Identification of New Small-Molecule Inducers of Estrogen-related Receptor α (ERRα) Degradation

Article abstract of DOI:10.1021/acsmedchemlett.9b00025

A series of (E)-3-(4-((2,4-bis(trifluoromethyl)benzyl)oxy)-3-methoxyphenyl)-2-cyanoacrylamide derivatives were designed and synthesized as new estrogen-related receptor α (ERRα) degraders based on the proteolysis targeting chimera (PROTAC) concept. One of the representative compounds 6c is capable of specifically degrading ERRα protein by >80% at a relatively low concentration of 30 nM, becoming one of the most potent and selective ERRα degraders to date. Compound 6c could be utilized as a new powerful research tool for further biological investigation of ERRα.

Full text of DOI:10.1021/acsmedchemlett.9b00025

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Letter
Identification of New Small-Molecule Inducers of
Estrogen-Related Receptor alpha (ERRa) Degradation
Lijie Peng, Zhensheng Zhang, Chong Lei, Shan Li, Zhang Zhang,
Xiaomei Ren, Yu Chang, Yan Zhang, Yong Xu, and Ke Ding
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00025 • Publication Date (Web): 12 Apr 2019
Downloaded from http://pubs.acs.org on April 12, 2019
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ACS Medicinal Chemistry Letters
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Identification of New Small-Molecule Inducers of Estrogen-Related
Receptor  (ERR) Degradation
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Lijie Peng, a,#,* Zhensheng Zhang, a,# Chong Lei, a Shan Li, a Zhang Zhang, a Xiaomei Ren,a Yu
Chang, a Yan Zhang, b Yong Xu, b Ke Ding, a,*
aInternational Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug
Development , Ministry of Education (MOE) of China, Guangzhou City Key Laboratory of Precision Chemical Drug
Development, School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China
bGuangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou
510530, China
KEYWORDS: Estrogen-Related Receptor (ERR), Proteolysis targeting chimera (PROTAC), protein degradation
ABSTRACT: A series of (E)-3-(4-((2,4-bis(trifluoromethyl)benzyl)oxy)-3-methoxyphenyl)-2-cyanoacrylamide derivatives
were designed and synthesized as new ERR degraders based on the proteolysis targeting chimera (PROTAC) concept.
One of the representative compounds 6c is capable of specifically degrading ERR protein by > 80% at a relatively low
concentration of 30 nM, becoming one of the most potent and selective ERR degraders to date. Compound 6c could be
utilized as new powerful research tool for further biological investigation of ERR.
Estrogen-Related Receptors (ERRs) belong to an
orphan nuclear receptor superfamily.1 Three isoforms of
ERRs (i.e., ERR,2  and 3) have been identified to date.
ERRβ is associated with early development,4 whereas
ERR obviously stimulate proliferation, migration,
angiogenesis, metastasis and drug resistance of human
breast cancer cells.16-18 ERR overexpression has also been
frequently detected in Her2+ and triple-negative breast
tumors and associated with poor clinical outcome of
patients.19 Pharmacological inhibition or gene knockout
of ERR were proven to mitigate breast cancer
progression both in vitro and in vivo.20, 21
ERR and ERR are being considered as novel potential
6
targets for metabolic disorders.5,
ERR is the first-
discovered and most-studied member of this subgroup,
and was named because of its relatively high homology
with Estrogen Receptor  (ER). In addition to the cross-
talk with ER signals,7 ERR also plays a critical role in
regulating metabolism and energy homeostasis8 by
interacting with multiple transcriptional cofactors, such
as the peroxisome proliferator-activated receptor  co-
activator 1 proteins (i.e., PGC-1 and PGC-1)9 and
receptor-interacting protein 140 co-repressor (RIP-140)10
etc.
To facilitate biological investigation on the target,
several ERR inverse agonists and small molecules
improving its transcriptional function have been
22-32
discovered.12,
Most recently, a proteolysis targeting
chimera (PROTAC) compound was also designed and
synthesized by Crews et. al. to induce the degradation of
ERR (Figure 1).33
PROTAC is a new “chemical-small-molecule-induced
protein knockdown” technology to recruit an E2/E3 ligase
to the target protein and promote polyubiquitination and
subsequent protein degradation via proteasome.34
Comparing with the classical activity inhibitors, PROTAC
can mimic the natural proteasome degradation process to
totally abolish functions of target protein. Additionally,
obvious advantages, such as superior target specificity35
and accessibility to “undruggable” targets,36 make
PROTAC as an attractive strategy for next generation
drug discovery.37, 38
A small molecule up-regulating function of ERR had
been suggested as a new potential therapeutic strategy for
type II diabetes and other metabolic disorders, based on
its
crucial
roles
in
mitochondrial
oxidative
phosphorylation (OXPHOS) and fatty acid oxidation
processes.6 However, whole-body ERR knockout animals
are unexpectedly lean and non-diabetic.11 Pharmacological
inhibition of ERR by using an inverse agonist also
exhibited in vivo antidiabetic efficacy.12 These paradoxical
results indicate complicated and diverse functions of
ERRα in metabolic regulation.
ERR/PGC-1() transcriptional axis also mediates
metabolic adaptions of various types of human cancer
cells.13-15 For instance, metabolic alterations driven by
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Page 2 of 7
N
N
O
O
determined by utilizing a well-established time-resolved
CF3
NC
MeO
O
MeO
S
N
NH
O
H
fluorescence resonance energy transfer (TR-FRET) assay.30
Under the experimental conditions, reference compounds
1 and 2 exhibited strong inhibitory potencies with IC50
values of 61.3 and 33.0 nM, respectively, which are
comparable to the reported data.12, 30 Encouragingly, the
structurally simplified molecule 4a displayed a ~11-fold
improved potency with an IC50 value of 5.67 nM.
Additionally, removal of the thiadiazoloacylamide group
also resulted in a significant solubility improvement of 4a
(data not shown). Highly consistent to our prediction, the
terminal carboxylic acid in 4a could be replaced by a
hydrophilic group, such as an unsubstituted (4c) or
substituted (4d, 4e and 4f) amide without affecting the
binding affinity with ERR. However, esterification of the
carboxylic acid (4b) caused a ~4-fold potency loss in the
TR-FRET assay. These data collectively suggested that the
terminal carboxylic acid group could be a feasible position
to introduce a linker for further PROTAC molecule design.
1
2
3
4
5
6
7
8
S
CN
O
CF3
F3
C
CF3
1 (XCT790)
2 (C29)
OH
O
O
N
O
N
N
H
O
S
N
H
O
MeO
O
N
NC
S
O
CF3
9
3 (C29_PROTAC)
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12
13
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Figure 1. Chemical structures of representative ERR inverse
agonists and previously reported ERR degrader.
Herein, we would like to report our effort to identify a
new series of PROTAC based ERR degradation inducers
with obviously improved potency.
Architecturally, PROTAC molecules possess two
distinct ligand moieties which are able to bind with the
target protein and E2/E3 ubiquitin ligase, respectively,
and a covalently connecting linker.34 Therefore, the first
step for designing a degrader is to identify a novel ERR
ligand.
Table 1. In Vitro binding affinities to ERR of inverse
agonists 4a-4f.a
O
H3CO
O
Compound 1(XCT790)23 is the first reported and widely
investigated ERR inverse agonist with an IC50 value of
0.37 μM in a GAL4-ERR cell-based transfection assay. A
preliminary computational study suggested that
compound 1 could fit into a ligand-induced pocket of
ERR (PDB code: 3K6P, Figure 2A). The -cyano-, β-
unsaturated carbonyl moiety is close to Cys325 residue
with a distance of 3.5Å, suggesting a covalent interaction
could be formed. The diaryl ether core was embedded in a
hydrophobic pocket formed by Phe382, Val369, Leu405,
Val366, Val491 and Phe495 residues. Whereas, the
R
CN
F3C
CF3
aTR-FRET
IC50 (nM)
Cpds
4a
4b
4c
4d
4e
4f
R
OH
OCH3
NH2
5.67±0.58
20.33±1.53
4.33±1.53
4.33±0.58
6.00±1.00
4.67±0.58
33.0±1.73
61.33±6.11
NHCH2CH2OCH3
thiadiazoloacylamide group of
1 seemed to extend
towards solvent exposing surface of the protein and did
not form any defined interaction with ERR. It was
hypothesized that the thiadiazoloacylamide group in
compound 1 could be removed without affecting the
binding with ERR. Compound 4a was thus designed and
synthesized based on this hypothesis (Figure 2B).
NHCH2CH2CH2CH2COOH
NHCH2CH2OCH2CH2COOH
2
1
aAffinity to ERR was determined by using a TR-FRET assay
(Invitrogen, USA). The data are means from at least three
independent experiments.
Several small-molecule E2/E3 ligase ligands,39 such as
Mouse Double Minute
2
(MDM2) ligand Nutlin
derivatives, cellular inhibitor of apoptosis protein 1
(cIAP1) inhibitor bestain, cereblon (CRBN) ligand
thalidomide derivatives, and the von-Hippel-Lindau
(VHL) ligand, have been incorporated into PROTAC
molecules to mediate degradation of the target proteins.
It is also clear that the E2/E3 ubiquitin ligase ligand
choice40 has significant impact on efficiency and target
specificity of a PROTAC molecule. The VHL ligand had
been previously proven to be highly efficient to induce
degradation of ERR protein.33 Therefore, VHL ligand 5
Figure 2. (A) Predicted binding mode of compound 1 with
ERR (PDB code: 3K6P). (B) Structure-activity relationship
(SAR) overview for compound 4a. (C) Chemical structure of
von-Hippel-Lindau (VHL) ligand 5. (D) Chemical structure of
designed PROTACs 6.
Activity of compound 4a to block the protein-protein
interaction of ERR with PGC-1 coactivator was
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(Figure 2C), which harbors a solvent-exposing acetamide
group for conjugating a linker,41 was first utilized for our
new ERR degrader design.
It was shown that length and chemical types of the
1
2
3
4
5
6
7
8
hybriding linker had minor influence on ERR binding
affinities of the PROTAC molecules. Compounds 6a~6j
exhibited strong binding with ERR, with IC50 values
ranged from 5.33 to 12.33 nM, which are comparable to the
parental molecule 4a. These data further supported that
our initial hypothesis that the terminal carboxylic acid
group in 4a is a feasible position for E2/E3 ligase ligand
linkage. However, the introduced linkers had significant
impact on ERR protein degradation capabilities of the
molecules. For instance, compound 6a and 6b, which
harbours a 2-carbon or 3-carbon linker, exhibited almost
the same ERR inhibitory potencies with IC50 values of 7.3
and 6.3 nM respectively. However, they displayed
significantly varied capabilities to induce degradation of
ERR protein, with D30nM and D100nM values of 28%, 50%
and 58%, 85%, respectively. Although a 4-carbon linker
caused an approximately 2-fold ERR binding affinity
loss, the resulting compound 6c exhibited the strongest
protein degradation potency, with D30nM and D100nM values
of 83% and 96%, respectively. It could even induce
Scheme 1. Synthesis of PROTACs 6a-6j.*
O
A.
O
O
COOH
CF3
N
COOtBu
CF3
H
a
CN
b
CN
H2
N
COOtBu
+
O
O
F3C
F3
C
4a
10
11
OH
O
O
O
O
O
O
O
S
N
COOH
+
N
N
9
H
a
N
H
H
N
H
N
CN
CN
13
O
O
10
11
12
13
14
15
16
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F3
C
CF3
F3C
CF3
12
6a-b, 6d, 6g-j
Linker
6a: (CH2
)
6b: (CH2
)
6d: (CH2
)
5
2
3
6g: CH2CH2OCH2
6h: CH2CH2OCH2CH2
6i: (CH2CH2O)2CH2CH2
6j: (CH2CH2O)3CH2CH2
OH
OH
B.
O
Boc
S
S
N
N
N
a
b
H2
N
H
N
H
H
N
H
N
BocHN
COOH
+
N
N
O
O
O
O
13
14
15
OH
OH
O
O
O
O
S
N
N
S
H2
N
N
N
H
H
N
H
a
N
N
4a
CN
H
+
N
O
O
N
H
O
O
O
F3C
CF3
16
6c, 6e-f
6e: (CH2
Linker
6c: (CH2
)
)
6f: (CH2
)
7
4
6
approximately 39% of ERR degradation at
a
a
concentration as low as 10 nM (D10nM). These results
strongly support the hypothesis that each PROTAC
causes a unique conformational change, and that protein
degradation requires certain plasticity in binding.42 The
compound with a 5-carbon linker (6d) also displayed
strong ERR degradation potency, whereas a further
longer carbon linker (e.g. 6- or 7- carbon linker) caused
an obvious protein-degrading potency loss (6e and 6f).
The investigation also revealed that the linker could be
tolerated by an oxygen atom-containing moiety to retain
the strong ERR degrading potency. Compounds 6g and
6h exhibited D100nM values of 92% and 89%, respectively.
It was noteworthy that compound 6h harbours the same
alkyl ether linker with that previously reported ERR
degrader 3,33 but its potency is significantly stronger than
that of compound 3. For instance, the ERR degrading
potency of compound 3 was almost abolished at 30 nM
(with a D30nM value of approximately 1%), but compound
6h induced 25% and 63% ERR degradation at
concentrations of 10 nM and 30 nM, respectively. Similar
to observation on the compounds with pure carbon
linkers, longer polyethylene glycol (PEG) linkers also
caused significant protein degrading potency loss of the
resulting compounds (6i and 6j). Not surprisingly,
although 6c-Epi exhibited strong binding with the target
protein ERR, its ERR degrading function was totally
abolished because the epi-VHL building block almost
completely prevented the binding with VHL E3 ligase.33
Reagents and conditions: (a) 1-[bis(dimethylamino) meth-
ylene]-1H-1,2,3-triazolo [4,5-b]-pyridinium 3-oxide hex-afluo-
rophosphate (HATU), N,N-diisopropylethylamine (DIPEA),
N,N-dimethylformamide (DMF), room temperature (rt), 39%
-
71%; (b) trifluoroacetic acid (TFA), dichloromethane
(DCM), rt.
A series of PROTAC molecules were first designed and
synthesized by hybriding 4a and VHL amino building
block 13 with different linkers (Scheme 1). Carboxylic acid
4a was coupled with amino ester 10 in the presence of
HATU and DIPEA at room temperature in DMF to give
the amide 11, which was deprotected with TFA to afford
the corresponding carboxylic acid intermediate 12. Upon
coupling with VHL amino building block 13 (prepared as
previously described, supporting information), the
designed PROTACs (6a-b, 6d, 6g-j) were generated.
Differently, ERR-PROTACs (6c, 6e-f) and the epimeric
6c (6c-Epi) were prepared by using an alternative
protocol. VHL amino ligand 13 was coupled with Boc-
protected amino acid 14, and the resulting compound 15
was treated with TFA to give 16. The final products were
obtained by condensation of 16 and 4a. Compound 6c-Epi
was synthesized by using an epi-VHL building block
harboring a reversed (S) stereochemistry at the proline 4-
position.33
Binding affinities of the new hybrids and their function
to degrade ERR were determined by utilizing in vitro
TR-FRET assay and western blotting analysis in the MDA-
MB-231 cells, respectively (Table
2 and Supporting
Table 2. Binding affinities and degradation Activities to
ERR of PROTACs 6a-6j.
Information). The first reported ERR PROTAC
3
(C29_PROTAC) was utilized as a positive control in both
assays. Compound 3 exhibited an IC50 value of 8.0 nM to
block the protein-protein interaction of ERR with PGC-
1 peptide, and induced approximately 40% protein
degradation at 100 nM (D100nM) after 4.0 hrs treatment,
which are similar to the previously reported data.33
OH
O
O
H3CO
N
linker
N
N
H
H
CN
S
H
N
O
O
O
N
F3C
CF3
PROTACs 6
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aTR-FRET
IC50 (nM)
7.33±3.06
6.33±1.15
hrs after treatment. However, the level of ERR could be
Cp
ds
Linker
bD30nM (%)
bD100nM (%)
1
2
3
4
5
6
7
8
recovered after 48 hrs (Figure 3C), which might be due to
resynthesis of the protein and/or other intracellular
feedback response. Further investigation also showed that
6c potently decreased protein levels of ERR downstream
target genes, e.g. ATP5B, medium-chain acyl CoA
dehydrogenase (MCAD), and pyruvate dehydrogenase
kinase 4 (PDK4) in the MDA-MB-231 cells after a 24-hr
treatment (Supporting Information).
6a
6b
(CH2)2
(CH2)3
28%
58%
50%
85%
83%
6c
(CH2)4
12.67±2.89
96%
(D10nM=39%)
58%
9
10
11
12
13
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6d
6e
6f
(CH2)5
(CH2)6
(CH2)7
5.67±1.53
12.33±2.52
7.67±0.58
78%
71%
9%
23%
3%
CH2CH2O
CH2
6g
6h
11.33±0.58
9.33±4.16
79%
92%
89%
Figure 3. Degradation effects of PROTAC 6c on ERR, ERRβ,
ERRγ proteins in MDA-MB-231 cells by Western blotting
analysis. (A) PROTAC 6c dose-dependently degrades ERR
protein. (B) PROTAC 6c has no effect on the levels of ERRβ
and ERRγ protein at indicated concentration; (C) Time-
points degradation analysis of PROTAC 6c at 30 nM.
63%
CH2CH2O
CH2CH2
(D10nM=25%)
15%
(CH2CH2O)2
CH2CH2
6i
6j
5.33±2.52
7.00±1.00
0
0
(D300nM=19%)
4%
Proteasome is importantly involved in the E2/E3 ligase
44
mediated protein degradation.43, In order to elucidate
(CH2CH2O)3
CH2CH2
the mechanism of 6c-induced ERR degradation, we
further investigated the contribution of proteasome in
ERR degrading process. MDA-MB-231 cells were
(D300nM=10%)
6c-
Epi
(CH2)4
5.33±1.15
6%
1%
cNT
preincubated with 1.0
M
proteasome inhibitor
Epoxomicin,45 then treated with 6c at 30 nM for 4.0 hrs.
Western blotting analysis suggested that pretreatment of
proteasome inhibitor successfully rescued the ERR
degradation induced by 6c, validating the crucial role of
proteasome system in VHL-mediated ERR degradation
(Figure 4A). However, no protein degradation was
observed for direct treatments of ERR ligand 4f and VHL
amino building block 13, either in a single agent or in a
combination (Figure 4A). Whereas, pretreatment with
ERR ligand 4f or VHL ligand 5 successfully abrogated
the ERR degradation mediated by 6c (Figure 4B). These
data collectively supported the necessity of ternary
complex formation for ERR degradation.
3
8.00±2.65
40%
aBinding affinity with ERR was determined by using a TR-
FRET assay. D (%): % degradation is measured at indicated
concentration by using western blot assay and generated by
ImageJ software analysis.46 The data are means from at least
three independent experiments. Representative western
blots can be found in the Supporting Information (Figure S2).
cNT: not tested.
b
Molecules by hybriding compound 4a with small
molecular ligands of other E2/E3 ligases (e.g. MDM2,
cIAP1 and CRBN) were also designed and synthesized.
However, none of them exhibited superior ERR
degradation potency than that of molecule 6c
(Supporting Information).
The selectivity of compound 6c to induce ERR
degradation was further validated in MDA-MB-231 breast
cancer cells (Figure 3 and 4). It was confirmed that
compound 6c dose-dependently induced ERR
degradation with an efficacious dose as low as 3.0 nM at
4.0 hrs (Figure 3A). Whereas, 6c did not show obvious
effect on ERRβ and ERR homologues at relatively high
concentrations (up to 10.0 μM, Figure 3B) at 4.0 hrs.
These data are highly consistent with the specific binding
of 6c with ERR protein (Supporting Information). A
time-course investigation further revealed that compound
6c could initiate ERR degradation after 30 min exposure,
and demonstrated a maximum effect at approximately 4.0
Figure 4. Mechanism study of PROTAC 6c in MDA-MB-
231 cells by Western blotting analysis. (A) PROTAC 6c-
mediated ERR degradation is abrogated by pretreatment
with proteasome inhibitor Epoxomicin. (B) PROTAC 6c-
mediated ERR degradation is abrogated by pretreatment
with ERR ligand 4f or VHL ligand 5.
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In
summary,
a
series
of
(E)-3-(4-((2,
4-
REFERENCES
(1) Giguère, V. Orphan Nuclear Receptors: From Gene to
Function. Endocr. Rev. 1999, 20 (5): 689–725.
(2) Giguère, V.; Yang, N.; Segui, P.; Evans, R. M. Identification
of a New Class of Steroid Hormone Receptors. Nature 1988, 331
(6151), 91−94.
(3) Eudy, J. D.; Yao, S.; Weston, M. D.; Ma-Edmonds, M.;
Talmadge, C. B.; Cheng, J. J.; Kimberling, W. J.; Sumegi, J.
Isolation of a Gene Encoding a Novel Member of the Nuclear
Receptor Superfamily from the Critical Region of Usher
Syndrome Type IIa at 1q41. Genomics 1998, 50 (3), 382−384.
(4) Luo, J.; Sladek, R.; Bader, J.-A.; Matthyssen, A.; Rossant, J.;
Giguère, V. Placental abnormalities in mouse embryos lacking
the orphannuclear receptor ERR-beta. Nature 1997, 388, 778-782.
(5) Audet-Walsh, E.; Giguère, V. The multiple universes of
estrogen-related receptor α and γ in metabolic control and
related diseases. Acta Pharmacol. Sin. 2015, 36 (1), 51-61.
(6) Handschin, C.; Mootha , V. K. Estrogen-related receptor 
(ERR): A novel target in type 2 diabetes. Drug Disc. Today:
Therapeutics Stategies. 2005, 2(2), 151-156.
1
2
3
4
5
6
7
8
bis(trifluoromethyl)benzyl)oxy)-3-methoxyphenyl)-2-
cyanoacrylamide derivatives were designed and
synthesized as new ERR degraders based on proteolysis
targeting chimera concept. Compounds 6c is capable of
specifically degrading ERR protein by > 80% at a
relatively low concentration of 30 nM, representing one of
the most potent and selective ERR degraders to date.
Mechanism investigation further validated 6c mediated
ERR degradation requires the ternary complex
formation and ubiquitin proteasome participation.
PROTAC 6c could be utilized as new powerful chemical
tool for further investigating functions of ERR in
physiological and pathological states.
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ASSOCIATED CONTENT
Supporting Information
Synthetic procedures and compound characterization,
(7) Giguère, V. To ERR in the estrogen pathway. Trends
Endocrinol. Metab. 2002, 13 (5), 220-225.
procedures, and in vitro TR-FRET assay, western blot
analysis, H NMR, 13C NMR spectra of compounds 6a-6j
1
(8) Giguère, V. Transcriptional control of energy homeostasis
by the estrogen-related receptors. Endocr. Rev. 2008, 29, 677–
696.
(9) Puigserver, P.; Spiegelman, B. M. Peroxisome proliferator-
activated receptor-γ coactivator 1 (PGC-1): transcriptional
coactivator and metabolic regulator. Endocr. Rev. 2003, 24, 78–
90.
(10) Christian, M.; White, R.; Parker, M. G. Metabolic regulation
by the nuclear receptor corepressor RIP140. Trends Endocrinol.
Metab. 2006, 17, 243–250.
(11) Luo, J.; Sladek, R.; Carrier, J.; Bader, J.; Richard, D.; Giguère,
V. Reduced fat mass in mice lacking orphan nuclear receptor
estrogen-related receptor . Mol. Cell. Biol. 2003, 23, 7947–7956.
(12) Patch, R. J.; Searle, L. L.; Kim, A. J.; De, D.; Zhu, X.; Askari,
H. B.; O'Neill, J. C.; Abad, M. C.; Rentzeperis, D.; Liu, J.;
Kemmerer, M.; Lin, L.; Kasturi, J.; Geisler, J. G.; Lenhard, J. M.;
Player, M. R.; Gaul, M. D. Identification of Diaryl Ether-Based
(PDF). This material is available free of charge via the
Internet.
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-85221523. Fax: +86-20-85224766. E-mail:
dingke@jnu.edu.cn (K. D.); elva_0916@ jnu.edu.cn (L. P.).
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors appreciate the financial support from National
Natural Science Foundation of China (81502907, 21572230,
81673285 and 81425021), Guangdong Province (2014TQ01R341,
2015A030306042, 2015A030312014, 2016A050502041 and
Nanyue-Baijie Award), China Postdoctoral Science
Foundation (2017M622660) and Jinan University. The
authors also thank Prof. Moshe Levi and Dr. Andrew Libby
from Georgetown University (USA) for the proof reading of
this manuscript.
Ligands for Estrogen-Related Receptor
Antidiabetic Agents. J. Med. Chem. 2011, 54, 788-808.
α
as Potential
(13) Ralph, J. D.; Julian, J. L.; Georgia, H,; Craig, B. T. The
Biology of Cancer: Metabolic reprogramming fuels cell growth
and proliferation. Cell Metab. 2008, 7, 11-20.
(14) Tam, I. S.; Giguère, V. There and back again: The journey of
the estrogen-related receptors in the cancer realm. J. Steroid
Biochem. Mol. Biol. 2016, 157, 13–19.
(15) Chang, Ch.-Y.; McDonnell, D.P. Molecular Pathways: The
metabolic regulator estrogen-related receptor as a therapeutic
target in cancer. Clin. Cancer Res. 2012, 18 (22), 6089-6095.
(16) Bianco, S.; Lanvin, O.; Tribollet, V.; Macari, C.; North, S.;
Vanacker, J. M. Modulating estrogen receptor-related receptor-
activity inhibits cell proliferation. J. Biol. Chem. 2009, 284,
23286–23292.
(17) Park, S.; Chang, Ch.-Y.; Safi, R.; Liu, X.; Baldi, R.; Jasper, J.S.;
Anderson, G.R.; Liu, T.; Rathmell, J.C.; Dewhirst, M.W.; Wood,
K.C.; Locasale, J.W.; McDonnell, D.P. ERR-regulated lactate
metabolism contributes to resistance to targeted therapies in
breast cancer. Cell Rep. 2016, 15, 323-335.
ABBREVIATIONS
ERR, estrogen-related receptor; ER, estrogen receptor;
OXPHOS, oxidative phosphorylation; PROTAC, proteolysis
targeting chimera; TR-FRET, time-resolved fluorescence
resonance energy transfer; VHL, von-Hippel-Lindau;
MDM2, Mouse Double Minute 2; cIAP1, cellular inhibitor of
apoptosis protein 1; CRBN, cereblon; DIPEA, N,N-
diisopropylethylamine;
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate;
trifluoroacetic acid; DMF, N,N-dimethylformamide; DCM,
dichloromethane; rt, room temperature; IC50, the half
maximal (50%) inhibitory concentration of a substance; PEG,
polyethylene glycol; MCAD, medium-chain acyl CoA
dehydrogenase; PDK4, pyruvate dehydrogenase kinase 4.
HATU,
1-[bis-
TFA,
(18) Deblois, G.; Smith, H.W.; Tam, I.S.; Gravel, S.P.; Caron, M.;
Savage, P.; Labbé, D.P.; Bégin, L.R.; Tremblay, M.L.; Park, M.;
Bourque, G.; St-Pierre, J.; Muller, W.J.; Giguère, V. ERR
mediates metabolic adaptations driving lapatinib resistance in
breast cancer. Nat. Commun. 2016, 7, 12156.
(19) Suzuki, T.; Miki, Y.; Moriya, T.; Shimada, N.; Ishida, T.;
Hirakawa, H.; Ohuchi, N.; Sasano, H. Estrogen-Related Receptor
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α in Human Breast Carcinoma as a Potent Prognostic Factor.
Cancer Res. 2004, 64 (13), 4670−4676.
(32) Wei, W.; Schwaid, A. G.; Wang, X.; Wang, X.; Chen, S.;
Chu, Q.; Saghatelian A.; Wan, Y. Ligand Activation of ERR by
Cholesterol Mediates Statin and Bisphosphonate Effects. Cell
Metab. 2016, 23, 479–491.
(33) Bondeson, D. P.; Mares, A.; Smith, I. E. D.; Ko, E.; Campos,
S.; Miah, A. H.; Mulholland, K.E.; Routly, N.; Buckley, D.L.;
Gustafson, J.L.; Zinn, N.; Grandi, P.; Shimamura, S.; Bergamini,
G.; Faelth-Savitski, M.; Bantscheff, M.; Cox, C.; Gordon, D.A.;
Willard, R.R.; Flanagan, J.J.; Casillas, L.N.; Votta, B.J.; den Besten,
W.; Famm, K.; Kruidenier, L.; Carter, P.S.; Harling, J.D.;
Churcher, I.; Crews, C. M. Catalytic in vivo protein knockdown
by small-molecule PROTACs. Nat. Chem. Biol. 2015, 11(8), 611–
617.
(34) Neklesa, T. K.; Winkler, J. D.; Crews, C. M. Targeted
protein degradation by PROTACs. Pharmacol. Ther. 2017, 174,
138−144.
(35) Adjei, A.A. What is the right dose? The elusive optimal
biologic dose in phase I clinical trials. J. Clin. Oncol. 2006, 24,
4054–4055.
(36) Lazo, J. S.; Sharlow, E. R. Drugging undruggable molecular
cancer targets. Annu. Rev. Pharmacol. Toxicol. 2016, 56, 23–40.
(37) Tinworth, C. P.; Lithgow, H.; Churcher, I. Small
moleculemediated protein knockdown as a new approach to
drug discovery. MedChemComm 2016, 7, 2206−2216.
(38) Lai, A. C.; Crews, C. M. Induced protein degradation: an
emerging drug discovery paradigm. Nat. Rev. Drug Discovery
2017, 16, 101−114.
(39) Bulatov, E.; Ciulli A. Targeting Cullin–RING E3 ubiquitin
ligases for drug discovery: structure, assembly and small-
molecule modulation. Biochem. J. 2015, 467 (3), 365−386.
(40) Lai A. C.; Toure, M.; Hellerschmied, D.; Salami, J.; Jaime-
Figueroa, S.; Ko, E.; Hines, J.; Crews, C. M. Modular PROTAC
Design for the Degradation of Oncogenic BCR-ABL. Angew.
Chem. Int. Ed. 2016, 55, 807 –810.
1
2
3
4
5
6
7
8
(20) Stein, R. A.; Chang, C. Y.; Kazmin, D. A.; Way, J.; Schroeder,
T.; Wergin, M.; Dewhirst, M. W.; McDonnell, D. P.; Estrogen-
related receptor  is critical for the growth of estrogen receptor-
negative breast cancer, Cancer Res. 2008, 68, 8805–8812.
(21) Dwyer, M. A.; Joseph, J. D.; Wade, H. E.; Eaton, M. L.;
Kunder, R. S.; Kazmin, D.; Chang, C. Y.; McDonnell, D. P.;
WNT11 expression is induced by estrogen-related receptor  and
-catenin and acts in an autocrine manner to increase cancer cell
migration, Cancer Res. 2010, 70, 9298–9308.
9
(22) Deuschle, U.; Heck, S.; Kober, l.; Bauer, U.; Balogh, I.
NR3B1 nuclear receptor binding 3-substituted pyrazole
derivatives. E. Patent 1398029, 2004.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(23) Busch, B. B.; Stevens, W. C. Jr.; Martin, R.; Ordentlich, P.;
Zhou, S.; Sapp, D. W.; Horlick, R. A.; Mohan, R. Identification of
a Selective Inverse Agonist for the Orphan Nuclear Receptor
Estrogen-Related Receptor α. J. Med. Chem. 2004, 47, 5593-5596.
(24) Kallen, J.; Lattmann, R.; Beerli, R.; Blechschmidt, A.;
Blommers, M. J. J.; Geiser, M.; Ottl, J.; Schlaeppi, J.-M.; Strauss,
A.; Fournier, B. Crystal Structure of Human Estrogen-related
Receptor α in Complex with a Synthetic Inverse Agonist Reveals
Its Novel Molecular Mechanism. J. Biol. Chem. 2007, 282, 23231-
23239.
(25) Chisamore, M. J.; Mosley, R. T.; Cai, S.-J.; Birzin, E. T.;
O’Donnell, G.; Zuck, P.; Flores, O.; Schaeffer, J.; Rohrer, S. P.;
Don Chen, J.; Wilkinson, H. A. Identification of Small Molecule
Estrogen-Related Receptor α-Specific Antagonists and
Homology Modeling To Predict the Molecular Determinants as
the Basis for Selectivity over ERRβ and ERRγ. Drug Dev. Res.
2008, 69 (4), 203−218.
(26) Wang, J.; Fang, F.; Huang, Z.; Wang, Y.; Wong, C.
Kaempferol Is an Estrogen-Related Receptor α and γ Inverse
Agonist. FEBS Lett. 2009, 583 (4), 643−647.
(41) Galdeano, C.; Gadd, MS.; Soares, P.; Scaffidi, S.; Van Molle,
I.; Birced, I.; Hewitt, S.; Dias, DM.; Ciulli, A. Structure-guided
design and optimization of small molecules targeting the
protein-protein interaction between the von Hippel-Lindau
(VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF)
alpha subunit with in vitro nanomolar affinities. J. Med. Chem.
2014, 57 (20), 8657-8663.
(42) Nowak, R. P.; DeAngelo, S. L.; Buckley, D.; He, Z.; Donovan,
K. A.; An, J.; Safaee, N.; Jedrychowski, M. P.; Ponthier, C. M.;
Ishoey, M.; Zhang, T.; Mancias, J. D.; Gray, N. S.; Bradner, J. E.;
Fischer, E. S. Plasticity in binding confers selectivity in ligand
induced protein degradation. Nat. Chem. Biol. 2018, 14 (7), 706-
714.
(43) Ciechanover, A. Proteolysis: from the lysosome to
ubiquitin and the proteasome. Nat. Rev. Mol. Cell Biol. 2005, 6,
79−87.
(44) Kleiger, G.; Mayor, T. Perilous journey: a tour of the
ubiquitin proteasome system. Trends Cell Biol. 2014, 24, 352−359.
(45) Meng, L.; Mohan, R.; Kwok B.; Elofsson, M.; Sin, N.;
Crews, C.M.; Epoxomicin, a potent and selective proteasome
inhibitor, exhibits in vivo antiinflammatory activity. Proc. Natl.
Acad. Sci. U S A. 1999, 96, 10403–10408.
(27) Xu, S.; Zhuang, X.; Pan, X.; Zhang, Z.; Duan, L.; Liu, Y.;
Zhang, L.; Ren, X.; Ding, K. 1-Phenyl-4-benzoyl-1H-1,2,3-triazoles
as orally bioavailable transcriptional function suppressors of
estrogen-related receptor α. J. Med. Chem. 2013, 56 (11), 4631-
4640.
(28) Patch, R.J.; Huang, H.; Patel, S.; Cheung, W.; Xu, G.; Zhao,
B.P.; Beauchamp, D.A.; Rentzeperis, D.; Geisler, J.G.; Askari,
H.B.; Liu, J.; Kasturi, J.; Towers, M.; Gaul, M.D.; Player, M.R.
Indazole-based ligands for estrogen-related receptor
 as
potential anti-diabetic agents. Eur. J. Med. Chem. 2017, 138, 830-
853.
(29) Zhang, L.; Liu, P.; Chen, H.; Li, Q.; Chen, L.; Qi, H.; Shi, X.;
Du, Y. Characterization of a selective inverse agonist for estrogen
related receptor α as a potential agent for breast cancer. Eur. J.
Pharmacol. 2016, 15 (789), 439-448.
(30) Du, Y.; Song, L.; Zhang, L.; Ling, H.; Zhang, Y.; Chen, H.;
Qi, H.; Shi, X.; Li, Q. The discovery of novel, potent ERR-alpha
inverse agonists for the treatment of triple negative breast
cancer. Eur. J. Med. Chem. 2017, 136, 457-467.
(31) Peng, L.; Gao, X.; Duan, L.; Ren, X.; Wu, D.; Ding, K.
Identification of Pyrido[1,2-α]pyrimidine-4-ones as New
Molecules Improving the Transcriptional Functions of Estrogen-
Related Receptor α. J. Med. Chem. 2011, 54 (21), 7729-7733.
(46) Schneider, G. A.; Rasband, W. S.; Eliceiri, K. W. NIH Image
to ImageJ: 25 years of image analysis. Nat. Methods 2012, 9 (7),
671-675.
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PROTAC
Proteasomal
degradation
OH
O
O
MeO
N
N
N
H
H
CN
O
N
O
O
H
N
S
F3C
CF3
D30nM = 83%
VHL
ERR
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