Jostling for position: Optimizing linker location in the design of estrogen receptor-targeting PROTACs

Article abstract of DOI:10.1002/cmdc.201000146

Estrogen receptor-α (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, hormone-sensitive ER-positive cancer cells eventually develop resistance to ER antagonists. It has been shown that in most of these resis

Full text of DOI:10.1002/cmdc.201000146

DOI: 10.1002/cmdc.201000146
Jostling for Position: Optimizing Linker Location in the
Design of Estrogen Receptor-Targeting PROTACs
Kedra Cyrus,[a] Marie Wehenkel,[a] Eun-Young Choi,[b] Hyosung Lee,[a] Hollie Swanson,[b] and
Kyung-Bo Kim*[a]
Estrogen receptor-a (ER) antagonists have been widely used
for breast cancer therapy. Despite initial responsiveness, hor-
mone-sensitive ER-positive cancer cells eventually develop re-
sistance to ER antagonists. It has been shown that in most of
these resistant tumor cells, the ER is expressed and continues
to regulate tumor growth. Recent studies indicate that tamoxi-
fen initially acts as an antagonist, but later functions as an ER
agonist, promoting tumor growth. This suggests that targeted
ER degradation may provide an effective therapeutic approach
for breast cancers, even those that are resistant to convention-
al therapies. With this in mind, we previously demonstrated
that proteolysis targeting chimeras (PROTACs) effectively
induce degradation of the ER as a proof-of-concept experi-
ment. Herein we further refined the PROTAC approach to
target the ER for degradation. The ER-targeting PROTACs are
composed of an estradiol on one end and a hypoxia-inducing
factor 1a (HIF-1a)-derived synthetic pentapeptide on the other.
The pentapeptide is recognized by an E3 ubiquitin ligase
called the von Hippel Lindau tumor suppressor protein (pVHL),
thereby recruiting the ER to this E3 ligase for ubiquitination
and degradation. Specifically, the pentapeptide is attached at
three different locations on estradiol to generate three differ-
ent PROTAC types. With the pentapeptide linked through the
C7a position of estradiol, the resulting PROTAC shows the
most effective ER degradation and highest affinity for the es-
trogen receptor. This result provides an opportunity to develop
a novel type of ER antagonist that may overcome the resist-
ance of breast tumors to conventional drugs such as tamoxifen
and fulvestrant (Faslodex).
Introduction
Breast cancer is the most common form of malignant disease
in women worldwide. The majority of breast tumors, about
two thirds of those initially diagnosed, are estrogen receptor-a
(ER)-positive. In most cases the ER plays a significant role in
the stimulation and growth of these breast tumors.[1,2] For ex-
ample, the protein level of the ER is elevated in pre-malignant
and malignant breast lesions relative to normal tissue.[1] In ad-
dition, the clear correlation between ER-positive breast tumors
and their response to anti-estrogen therapy has been demon-
strated.[3]
safety and significant anti-neoplastic activities of these anti-es-
trogens, most initially responsive breast tumors acquire resist-
ance.[10–14] As such, the major challenge is to develop novel
therapeutic agents that not only inhibit the growth of hor-
mone-sensitive breast tumors, but also prevent the develop-
ment of drug resistance.
Although the mechanism by which ER-positive cancer cells
acquire drug resistance is not clearly understood, it is unlikely
that any single mechanism or gene confers anti-estrogen re-
sistance. Given that less than 25% of tumors which recur fol-
lowing treatment with fulvestrant or tamoxifen lack the
ER,[15,16] ER loss does not seem to be the major mechanism
that drives acquired resistance. It has also been shown that a
loss of anti-estrogen responsiveness by initially sensitive
tumors is unlikely due to mutations or deletion of the ER
gene.[1,14] In fact, most tumors at recurrence retain levels of ER
Most ER-positive tumors initially respond well to anti-estro-
gens, which block the site at which estrogen can bind, thereby
halting the growth of cancer cells. Tamoxifen, a non-steroid se-
lective ER modulator (SERM), is an anti-estrogen therapeutic
which has been widely used for more than 20 years in all
stages of ER-positive breast cancers.[4,5] Specifically, tamoxifen
induces a conformational change of the ER that blocks the re-
ceptor’s function.[6] Another example is fulvestrant (Faslo-
dex),[7,8] which is a pure steroidal anti-estrogen approved for
the treatment of post-menopausal women with hormone-sen-
sitive, advanced, or metastatic breast cancers. Like tamoxifen,
fulvestrant competitively binds the ER and blocks ER-promoted
cell proliferation. Whereas tamoxifen causes accumulation of
the ER, fulvestrant, like ER-targeting PROTAC molecules, indu-
ces selective proteasome-mediated degradation of the ER and
therefore does not exhibit the agonistic effects commonly as-
sociated with SERMs.[9] Unfortunately, despite the relative
[a] Dr. K. Cyrus,+ M. Wehenkel,+ Dr. H. Lee, Prof. Dr. K.-B. Kim
Department of Pharmaceutical Sciences, University of Kentucky
789 South Limestone, Lexington, KY 40536-0596 (USA)
Fax: (+1)859-257-7564
E-mail: kbkim2@uky.edu
[b] Dr. E.-Y. Choi, Prof. Dr. H. Swanson
Department of Molecular and Biomedical Pharmacology
University of Kentucky, 800 Rose St., Lexington, KY 40536 (USA)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000146.
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expression that would define them as ER posi-
tive.[17–21] In addition, the ER appears to maintain the
ability to continuously regulate tumor growth in
most anti-estrogen-resistant tumors.[20] It has been
suggested that one likely cause of tamoxifen resist-
ance is the ability of tumors to switch from recogniz-
ing an anti-estrogen as an antagonist to recognizing
it as an agonist, perhaps through differential co-regu-
lator recruitment to the ER.[22–26] For fulvestrant, a
conformational change in the ER is suggested to
affect the binding of co-regulator(s) and to influence
the stability of the ER, thereby causing its rapid deg-
radation. Additionally, fulvestrant causes a loss of the
ER protein without affecting ER mRNA levels.[27,28]
Consistent with this observation, it has been reported
that resistance of human breast cancer cells to fulves-
trant is not associated with a general loss of ER ex-
pression or lack of estrogen responsiveness.[29] Taken
together, it seems that tamoxifen and fulvestrant
maintain their ER binding affinity despite the loss of
their antiproliferative or ER-antagonistic effects, and
that the ER continues to regulate the growth of re-
sistant ER-positive breast cancer cells. Consequently,
a small molecule that targets the ER for degradation,
regardless of changes that induce drug resistance,
may prove useful as an alternative therapeutic option
for ER-positive breast cancer therapy.
Figure 1. PROTAC-induced targeted degradation of the ER. A) The PROTAC is composed
of three components: an E3 ligase recognition motif, linker, and ligand (in this case, tar-
geting the ER). B) The PROTAC recruits the ER to the pVHL E3 ligase complex for ubiquiti-
nation and subsequent degradation by the 26S proteasome; this degradation of the ER
should decrease the amount of dimerized ER bound to estradiol available to drive gene
transcription, a major factor in the growth and survival of breast cancers.
Toward this end, we used a novel small-molecule-
based technology termed PROteolysis TArgeting Chi-
meras (PROTACs) to target the ER for degradation at
the posttranslational level. PROTAC molecules are
composed of three major components (Figure 1A): a
small-molecule ligand for a targeted protein, an E3 ubiquitin
ligase recognition motif, and a linker. For the E3 ligase recogni-
tion domain, a 12-mer polypeptide containing two phospho-
serines (derived from IkBa) was initially used as a proof of con-
cept.[30] However, the poor bioavailability of the 12-mer
PROTAC necessitates microinjection for delivery, hence limiting
its use as a molecular probe. To circumvent this, an alternative
E3 recognition motif was required, so HIF-1a polypeptides
were developed. A pentapeptide was found by our research
group and others to be sufficient for HIF-1a-based PROTACs to
cross cell membranes and to effectively degrade target pro-
teins.[31–33]
Other potential positions for derivation from 17b-estradiol
have been reported. A geldanamycin-tagged estradiol com-
pound derivatized at the C16 position on estradiol was report-
ed to maintain its interaction with the ER.[35,36] Similarly, deriva-
tization at the C7a position of estradiol has been shown to
preserve its binding affinity with the ER.[7,8,37] For instance, ful-
vestrant has a high ER binding affinity, and it is an E2 deriva-
tive with a long hydrocarbon tail at the C7a position. Thus, we
endeavored to create a more stable linker by attaching the
pentapeptide at the C16 and C7 positions of estradiol to pre-
pare PROTACs (Scheme 1). Herein we report the rational
design and optimization of PROTACs that target the ER for
ubiquitination and degradation. The information contained
herein also provides an example for PROTAC design that will
produce molecules with superior effects in cell-based systems.
Previously, we developed PROTACs that target the ER by
using estradiol as the ligand and the optimized pentapeptide
as the E3 recognition motif. These proof-of-concept PROTACs
used an ester bond to attach the linker moiety to the O17 po-
sition of estradiol. A significant drawback to this attachment
site is its vulnerability to esterases, which would inactivate the Results and Discussion
PROTAC as well as release estradiol. Another critical issue with
PROTAC synthesis
the O17-linked PROTAC was the loss of the 17-OH group,
which is reported to be important for ER binding of E2.[34]
Therefore, E2-based PROTACs that can spare the 17-OH group
are desirable for the design of improved PROTACs that target
the ER.
The parent ER-targeting PROTAC 2 was synthesized as de-
scribed.[38] In addition, PROTAC 2 was deprotected at the C ter-
minus of the pentapeptide to give PROTAC 2*, using the same
procedure as described below (13 and 14). To synthesize
PROTAC 3, a “free amine handle” was introduced at the C16a
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was activated to the succinimid-
yl ester 12 by using disuccini-
midyl suberate (DSS). Compound
12 was finally coupled with the
HIF-1a pentapeptide, and the
TBDMS group was deprotected
with tetrabutylammonium fluo-
ride (TBAF) to yield PROTAC 13.
The benzyl group of PROTAC 13
was removed by hydrogenolysis
to obtain PROTAC 14.
As was the case for PROTAC
13, the most critical step for the
synthesis of PROTAC 24 is the in-
troduction of a primary amine
“handle” at the C7a position of
estradiol (Scheme 3). A proce-
dure similar to that reported by
Skaddan and Katzenellenbo-
Scheme 1. Synthetic strategy for next-generation ER-targeting PROTACs. Attaching the linker by a carbon–carbon
bond should produce PROTACs with higher ER binding affinity and stability in living cells than the parent ER-tar-
geting PROTAC 2.
position of E2 (17b-estradiol) via a carbon–carbon linkage, by
following a procedure similar to that previously reported
(Scheme 2).[36,39,40] First, estrone 5 was treated with tert-butyldi-
methylsilyl chloride (TBDMSCl) to yield compound 6. Alkylation
at the C16 position of 6 was carried out by using lithium diiso-
propylamide (LDA) and 1,4-dibromo-2-butene. The resulting
alkyl bromo residue was converted into the azido compound 8
by treatment with sodium azide. Sequential reduction of the
keto and azide groups with sodium borohydride and lithium
aluminum hydride yielded the free amine “handle”, 9. The
handle was further extended with glycine to give 11, which
gen[41,42] was followed to introduce this handle. As a first step,
tetrahydropyran (THP)-protected 15 was synthesized by follow-
ing a procedure similar to that previously described.[43–45]
Deprotonation at the C6 position under “superbase” condi-
tions followed by treatment with trimethyl borate yielded an
intermediate borate ester, which was oxidized with hydrogen
peroxide to provide 16 as epimers. Further oxidation of 16
with pyridinium chlorochromate (PCC) afforded the keto func-
tionality at the C6 position in 17. Treatment of 17 with potassi-
um tert-butoxide and allyl iodide afforded 18. Simultaneous
deprotection of the THP groups and the C6 keto functionality
Scheme 2. Synthesis of the C16-derivatized estradiol-based PROTACs: a) TBDMSCl, imidazole, 99%; b) LDA, C4H6Br2, THF, 45%; c) NaN3, THF, DMSO, H2O, 96%;
d) 1. NaBH4, MeOH; 2. LiAlH4, THF, 35%; e) Fmoc-Gly-OH, HBTU, DIPEA, CH2Cl2, 47%; f) 20% piperidine in DMF, 67%; g) DSS, DMF, 38%; h) 1. HIF-1a pentapep-
tide, DMAP, DMF; 2. TBAF, THF, 47%; i) H2, Pd/C, EtOAc, MeOH, 89%.
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Scheme 3. Synthesis of an estradiol containing an amine functional group at the C7 position: a) DHP/TsOH, 94%; b) 1. LiDAKOR; 2. B(OMe)3; 3. H2O2, 66%;
c) PCC, 80%; d) 1. KOtBu, C3H5I, 2. NaOMe, 36%; e) Et3SiH, BF3·Et2O, 90%; f) TBDMSCl, imidazole, 85%; g) 1. 9-BBN; 2. H2O2, KOH, 66%; h) PPh3, DIAD, phthali-
mide, 83%; i) NH2NH2, 76%; j) disuccinimidyl glutarate, 37%; k) 1. HIF-1a pentapeptide; 2. TFA, 25% over two steps. HIF-1a pentapeptide=Leu-Ala-ProOH-Tyr-
Ile-Bzl.
was carried out with triethylsilane and boron trifluoride diethyl
etherate to give 19. The free OH groups were re-protected
with TBDMS groups to give 20, which was then treated under
hydroboration–oxidation conditions to produce alcohol 21.
Treatment of 21 with phthalimide under Mitsunobu conditions
followed by hydrazinolysis provided the primary amine
“handle” 23. Compound 23 was further extended and activat-
ed using DSS. Sequential coupling of HIF-1a pentapeptide and
deprotection of TBDMS groups yielded PROTAC 24.
Biological characterization of PROTACs
PROTACs 2, 2*, 13, 14, and 24 were tested for their ability to
degrade the endogenous ER in MCF7 breast cancer cells. After
Figure 2. C-terminal protection of the HIF-1a pentapeptide provides im-
proved degradation of the ER by PROTACs linked at any position on estra-
diol. A) O17 (2 and 2*) and C16 (13 and 14) derivatives of E2-based PROTACs
incubation with PROTACs for 48 h, their effects on ER protein
levels were evaluated by western blotting analysis and immu-
nofluorescence.
with the benzyl-protected HIF-1a pentapeptide (2 and 13) are more effec-
tive in degrading the endogenous ER than unprotected PROTACs (2* and
14). B) A similar pattern was observed for the C7 derivative of E2-based
PROTAC, as the benzyl-protected compounds 13 and 24 showed greater ER
degradation than the deprotected compound 14.
Previous reports indicated that the O17-linked PROTAC 2
would degrade the ER in
a
proteasome-dependent
manner.[31,32,38] Our initial data confirmed this finding (Fig-
ure 2A) while demonstrating that the C-terminal-protected
PROTAC 2 is superior to the deprotected PROTAC 2*. A major
drawback to these compounds is their susceptibility to endog-
enous esterases, so we moved forward with the testing of
carbon-based linkages to confirm the results from these proof-
of-concept PROTACs. A similar trend in ER degradation was ob-
served for the C16a-based PROTACs 13 and 14, with the pro-
tected pentapeptide providing superior degradation (Fig-
ure 2B). The C7a-based PROTAC 24 showed a similar ability to
degrade the ER when compared with the C16a-based PROTAC
13 (Figure 2B).
Next, we further confirmed the PROTAC-mediated degrada-
tion of the ER in MCF7 cells by immunofluorescence studies.
Estradiol (E2) was used as a positive control. E2 induces protea-
some-mediated degradation of the ER as a result of transcrip-
tional activation, resulting in cell proliferation. ER degradation
by E2 was blocked by co-treatment with epoxomicin, a natural
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Figure 3. PROTACs bind to the ER and cause its proteasome-mediated degradation. A) Immunofluorescence images of MCF7 cells after 48 h treatment with
25 mm PROTACs, 10 nm E2, or DMSO vehicle. The ER is visualized by conjugation with FITC (green), and DAPI staining (blue) indicates the nucleus. Co-treat-
ment with epoxomicin causes accumulation of the ER. B) Western blot data demonstrating the proteasome dependence of PROTAC-mediated ER degradation.
C) Competitive binding assay indicating that C7 is a superior position for PROTAC derivatization due to its enhanced binding to the ER relative to the O17-
and C16a-based PROTACs.
product proteasome-specific inhibitor. Compared with DMSO Conclusions
control, the attenuation of ER signal (green) was observed with
the treatment of 13 and 24 (Figure 3A, top row). These com-
pounds showed an accumulation of the green signal when co-
treated with epoxomicin, which was also observed in control/
epoxomicin treated cells (Figure 3A, bottom row). This result
was confirmed by western blotting using 24 and E2 (Fig-
ure 3B). To validate the superior position for PROTAC linkage, a
competitive ligand binding affinity assay was performed. These
in vitro data indicate that the C7a-based PROTAC 24 has the
highest affinity of the E2-based PROTAC compounds tested, su-
perior even to tamoxifen. The binding data for the C16a-based
PROTACs 13 and 14 also support the data from Figure 2 which
indicates that benzyl protection of the C terminus of the pen-
tapeptide gives a superior compound. Because a PROTAC with
a higher binding affinity for the target protein should induce
optimal ubiquitination and subsequent degradation by the
proteasome, further optimization of the PROTAC approach for
the ER should use the C7a linkage.
The ubiquitin–proteasome degradation pathway is a destruc-
tive, irreversible process and controls many important biologi-
cal processes such as cell-cycle progression, differentiation,
and inflammation. We have shown previously that the cellular
ubiquitination machinery can be hijacked by a PROTAC con-
taining a pentapeptide that is recognized by the E3 ligase
pVHL, and that a PROTAC containing a ligand of a target pro-
tein induces ubiquitination and degradation of that protein in
living cells.[31,38,46] This “chemical knockout” approach provides
flexible spatial and temporal control, which is critical to dissect
complex signaling pathways in cells.
As many diseases are driven by the expression of a few pro-
teins, a potential therapeutic strategy is to remove these es-
sential proteins. Small interfering RNAs (siRNAs), which knock
down the expression of a gene of interest by destroying its
mRNAs, have drawn considerable attention as a potential ther-
apeutic approach.[47,48] However, this siRNA knock-down strat-
egy has some inherent problems,[47–49] such as off-target ef-
fects, sequence-independent effects, activation of unrelated
signaling pathways, and drug delivery. The PROTAC approach
does not appear to suffer these same limitations and thus may
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maldehyde (PFA) was purchased from Fisher Scientific, while mon-
obasic and dibasic potassium phosphate were acquired from Mal-
linckrodt Baker (Phillipsburg, NJ, USA). Antibody Dilutant with
Background Reducing Components was purchased from DAKO
(Glostrup, Denmark), while [6,7-3H]17b-estradiol was purchased
from Amersham Biosciences (Buckinghamshire, UK).
provide an additional strategy to treat diseases by destroying
disease-promoting proteins.
As ER-positive breast cancers require ER-promoted cell pro-
liferation, the current therapeutic paradigm relies on the block-
age of this hormone-mediated cell growth. Thus, a strategy
which allows selective degradation of the ER could provide a
valuable and novel therapeutic option for many breast cancer
patients. Additionally, most breast cancers that develop resist-
ance to current treatments retain functional ERs. Optimized
PROTACs, which induce the degradation of the ER, may thus
provide an additional therapeutic option for the treatment of
tamoxifen- or fulvestrant-resistant breast tumors.
Cell culture: The MCF7 human breast cancer cell line was pur-
chased from the American Type Culture Collection (Manassas, VA,
USA). MCF7 cells were maintained in RPMI 1640 medium contain-
ing FBS (10% v/v), penicillin (100 UmLÀ1), and streptomycin
(100 ugmLÀ1) (Gibco, Carlsbad, CA, USA). All experiments were per-
formed when the cells were 70% confluent and had been main-
tained in 5% (v/v) charcoal–dextran-treated FBS RPMI with antibi-
otics for at least 24 h. Compounds were treated in a DMSO vehicle
at the appropriate dilutions for 48 h unless noted otherwise.
In summary, we have shown that a chimeric small molecule
induces the proteasome-dependent degradation of the ER in
living cells. Whether the targeted degradation of ER is useful in
treating hormone-sensitive or anti-estrogen-resistant breast
tumors remains to be determined. Generally, PROTACs should
be optimized by determining a position for derivatization that
retains a maximal binding affinity. Additionally, pentapeptide-
based PROTACs benefit from C-terminal protection of the pep-
tide regardless of the linker position. Finally, the small-mole-
cule strategy reported herein presents a generic approach to
target any cancer-promoting protein for degradation.
Western blotting: Whole-cell lysates were prepared by incubating
cells in non-denaturing lysis buffer (50 mm Tris·HCl, 150 mm NaCl,
1% NP40, 1% Triton X-100, and 1% protease inhibitor cocktail) on
ice for 1 h. Cells were then centrifuged (14000 rpm, microliter rotor
24ꢁ1.5/2 mL, Biofuge Primo R) with supernatants collected and
subjected to protein assay by the Bradford method. The sample
was mixed with an equal volume of 2ꢁ Laemmli sample buffer
and heated in boiling water for 10 min. Equal protein concentra-
tions of the samples were subjected to 10% SDS polyacrylamide
gel electrophoresis and blotted onto a PVDF membrane. After
blocking, the membranes were incubated overnight at 48C in pri-
mary antibody and for 1 h at room temperature with secondary
antibody. Antibody binding was detected using ECL and film. All
membranes were then re-probed with mouse anti-b-actin to
ensure equal protein loading.
Experimental Section
Chemistry
All reagents were purchased from Aldrich. PROTAC 2 was synthe-
sized by following a procedure similar to that previously report-
ed.[50] Epoxomicin was synthesized as previously reported.[51] See
the Supporting Information for more detailed information on the
chemical syntheses.
Cell viability assay by MTS: MCF7 cells were plated at a density of
5ꢁ103 cells per well in 96-well plates in RPMI 1640 with 10% FBS
and left overnight at 378C. The media was changed to 5% char-
coal RPMI for 24 h prior to the addition of compounds. The prolif-
eration rate of the cells was determined after 48 h by using the
CellTiter 96 Aqueous One Solution Cell Proliferation Assay accord-
ing to the supplier’s instructions. Absorbance was measured at
l 490 nm on a microplate reader using the KC4 program. IC50
values were obtained from at least triplicate results via a sigmoid
dose–response curve using nonlinear regression to a logarithmic
function (GraphPad Prism software, San Diego, CA, USA).
Biological assays
Reagents: Fetal bovine serum (FBS), RPMI 1640, phenol-red-free
RPMI, conjugated secondary antibody Alexa Fluor 488 (FITC), anti-
biotics, Hank’s balanced salt solution (HBSS), goat serum, Prolong
Gold antifade with DAPI, recombinant human estrogen receptors
(ER-a), and trypsin–EDTA were purchased from Invitrogen (Carls-
bad, CA, USA). 17b-Estradiol, tamoxifen, Kodak XAR film, NaCl, Non-
idet-P40 (NP40), protease inhibitors cocktail, Tween-20, ethanol,
bovine serum albumin (BSA), and 2ꢁ Laemmli sample buffer were
obtained from Sigma–Aldrich (St. Louis, MO, USA). Charcoal–dex-
tran-treated FBS was purchased from Hyclone (Logan, UT, USA).
Anti-ER antibodies were acquired from Santa Cruz Biochemical
(Santa Cruz, CA, USA) for western blotting, and Abcam (Cambridge,
MA, USA) for immunofluorescence, while the anti-b-actin antibody
was purchased from Novus Biologicals (Littleton, CO, USA). The
anti-mouse IgG–horseradish peroxidase (HRP) conjugate was ob-
tained from Zymed Laboratories (South San Francisco, CA, USA).
Anti-rabbit IgG–HRP and enhanced chemiluminescence (ECL) de-
tection reagents were acquired from GE Healthcare (Piscataway, NJ,
USA). Protein Assay Dye Reagent Concentrate, Tris·HCl, Triton X-
100, sodium dodecyl sulfate (SDS), and PVDF membranes were
purchased from Bio-Rad (Hercules, CA, USA). Dimethyl sulfoxide
(DMSO) and KCl were obtained from EMD (Darmstadt, Germany),
while the CellTiter 96 Aqueous One Solution Cell Proliferation
Assay was purchased from Promega (Madison, WI, USA). Parafor-
ER binding affinity assay: Competitive ligand binding assays were
performed according to the manufacturer’s protocol (Invitrogen).
3
Purified human recombinant ER (10 nm) was added to H-labeled
estradiol (20 nm) and the indicated concentrations of estradiol or
PROTACs. After incubation for 2 h at room temperature or over-
night at 48C, a 50% hydroxyapatite slurry was added to bind the
receptor–ligand complex. The sample was centrifuged (14000 rpm,
microliter rotor 24ꢁ1.5/2 mL, Biofuge Primo R), and the pellet re-
suspended for analysis of tritium activity by scintillation counting.
The percent specific binding was calculated, and IC50 values were
obtained using one-site competition curve analyses in GraphPad
Prism. Relative binding affinity (RBA) was calculated by the follow-
ing equation: RBA=(IC50 E2/IC50 sample)ꢁ100.
Immunofluorescence: Cover slips were sterilized with ethanol and
UV light exposure. MCF7 cells were added directly to the cover slip
and allowed to attach for 24 h. The media was then changed to
phenol-red-free RPMI with 5% charcoal–dextran-treated FBS until
treatment with compounds. Compounds were diluted in the
phenol-red-free media and treated as before. Cells were then fixed
with 4% PFA, washed with phosphate-buffered saline (PBS), and
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permeabilized with 0.2% Triton X-100 in PBS. Between all subse-
quent steps, cover slips were washed in PBST (PBS with 0.05%
Tween-20). Cover slips were blocked in 10% goat serum with 0.1%
BSA in PBST. Next, primary antibody was added in the antibody di-
lutant directly to the cover slip, then secondary antibody was
added in the same way. Prolong Gold with DAPI was added to
clean slides to mount the cover slips, and the mounted slides were
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Acknowledgements
This work was supported by Graduate School Academic Year,
Kentucky Opportunity, and American Foundation for Pharma-
ceutical Education Fellowships (M.W.). K.B.K. gratefully acknowl-
edges the support of the Markey Cancer Foundation, NIH
(CA131059), and DoD (W81XWH-08-1-0092). H.S. thanks the NIH
for financial support (ES014849). We thank members of the
Swanson, Lee, and Kim research groups for their helpful com-
ments on the manuscript. We are grateful to Dr. Wooin Lee for
helping with the collection of fluorescent images.
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Keywords: E3 ubiquitin ligase · PROTACs · proteasome ·
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Received: April 7, 2010
Revised: April 28, 2010
Published online on May 28, 2010
ChemMedChem 2010, 5, 979 – 985
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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