Multivalent peptidomimetic conjugates: A versatile platform for modulating androgen receptor activity

Article abstract of DOI:10.1021/ja300170n

We introduce a family of multivalent peptidomimetic conjugates that modulate the activity of the androgen receptor (AR). Bioactive ethisterone ligands were conjugated to a set of sequence-specific peptoid oligomers. Certain multivalent peptoid conjugates enhance AR-mediated transcriptional activation. We identify a linear and a cyclic conjugate that exhibit potent anti-proliferative activity in LNCaP-abl cells, a model of therapy-resistant prostate cancer. The linear conjugate blocks AR action by competing for ligand binding. In contrast, the cyclic conjugate is active despite its inability to compete against endogenous ligand for binding to AR in vitro, suggesting a non-competitive mode of action. These results establish a versatile platform to design competitive and non-competitive AR modulators with potential therapeutic significance.

Full text of DOI:10.1021/ja300170n

Communication
pubs.acs.org/JACS
Multivalent Peptidomimetic Conjugates: A Versatile Platform for
Modulating Androgen Receptor Activity
Paul M. Levine,† Keren Imberg,‡ Michael J. Garabedian,§ and Kent Kirshenbaum*,†
†Department of Chemistry, New York University, New York, New York 10003, United States
‡Department of Pharmacology and §Department of Microbiology, NYU Langone School of Medicine, New York, New York 10016,
United States
S
* Supporting Information
ABSTRACT: We introduce a family of multivalent
peptidomimetic conjugates that modulate the activity of
the androgen receptor (AR). Bioactive ethisterone ligands
were conjugated to a set of sequence-specific peptoid
oligomers. Certain multivalent peptoid conjugates enhance
AR-mediated transcriptional activation. We identify a
linear and a cyclic conjugate that exhibit potent anti-
proliferative activity in LNCaP-abl cells, a model of
therapy-resistant prostate cancer. The linear conjugate
blocks AR action by competing for ligand binding. In
contrast, the cyclic conjugate is active despite its inability
to compete against endogenous ligand for binding to AR in
vitro, suggesting a non-competitive mode of action. These
results establish a versatile platform to design competitive
and non-competitive AR modulators with potential
therapeutic significance.
Figure 1. Peptoid scaffolds permit design of oligomers to modulate
androgen receptor activity through multivalent interactions. Colored
circles represent sites amenable to chemical modification and ligand
display. Homology model of the AR ligand binding domain dimer
(green ribbon, PDB 1I37) bound to native ligand (DHT,
magenta).14,15
here is a critical need to develop potent and selective
compounds that can modulate androgen receptor (AR)
T
activity to provide a therapeutic modality for prostate cancer.
Androgens are steroid hormones that can interact with the AR
to play an important role in human endocrinology and disease.1
AR is a ligand-dependent transcription factor capable of binding
the native androgen dihydrotestosterone (DHT).2 The classical
mechanism of AR activation involves DHT displacing a
chaperone protein, thus inducing a conformational change
that promotes receptor dimerization.3 Upon phosphorylation
and translocation into the nucleus, AR binds to specific DNA
sequences and recruits necessary transcriptional co-factors to
regulate gene expression.4,5
Androgens functioning through the AR can also promote
prostate cancer development, growth and progression.6 The
advancement of prostate cancer from an androgen-dependent
disease state to one that is androgen-independent represents
the disease’s lethal transformation, as limited therapeutic
options exist for patients with advanced disease.7 The standard
approach for treating androgen-dependent prostate cancer is
androgen ablation by suppression of testosterone production.
This treatment option is typically accompanied by competitive
DHT antagonists, such as bicalutamide, to block AR signaling.8
While initially effective at suppressing tumor growth, these
therapies often evoke castrate-resistant (or androgen-independ-
ent) prostate cancer progression.9
AR is expressed in both androgen-dependent and androgen-
independent prostate cancer cells.10 Two cell model systems,
LNCaP (androgen-dependent) and LNCaP-abl (androgen-
independent) have been established to study AR function in
these different disease states. AR-based drug discovery typically
focuses on the development of competitive DHT antagonists
that bind in the ligand-binding domain (LBD) of the AR.11
Drug resistance can therefore arise through mutations within
the AR-LBD.12 Recent evidence suggests that allosteric binding
sites on AR can also regulate receptor activity through non-
competitive mechanisms, providing additional targets for
pharmacology.13 Thus, the development of non-competitive
modulators that act independently or synergistically with
competitive antagonists could shift the paradigm for prostate
cancer therapy. The approach described herein introduces a
versatile multivalent scaffold to design competitive or non-
competitive AR modulators with potential therapeutic signifi-
cance (Figure 1).
Received: January 16, 2012
Published: April 17, 2012
© 2012 American Chemical Society
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a
Scheme 1. General Synthesis of Linear and Cyclic Multivalent Peptoid Conjugates
a
Rink Amide and 2-chlorotrityl chloride resin were used to generate linear and cyclic peptoid oligomers, respectively. Rink Amide resin was cleaved
with 95% trifluoroacetic acid following conjugation reactions.
Multivalent pharmacological strategies have several advan-
tages over traditional small molecule approaches. Multivalent
constructs have been shown to enhance binding to target
receptors through avidity effects.16 The assembly of a
multivalent display on a modular oligomer framework can
also enable control over important physico-chemical features of
the products.17 In particular, control over size, charge and
branching of the scaffold can influence solubility, cellular uptake
and other desirable pharmacological characteristics.
We previously introduced the use of a N-substituted glycine
oligomer scaffold, termed a “peptoid”, to establish multivalent
displays of diverse bioactive ligands.18 Peptoids are a class of
peptidomimetics in which the side chains are appended to the
amide nitrogen atoms, engendering proteolytic stability and
enhanced cellular permeability.19,20 The ability to incorporate
highly diverse side chain groups facilitates the discovery of
peptoid sequences that exhibit a wide variety of sophisticated
functions ranging from enantioselective catalysis to modulation
of protein−protein interactions.21−23 Peptoids can be synthe-
sized on solid support to allow generation of monodisperse
products, offering distinct advantages over other multivalent
display approaches (e.g., random copolymers or dendrimers).24
The sequence-specific assembly of peptoids enables precise
tuning of ligand valency and spacing to potentially enhance
affinity and specificity for corresponding biomolecular targets.17
In addition, the peptoid scaffold can be used as a versatile
platform for optimizing other properties critical for biological
activity, such as the ability to alter the topology of the scaffold
to achieve optimal ligand−receptor interactions.25
powerful synthetic tool due to its bioorthogonality, high yields
and mild reaction conditions.26
Using modified solid-phase peptoid synthesis protocols, we
synthesized linear and cyclic peptoid oligomers containing
azido-alkyl functionalized side chains at specific positions in the
oligomer sequence.18 Following oligomerization (and cycliza-
tion as required), the peptoid scaffolds were used as substrates
for CuAAC-mediated conjugation of ethisterone ligands
(Scheme 1). In order to alleviate steric congestion, the
ethisterone moieties were conjugated at least three residues
apart (i, i+3) in the peptoid oligomer sequence. It is important
to note that the distance between the hormone binding pockets
of dimerized AR is approximately 26 Å (Figure S6). Increasing
the spacing between the steroid ligands could potentially span
the AR dimer and allow simultaneous binding to the two
pockets. To enhance overall water solubility, all other
intervening positions in the peptoid sequence included the
hydrophilic monomer N-(methoxyethyl)glycine.
We synthesized a set of linear peptoid conjugates with one,
two, three, or six ethisterone ligands (conjugates 1−4)
displayed along the peptoid backbone. Additional divalent
peptoid conjugates containing ethisterone ligands separated by
five (conjugate 5) or eight (conjugate 6) intervening monomer
units were synthesized (Scheme 1). We also generated a cyclic
divalent construct (conjugate 7) to constrain the spatial
disposition of the ethisterone moieties.
An in vitro ligand-binding assay was used to evaluate binding
of the multivalent peptoid conjugates to the AR. In this assay,
competitive binding was detected by a change in fluorescence
polarization that occurs upon displacement of a fluorescently
labeled hormone ligand (Fluormone) from AR by a
competitive ligand.29 Conjugates 1−3 and 7 do not compete
against Fluormone for binding at concentrations up to 10 μM
(Figure 2A and Figure S7). Conjugates 4−6 compete for
binding, indicated by a decrease in fluorescence polarization
relative to vehicle treatment. To differentiate effects due to
multivalency and not hydrophobicity, a control peptoid
conjugate was synthesized (8, see SI). Conjugate 8, a divalent
dodecamer containing one inactive hydrophobic moiety and
one bioactive ethisterone ligand, did not compete for ligand
We designed a series of peptoid-based multivalent conjugates
to evaluate the influence of valency, spacing and conformational
ordering on AR activity. We used Cu-catalyzed azide−alkyne
[3+2] cycloaddition (CuAAC) “click” reactions to conjugate
ethisterone, a 17α-ethynyl homologue of DHT, to peptoid side
chains, thus generating a family of multivalent conjugates.26
Ethisterone was chosen as a ligand because it is known to
compete for AR binding and suppresses levels of AR
transcriptional activation relative to DHT.27,28 Additionally,
the ethynyl moiety provides accessibility to CuAAC reactions, a
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CyQUANT assay (Figure S9).32 Conjugates 1 and 2, which fail
to competitively bind AR or activate AR gene expression, had
little impact on the proliferation of the LNCaP-abl cells. In
contrast, conjugate 3, which elicited only a 3-fold induction of
AR reporter gene activity, suppressed the proliferation of the
LNCaP-abl cells. An agonist-induced transcription assay
confirms conjugate 3 is functioning as an AR antagonist
(Figure S10). Conjugate 4, which induced a potent AR
transcriptional response, inhibited cell proliferation to the same
extent as conjugate 3. Conjugates 6 and 7 displayed the greatest
levels of inhibition of cell proliferation. Although conjugates 3−
7 suppress the proliferation of LNCaP-abl cells, they do so
through different mechanisms, given the distinctive profiles for
competitive binding elicited by the conjugates.
Conjugates 6 and 7 were further evaluated for their ability to
inhibit LNCaP-abl cell proliferation. At concentrations of 1 μM
or 10 nM, conjugates 6 and 7 evoke a potent reduction in cell
proliferation, relative to vehicle treatment (Figure 3A and
Figure S11). To demonstrate that suppression of proliferation
can be specifically attributed to the presence of the steroid
Figure 2. (A) Multivalent peptoid conjugates can compete with
fluorescetly labeled hormone ligand and bind to AR (Veh.,
fluorescence polarization in absence of any competitor; DHT, 1 μM;
conjugates 1−3 and 5−7, 10 μM; conjugate 4, 100 nM). (B)
Transcriptional activation by AR in the presence of multivalent
peptoid conjugates quantified by luciferase activity in LB1 cells (Veh.,
EtOH treated cells; DHT, 10 nM; conjugates 1−7, 1 μM). All data
presented as mean + SD of triplicates.
binding (Figure S8). Increasing the valency and spacing of the
ethisterone moieties along the peptoid backbone enhances AR
binding, consistent with multivalent interactions.30
To determine if the conjugates function as AR agonists, we
evaluated the ability of the conjugates to induce AR-mediated
transcriptional activation in LNCaP cells. For this study, we
used a LNCaP cell line that stably expresses the AR-responsive
luciferase reporter gene under the probasin promoter.31 These
cells, termed LB1, were treated with conjugates 1−7 at a
concentration of 1 μM for 24 h, and AR-mediated transcrip-
tional activation was measured (Figure 2B). As a positive
control for full AR-mediated transcriptional activation, cells
were also treated with DHT. Compared to DHT, which
resulted in a 23.5-fold induction of the reporter gene over
baseline, conjugates 1−3 were weak activators of AR, displaying
only a 1.5- to 3-fold induction of the reporter gene. In contrast,
hexavalent conjugate 4 produced a robust induction of the
reporter gene to levels similar to DHT. For the linear divalent
conjugates, dodecamer conjugate 6 showed greater reporter
gene induction as compared to nonamer conjugate 5 (6- vs 4.5-
fold). This is consistent with the competitive AR binding data
observed for conjugates 1−6. Cyclic conjugate 7 displayed a
strong 11-fold induction of reporter gene activity despite its
inability to compete with DHT for binding to the AR. These
results indicate that different multivalent conjugates can operate
through either competitive or non-competitive mechanisms to
regulate AR-mediated transcriptional activation.
Figure 3. Effect of multivalent peptoid conjugates on cell proliferation
in LNCaP-abl (A,B) and HEK293 (C) cells (Veh., EtOH treated cells;
Bic., Bicalutamide, 1 μM; conjugates 6−8, 1 μM; control peptoid
conjugate 9, 1 μM; Doxo., positive cytotoxic control Doxorubicin, 1
μM.33 All data presented as mean + SD of triplicates. *Similar results
observed for conjugate 7 (Figure S13).
We next evaluated if conjugates 1−7 were able to suppress
the proliferation of LNCaP-abl cells. These cells are models of
advanced disease that express AR, and proliferate in the absence
of hormone. LNCaP-abl cells were treated with conjugates 1−7
for 72 h, and cellular proliferation was measured utilizing the
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(5) Gioeli, D.; Ficarro, S. B.; Kwiek, J. J.; Aaronson, D.; Hancock, M.;
Catling, A. D.; White, F. M.; Christian, R. E.; Settlage, R. E.;
Shabanowitz, J.; Hunt, D. F.; Weber, M. J. J. Biol. Chem. 2002, 277,
29304.
(6) Miyamoto, H.; Yeh, S.; Wilding, G.; Chang, C. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 7379.
(7) Bubley, G. J.; Balk, S. P. Oncologist 1996, 1, 30.
(8) McGinley, P. L.; Koh, J. T. J. Am. Chem. Soc. 2007, 129, 3822.
(9) Chen, C. D.; Welsbie, D. S.; Tran, C.; Baek, S. H.; Chen, R.;
Vessella, R.; Rosenfeld, M. G.; Sawyers, C. L. Nat. Med. 2004, 10, 33.
(10) Culig, Z.; Hoffmann, J.; Erdel, M.; Eder, I. E.; Hobisch, A.;
Hittmair, A.; Bartsch, G.; Utermann, G.; Schneider, M. R.; Parczyk, K.;
Klocker, H. Br. J. Cancer 1999, 81, 242.
ligands, two additional control compounds (conjugates 9 and
10, see SI) were synthesized. Conjugate 9, lacking the
ethisterone moieties, and conjugate 10, containing bioactive
progesterone receptor ligands, had no effect on cell
proliferation (Figure 3A and Figure S12). The standard AR
monotherapy bicalutamide also had no effect on cell
proliferation. In addition, LNCaP-abl cells were treated with
conjugates 6 and 7 at a concentration of 1 μM for 14 days, and
stained with crystal violet to detect differences on long-term
proliferation and colony-formation. Conjugates 6 and 7
strongly diminished the number and size of colonies formed
relative to vehicle treatment (Figure 3B).
To evaluate the general cell toxicity of conjugates 6 and 7, a
cell viability assay was utilized. Conjugates 6 and 7 did not
exhibit cytotoxic effects in human embryonic kidney (HEK293)
cells or AR-deficient prostate cancer (PC3) cells (Figures 3C
and S14). These results suggest that conjugates 6 and 7 are
selectively active and exert anti-proliferative activity in LNCaP-
abl cells. Our findings highlight the potential biomedical
significance of multivalent peptoid conjugates for advanced
prostate cancer.
The peptoid-based conjugates represent the first multivalent
constructs designed to specifically target the AR. The
oligomeric scaffold provides a versatile platform that can be
utilized to modulate AR activity. This study shows that
multivalent ethisterone conjugates can compete for AR binding
and modulate AR-mediated transcription. A linear and a cyclic
conjugate exhibit potent anti-proliferative activity in therapy-
resistant prostate cancer cells through competitive and non-
competitive mechanisms, respectively. Cytotoxicity was not
observed in non-AR expressing HEK293 or PC3 cells. Future
studies will evaluate the mechanism of action of these
compounds, and explore their potential applications in AR
pharmacology and chemical biology.
(11) Tran, C.; et al. Science 2009, 324, 787.
(12) Andersen, R. J.; Mawji, N. R.; Wang, J.; Wang, G.; Haile, S.;
Myung, J.-K.; Watt, K.; Tam, T.; Yang, Y. C.; Banuelos, C. A.;
Williams, D. E.; McEwan, I. J.; Wang, Y.; Sadar, M. D. Cancer Cell
2010, 17, 535.
(13) Estebanez-Perpina, E.; Arnold, L. A.; Nguyen, P.; Rodrigues, E.
D.; Mar, E.; Bateman, R.; Pallai, P.; Shokat, K. M.; Baxter, J. D.; Guy,
R. K.; Webb, P.; Fletterick, R. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
16074.
(14) Sack, J. S.; Kish, K. F.; Wang, C.; Attar, R. M.; Kiefer, S. E.; An,
Y.; Wu, G. Y.; Scheffler, J. E.; Salvati, M. E.; Krystek, S. R.; Weinmann,
R.; Einspahr, H. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4904.
(15) Homology model generated via sequence alignment with the
glucocorticoid receptor ligand binding domain dimer (teal ribbon,
Dexamethasone; orange, PDB 1M2Z). (a) Bledsoe, R. K.; Montana, V.
G.; Stanley, T. B.; Delves, C. J.; Apolito, C. J.; McKee, D. D.; Consler,
T. G.; Parks, D. J.; Stewart, E. L.; Willson, T. M.; Lambert, M. H.;
Moore, J. T.; Pearce, K. H.; Xu, H. E. Cell 2002, 110, 93.
(16) Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Curr. Opin. Chem.
Biol. 2000, 4, 696.
(17) Childs-Disney, J. L.; Tsitovich, P. B.; Disney, M. D.
ChemBioChem 2011, 12, 2143.
(18) Holub, J. M.; Garabedian, M. J.; Kirshenbaum, K. QSAR Comb.
Sci. 2007, 26, 1175.
(19) Miller, S. M.; Simon, R. J.; Ng, S.; Zuckermann, R. N.; Kerr, J.
M.; Moos, W. H. Drug Dev. Res. 1995, 35, 20.
(20) Tan, N. C.; Yu, P.; Kwon, Y.-U.; Kodadek, T. Bioorg. Med. Chem.
2008, 16, 5853.
(21) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J.
Am. Chem. Soc. 1992, 114, 10646.
(22) Maayan, G.; Ward, M. D.; Kirshenbaum, K. Proc. Natl. Acad. Sci.
U.S.A. 2009, 106, 13679.
ASSOCIATED CONTENT
■
S
* Supporting Information
Complete ref 11; detailed peptoid and submonomer synthesis;
mass spectra analysis; and assay methods. This material is
available free of charge via the Internet at http://pubs.acs.org.
(23) Udugamasooriya, D. G.; Dineen, S. P.; Brekken, R. A.; Kodadek,
T. J. Am. Chem. Soc. 2008, 130, 5744.
(24) Lee, Y.; Sampson, N. S. Curr. Opin. Struct. Biol. 2006, 16, 544.
(25) Yoo, B; Shin, S. B. Y.; Huang, M. L.; Kirshenbaum, K. Chem.
Eur. J. 2010, 16, 5528.
(26) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004.
(27) Lemus, A. E.; Enriquez, J.; Garcia, G. A.; Grillasca, I.; Perez-
Palacios, G. J. Steroid Biochem. 1997, 60, 121.
(28) Chang, H.-C.; Miyamoto, H.; Marwah, P.; Lardy, H.; Yeh, S.;
Huang, K.-E.; Chang, C. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11173.
(29) Kolev, M. V.; Ruseva, M. M.; Morgan, B. P.; Donev, R. M. J.
Immunol. Res. 2010, 184, 6035.
AUTHOR INFORMATION
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Corresponding Author
kent@nyu.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the NSF (CAREER
Award CHE-0645361 to K.K.). We thank Justin Holub and
Bishwajit Paul for their helpful discussions and suggestions.
(30) Note that recombinant AR-LBD does not contain N/C terminal
interactions that are present in full length AR or AR-LBD mutants.
(a) He, B.; Minges, J. T.; Lee, L. W.; Wilson, E. M. J. Biol. Chem. 2002,
277, 10226. (b) Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton,
J. T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6201.
(31) Zhang, J.; Gao, N.; DeGraff, D. J.; Yu, X.; Sun, Q.; Case, T. C.;
Kasper, S.; Matusik, R. J. The Prostate 2010, 70, 934.
(32) Jones, L. J.; Gray, M.; Yue, S. T.; Haugland, R. P.; Singer, V. L. J.
Immunol. Methods 2001, 254, 85.
REFERENCES
■
(1) Bhasin, S.; Cunningham, G. R.; Hayes, F. J.; Matsumoto, A. M.;
Snyder, P. J.; Swerdloff, R. S.; Montori, V. M. J. Clin. Endocrinol. Metab.
2010, 95, 2536.
(2) Heinlein, C. A.; Chang, C. Endo. Rev. 2004, 25, 276.
(3) Schaufele, F.; Carbonell, X.; Guerbadot, M.; Borngraeber, S.;
Chapman, M. S.; Ma, A. A. K.; Miner, J. N.; Diamond, M. I. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 9802.
(33) David, A. G. Biochem. Pharmacol. 1999, 57, 727.
(4) Centenera, M. M.; Harris, J. M.; Tilley, W. D.; Butler, L. M. Mol.
Endocrinol. 2008, 22, 2373.
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