) caused a

small (7.1 ± 3.2%) increase of cell death (Fig. 6A).

Next, the possibility of antagonistic action of R2C16

was tested. Colo205 cells were treated with increasing

concentration of monomeric or dimeric R2C16 for 1 h

before treatment with 20 ngÆmL)1 TRAIL for 2 h and

annexin V staining was used to quantify cell death.

Both forms of R2C16 were able to inhibit TRAIL-

induced cell death, with the dimeric form being more

efficient than the monomer. In addition, the dimeric

A

B

Fig. 2. (A) Competition ELISAs of peptide R2C16, in both mono-

meric and dimeric forms, and control peptide WP9QY with phage

77-R2C16 (1 · 1010 pfuÆmL)1). Increasing amounts of monomer (•)

and dimer ( ) competed with phage 77-R2C16 for binding to

DR5-Ig, whereas peptide WP9QY ( ) did not. This showed that the

monomeric form of the dimeric peptide can bind to DR5. (B)

Competition ELISA of peptide R2C16, in both monomeric and

dimeric forms, with sTRAIL (10 ngÆmL)1). The amount of sTRAIL

binding was measured. Increasing amounts of monomer (•) and

dimer ( ) competed with sTRAIL for binding to DR5-Ig, not only

confirming that the R2C16 peptide is indeed a DR5-binding peptide,

but also suggesting that R2C16 and TRAIL bind in an overlapping

area on DR5.



form of R2C16 acted as an antagonist best in the con-

centration range 1–5 lgÆmL)1 (equal to 0.36–1.8 lm).

Above this concentration, the efficiency of the peptide

to inhibit TRAIL-induced cell death was reduced

(Fig. 6B). This reduction may be caused by aggrega-

tion of the peptide.

Discussion

TRAIL and its receptors (DR4, DR5, DcR1, DcR2

and OPG) are of high interest because of the potential

of TRAIL to specifically induce apoptosis in cancer

cells, as well as its involvement in many diseases with

dysregulated apoptosis. Using phage display, we identi-

fied peptides that share a homologous consensus,

C(K ⁄ I ⁄L)V(Y ⁄ I ⁄A)LT(Q ⁄H ⁄L)(K ⁄R)C, and, when dis-

played on a phage, bind to DR5. The phage displayed

peptide that showed the highest affinity for DR5 was

further characterized and shown to bind exclusively to

DR5, with no affinity towards any of the other four

TRAIL receptors, receptor activator of nuclear factor-

jB or TNF-receptor I. The synthetic constrained

peptide, YCKVILTHRCY, in both monomeric and

dimeric forms, competed with the phage 77-R2C16

and with sTRAIL for binding to DR5 in a concentra-

tion-dependent manner and retained DR5 selectivity.

The dimeric form of the peptide displayed higher affin-

ity for DR5 compared to the monomeric form with an

apparent Kd value of 40 nm versus 272 nm, respec-

tively. This is in accordance with the reported observa-

tions in the literature of higher affinities of dimeric

and even multimeric peptides compared to their mono-

meric form as a result of an avidity effect [30,37–40].

A similar phenomenon was seen for the biotin-labelled

synthetic constrained peptide. Both the monomeric

and the dimeric form were able to bind to Jurkat cells

in a concentration-dependent manner. The dimeric

form of the biotin-labelled peptide appeared to have a

higher affinity for the Jurkat cells, although part of

this effect can be attributed the double biotin label.

The higher avidity for the dimeric peptide, which was

observed in all binding studies, might also be a conse-

quence of the reduced rigidity compared to the mono-

meric peptide. This would allow the dimeric peptides

to adapt an improved confirmation for DR5-binding.

Co-incubation of the Jurkat cells with biotinylated

A B

C D

Fig. 3. BIAcore curves of peptide R2C16, in both monomeric and dimeric forms, binding to immobilized DR5-Ig (A, B) or DR4-Ig (C, D).

(A) Increasing concentrations of the monomeric peptide (4086, 2043, 1021, 511, 255, 128, 63.8, 31.9, 16.0 and 8.0 nM) were injected,

demonstrating an increased signal. The curves shown are corrected for the signal obtained in the lane coated with DR4-Ig. (B) Increasing

concentrations of dimeric peptide (179, 119, 89.5, 59.7, 44.8, 29.8, 22.4, 14.9, 11.2 and 7.5 nM) were injected, demonstrating an increased

signal. The curves shown are corrected for the signal obtained in the lane coated with DR4-Ig. (C, D) Increasing concentrations of

monomeric (C) or dimeric peptide (D) demonstrated some binding to DR4-Ig at higher concentrations. This binding is most likely caused by

aggregation of the peptide as a result of its hydrophobic nature.



peptide and sTRAIL showed a decrease in peptide

binding with increasing sTRAIL concentrations. This

suggests that the peptides bind at a similar patch on

DR5. Furthermore, both the monomeric and the

dimeric peptide acted as DR5 antagonists because they

were able to inhibit TRAIL-mediated apoptosis in

Colo205 cells, with the dimeric form of R2C16 demon-

strating the most efficient antagonistic effect. This is

the first DR5-specific antagonistic peptide described.

In 2004, Kajiwara et al. [41] described synthetic pep-

tides inhibiting TRAIL-induced cell death, although

these peptides bound to TRAIL instead of DR5. More

recently, Li et al. [32] described peptides binding to

DR5, although none of these were antagonistic. We

hypothesize that, at higher concentrations, the peptide

might aggregate in an aqueous environment based on

the fact that the amino acids in the sequences of pep-

tide are rather hydrophobic. In addition, we observed

that biotinylated was more prone to aggregation than

the label-free peptide, which is in accordance with

biotin being well known for its hydrophobic character-

istics.

One mechanism of DR5 that is not yet fully under-

stood is the ability to mediate distinct cell signals

when interacting with different receptor agonists. An

agonistic DR5 monoclonal antibody could induce

both caspase-dependent and caspase-independent cell

death in Jurkat cells, whereas TRAIL could only trig-

ger the caspase-dependent cell death [20]. Thomas

et al. [19] found that TRAIL and some agonistic anti-

bodies required the C-terminal tail of DR5 for

recruitment of Fas-associated death domain, whereas

other agonistic antibodies could function in the

absence of this C-terminal tail. Thus, different recep-

tor agonists can use distinct molecular mechanisms to

activate signalling from the same receptor. It is postu-

lated that the binding of different agonists to the

extracellular domain causes different conformational

changes in the intracellular domain, which may inter-

act with different cytoplasmic adaptor proteins and

trigger different cell signals [20]. To address these

questions, the R2C16 antagonistic peptide described

in the presrent study comprises a useful tool for eluci-

dating the mechanisms of binding and signalling initi-

ation of DR5. Accordingly, the manner in which the

peptide is able to antagonize TRAIL should be fur-

ther elucidated by studying the trimerization of DR5.

In addition, the effects of the peptide on the different

cell signals that it can trigger or block should be

investigated.

Up to now, the focus of TRAIL research has been

mainly on its therapeutic value in cancer, as a result of

the quality of TRAIL that leads to the induction of

apoptosis in a broad range of cancer cells but not in

most untransformed cells [1]. However, the involve-

ment of TRAIL in neurodegenerative diseases has not

received much attention, despite the mounting evidence

emphasizing the role of TRAIL in these disorders.

Recent data show that, although TRAIL is absent in

normal brain, it is upregulated under pathological con-

ditions such as Alzheimer’s disease. Human brain cells

express all four TRAIL receptors and are sensitive to

TRAIL-induced apoptosis. TRAIL is also suggested to

be involved in the pathogenesis of HIV-1-associated

dementia [2]. HIV infection triggers TRAIL expression

in macrophages and these TRAIL expressing macro-

phages can initiate neuronal injury. The involvement

of TRAIL in Alzheimer’s disease was shown by

A

B

Fig. 4. Dose–response curves of increasing concentration of pep-

tide R2C16, (A) monomeric (•) or (B) dimeric ( ), binding to DR5-Ig

measured with surface plasmon resonance. The amount of binding

is depicted as the RU measured at 176 s. Graphs were fit with

four-parameter sigmoid curves. Apparent Kd values were calculated

as the concentration of peptide that gives a signal of 50% of the

maximum RU.



neutralization of the TRAIL death pathway, which

protected a human neuronal cell line from b-amyloid

toxicity [2]. TRAIL plays a dual role in T cell-induced

experimental autoimmune encephalomyelitis (EAE), an

animal model of multiple sclerosis. Blockade of

TRAIL within the periphery exacerbates EAE, whereas

blockade of TRAIL in the central nervous system sup-

presses EAE by inhibiting brain cell apoptosis [42].

Inhibition of TRAIL-induced apoptosis within the cen-

tral nervous system may represent a possible therapeu-

tic strategy for preventing neuronal damage in patients

with neurodegenerative diseases. The R2C16 peptide,

with its DR5 antagonistic activity and lipophilic prop-

erties, has the potential to act as a lead peptide in

studies aiming to block TRAIL and reducing its toxic-

ity in neurodegenerative diseases.

Overall, the peptides described in the present study,

or their derivatives, may have various applications in

the field of TRAIL-mediated signalling and diseases

caused by dysregulated TRAIL signalling. The small

size of this peptide offers the possibility of designing

structurally mimetic nonpeptidic molecules.

A1 B1

A2 B2

A3 B3

Fig. 5. Dose– response histograms of

biotinylated peptide R2C16, (A1) monomeric

or (B1) dimeric, on Jurkat cells. Increasing

amounts of both monomeric (m1 = 0.57 nM,

m2 = 2.86 nM, m3 = 11.42 nM) and dimeric

(d1 = 0.14 nM, d2 = 1.43 nM, d3 = 5.71 nM)

biotinylated peptide give rise to a right shift

in the fluorescent PE signal compared to

the control (C, filled grey). Competition his-

tograms of 5.71 nM monomeric biotinylated

peptide (m, tinted) (A2) and 1.43 nM dimeric

biotinylated peptide (d, tinted) (B2) show a

strong right shift compared to control

(C, filled grey). Co-incubation of the biotiny-

lated peptides with 2.1 nM sTRAIL [m + T

(A2) and d + T (B2), respectively] show a

decrease in the right shift that they were

initially able to cause. The curves represent

the relative binding signal binding of the

(A3) monomer and (B3) dimer upon co-

incubation with sTRAIL compared to the

signal obtained when no sTRAIL was used.



Experimental procedures

TRAIL purification

cDNA corresponding to soluble human TRAIL (C-terminal

amino acids 114–281) was cloned into the NcoI and BamHI

sites of a pET15b vector and transformed to and expressed

in Escherichia coli BL21 (DE3). The trimeric soluble protein

was purified as described previously [43].

Biopanning

The Ph.D.–C7C phage display peptide library (New Eng-

land Biolabs, Hitchin, UK), consisting of randomized hept-

amer constrained peptides (1.2 · 109 individual clones), was

used to identify peptides binding to DR5. The disulfide-

constrained heptapeptides are expressed at the N-terminus

of g3p, with the first cysteine preceded by an alanine

residue, and the second cysteine followed by a short spacer

(Gly-Gly-Gly-Ser). DR5-Fc, fusion of the ecto-domain of

the receptor to the Fc-portion of human IgG1 (R&D Sys-

tems, Minneapolis, MN, USA), was used to coat Protein A

magnetic dynabeads (Dynal, Hammerfest, Norway). To

6 lL of beads, 1 lg of DR5-Fc was added in 0.1 m NaH-

CO3 (pH 8.6) and incubated overnight at 4 �C. The beads

were then blocked using 0.1 m NaHCO3 (pH 8.6),

5 mgÆmL)1 BSA and 0.02% NaN3. Phage library was

added to the beads at 2 · 1011 pfu and incubated for

45 min. Unbound phages were removed by washing ten

times with washing buffer (NaCl ⁄Tris containing 0.1%

Tween 20). Bound phages were eluted with 1 mL of 0.2 m

glycine ⁄HCl (pH 2.2), 1 mgÆmL)1 BSA for no more then

10 min, and immediately neutralized with 150 lL of 1 m

Tris-HCl (pH 9.1). The eluted phages were amplified in

E. coli ER2738 and titred according to the manufacturer’s

instructions (New England Biolabs). Another two rounds

of biopanning were then performed. The incubation time

was decreased to 30 min in the second round and to 15 min

in the third round. In both rounds, the concentration of

Tween 20 in the washing buffer was increased to 0.5%, and

1 lm sTRAIL in NaCl ⁄Tris was used for competitive elu-

tion. Before the third round, a subtractive round was per-

formed, by incubating the amplified phage of round two

with human IgG Fc fragment (Rockland Immunochemi-

cals, Inc., Gilbertsville, PA, USA) bound to protein A

beads. To 20 lL of beads, 10 lg of Fc fragment was added,

incubated and blocked as described above. Phages were

incubated for 30 min with the beads, and unbound phages

were subsequently used in the third positive panning round.

After round three, 25 individual phage clones were picked

from agar plates and amplified. The single-stranded DNA

was isolated and sequenced according to the manufacturer’s

instructions. Supernatants were screened using ELISA, as

described below. Samples that showed a high binding signal

were further purified using poly(ethylene glycol) precipita-

tion according to the manufacturer’s instructions (New

England Biolabs).

ELISA with phage

Maxisorp 96-wells plates (Nunc, Roskilde, Denmark) were

coated for 1–2 h with 100 lL of 1 ngÆlL)1 receptor-Fc

A

B

Fig. 6. Annexin V cell assays with peptide R2C16 using Colo205

cells. (A) Treatment of cells with increasing concentrations of the

monomeric R2C16 peptide (•) caused no cell death. Treatment with

the dimeric form of R2C16 ( ) gave similar results. Only the high-

est concentration (8 lM) caused a small (7.1 ± 3.2%) increase of

cell death, suggesting that the R2C16 peptide is not a DR5 agonist.

(B) Reduction of TRAIL-induced cell death in Colo205 cells by pep-

tide R2C16. Colo205 cells were treated with increasing concentra-

tion of monomeric (•) or dimeric ( ) R2C16 before treatment with

20 ngÆmL)1 TRAIL and annexin V staining was used to quantify the

dying cells. Treatment of Colo205 cells with only 20 ngÆmL)1 TRAIL

induced 80% cell death. Both forms of R2C16 were able to reduce

TRAIL-induced cell death, with the dimeric form being more

efficient than the monomer. The dimeric form of R2C16 acted as

an antagonist best in the concentration range 1–5 lgÆmL)1 (equal

to 0.76–3.8 lM). Above this concentration, the efficiency of the

peptide to inhibit TRAIL-induced cell death was reduced, most likely

as a result of aggregation of the peptide.



(R&D Systems) at 4 �C in 0.1 m NaHCO3 (pH 8.6). The

wells were blocked for 1–2 h with 200 lL of 2% BSA in

0.1 m NaHCO3 and washed three times with NaCl ⁄Tris,0.5% Tween 20 (TBST). Phage supernatant, purified phage

diluted in TBST with 0.5% BSA or a premix of different

concentration of synthesized peptides with 1 · 1010

pfuÆmL)1 of phage was added at 100 lL per well and incu-

bated for 30 min. After washing the wells six times with

TBST, 200 lL of 1 : 5000 diluted HRP ⁄ anti-M13 monoclo-

nal conjugate (Amersham Pharmacia Biotech, Little Chal-

font, UK) in TBST was added for 1 h. Wells were washed

six times with TBST and bound phages were detected with

100 lL of tetramethylbenzidine (TMB; one-step Turbo TMB-

ELISA) (Pierce Biotechnology, Rockford, IL, USA). The

reaction was stopped with 100 lL of approximately 1.8 m

H2SO4. The signal was read at 450 nm in a multiscan Ascent

plate reader (Thermo Labsystems, Helsinki, Finland).

Curves were fitted with four-parameter sigmoid curves.

Competition ELISA with peptides and sTRAIL

Maxisorp plates were coated for 1–2 h with 100 lL of

1 ngÆlL)1 DR5-Fc at 4 �C in 0.1 m NaHCO3 (pH 8.6). The

wells were blocked for 1–2 h with 200 lL of 2% BSA in

0.1 m NaHCO3 (pH 8.6) and washed three times with

TBST. Different concentrations of synthesized peptides

were premixed with 10 ngÆmL)1 sTRAIL in TBST and

100 lL of this premix was added to each well, and incu-

bated for 30 min. After washing the wells six times with

TBST, 200 lL of 1 : 200 diluted anti-human TRAIL sera

(AF375; R&D Systems) in TBST was added to the wells

for 1 h, washed six times with TBST and 200 lL of

1 : 25 000 diluted HRP conjugated swine anti-goat (Bio-

Source International, Camarillo, CA, USA) was added for

1 h. After washing wells six times with TBST, 100 lL of

TMB was added to measure the amount of bound sTRAIL.

The reaction was stopped with 100 lL of approximately

1.8 m H2SO4. The signal was read at 450 nm. Curves were

fitted with four-parameter sigmoid curves.

Synthetic peptides

The constrained peptide (YCKVILTHRCY), in both mono-

meric and dimeric forms, was synthesized by Pepscan (Lelys-

tad, the Netherlands). Both peptides had a free amine at the

N-terminal and a free acid at the C-terminal. The lyophilized

peptides were solubilized in acetonitril ⁄water (1 : 1) to give a

stock concentration of 20 mgÆmL)1. A control peptide

(YCWSQYLCY) was purchased from Bachem AG (Buben-

dorf, Switzerland). The lyophilized peptide was solubilized in

50% acetic acid to give a stock concentration of 10 mgÆmL)1.

The stock of each peptide was stored at )20 �C.The constrained peptide with biotin label (YCK-

VILTHRCY-K[biotin]), in both monomeric and dimeric

forms, was also synthesized by Pepscan Systems, again with

a free amine at the N-terminal and the biotin label at the

C-terminus. The biotin label was coupled to the C-terminal

tyrosine, resulting in two biotin labels for the dimeric

peptide. The lyophilized peptides were solubilized in

acetonitril ⁄water (1 : 1) to give a stock concentration of

20 mgÆmL)1. The stock of each peptide was stored at

)20 �C.

Interaction studies by surface plasmon resonance

To evaluate the binding of the peptides to DR5, BIAcore

2000 (BIAcore AB, Uppsala, Sweden) was used. All

reagents used were also purchased from BIAcore AB.

Immobilization of the receptor DR4-Fc and DR5-Fc on

the sensor surface of a CM5-chip was performed in accor-

dance with a standard amine coupling procedure at a flow

rate of 10 lLÆmin)1. To activate carboxyl groups on the

sensor surface, 70 lL of a solution containing 0.2 m

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-

ride and 0.05 m N-hydroxysuccinimide was injected. Recep-

tors (6.7 lgÆmL)1 in 10 mm NaAc buffer, pH 5.0) were

flowed over the chip surface until a surface density of

approximately 3500 RU was reached. Remaining active

groups were blocked by injecting 70 lL of 1.0 m ethanol-

amine-HCl (pH 8.5). Assays were performed at 25 �C with

NaCl ⁄Po containing 0.005% (v ⁄ v) P20 surfactant as

running buffer and a flow rate of 70 lLÆmin)1. Peptides

were injected at variable concentrations for 3 min followed

by 4 min of running buffer. The surface was regenerated

after each binding step by removing all bound peptide by

injecting 35 lL of 10 mm glycine (pH 2.0). Sensorgrams

were evaluated with BIAevaluation software, version 4.1

(Biacore, GE Healthcare, Chalfont St Giles, UK). The lane

coated with DR4-Fc was used as a reference. The signal

obtained at equilibrium (176 s after start injection) was

plotted against the concentration of the peptide and fitted

with four-parameter sigmoid curves. From these curves, the

apparent Kd values were calculated.

Interaction studies on Jurkat cells by FACS

analysis

Jurkat cells were maintained in RPMI 1640

medium + GlutaMAX-I supplemented with 10% fetal

bovine serum, 100 UÆmL)1 penicillin and 100 lgÆmL)1 strep-

tomycin (all from Gibco, Gaithersburg, MD, USA) at 37 �Cin 5% CO2 in a humidified environment. Cells were

harvested at 106 cells per sample and washed in ice-cold

NaCl ⁄Pi ⁄ 2% fetal bovine serum to prevent peptide reduc-

tion by the reducing agent glutathione present in the RPMI

medium. Increasing concentrations of biotin-labelled pep-

tides were added to the cells and incubated for 1 h on ice.

The cells were washed twice in ice-cold NaCl ⁄Pi ⁄ 2% fetal

bovine serum. The cells were incubated for an additional

1 h with streptavidin-phycoerythrin (PE) (BD Pharmingen,



San Diego, CA, USA) on ice, in the dark. The cells were

washed twice and then resuspended in 300 lL of ice-cold

NaCl ⁄Pi ⁄ 2% fetal bovine serum. Finally, the samples were

measured on a FACSCalibur Flow cytometer (Becton-Dick-

inson, Franklin Lakes, NJ, USA) and analysed with FlowJo

(Tree Star Inc., Ashland, OR, USA). The PE signals

obtained are displayed as histograms.

For the competition assays with sTRAIL, a similar pro-

tocol was used. Cells were harvested 106 cells ⁄ sample and

washed in ice-cold NaCl ⁄Pi ⁄ 2% fetal bovine serum. Cells

were co-incubated with 5.71 nm monomeric or 1.43 nm

dimeric peptide and increasing concentrations sTRAIL for

1 h on ice. The cells were washed twice in ice-cold

NaCl ⁄Pi ⁄ 2% fetal bovine serum. The cells were incubated

for another hour with Streptavidin-PE (BD Pharmingen)

on ice, in the dark. The cells were washed twice and then

resuspended in 300 lL of ice-cold NaCl ⁄Pi ⁄ 2% fetal bovine

serum. Finally, the samples were measured on a FACSCali-

bur Flow cytometer (Becton-Dickinson) and analysed with

Flowjo (Tree Star Inc.). The PE signals obtained are

displayed as histograms. In addition, the signals obtained

in triplo were expressed as a percentage of the PE signal

related to the signal obtained with no sTRAIL.

Apoptosis assay

Colo205 cells were maintained in RPMI 1640 medium

(Sigma, St Louis, MO, USA) supplemented with 10% fetal

bovine serum (Sigma), 100 UÆmL)1 penicillin, 100 lgÆmL)1

streptomycin (Sigma), 2 mm l-glutamine (Sigma) and 1 mm

sodium pyruvate (Gibco) at 37 �C in 5% CO2 in a humidi-

fied environment. One hour before treatment, the cells were

seeded in a 24-well plate at a concentration of 300 000 cell-

sÆmL)1 in NaCl ⁄Pi ⁄ 1% BSA to prevent peptide reduction

by the reducing agent glutathione present in the RPMI

medium. Varying concentrations of peptides were added to

the cells and incubated for 1 h before the addition of

20 ngÆmL)1 sTRAIL for 2 h. After treatment, the cells were

transferred into Eppendorf tubes and collected by centrifu-

gation at 3000 g for 5 min. The cell pellets were resus-

pended in 50 lL of calcium buffer [10 mm Hepes, pH 7.5

(set with NaOH), 140 mm NaCl and 2.5 mm CaCl2] con-

taining 3 lL of annexin V (IQ Corporation, Groningen, the

Netherlands) and incubated for 15 min on ice. The staining

was terminated by diluting the annexin V solution to

300 lL with calcium buffer and the samples were analysed

immediately on a FACSCalibur Flow cytometer (Becton

Dickinson). The results were expressed as percentage of

annexin V positive cells.

Acknowledgements

We thank Almer van der Sloot for providing sTRAIL

and for his support with the BIAcore. We also thank

Geert Mesander and Henk Moes for their technical

assistance with the FACS analysis. Finally, we thank

the EU Sixth Framework Program LSH-2005-2.2.0-2

(TRIDENT) and TIPharma PROJECT T3-112 (TNF

ligands in cancer) for providing financial support.

References

1 Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA,

Marsters SA, Blackie C, Chang L, McMurtrey AE,

Hebert A et al. (1999) Safety and antitumor activity of

recombinant soluble Apo2 ligand. J Clin Invest, 104,

155–162.

2 Huang Y, Erdmann N, Peng H, Zhao Y & Zheng J

(2005) The role of TNF related apoptosis-inducing

ligand in neurodegenerative diseases. Cell Mol Immunol,

2, 113–122.

3 Aktas O, Waiczies S & Zipp F (2007) Neurodegenera-

tion in autoimmune demyelination: recent mechanistic

insights reveal novel therapeutic targets. J Neuro-

immunol, 184, 17–26.

4 Uberti D, Ferrari-Toninelli G, Bonini SA, Sarnico I,

Benarese M, Pizzi M, Benussi L, Ghidoni R, Binetti G,

Spano P et al. (2007) Blockade of the tumor necrosis

factor-related apoptosis inducing ligand death receptor

DR5 prevents beta-amyloid neurotoxicity. Neuropsycho-

pharmacology, 32, 872–880.

5 PanG, O’Rourke K, Chinnaiyan AM,Gentz R, Ebner R,

Ni J & Dixit VM (1997) The receptor for the cytotoxic

ligand TRAIL. Science, 276, 111–113.

6 Pan G, Ni J, Wei YF, Yu G, Gentz R & Dixit VM

(1997) An antagonist decoy receptor and a death

domain-containing receptor for TRAIL. Science, 277,

815–818.

7 Sheridan JP, Marsters SA, Pitti RM, Gurney A,

Skubatch M, Baldwin D, Ramakrishnan L, Gray CL,

Baker K, Wood WI et al. (1997) Control of TRAIL-

induced apoptosis by a family of signaling and decoy

receptors. Science, 277, 818–821.

8 Walczak H, gli-Esposti MA, Johnson RS, Smolak PJ,

Waugh JY, Boiani N, Timour MS, Gerhart MJ, Schoo-

ley KA, Smith CA et al. (1997) TRAIL-R2: a novel

apoptosis-mediating receptor for TRAIL. EMBO J, 16,

5386–5397.

9 Budihardjo I, Oliver H, Lutter M, Luo X & Wang X

(1999) Biochemical pathways of caspase activation

during apoptosis. Annu Rev Cell Dev Biol, 15, 269–290.

10 LeBlanc HN & Ashkenazi A (2003) Apo2L ⁄TRAIL

and its death and decoy receptors. Cell Death Differ,

10, 66–75.

11 Pan G, Ni J, Yu G, Wei YF & Dixit VM (1998)

TRUNDD, a new member of the TRAIL receptor

family that antagonizes TRAIL signalling. FEBS Lett,

424, 41–45.



12 Emery JG, McDonnell P, Burke MB, Deen KC, Lyn S,

Silverman C, Dul E, Appelbaum ER, Eichman C,

DiPrinzio R et al. (1998) Osteoprotegerin is a receptor

for the cytotoxic ligand TRAIL. J Biol Chem, 273,

14363–14367.

13 Hymowitz SG, Christinger HW, Fuh G, Ultsch M,

O’Connell M, Kelley RF, Ashkenazi A & de Vos AM

(1999) Triggering cell death: the crystal structure of

Apo2L ⁄TRAIL in a complex with death receptor 5.

Mol Cell, 4, 563–571.

14 Mongkolsapaya J, Grimes JM, Chen N, Xu XN, Stuart

DI, Jones EY & Screaton GR (1999) Structure of the

TRAIL-DR5 complex reveals mechanisms conferring

specificity in apoptotic initiation. Nat Struct Biol, 6,

1048–1053.

15 Cha SS, Sung BJ, Kim YA, Song YL, Kim HJ, Kim S,

Lee MS & Oh BH (2000) Crystal structure of TRAIL-

DR5 complex identifies a critical role of the unique

frame insertion in conferring recognition specificity.

J Biol Chem, 275, 31171–31177.

16 Papoff G, Hausler P, Eramo A, Pagano MG, Di LG,

Signore A & Ruberti G (1999) Identification and char-

acterization of a ligand-independent oligomerization

domain in the extracellular region of the CD95 death

receptor. J Biol Chem, 274, 38241–38250.

17 Chan FK, Chun HJ, Zheng L, Siegel RM, Bui KL &

Lenardo MJ (2000) A domain in TNF receptors that

mediates ligand-independent receptor assembly and sig-

naling. Science, 288, 2351–2354.

18 Siegel RM, Frederiksen JK, Zacharias DA, Chan FK,

Johnson M, Lynch D, Tsien RY & Lenardo MJ (2000)

Fas preassociation required for apoptosis signaling and

dominant inhibition by pathogenic mutations. Science,

288, 2354–2357.

19 Thomas LR, Johnson RL, Reed JC & Thorburn A

(2004) The C-terminal tails of tumor necrosis factor-

related apoptosis-inducing ligand (TRAIL) and Fas

receptors have opposing functions in Fas-associated

death domain (FADD) recruitment and can regulate

agonist-specific mechanisms of receptor activation.

J Biol Chem, 279, 52479–52486.

20 Guo Y, Chen C, Zheng Y, Zhang J, Tao X, Liu S,

Zheng D & Liu Y (2005) A novel anti-human DR5

monoclonal antibody with tumoricidal activity induces

caspase-dependent and caspase-independent cell death.

J Biol Chem, 280, 41940–41952.

21 Kim K, Fisher MJ, Xu SQ & El-Deiry WS (2000)

Molecular determinants of response to TRAIL in killing

of normal and cancer cells. Clin Cancer Res, 6, 335–

346.

22 Kurbanov BM, Geilen CC, Fecker LF, Orfanos CE &

Eberle J (2005) Efficient TRAIL-R1 ⁄DR4-mediated

apoptosis in melanoma cells by tumor necrosis factor-

related apoptosis-inducing ligand (TRAIL). J Invest

Dermatol, 125, 1010–1019.

23 MacFarlane M, Kohlhaas SL, Sutcliffe MJ, Dyer MJ &

Cohen GM (2005) TRAIL receptor-selective mutants

signal to apoptosis via TRAIL-R1 in primary lymphoid

malignancies. Cancer Res, 65, 11265–11270.

24 Kelley RF, Totpal K, Lindstrom SH, Mathieu M,

Billeci K, Deforge L, Pai R, Hymowitz SG & Ashke-

nazi A (2005) Receptor-selective mutants of apoptosis-

inducing ligand 2 ⁄ tumor necrosis factor-related apopto-

sis-inducing ligand reveal a greater contribution of

death receptor (DR) 5 than DR4 to apoptosis signaling.

J Biol Chem, 280, 2205–2212.

25 van der Sloot AM, Tur V, Szegezdi E, Mullally MM,

Cool RH, Samali A, Serrano L & Quax WJ (2006)

Designed tumor necrosis factor-related apoptosis-induc-

ing ligand variants initiating apoptosis exclusively via

the DR5 receptor. Proc Natl Acad Sci USA, 103,

8634–8639.

26 Reis CR, van der Sloot AM, Szegezdi E, Natoni A,

Tur V, Cool RH, Samali A, Serrano L & Quax WJ

(2009) Enhancement of antitumor properties of

rhTRAIL by affinity increase toward its death receptors

(dagger). Biochemistry, 48, 2180–2191.

27 Tur V, van der Sloot AM, Reis CR, Szegezdi E, Cool

RH, Samali A, Serrano L & Quax WJ (2008)

DR4-selective tumor necrosis factor-related apoptosis-

inducing ligand (TRAIL) variants obtained by

structure-based design. J Biol Chem, 283, 20560–20568.

28 Ladner RC, Sato AK, Gorzelany J & de SM (2004)

Phage display-derived peptides as therapeutic alterna-

tives to antibodies. Drug Discov Today, 9, 525–529.

29 Wrighton NC, Farrell FX, Chang R, Kashyap AK,

Barbone FP, Mulcahy LS, Johnson DL, Barrett RW,

Jolliffe LK & Dower WJ (1996) Small peptides as

potent mimetics of the protein hormone erythropoietin.

Science, 273, 458–464.

30 Cwirla SE, Balasubramanian P, Duffin DJ, Wagstrom

CR, Gates CM, Singer SC, Davis AM, Tansik RL,

Mattheakis LC, Boytos CM et al. (1997) Peptide

agonist of the thrombopoietin receptor as potent as the

natural cytokine. Science, 276, 1696–1699.

31 Sato A & Sone S (2003) A peptide mimetic of human

interferon (IFN)-beta. Biochem J, 371, 603–608.

32 Li B, Russell SJ, Compaan DM, Totpal K, Marsters

SA, Ashkenazi A, Cochran AG, Hymowitz SG & Sidhu

SS (2006) Activation of the proapoptotic death receptor

DR5 by oligomeric peptide and antibody agonists.

J Mol Biol 361, 522–536.

33 Yanofsky SD, Baldwin DN, Butler JH, Holden FR,

Jacobs JW, Balasubramanian P, Chinn JP, Cwirla SE,

Peters-Bhatt E, Whitehorn EA et al. (1996) High affin-

ity type I interleukin 1 receptor antagonists discovered

by screening recombinant peptide libraries. Proc Natl

Acad Sci USA, 93, 7381–7386.

34 Binetruy-Tournaire R, Demangel C, Malavaud B, Vassy

R, Rouyre S, Kraemer M, Plouet J, Derbin C, Perret G



& Mazie JC (2000) Identification of a peptide blocking

vascular endothelial growth factor (VEGF)-mediated

angiogenesis. EMBO J, 19, 1525–1533.

35 England BP, Balasubramanian P, Uings I, Bethell S,

Chen MJ, Schatz PJ, Yin Q, Chen YF, Whitehorn EA,

Tsavaler A et al. (2000) A potent dimeric peptide

antagonist of interleukin-5 that binds two interleukin-5

receptor alpha chains. Proc Natl Acad Sci USA, 97,

6862–6867.

36 Takasaki W, Kajino Y, Kajino K, Murali R & Greene

MI (1997) Structure-based design and characterization

of exocyclic peptidomimetics that inhibit TNF alpha

binding to its receptor. Nat Biotechnol, 15, 1266–1270.

37 Terskikh AV, Le Doussal JM, Crameri R, Fisch I,

Mach JP & Kajava AV (1997) ‘‘Peptabody’’: a new type

of high avidity binding protein. Proc Natl Acad Sci

USA, 94, 1663–1668.

38 Janssen M, Oyen WJ, Massuger LF, Frielink C, Di-

jkgraaf I, Edwards DS, Radjopadhye M, Corstens FH

& Boerman OC (2002) Comparison of a monomeric

and dimeric radiolabeled RGD-peptide for tumor

targeting. Cancer Biother Radiopharm, 17, 641–646.

39 O’Leary PD & Hughes RA (2003) Design of potent

peptide mimetics of brain-derived neurotrophic factor.

J Biol Chem, 278, 25738–25744.

40 Aggarwal S, Harden JL & Denmeade SR (2006) Syn-

thesis and screening of a random dimeric peptide library

using the one-bead-one-dimer combinatorial approach.

Bioconjug Chem, 17, 335–340.

41 Kajiwara K, Saito A, Ogata S & Tanihara M (2004)

Synthetic peptides corresponding to ligand-binding

region of death receptors, DR5, Fas, and TNFR, specif-

ically inhibit cell death mediated by the death ligands,

respectively. Biochim Biophys Acta, 1699, 131–137.

42 Aktas O, Smorodchenko A, Brocke S, Infante-Duarte

C, Topphoff US, Vogt J, Prozorovski T, Meier S,

Osmanova V, Pohl E et al. (2005) Neuronal damage in

autoimmune neuroinflammation mediated by the death

ligand TRAIL. Neuron, 46, 421–432.

43 van der Sloot AM, Mullally MM, Fernandez-Ballester

G, Serrano L & Quax WJ (2004) Stabilization of

TRAIL, an all-beta-sheet multimeric protein, using

computational redesign. Protein Eng Des Sel, 17,

673–680.



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