On the intractability of estrogen-related receptor α as a target for activation by small molecules

Article abstract of DOI:10.1021/jm7012387

The estrogen-related receptor α (ERRα) is a potential target for activation in the treatment of metabolic disease. To date, no small-molecule agonists of ERRα have been identified despite several high-throughput screening campaigns. We describe the synthesis and profiling of a small array of compounds designed on the basis of a previously reported agonist-bound crystal structure of the closely related receptor ERRγ. The results suggest that ERRα may be intractable as a direct target for pharmacologic activation.

Full text of DOI:10.1021/jm7012387

6722
J. Med. Chem. 2007, 50, 6722–6724
On the Intractability of Estrogen-Related Receptor r as a Target for Activation by Small
Molecules
Stephen M. Hyatt,† Elizabeth L. Lockamy,†,‡ Rebecca A. Stein,§ Donald P. McDonnell,§ Aaron B. Miller,†
Lisa A. Orband-Miller,† Timothy M. Willson,† and William J. Zuercher*,†
Molecular DiscoVery Research, GlaxoSmithKline, FiVe Moore DriVe, Research Triangle Park, North Carolina 27709, and Department of
Pharmacology and Cancer Biology, LeVine Science Research Center, Duke UniVersity, Research DriVe, Durham, North Carolina 27710
ReceiVed October 1, 2007
The estrogen-related receptor R (ERRR) is a potential target for activation in the treatment of metabolic
disease. To date, no small-molecule agonists of ERRR have been identified despite several high-throughput
screening campaigns. We describe the synthesis and profiling of a small array of compounds designed on
the basis of a previously reported agonist-bound crystal structure of the closely related receptor ERRγ. The
results suggest that ERRR may be intractable as a direct target for pharmacologic activation.
Introduction
The estrogen-related receptor R (ERRR,a NR3B1) is an
orphan member of the nuclear receptor superfamily that is
broadly expressed in adult tissues.1,2 Genetic and functional
analyses have implicated the receptor as a critical regulator of
oxidative metabolism in muscle, a pathway that contributes to
the pathology of type 2 diabetes.3 As such, small-molecule
ERRR agonists have the potential to increase expression of
mitochondrial OXPHOS genes that would ameliorate metabolic
diseases of poor energy balance. Despite its evolutionary
relationship to the classical estrogen receptor R (ERR), the
orphan ERRR does not bind to any of the known steroidal
hormones.4 Instead, the activity of ERRR has been shown to
be regulated by coactivator proteins, such as the peroxisome
proliferator-activated receptor γ coactivators (PGC-1R and PGC-
1ꢀ) and the nuclear receptor corepressor-interacting protein 140
(RIP-140).5–8 X-ray crystallography has shown that the ligand
Figure 1. Overlay of the X-ray crystal structures of the 1-ERRγ
binding domain (LBD) of ERRR contains a small hydrophobic
pocket, suggesting that the receptor may be able to accommodate
a synthetic small-molecule ligand.9 Although several ERRR
inverse agonists have been identified,10,11 the tractability of the
receptor for small-molecule activation remains an open question.
Having failed to identify ERRR agonists through random
screening of the GSK compound collection, we utilized
structure-guided design to address the issue of the chemical
tractability of this orphan receptor as a molecular target for
metabolic diseases.
complex and apo-ERRR. 1 is shown in stick representation colored by
atom type with carbon in green, nitrogen in blue, and oxygen in red.
The protein backbone is shown in ribbon view with ERRγ in cyan
and ERRR in yellow. The helix 3 backbones have been removed
from the view. The side chains of key residues in the ligand binding
pocket are shown as sticks with carbon atoms colored to match their
respective proteins. Two water molecules that make contact with 1
are also shown. A272 in ERRγ is replaced by F330 in ERRR, which
occludes the binding pocket.
crystallography of 1 bound to the ERRγ LBD revealed a
rearrangement of a loop between helices 1 and 3, which allowed
the agonist ligand to access a lipophilic pocket that was not
available in the unliganded receptor (Figure 1).13 Because of
this rearrangement, the phenol of 1 did not interact with E275
and R316 that are analogous to the classical phenol-binding
residues in ERR. Instead, the phenolic hydroxyl formed a
hydrogen bond with D328 near the surface of the receptor. E275
was rotated into a conformation to make contact with E247,
while R316 interacted with the acyl hydrazone carbonyl of 1
through a bridging water molecule. The agonist profile of 1 was
ascribed to global stabilization of the receptor rather than direct
interaction with the C-terminal activation helix, AF-2.
Results and Discussion
We have previously identified and characterized a phenolic
acyl hydrazone 1 (GSK4716) as a small-molecule agonist of
the closely related ERRꢀ (NR3B2) and ERRγ (NR3B3).12 X-ray
* To whom correspondence should be addressed. Phone: 919-483-5275.
Fax: 919-315-0430. E-mail: william.j.zuercher@gsk.com.
†
GlaxoSmithKline.
GlaxoSmithKline Women in Science Scholar. Current address: Depart-
‡
ment of Biochemistry, Molecular Biology, and Biophysics, University of
Minnesota, Minneapolis, MN 55455.
§
Although 1 and close analogues showed no measurable
binding affinity to ERRR, an analysis of the apo-crystal structure
suggested that a parallel ligand-induced conformational change
might be possible, since all of the critical polar residues present
in ERRγ are conserved (Figure 1). However, the comparison
Duke University.
aAbbreviations: ERR, estrogen receptor-related receptor; PGC-1, per-
oxisome proliferator-activated receptor γ coactivator; RIP-140, nuclear
receptor corepressor-interacting protein 140; LBD, ligand-binding domain;
FRET, fluorescence resonance energy transfer; ERE, estrogen response
element; 4-OHT, 4-hydroxytamoxifen.
10.1021/jm7012387 CCC: $37.00
2007 American Chemical Society
Published on Web 12/04/2007

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6723
Table 1a
compd
R
compd
R
Figure 2. Normalized luciferase activity after treatment with vehicle,
10 µM 3h and 4 and µM 5.
2a
2b
2c
2d
2e
2f
2g
3a
3b
ethyl
3c
3d
3e
3f
3g
3h
3i
ethyl
isopropyl
2-butyl
3-buten-1-yl
3-methyl-2-pentyl
3-buten-3-yl
n-pentyl
1-propyl
2-propyl
methallyl
1-butyl
isobutyl
neopentyl
1-pentyl
3-methyl-1-butyl
previous reports, the level of constitutive activity in cells of
the ERRR F272A mutant was greatly attenuated relative to wild
type ERRR.15 Unexpectedly, there was no observed increase
in luciferase activity upon exposure of 1 relative to vehicle
treated cells. Moreover, none of the truncated analogues of 1
showed any effect on the activity of the point mutant receptor;
representative data are shown for 3h (Figure 2). In contrast to
the inability of the phenolic acyl hydrazones and amides to
activate the mutant receptor, we were surprised to find that
4-hydroxytamoxifen (4-OHT, 5) profiled as an agonist. It had
been previously reported that 4-OHT could bind the point mutant
receptor.16 Our observation that 4-OHT not only binds, but is
also an agonist of the ERRR F272A mutant demonstrates that
the mutant receptor is not inert to activation by small-molecule
ligands. Although 4-OHT has been shown to function as an
inverse agonist of ERRꢀ and ERRγ, the compound has no
activity on wild type ERRR.12
H
methyl
3j
3k
a Reagents and conditions for chemical conversion: (a) RCHO, MeOH,
then PS-NHNH2, PS-CHO; (b) (i) SOCl2, ∆; (ii) RNH2, Et3N, CH2Cl2.
also showed that F272 in ERRR occludes the region of the
pocket where the 4-isopropylphenyl group of 1 sits in ERRγ.
The alanine to phenylalanine switch explained the selectivity
profile of 1 across the ERR subtypes but also suggested that
new analogues could be designed to fit the smaller ERRR
pocket. Specifically, molecular docking studies predicted that
secondary phenolic acyl hydrazones and amides with small
N-alkyl groups would be accommodated within the volume of
the ERRR LBD.
Two small arrays of compounds were prepared as potential
ERRR agonists (Table 1). Phenolic acyl hydrazones (2) were
formed in a single step by condensation of 4-hydroxybenzoic
hydrazide and alkylaldehydes followed by reagent scavenging
with PS-NHNH2 and PS-CHO. Secondary phenolic amides (3)
were synthesized from 4-acetoxybenzoic acid through formation
of the acid chloride with SOCl2 followed by exposure to an
amine and subsequent cleavage of the aryl acetate. The resulting
compounds were screened in biochemical and cell-based reporter
assays for activation of ERRR. By use of a fluorescence
resonance energy transfer (FRET) based assay to measure the
interaction of the ERRR LBD with a peptide fragment of the
cofactor RIP-140, none of the compounds showed activity up
to 30 µM, and select compounds were assayed up to 100 µM
with no observable effect on RIP-140 interaction. In addition
to being inactive in the ERRR FRET assay, the compounds also
failed to show activity in the ERRγ FRET assay, consistent
with the previously observed structure–activity. A heterologous
reporter assay was also developed in which HeLa cells were
transfected with an expression vector for the human ERRR and
a reporter construct engineered from a triple repeat of an
estrogen response element (ERE) fused to luciferase. Coex-
pression of ERRR and the reporter led to an increase in
normalized luciferase signal. The luciferase activity could be
fully ablated by exposure to the ERRR inverse agonist 414 or
further increased by coexpression of PCG-1R, but the activity
was unaffected by any of the phenolic hydrazones (2a–h) or
amides (3a–k) up to 50 µM.
Conclusions
We have utilized a cocrystal structure of 1 bound to ERRγ
to design potential ERRR agonists. However, the compounds
neither increase nor decrease the activity of ERRR, raising doubt
about the ability of small molecules to induce a conformational
change that would lead to activation of the receptor. The
prospects for ERRR agonists are further diminished by the
inability of 1 or its analogues to activate the ERRR F272A point
mutant. Whether the poor tractability of ERRR is due to
occlusion of a critical part of the ligand binding pocket by F232
or an inability to expand the apo-receptor to accommodate
synthetic activating molecules remains unclear. However, the
accumulated evidence from extensive high-throughput screening
combined with the failure of structure-guided design demon-
strates that ERRR is a low tractability molecular target for
metabolic diseases.
Experimental Section
General Procedure for the Preparation of Phenolic Acyl
Hydrazones 2. N′-[(1E)-Hexylidene]-4-hydroxybenzohydrazide
(2g). To a solution of 4-hydroxybenzoic hydrazide (152 mg, 1.0
mmol) in absolute ethanol (2 mL) containing a catalytic amount of
acetic acid was added hexanal (100 mg, 1.0 mmol). The mixture
was stirred at room temperature for 2 h, and precipitation was
observed. The mixture was then neutralized by pouring into 10%
aqueous sodium bicarbonate solution. The precipitate formed was
filtered and dried, furnishing 2g (149 mg, 64%): 1H NMR (MeOD-
d4) δ 0.92 (t, J ) 7.0 Hz, 3H), 1.34–1.39 (m, 4H), 1.53–1.60 (m
2H), 2.34 (q, J ) 6.9 Hz, 2H), 6.84 (d, J ) 8.9 Hz, 2H), 7.64 (t,
J ) 5.8 Hz, 1H), 7.75 (d, J ) 8.9 Hz, 2H); 13C NMR (MeOD-d4)
δ 13.12, 22.31, 26.21, 31.37, 32.22, 115.03, 123.54, 129.49, 153.36,
161.40, 165.37; MS (ESI) m/z 217 (M - H)-, 219 (M + H)+.
General Procedure for the Preparation of Phenolic Ami-
des 3. 4-Hydroxy-N-(2-methylpropyl)benzamide (3h). To 4-ac-
etoxybenzoic acid (0.5 mmol) was added SOCl2 (2 mmol). The
The lack of observed activity in biochemical and cellular
assays called into question the hypothesis that ERRR could
undergo a conformational rearrangement similar to that observed
with in ERRγ. To examine the validity of the structure-based
design, we prepared a point mutant of ERRR where F272 was
converted to the alanine found in ERRγ. Consistent with

6724 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Chart 1. ERRγ Agonist Acyl Hydrazone 1
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mixture was stirred at 80 °C for 30 min. The volatiles were then
removed under reduced pressure, and isobutylamine (1 mmol) and
Et3N (1 mmol) in CH2Cl2 (2 mL) were added. The mixture was
allowed to stir at room temperature overnight, after which time
amide formation and acetate cleavage were checked for completion.
After removal of volatiles and dissolution in MeOH, the phenolic
1
amide product 3h was purified by reverse-phase HPLC. H NMR
(DMSO-d6) δ 0.84 (d, J ) 6.7 Hz, 6H), 1.79 (m, 1H), 3.01 (m,
2H), 6.76 (d, J ) 8.8 Hz, 2H), 7.68 (d, J ) 8.9 Hz), 8.17 (m, 1H),
9.89 (m, 1H); 13C NMR (DMSO-d6) δ 20.92, 28.84, 47.28, 115.34,
126.15, 129.70, 160.56, 166.56; MS (ESI) m/z 192 (M - H)-, 194
(M + H)+.
Acknowledgment. The authors thank Carrow Wells for
postsynthesis compound processing and Matt Lochansky for
HRMS data collection. R.A.S. and D.P.M. acknowledge the
support of the NIH (Grant DK074652).
Supporting Information Available: General experimental
procedures, FRET and cell-based assay protocols, and compound
characterization data. This material is available free of charge via
the Internet at http://pubs.acs.org.
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JM7012387