Small-Molecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging

Article abstract of DOI:10.1002/anie.201503720

Androgen receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Consequently, it is the target of several antitumor chemotherapeutic agents, including the AR antagonist MDV3100/enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts MDV3100 action from an antagonist to an agonist. Here we describe the generation of a novel class of selective androgen receptor degraders (SARDs) to address this resistance mechanism. Molecules containing hydrophobic degrons linked to small-molecule AR ligands induce AR degradation, reduce expression of AR target genes and inhibit proliferation in androgen-dependent prostate cancer cell lines. These results suggest that selective AR degradation may be an effective therapeutic prostate tumor strategy in the context of AR mutations that confer resistance to second-generation AR antagonists.

Full text of DOI:10.1002/anie.201503720

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201503720
German Edition: DOI: 10.1002/ange.201503720
Antiproliferation
Hot Paper
Small-Molecule-Mediated Degradation of the Androgen Receptor
through Hydrophobic Tagging**
Jeffrey L. Gustafson, Taavi K. Neklesa, Carly S. Cox, Anke G. Roth, Dennis L. Buckley,
Hyun Seop Tae, Thomas B. Sundberg, D. Blake Stagg, John Hines, Donald P. McDonnell,
John D. Norris, and Craig M. Crews*
Abstract: Androgen receptor (AR)-dependent transcription is
a major driver of prostate tumor cell proliferation. Conse-
quently, it is the target of several antitumor chemotherapeutic
agents, including the AR antagonist MDV3100/enzalutamide.
Recent studies have shown that a single AR mutation (F876L)
converts MDV3100 action from an antagonist to an agonist.
Here we describe the generation of a novel class of selective
androgen receptor degraders (SARDs) to address this resist-
ance mechanism. Molecules containing hydrophobic degrons
linked to small-molecule AR ligands induce AR degradation,
reduce expression of AR target genes and inhibit proliferation
in androgen-dependent prostate cancer cell lines. These results
suggest that selective AR degradation may be an effective
therapeutic prostate tumor strategy in the context of AR
mutations that confer resistance to second-generation AR
antagonists.
approaches for post-translational targeting of specific pro-
teins to the ubiquitin–proteasome system (UPS).[6–9] For
instance, we recently reported a strategy for post-translational
protein degradation whereby
a
hydrophobic moiety
appended to the surface of a target protein engages the
cellular quality control machinery. This “hydrophobic tag”
may mimic a partially denatured protein folding state, leading
to degradation by the UPS. We demonstrated the feasibility of
this approach by covalently coupling hydrophobic tags to
engineered dehalogenase HaloTag-2[10–12] fusion proteins.
Recently, a similar approach was applied to degradation of
E. coli DHFR by non-covalent appendage of a hydrophobic
tag.[13] A key next step in the development of this nascent
technology is to degrade clinically relevant target proteins
with a small drug-like molecule. To this end, here we show
that coupling a hydrophobic tag to an androgen receptor
agonist converts it to a potent selective androgen receptor
degrader (SARD) capable of inducing > 50% of AR
degradation (DC50) at 1 mm. Remarkably, this SARD retained
anti-proliferative activity in cell lines resistant to current
standard-of-care drugs for castration-resistant prostate cancer
(CRPC).
T
argeted degradation represents an intriguing strategy to
regulate the function of therapeutically relevant proteins
(e.g., transcription factors and scaffolding proteins) not
amenable to traditional small-molecule approaches.[1,2] More-
over, targeted protein degradation could overcome resistance
mechanisms that modulate the activity of small-molecule
drugs following target engagement. For instance, point
mutants conferring agonist activity to antagonists limit
efficacy of androgen receptor (AR) antagonists for treatment
of prostate cancer.[3] While deletion of the disease-causing
protein offers a direct solution to this problem, strategies for
doing so through genome editing or RNAi remain clinically
challenging.[4,5] As an alternative, we have developed several
The androgen receptor (AR)[14] is a ligand-dependent
transcription factor that upon binding to the androgen
dihydrotestosterone (DHT) undergoes a conformational
change leading to homodimerization, nuclear translocation
and upregulation of gene transcription. While vital for the
normal development and maintenance of the prostate, AR-
mediated gene expression remains an important driver
throughout prostate cancer progression. Many therapeutic
strategies focus on regulating AR activity. For example,
androgen deprivation therapy[15] combined with AR antago-
nists (i.e., anti-androgens) such as bicalutamide[16] has been
used as a first-line treatment for early stage prostate cancer
for decades. While initially effective at suppressing tumor
growth, this strategy in most cases leads to the progression of
an AR-dependent yet androgen-independent form of the
disease (i.e., CRPC),[17] which is responsible for the vast
majority of prostate cancer deaths. Moreover, in CRPC, the
first-generation anti-androgen drugs, such as flutamide[18] and
bicalutamide,[19] can display AR agonist activity. While the
mechanisms responsible for the progression to CRPC are not
entirely known, it has become clear that an increased level of
AR protein is present in the majority of CRPC and that
agents targeting androgen synthesis and/or AR signaling, such
as abiraterone and MDV3100/enzalutamide, respectively,
demonstrate clinical benefit to CRPC patients.[20–22]
[*] Dr. J. L. Gustafson, Dr. T. K. Neklesa, C. S. Cox, Dr. A. G. Roth,
Dr. D. L. Buckley, Dr. H. S. Tae, Dr. T. B. Sundberg, Dr. J. Hines,
Prof. C. M. Crews
Departments of Molecular, Cellular, and Developmental Biology,
Chemistry, and Pharmacology, Yale University
New Haven, CT 065111 (USA)
E-mail: craig.crews@yale.edu
D. B. Stagg, Prof. D. P. McDonnell, Dr. J. D. Norris
Department of Pharmacology and Cancer Biology
Duke University School of Medicine
Durham, NC 27710 (USA)
[**] We are grateful to GlaxoSmithKline for continued support and
helpful discussion. This work was supported by the NIH
(T32GM067543, F32GM10052101, R01CA139818, R01AIO84140)
and the DoD W81XWH-12-1-0484). A.G.R. was a Leopoldina–
Nationale Akademie der Wissenschaften Postdoctoral Fellow.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201503720.
Angew. Chem. Int. Ed. 2015, 54, 9659 –9662
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
9659

.
Angewandte
Communications
Hypothesizing that increased AR levels may drive the
development of CRPC and considering the clinical success of
the selective estrogen receptor degrader (SERD) fulves-
trant[23] we sought to induce AR degradation through our
hydrophobic tagging approach. To accomplish this, we
designed a series of SARDs based on the high-affinity AR
agonist RU59063[24] connected through a short PEG linker to
an adamantyl group (Figure 1A), a hydrophobic degron
shown to be effective in our previous work with Halotag
fusion proteins.
Predictably, target degradation by the SARDs is selective
for the AR; the glucocorticoid receptor (GR), another steroid
receptor not recognized by the parent ligand RU59063, is not
degraded in LNCaP cells under conditions that result in near-
complete degradation of the androgen receptor (Figure S2).
Futhermore, consistent with our initial report on protein
hydrophobic-tagging,[10] degradation of the AR is dependent
on the UPS—pretreatment/co-treatment of LNCaP cells with
the proteasome-specific inhibitor, epoxomicin, prevents
SARD-mediated degradation of the AR. To further explore
the mechanism of SARD-mediated AR degradation, we
investigated the possible involvement of heat shock proteins
(HSPs), given their known role in stabilizing misfolded
proteins or targeting them for degradation by the UPS. We
found that incubating cells with the potent Hsp90 inhibitor
geldanamycin, at concentrations that did not affect AR levels,
enhanced AR degradation at sub-DC50 concentrations of
SARD279 (Figure S3). Immunoblotting revealed a geldana-
mycin-dependent increase in Hsp70 levels, consistent with the
finding that Hsp90 inhibition leads to activation of heat shock
factor 1 (HSF1) and its target genes, including HSP70.[25]
Hsp70 and the associated E3 ubiquitin ligase CHIP play a key
role in targeting intractably misfolded proteins to the UPS,[26]
and we have shown that labeling HaloTag with hydrophobic
tags leads to enhanced association with Hsp70.[27] Together,
this suggests that the Hsp70/CHIP complex mediates SARD-
induced AR degradation and that elevated Hsp70 levels
underlie the increase in SARD279 activity in the context of
Hsp90 inhibition.
We next examined the effects of SARDs on AR-
dependent gene expression in AR overexpressing LNCaP-
AR cells, a validated cellular model of CRPC. Consistent with
the documented AR agonist activity of RU59063 in CRPC,
we observed increasing expression of a number of AR target
genes such as FKBP52, PMPEA1 and NKX3.1 (Figure S4).
Despite the agonist activity of RU59063, no such agonist
activity was observed for SARD279- and SARD033-treated
cells. Moreover, both SARDs antagonized gene expression
induced by the synthetic androgen R1881. Importantly, with
respect to this latter activity, both SARDs compared favor-
ably to the recently FDA-approved selective androgen
receptor modulator (SARM) MDV3100/enzalutamide.[21]
Given that both SARDs reduced expression of the AR
target genes, we directly evaluated their pharmacological
properties in androgen responsive element (ARE)-driven
luciferase reporter assay in 293 cells. We determined that,
despite their construction from an AR agonist, both SARDs
blocked transactivation of the androgen receptor by the
agonist R1881 (Figure S5). That the IC50 values of the SARDs
in reporter assay—156 nm for SARD279 and 293 nm for
SARD033—were 10-fold lower than their DC50 values to
degrade AR suggests that their ability to block expression of
AR target genes may be multimodal: partly due to pharma-
cological antagonism of AR signaling and partly due to
degradation of the AR itself.
Figure 1. A) Structures of selective androgen receptor degraders
(SARDs) based on the androgen receptor agonist RU59063. B) Immu-
noblot analyses of LNCaP human prostate tumor cells incubated with
SARDs or parent ligand for 24 h.
Gratifyingly, such heterodimeric molecules retained the
ability to bind directly to the AR (Figure S1 in the Supporting
Information): competition radioligand binding assay using
[3H]-R1881 showed that appending of the adamantyl group to
RU59063 reduced affinity for the AR approximately 37-fold
in the case of SARD279, and nearly 300-fold for SARD033.
In accordance with their binding affinities, the synthesized
SARDs induced AR degradation at sub-micromolar concen-
trations. For example, SARD279, in which the adamantyl
moiety is coupled to RU59063 through an 8-atom ester
linkage reduced AR protein levels by 50% at 1 mm (DC50)
(Figure 1B), while no degradation was detected in cells
treated with the parental AR ligand. SARD033, possessing an
adamantyl moiety attached through a longer ether linkage
induced AR degradation with a ca. 2 mm DC50 value (Fig-
ure 1B). SARD-mediated AR degradation requires direct
interaction with AR since co-incubation with the competitive
AR agonist RU59063 blocked the activity of SARD279 (not
shown).
We next asked whether SARD-induced AR degradation
reduces proliferation of prostate cancer cells. We incubated
androgen-dependent LnCAP cells with SARD279,
SARD033, RU59063, or MDV3100 and measured cell
9660 www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 9659 –9662

Angewandte
Chemie
Figure 2. SARDs block human prostate cancer cells (LNCaP) as well as
MDV3100. LNCaP cell proliferation assay in the presence of indicated
compounds at 3 mm. The assay was performed in 10% FBS, and viable
cells were counted at each indicated day.
Figure 3. SARD279 has more antiproliferative activity than MDV3100
in the presence of low androgen levels. LNCaP cells supplemented
with 1 nm of the AR agonist R1881 were incubated for 7 days with
MDV3100 or SARD279 after which cell proliferation was measured.
The growth media contained 5% charcoal stripped FBS to eliminate
exogenous androgens.
numbers by vital dye staining and hemocytometry over the
course of 9 days. In LNCaP cells, SARD279 and SARD033
both suppressed proliferation with similar efficacy to
MDV3100 within 2 days (Figure 2). In contrast, proliferation
of the AR-independent HEK293T and PC3 human prostate
cancer cell lines was unaffected by SARD279, SARD033 or
MDV3100 (Figure S6). The ability of these SARDs to
specifically suppress AR-dependent proliferation argues
that their activity results from targeted AR degradation
rather than a non-specific cytotoxicity.
Despite the initial efficacy of MDV3100 against AR-
driven CRPC, within a year patients usually progress with an
apparent reactivation of AR signaling, as exhibited by an
increase in circulating PSA levels and radiographic tumor
progression.[28] Numerous mechanisms have been proposed to
explain this resistance, including intratumoral androgen syn-
thesis that competes with MDV3100 binding as well as the
F876L mutation in AR that enables the receptor to recognize
MDV3100 as an agonist.[3,29–31] We sought to determine
whether SARDs can circumvent resistance associated with
these two mechanisms.
like from antagonists to agonists. Indeed, in LNCaP cells
engineered to express the AR-F876L mutation (LNCaP/AR-
F876L) MDV3100 functions as a robust agonist inducing the
expression of PSA mRNA in the absence of androgens
(Figure S7). Not surprisingly, eliminating AR with a SARD
demonstrates no such activation. Consistent with the AR
activity data, the proliferation of these cells can be induced
with MDV3100 whereas SARD279 exhibits antiproliferative
effects (Figure 4). Together, these data demonstrate that
eliminating AR with a SARD can overcome resistance
mechanisms associated with MDV3100.
While MDV3100 is capable of antagonizing AR activity at
castration levels of testosterone (less than 0.7 nm), studies
have demonstrated that prostate cancer cells themselves
upregulate de novo dihydrotestosterone (DHT) synthe-
sis.[31,32] In fact, 1 nm R1881 can abolish the antiproliferative
activity of MDV3100 when assayed in cellular models of
prostate cancer.[21] Hypothesizing that a SARD only needs to
make a transient interaction with AR to enable its ubiquiti-
nation, we tested the activity of SARD279 and MDV3100
against proliferating LNCaP cells supplemented with 1 nm
R1881. As expected, this level of R1881 blocked MDV3100
from antagonizing AR, whereas SARD279 exhibited anti-
proliferative activity (Figure 3). These data show that
MDV3100 is ineffective in the presence of higher androgen
levels whereas a SARD is still capable of degrading AR and
inhibiting proliferation.
Figure 4. SARD-mediated antiproliferative activity in MDV3100-resist-
ant cells. LNCaP/AR-F876L cells were incubated with indicated doses
of MDV3100 or SARD279 for 7 days in the presence of 5% charcoal
stripped FBS.
Another emerging mechanism of MDV3100 resistance is
the F876L mutation in the AR, where the resistance is
achieved by shifting AR responsiveness to MDV3100 and the
Angew. Chem. Int. Ed. 2015, 54, 9659 –9662
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 9661

.
Angewandte
Communications
[11] H. S. Tae, T. B. Sundberg, T. K. Neklesa, D. J. Noblin, J. L.
Gustafson, A. G. Roth, K. Raina, C. M. Crews, ChemBioChem
2012, 13, 538 – 541.
[12] G. V. Los, L. P. Encell, M. G. McDougall, D. D. Hartzell, N.
Karassina, C. Zimprich, M. G. Wood, R. Learish, R. F. Ohana,
M. Urh, et al., ACS Chem. Biol. 2008, 3, 373 – 382.
[13] M. J. C. Long, D. R. Gollapalli, L. Hedstrom, Chem. Biol. 2012,
19, 629 – 637.
[14] C. A. Heinlein, C. Chang, Endocr. Rev. 2004, 25, 276 – 308.
[15] C. Huggins, C. V. Hodges, Cancer Res. 1941, 1, 293 – 297.
[16] P. F. Schellhammer, Expert Opin. Pharmacother. 2002, 3, 1313 –
1328.
[17] B. J. Feldman, D. Feldman, Nat. Rev. Cancer 2001, 1, 34 – 45.
[18] H. I. Scher, W. K. Kelly, J. Clin. Oncol. 1993, 11, 1566 – 1572.
[19] E. J. Small, P. R. Carroll, Urology 1994, 43, 408 – 410.
[20] H. Miyamoto, M. M. Rahman, C. Chang, J. Cell. Biochem. 2004,
91, 3 – 12.
[21] C. Tran, S. Ouk, N. J. Clegg, Y. Chen, P. Watson, V. Arora, J.
Wongvipat, P. M. Smith-Jones, D. Yoo, A. Kwon, et al., Science
2009, 324, 787 – 790.
[22] G. Attard, A. H. Reid, T. A. Yap, F. Raynaud, M. Dowsett, S.
Settatree, M. Barrett, C. Parker, V. Martins, E. Folkerd, et al., J.
Clin. Oncol. 2008, 26, 4563 – 4571.
[23] J. D. Croxtall, K. McKeage, Drugs 2011, 71, 363 – 380.
[24] G. Teutsch, F. Goubet, T. Battmann, A. Bonfils, F. Bouchoux, E.
Cerede, D. Gofflo, M. Gaillard-Kelly, D. Philibert, J. Steroid
Biochem. Mol. Biol. 1994, 48, 111 – 119.
[25] H. R. Kim, H. S. Kang, H. D. Kim, IUBMB Life 1999, 48, 429 –
433.
Here, we show that linking a hydrophobic tag-based
degron to an AR agonist creates a novel class of anti-
androgens (termed SARDs) that induce targeted AR degra-
dation and override some resistance mechanisms to tradi-
tional prostate cancer drugs. Thus, SARDs may prove to be
a novel therapeutic approach to combat CRPC: since they
remove AR, and thus block a key mitogenic pathway in
prostate cancer cells.
This SARD approach is not expected to address drug-
resistant prostate cells expressing AR splice variant 7 lacking
the ligand binding domain. However, our data suggest that in
cells with an intact AR, if the residency time of an antagonist
is insufficient—as is the case with MDV3100 in the milieu of
increased androgens—SARD-mediated degradation of AR
can be achieved with essentially the same AR binding ligand.
The hydrophobic tagging strategy described here adds to the
emerging concept of induced protein degradation as a ther-
apeutic strategy[9] and may prove useful for the manipulation
of disease-relevant proteins, and related drug-resistant
mutants, that have proven intractable to traditional small-
molecule approaches.
Keywords: antiproliferation · cancer · drug design · hormones ·
protein degradation
How to cite: Angew. Chem. Int. Ed. 2015, 54, 9659–9662
Angew. Chem. 2015, 127, 9795–9798
[26] H. McDonough, C. Patterson, Cell Stress Chaperones 2003, 8,
303 – 308.
[27] T. K. Neklesa, D. J. Noblin, A. Kuzin, S. Lew, J. Seetharaman,
T. B. Acton, G. Kornhaber, R. Xiao, G. T. Montelione, L. Tong,
et al., ACS Chem. Biol. 2013, 8, 2293 – 2300.
[28] N. J. Clegg, J. Wongvipat, J. D. Joseph, C. Tran, S. Ouk, A. Dilhas,
Y. Chen, K. Grillot, E. D. Bischoff, L. Cai, et al., Cancer Res.
2012, DOI: 10.1158/0008-5472.CAN-11-3948.
[29] A. Egan, Y. Dong, H. Zhang, Y. Qi, S. P. Balk, O. Sartor, Cancer
Treat. Rev. 2014, 40, 426 – 433.
[30] M. Korpal, J. M. Korn, X. Gao, D. P. Rakiec, D. A. Ruddy, S.
Doshi, J. Yuan, S. G. Kovats, S. Kim, V. G. Cooke, et al., Cancer
Discov. 2013, 3, 1030 – 1043.
[1] Y. H. Lin, M. R. Pratt, ChemBioChem 2014, 15, 805 – 809.
[2] C. M. Crews, Chem. Biol. 2010, 17, 551 – 555.
[3] M. D. Balbas, M. J. Evans, D. J. Hosfield, J. Wongvipat, V. K.
Arora, P. A. Watson, Y. Chen, G. L. Greene, Y. Shen, C. L.
Sawyers, Elife 2013, 2, e00499.
[4] D. H. Kim, J. J. Rossi, Nat. Rev. Genet. 2007, 8, 173 – 184.
[5] B. L. Davidson, P. B. McCray, Nat. Rev. Genet. 2011, 12, 329 –
340.
[6] T. W. Corson, N. Aberle, C. M. Crews, ACS Chem. Biol. 2008, 3,
677 – 692.
[7] J. S. Schneekloth, F. N. Fonseca, M. Koldobskiy, A. Mandal, R.
Deshaies, K. Sakamoto, C. M. Crews, J. Am. Chem. Soc. 2004,
126, 3748 – 3754.
[8] A. R. Schneekloth, M. Pucheault, H. S. Tae, C. M. Crews,
Bioorg. Med. Chem. Lett. 2008, 18, 5904 – 5908.
[9] D. L. Buckley, C. M. Crews, Angew. Chem. Int. Ed. 2014, 53,
2312 – 2330; Angew. Chem. 2014, 126, 2344 – 2363.
[10] T. K. Neklesa, H. S. Tae, A. R. Schneekloth, M. J. Stulberg, T. W.
Corson, T. B. Sundberg, K. Raina, S. Holley, C. M. Crews, Nat.
Chem. Biol. 2011, 7, 538 – 543.
[31] P. R. Dillard, M. F. Lin, S. A. Khan, Mol. Cell. Endocrinol. 2008,
295, 115 – 120.
[32] F. Ishizaki, T. Nishiyama, T. Kawasaki, Y. Miyashiro, N. Hara, I.
Takizawa, M. Naito, K. Takahashi, Sci. Rep. 2013, 3, 1528.
Received: April 23, 2015
Published online: June 17, 2015
9662 www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 9659 –9662