This journal is c The Royal Society of Chemistry 2011 Mol. BioSyst., 2011, 7, 337–345 337

Cite this: Mol. BioSyst., 2011, 7, 337–345

Modulation of human estrogen receptor a activity by multivalent

estradiol–peptidomimetic conjugateswzJustin M. Holub,

aMichael J. Garabedian

band Kent Kirshenbaum*

a

Received 6th September 2010, Accepted 24th November 2010

DOI: 10.1039/c0mb00189a

Estradiol–peptidomimetic conjugates (EPCs) are linear, sequence-specific peptoid oligomers that

site-specifically display multiple copies of 17b-estradiol (E2), a ligand for the human estrogen

receptor a (hERa). We evaluate the ability of multivalent EPCs to activate hERa-mediated

transcription. EPCs activated the hERa in both a length- and valence-dependent manner, with the

highest levels of activation generated by divalent peptoid 6-mers, divalent 18-mers, and trivalent

9-mers. Hexavalent EPCs did not activate hERa, but instead blocked E2-mediated hERaactivation. The physicochemical features of EPCs can be precisely tuned, which may allow

the generation of a library of chemical tools for modulating specific effects of estrogens.

Introduction

Multivalent ligands have attracted substantial interest for their

potential to modulate molecular interactions of biomedical

significance. Multivalent displays enhance the effective local

concentration of low affinity ligands to promote strengthened

binding to target receptors. Conjugating multiple ligands onto

an individual scaffold molecule yields products with strong

binding interactions, termed avidities.1–4 In principle, such

chemical constructs can target multimeric protein receptors

via multi-site binding contacts. The ability of multivalent

ligands to engage cell-surface receptor arrays can enable

modulation of a variety of biochemical processes, including

chemotaxis, adhesion, and immune responses. The targets of

synthetic multivalent ligands include RNA, enzymes, bacterial

toxins, and lectins.5–9

One potential benefit in creating multivalent displays is the

ability to tune the physico-chemical features of the scaffold.

For example, variation in the spacing between sites of

conjugation can be used to influence the density of displayed

ligands. Alteration of size, charge, and branching of the

scaffold can influence solubility and other pharmacological

characteristics, along with cellular uptake.

This report describes the characterization of multivalent

ligands targeting a steroid hormone receptor. Estrogen is a

steroid hormone that mediates its effect primarily thorough

the intracellular action of the estrogen receptor. The human

estrogen receptor (hER) belongs to a superfamily of ligand-

mediated transcription factors.10 In the classic model for

hER-mediated gene transcription, nuclear-localized hER

binds its natural ligand 17b-estradiol (E2), undergoes a

conformational rearrangement, dimerizes, and binds estrogen

response elements (ERE) on DNA, where it participates in the

recruitment of additional transcriptional cofactors.11–13 The

hER is known to exist in two isoforms: the widely-studied

hERa, and the hERb.14,15 Compounds that modulate hERaactivity are a major target for development of therapeutics

that address a range of human diseases, including cancer and

osteoporosis.16–18

In addition to nuclear-localized estrogen receptor, discrete

cellular pools of ER at the plasma membrane have been

implicated in the rapid ‘‘non-genomic’’ activation of MAPK,

contributing to the overall effects of estrogen action.19,20

Accordingly, several groups have focused on developing

macromolecular conjugates that display multiple copies of

E2 in an attempt to specifically modulate extranuclear

hER.21–25 These macromolecular constructs can be challenging

to synthesize, however, and can exhibit polydispersity in the

product composition, uneven loading of bioactive ligand,

aggregation, or cleavage of biologically labile linkers.

We have previously described the synthesis of multivalent

estrogen ligands via conjugation of estradiol groups at multiple

sites on a linear peptoid scaffold.26 Peptoids are oligomers

composed of N-substituted glycine units.27 Step-wise peptoid

oligomer synthesis on solid phase support allows the genera-

tion of monodisperse, sequence-specific products, and enables

precise control over the spacing of reactive groups along

the oligomer backbone.28 Peptoids show substantial potential

for the design of bioactive compounds.29,30 Owing to their ease

of synthesis, high proteolytic stability,31 and favorable cell

aDepartment of Chemistry, New York University,100 Washington Square East, New York, New York 10003, USA.E-mail: kent@nyu.edu

bDepartment of Microbiology, NYU Langone School of Medicine,550 First Avenue, New York, New York 10016, USA

w This article is part of aMolecular BioSystems ‘Emerging Investigators’issue highlighting the work of outstanding young scientists at thechemical- and systems-biology interfaces.z Electronic supplementary information (ESI) available: Peptoid synthesisprotocols, compound characterization and Flu-EPC emission spectra. SeeDOI: 10.1039/c0mb00189a

MolecularBioSystems

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338 Mol. BioSyst., 2011, 7, 337–345 This journal is c The Royal Society of Chemistry 2011

permeability,32 a variety of peptoid oligomers are being

evaluated as molecular tools for chemical biology and other

biomedical applications. These include use as transfection

reagents, modulators of protein activity and protein capture

agents.33–36

We have developed synthetic protocols to expand the

chemical diversity and potential biomedical applications of

peptoid oligomers by conjugating bioactive groups, such as

estradiol, to peptoid scaffolds using the Cu-catalyzed

azide–alkyne [3+2] cycloaddition (CuAAC) ‘‘click’’ reaction.37,38

We determined that multivalent estradiol–peptidomimetic

conjugates (EPCs) are capable of binding the hERain vitro.26 In addition, we successfully demonstrated that

EPC–hERa affinity is enhanced as the valency of ligands

along the oligomer backbone is increased. Here, we investigate

whether EPCs are capable of modulating the activity of the

hERa in vivo. HEK293 cells were chosen as a model because

they lack endogenous hERa, allowing us to express plasmid-

encoded hERa and examine the ability of multivalent EPCs to

modulate hERa-dependent transcription. We used this system

to evaluate libraries of monodisperse estrogen conjugates in

which the length and valency of the ligand presentation is

precisely controlled.

Results and discussion

Synthesis of estradiol–peptidomimetic conjugates

Peptoid oligomers incorporating site-specific azide-functionalized

side chains were synthesized on solid support using a modifi-

cation of the submonomer synthesis protocol established by

Zuckermann et al.28 Following oligomerization, the resin-bound

peptoid oligomers were allowed to react with 17a-ethynyl-estradiol in the presence of a copper catalyst to generate

multivalent EPCs (Fig. 1, ESIz, Scheme S1 and Table S1).

This reaction resulted in a stable triazole linkage formed

between the alkyne-containing estradiol moiety and the

azide-functionalized peptoid side chain. To mitigate steric

hindrance during conjugation and to facilitate receptor

binding, multivalent EPCs 1–4 (Fig. 1a) were designed with

the pendant groups conjugated at every third residue. It

should be noted that the distance between the binding pockets

of dimerized hERa is approximately 24 A (determined from

PDB ID: ERE1). Placement of the E2 moieties at every third

residue along the EPC scaffolds could potentially bridge this

distance when the EPC is in an extended conformation.

Methoxyethyl moieties were included at all other side chain

positions to enhance overall EPC hydrophilicity.

Given that the hERa activates transcription as a homo-

dimer, and that our EPC synthetic protocol enables site-

specific conjugation of bioactive ligands to the peptoid scaffold,

we expanded our multivalent EPC library and generated a

set of divalent EPCs 5–7 (Fig. 1b). To enhance overall

hydrophilicity and facilitate receptor binding, EPCs 5, 6, and

7 were designed and synthesized with the E2 moieties spaced

between the N and C termini of the peptoid oligomer by 5, 8,

and 14 intervening monomer units, respectively. Mass spectra

analysis for all peptoids described herein can be found in the

ESIz, Table S2.

Synthesis of fluorescently labeled EPCs

To evaluate the cell permeability of our EPCs, we synthesized

fluorescently labeled EPCs (Flu-EPCs, Fig. 2) on solid

support using a modification of previously described synthetic

methods39,40 (ESIz, Scheme S2). To mitigate intramolecular

steric clash between the relatively bulky tracer and E2 ligand,

we inserted a six-carbon aliphatic linker between the fluores-

cein and the E2-functionalized peptoid scaffold (Fig. 2).

Estrogenic compounds containing phenol rings are known to

quench fluorescence,41 thus we were aware that the presence of

multiple E2 pendant groups could potentially diminish the

fluorescence emission intensity of our Flu-EPCs. The inserted

aliphatic linker spaces the fluorophore from the E2 ligand and

should mitigate intramolecular quenching. We did observe,

however, that increasing E2 valency diminishes fluorescence

emission intensity of Flu-EPCs (ESIz, Fig. S1).

Toxicity of estradiol–peptidomimetic conjugates

Prior to examining cellular localization, we evaluated the cell

toxicity of the EPCs. We employed a standard MTT cell

Fig. 1 Estradiol–peptidomimetic conjugates for use as estrogen receptor modulators. (a) Compound 1 (n=1); compound 2 (n=2); compound 3

(n = 3); compound 4 (n = 6). (b) Compound 5 (n = 5); compound 6 (n = 8); compound 7 (n = 14).

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viability assay on HEK293 cells treated with 1.0 mMEPCs 1–7

for 3 days. Relative EPC concentrations were based on molar

concentration of the conjugate molecule and not E2 equiv-

alencies. As a control for cell toxicity, cells were treated with

Doxorubicin, a known cytotoxic agent.42 Following incuba-

tion and workup (see Experimental section), the relative UV

absorbance was plotted as a function of cell viability (Fig. 3).

From these data, we determined that EPC constructs 1–7 were

not toxic to HEK293 cells (at 1.0 mM) over the treatment times

tested.

Cell permeability of estradiol–peptidomimetic conjugates

Previous reports indicate that peptoids exhibit enhanced cell

permeability compared to corresponding peptide analogues.32

To investigate the cell uptake efficiency of our EPCs, we

incubated HEK293 cells in the presence of Flu-EPCs 8–13

and used qualitative live-cell fluorescence imaging analysis to

visualize EPC internalization (Fig. 4 and 5). As a control, we

treated the cells with 5,6 carboxyfluorescein using similar

conditions and it was verified that the free fluorophore was

not internalized by the cells (data not shown). It should be

noted that hexavalent Flu-EPCs were not evaluated in this

context because of inadequate solubility at the concentrations

tested.

Interestingly, the smallest Flu-EPC construct, monovalent

compound 8, did not show substantial cell uptake (Fig. 4a). Of

the Flu-EPC subset shown in Fig. 4, the highest levels

of cell permeability were observed with divalent 9 and

trivalent 10 (Fig. 4b and c). Compound 8 has substantially

greater fluorescence emission intensity when compared to

Flu-EPCs 9 and 10 (ESIz, Fig. S1), indicating that the lack

of intracellular visualization of 8 is not a result of EPC

self-quenching. The enhanced hydrophobic character of

compounds 9 and 10 may lead to improved cell permeability.

Additionally, divalent compound 9 distributes more evenly

throughout the cell (Fig. 4b) when compared to trivalent 10

(Fig. 4c).

Following confirmation that multivalent Flu-EPCs 9 and 10

were cell permeable, we used similar techniques to evaluate

the uptake efficiency of divalent Flu-EPCs 11–13 (Fig. 5).

Surprisingly, divalent 9-mer Flu-EPC 11 was not detected in

Fig. 2 Fluorescently-labeled estradiol–peptidomimetic conjugates (Flu-EPCs). (a) Compound 8 (n = 1); compound 9 (n = 2); compound 10

(n = 3). (b) Compound 11 (n = 5); compound 12 (n = 8); compound 13 (n = 14).

Fig. 3 EPC toxicity in HEK293 cells. (a) Relative viability of cells treated with multivalent EPCs 1–4. (b) Relative viability of cells treated with

divalent EPCs 2, 5–7. Veh., EtOH (0.1% in DMEM); Doxo, Doxorubicin, 1.0 mM.Publ

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340 Mol. BioSyst., 2011, 7, 337–345 This journal is c The Royal Society of Chemistry 2011

the intracellular environment of HEK293 cells by fluorescence

microscopy (Fig. 5a). However, further separation of the two

estradiol moieties by increasing the length of the peptoid

backbone rescues cell permeability, as seen with compounds

12 (Fig. 5b) and 13 (Fig. 5c).

To rule out the possibility that the Flu-EPC constructs 8

and 11 were being internalized and then subsequently expelled

from the cells before imaging at 18 h, HEK293 cells were

treated with compounds 8 and 11 as described above and

imaged at 0.5, 1, 1.5, 2, 3, 6, 9, 12, and 15 h. Compounds 8 and

Fig. 4 Live-cell imaging showing uptake of multivalent Flu-EPCs in HEK293 cells. All cells were treated with 50 mM Flu-EPC for 18 h, washed

and subsequently counter-stained with Hoechst 33342 to visualize the nucleus. Headings above image columns indicate fluorescence filters used.

Scale bar = 20 mm. (a) Monovalent 8; (b) Divalent 9; (c) Trivalent 10.

Fig. 5 Live-cell imaging showing uptake of divalent Flu-EPCs in HEK293 cells. All cells were treated with 50 mMFlu-EPC for 18 h, washed, and

subsequently counter-stained with Hoechst 33342 to visualize the nucleus. Headings above image columns indicate fluorescence filters used. Scale

bar = 20 mm. (a) EPC 9-mer 11; (b) EPC 12-mer 12; (c) EPC 18-mer 13.

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11 were not taken up by the cell at these time points (data not

shown), suggesting that these particular Flu-EPCs were not

internalized by the cells at any time during the treatment.

Comparison of the uptake efficiency of 9-mers 10 and

11 (Fig. 4c and 5a) reveals that peptoid oligomer chain

length may not be the primary determinant of Flu-EPC cell

permeability. These data suggest that the presence of one

additional E2 equivalent along the oligomeric backbone signifi-

cantly increases the likelihood of transport across the cell

membrane. Enhanced hydrophobicity may facilitate the cell

permeability of these oligomeric molecules.43,44 Notably, the

smallest divalent Flu-EPC 9 shows the highest degree of

cell permeability when compared to the longer divalent counter-

parts, 11, 12 and 13. The divalent 12-mer Flu-EPC 12 shows

some degree of cell uptake while the shorter, divalent 9-mer

Flu-EPC 11, shows no internalization (compare Fig. 6a and b).

Specific scaffold lengths and ligand valencies may be required

for optimized cell permeability of steroid–peptoid conjugates.32

In addition, all cell permeable Flu-EPC constructs appear to

localize extensively within the cytosol.

Multivalent EPCs modulate hERa-mediated transcription

We evaluated the ability for the EPC constructs to activate the

hERa in vivo. HERa-mediated luciferase reporter assays were

used to determine relative levels of EPC-induced hERa activa-

tion (Fig. 6). HEK293 cells were stably transfected with

plasmids containing a hERa regulatory region luciferase

reporter and full-length hERa (hERa(+)). Expression of

hERa in transfected hERa(+) HEK293 cells was confirmed

by Western blotting (ESIz, Fig. S2). To optimize treatment

times and dosing concentrations for maximal ligand-mediated

hERa activation, time courses and dose response experiments

were performed with E2. Maximal E2-mediated activation of

the hERa occurred at a treatment concentration of 100 nM E2

at 18 h (ESIz, Fig. S3). Unless otherwise indicated, all E2 and

EPC treatments reported herein were performed at 100 nM

concentrations for 18 h. Relative EPC concentrations are

based on the overall molarities of the conjugate molecule

and not individual E2 equivalencies.

To determine if EPCs activate the estrogen receptor in vivo,

we first treated hERa(+) HEK293 cells with EPCs 1–4.

Following treatment, relative luciferase activity was quantified

(Fig. 6a). In accordance with our previous in vitro studies

involving EPC–hERa binding,26 in which increased valencies

yielded enhanced binding affinities (up to 10.9 nM for

hexavalent EPC 4), we hypothesized that increased ligand

valency would lead to strengthened binding avidities in vivo

and may result in enhanced hERa activation. Indeed, mono-

valent EPC 1 did not activate the hERa over background. This

null-effect may be due to either the low binding affinity of 1 for

the hERa,26 or cell impermeability of the monovalent EPC

construct (see Fig. 4a). We observed, however, that increasing

the valency of receptor ligand along the oligomer scaffold

enhanced EPC-mediated hERa activation (Fig. 6a), with

divalent compound 2 showing enhanced hERa activation

over monovalent compound 1, and maximal activation

attained with trivalent EPC 3 (Fig. 6a). Notably, there was

no activation of the hERa seen with hexavalent compound 4

(vida infra).

Divalent EPCs modulate hERa-mediated transcription

Due to steric crowding of bioactive ligands on multivalent

EPCs, and the fact that the hERa is most active in its

homodimeric form,45 we hypothesized that divalent EPCs

may activate the hERa as potently as their similar-length

multivalent counterparts. Transfected hERa(+) HEK293

cells were treated with 100 nM concentrations of divalent

EPCs 5, 6 or 7 for 18 h. Following incubation, relative

luciferase activity was quantified (Fig. 6b). Luciferase reporter

assays demonstrated that compounds 5 and 6 do not activate

the hERa above background. These data correlate with

results from our cell uptake assays, which showed minimal

cell-permeability by corresponding Flu-EPCs 11 and 12

(Fig. 5a and b). The overall lack of hERa activation by

Fig. 6 Modulation of hERa by (a) variable valency and (b) variable length divalent estradiol–peptidomimetic conjugates. All compounds were

administered at a concentration of 100 nM for 18 h. Relative luciferase activities were normalized to vehicle treatment in pcDNA3-containing

transfectants. Veh., vehicle (EtOH, 0.1% in media); E2, 17b-estradiol.

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compounds 5 and 6 may be due to the inability of these

constructs to be internalized by the cell. Cell-permeable

divalent 18-mer 7, however, demonstrated significant activa-

tion of the hERa in this system and showed a similar increase

in activation as trivalent 18-mer 3 (compare Fig. 6a and b).

Non-steroidal peptidomimetic conjugates do not modulate

hERa-mediated transcription

To establish the role of the E2 pendant groups on mediating

hERa action, we designed a trivalent non-steroidal control

peptoid conjugate 14 (ESIz, Fig. S4). These constructs displayedpentylbenzene units in the place of estradiol groups. Using

similar hERa-mediated luciferase reporter assays as described

above, we compared the ability for 14 to activate the hERawith its trivalent EPC counterpart 3 (ESIz, Fig. S5). Trivalent14 does not activate hERa over background, indicating that

the estradiol pendant groups are required for EPC bioactivity.

A hexavalent estradiol–peptidomimetic conjugate blocks

estrogenic activity in transfected HEK293 cells

Interestingly, hexavalent EPC 4, which has previously been

shown in vitro to bind the hERa with 10.9 nM affinity,26

did not activate hERa-mediated transcription in the cell

based reporter assay (Fig. 6a). Due to its relatively large size

(>4000 Da) and inability to activate the hERa in vivo, we were

concerned that compound 4 was cell-impermeable and

therefore not active in this system. Alternatively, hexavalent

EPC 4 could be binding hERa in vivo, but fail to elicit a

transcriptional response, and thus antagonize hERa activation

by E2. To test whether compound 4 was able to block

estrogenic activity in vivo, we treated transfected hERa(+)

HEK293 cells with 100 nM compound 4 for 8 h, followed by

the administration of E2 (100 nM) for an additional 10 h.

Relative luciferase activity was then measured (Fig. 7). It

was seen from these data that 4 blocks E2-mediated hERaactivation in this system. Similar experiments were performed

with EPCs 1–3 and 5–7 and showed that these constructs were

not capable of blocking E2-mediated activation of the hERa(ESIz, Fig. S6). Therefore, hexavalent EPC 4 binds to

ER in vivo and functions as an ER antagonist. Furthermore,

it is likely that EPC 4 can bind multiple hERa molecules,

potentially disrupting the transcriptional activity and function

of the active dimeric receptor.

Hexavalent EPCs do not promote hERa degradation

The potency and mechanism through which hERa antagonists,

such as tamoxifen and fulvestrant (ICI 182,780), exert their

actions vary widely.46–48 For example, tamoxifen competes for

the ligand binding pocket within the hERa and causes a subtle

conformational change to the receptor, resulting in effects on

co-factor binding and down-regulation of estrogen responsive

genes.49 On the other hand, the hERa antagonist fulvestrant is

thought to influence substantial misfolding of the receptor

upon binding, causing the protein to be degraded in vivo. Due

to its relatively large size, high ligand valency, and ability to

block E2-mediated transcription, we evaluated whether EPC 4

induces hERa degradation within the cell. Transfected

hERa(+) HEK293 cells were treated with E2 (10 nM), ICI

182,780 (1.0 mM), or 4 (1.0 mM) for 6, 12 or 24 h. Intracellular

proteins were then extracted, subjected to SDS-PAGE, and

analyzed by Western Blot (Fig. 8). When compared to ICI

182,780, which induces hERa degradation, hexavalent

compound 4 does not influence intracellular hERa levels

over the treatment times and concentrations tested. These

results indicate that compound 4 is capable of disrupting the

transcriptional activity of E2-mediated hERa though a

distinct mechanism from that of the ICI compound.

Conclusions

We have designed a series of bioactive peptidomimetic

oligomers that modulate the hERa. We reported previously

that multivalent EPCs competitively bind the estrogen

receptor in whole cell lysates and that EPC–hERa affinity is

enhanced as the valency of ligand along the oligomer back-

bone is increased.26 Herein, we have augmented our library of

previously reported multivalent EPC constructs to include

variable length divalent EPCs and fluorescently labeled EPC

constructs. In addition, we have expanded our earlier studies

Fig. 7 Estrogen-mediated activation of the hERa is blocked by

hexavalent EPC 4. All compounds were administered at a concentra-

tion of 100 nM. Relative luciferase activities were normalized to

vehicle treatment in pcDNA3-containing transfectants. Veh., vehicle

(EtOH, 0.1% in media); E2, 17b-estradiol.

Fig. 8 Hexavalent EPC 4 does not cause degradation of the hERa in

transfected HEK293 cells. Treatment concentrations: E2, 10 nM; ICI,

1.0 mM; EPC 4, 1.0 mM. Treatment times (h) are shown above

respective lanes. +, Positive control (untreated hERa(+) HEK293

cell lysate); �, negative control (untreated hERa(�) HEK293 cell

lysate); E2, 17b-estradiol; ICI, ICI 182,780.

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by demonstrating that EPCs are capable of modulating the

hERa in vivo.

Qualitative live-cell imaging experiments indicated that

there is a marked length and valency dependence on cell

permeability of Flu-EPCs, with divalent 6-mer 9, trivalent

9-mer 10, and divalent 18-mer 13 having the highest levels of

internalization. EPCs are intrinsically amphipathic, consisting

of a relatively hydrophilic backbone and large, bulky, hydro-

phobic pendant groups. The low levels of cell permeability that

are seen with Flu-EPCs monovalent 3-mer 8, divalent 9-mer

11, and divalent 12-mer 12 may be due to a number of factors:

the presence of an anionic fluorophore, overall hydrophilicity,

or folded conformation in solution.50 Nevertheless, an obvious

advantage of working with EPCs is their intrinsic length and

valence ‘‘tunability’’, which affords precise control over ligand

spacing and can facilitate physicochemical optimization of

these bioactive ligands for enhanced cell permeability.

In addition, we have shown that our EPC constructs are

capable of modulating the hERa in a living system, with

maximal activity coming from the most cell-permeable

constructs: divalent 6-mer 2, trivalent 9-mer 3, and divalent

18-mer 7. The minimal activation of monovalent 3-mer 1,

divalent 9-mer 5, and divalent 12-mer 6 may be due to their

inability to traverse the cell membrane. The ability for

compounds 2, 3, and 7 to activate nuclear actions of the hERais apparent despite the fact that their fluorescently labeled

counterparts do not seem to penetrate to the nucleus. This

result may be due the presence of the fluorophore perturbing

the ability for these constructs to cross the nuclear envelope.

Surprisingly, hexavalent 18-mer 4, which has the highest

receptor affinity of any EPC tested,26 did not activate the

hERa in vivo. Conversely, divalent 18-mer 7, which is identical

in length but contains substantially fewer E2 equivalents than

EPC 4, shows enhanced activation of the hERa over

background levels. This result indicates that increased local

concentration of receptor ligand and high EPC-receptor

binding affinity does not necessarily translate to enhanced

hERa activation. To the contrary, hexavalent EPC 4 is able

to block estrogenic-mediated activation of the hERa when

co-administered with E2, indicating that the characteristics of

EPC activity are markedly dependent on the valency of the

displayed ligands.

The ability to tune precisely the length and valency of our

EPCs allows for the generation of novel bioactive constructs

that may be optimized for use in a variety of experimental

settings. There is now evidence that pools of estrogen receptor

localize at the cell membrane, in the cytosol, and in the

nucleus, and that each of these receptor pools are capable

of independently activating various ER-mediated pathways

within the cell.20,51–53 The EPC synthetic model outlined

herein affords us the capacity to tailor the physicochemical

properties of these bioactive oligomer constructs. We are

currently exploring the ability for EPC conjugates to activate

specific subsets of estrogen receptors based on subcellular

localization in hERa(+) breast cancer cell lines. Importantly,

the approach outlined herein can be extended to display a

variety of steroid hormones or other ligands,5,26,37 suggesting a

general strategy for modulating protein receptor mediated

signaling pathways.

Experimental

Detailed peptoid synthesis protocols, compound characteriza-

tion and Flu-EPC fluorescence emission spectra are outlined in

the ESI.z

Cell culture

HEK293 cells were maintained at 5% CO2 in a 37 1C

incubator and cultured in Dulbecco’s Modification of Eagles

Medium (DMEM) supplemented with 10% fetal bovine

serum (FBS) and penicillin/streptomycin (PS). Typically, cells

were grown on 10 cm tissue-culture dishes (BD/Falcon) to

approximately 80% confluence before subculture.

EPC toxicity assays

HEK293 cells were plated (5000 cells/well) in triplicate on

treated 96-well plates (BD Falcon) in DMEM supplemented

with 10% FBS and PS, and allowed to attach overnight. The

cells were then switched to media supplemented with either

vehicle (0.1% EtOH), doxorubicin (1.0 mM) or EPCs (1.0 mM).

The final EtOH concentration in the treatment media was

0.1%. The cells were allowed to incubate in a humidified

atmosphere at 37 1C for 3 d. Following incubation, relative

cell viability was quantified using a colorimetric MTT assay

(Promega) performed according to the manufacturer’s

instructions.

Cell uptake of fluorescently labeled EPCs

HEK293 cells were seeded on poly-D-lysine-coated clear-

bottom 96-well plates (Corning) at a density of 5000 cells

per well in DMEM supplemented with 10% FBS and PS.

Following overnight attachment, the cells were switched to

phenol red-free DMEM supplemented with 5% charcoal-

stripped FBS (CS-FBS) and L-Glutamine (L-Gln). The cells

were allowed to starve for 48 h before treatment. Purified

Flu-EPCs (10 mM in EtOH) or 5,6 carboxyfluorescein

(10 mM in EtOH) were dissolved to a final concentration of

50 mM in phenol red-free DMEM supplemented with 5%

CS-FBS and L-Gln. The final EtOH concentration in the

treatment media was 0.5%. The cells were allowed to incubate

in the treatment solution for 18 h at 37 1C in the dark.

Following incubation, cells were counter-stained in phenol

red-free DMEM supplemented with 5% CS-FBS and L-Gln

containing Hoechst 33342 (Invitrogen) according to the

manufacturer’s instructions. Live cell imaging was conducted

on a DMIRE-2 fluorescence microscope (Leica) outfitted with a

digital camera (Hamamatsu) using appropriate filters. Images

were processed using FW4000 imaging software (Leica).

EPC-mediated hERa activation

HEK293 cells were seeded in triplicate on poly-D-lysine-coated

24-well plates (Corning) at a density of 15 000 cells per well in

DMEM supplemented with 10% FBS and PS. Following

overnight attachment, the cells were transfected with appropriate

plasmids using the ExGen 500 transfection reagent (Fermentas)

according to the manufacturer’s instructions. Transfectable

plasmids were suspended in phenol red-free DMEM and

administered to cells. Each well received 100 ng ERE-containing

XETL regulatory region-luciferase reporter plasmid,54 3 ng

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344 Mol. BioSyst., 2011, 7, 337–345 This journal is c The Royal Society of Chemistry 2011

pcDNA3-hERa or empty vector (pcDNA3), 5 ng pcMV-lacZ,

and 142 ng pBluescript II SK (Stratagene). Following a 5 h

incubation at 37 1C, the transfection solutions were aspirated

from the wells and the cells were allowed to recover overnight

in DMEM supplemented with 10% FBS and PS. The

transfectants were then switched to phenol red-free DMEM

supplemented with 5% CS-FBS and L-Gln and the cells were

allowed to starve for 48 h. E2 or EPCs 1–7 (10 mM in EtOH)

were dissolved to a final concentration of 100 nM in phenol

red-free DMEM supplemented with 5% CS-FBS and L-Gln.

The final EtOH concentration in each treatment solution was

0.1%. Treatment solutions were added and the cells were

allowed to incubate at 37 1C for 18 h. Following incubation,

the cells were washed with PBS and lysed in 1� luciferase cell

culture lysis reagent (Promega) according to the manufacturer’s

instructions. Luciferase activity was quantified in a reaction

mixture containing 10 mL cell lysate and 100 mL 1� luciferase

assay reagent (Promega) using a microplate luminometer

(LMax). Relative luciferase activity values were individually

normalized to b-galactosidase activity and collectively normalized

to vehicle treatment in pcDNA3-containing transfectants. Data

were processed using Microsoft Excel.

hERa degradation assays

Transfected hERa(+) HEK293 cells were grown to approxi-

mately 60% confluence on 6 cm culture dishes (BD Falcon) in

DMEM supplemented with 10% FBS and PS. The cells were

then switched to phenol red-free DMEM supplemented with

5% CS-FBS and L-Gln and the cells were allowed to starve for

48 h before treatment. E2 (10 mM in EtOH) was dissolved to a

final concentration of 10 nM in phenol red-free DMEM

supplemented with 5% CS-FBS and L-Gln. ICI 182,780

or EPC 4 (each 10 mM in EtOH) were dissolved to a final

concentration of 1.0 mM in phenol red-free DMEM

supplemented with 5% CS-FBS and L-Gln. The final EtOH

concentration in each treatment solution was 0.1%. Treatment

solutions containing appropriate concentrations of E2, ICI

182,780, or EPC 4 were added and the cells were allowed to

incubate at 37 1C for 6, 12 or 24 h. Following incubation, the

cells were harvested into ice-cold PBS and pelleted. Whole cells

were lysed in SKL lysis buffer in the presence of 1 mM

Na3VO4 and 1� protease-inhibitor cocktail. Protein concen-

trations were quantified using a standard colorimetric

Bradford assay (Bio-Rad). Samples were loaded onto 10%

acrylamide gels at a concentration of 50 mg protein per well

and subjected to SDS-PAGE. The separated proteins were

then transferred onto Immobilon membranes (Millipore)

and probed with rabbit affinity-purified anti-hERa (HC-20)

(Santa Cruz Biotechnology) or anti-ERK1/2 (Cell Signaling

Technology) primary antibodies. Membranes were then

incubated with anti-rabbit, horseradish peroxidase-conjugated

secondary antibodies (KPL). Signals were detected on X-ray

film (Denville) using an ECL detection kit (GE Health

Sciences) according to the manufacturer’s instructions.

Acknowledgements

The authors gratefully acknowledge the support of the

NSF (CAREER Award #CHE-0645361) and the NIH

(Research Facilities Improvement Grant C060RR-165720).

We thank Jerome Nwachukwu for valuable technical

assistance.

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