Disulfide-trapping identifies a new, effective chemical probe for activating the nuclear receptor human LRH-1 (NR5A2)

Article abstract of DOI:10.1371/journal.pone.0159316

Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, a

Full text of DOI:10.1371/journal.pone.0159316

RESEARCH ARTICLE
Disulfide-Trapping Identifies a New, Effective
Chemical Probe for Activating the Nuclear
Receptor Human LRH-1 (NR5A2)
Felipe de Jesus Cortez1, Miyuki Suzawa2, Sam Irvy3, John M. Bruning4, Elena Sablin5,
Matthew P. Jacobson1, Robert J. Fletterick5, Holly A. Ingraham2*, Pamela M. England1,2
*
1 Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco,
California 94158, United States of America, 2 Department of Cellular and Molecular Pharmacology,
University of California San Francisco, San Francisco, California 94158, United States of America,
3 Chemistry and Chemical Biology Graduate Program, University of California San Francisco, San
Francisco, California 94158, United States of America, 4 Pharmaceutical Sciences and Pharmacogenomics
Graduate Program, University of California San Francisco, San Francisco, California 94158, United States of
America, 5 Department of Biochemistry and Biophysics, University of California San Francisco, San
Francisco, California 94158, United States of America
a11111
* holly.ingraham@ucsf.edu (HAI); pamela.england@ucsf.edu (PME)
OPEN ACCESS
Abstract
Citation: de Jesus Cortez F, Suzawa M, Irvy S,
Bruning JM, Sablin E, Jacobson MP, et al. (2016)
Disulfide-Trapping Identifies a New, Effective
Chemical Probe for Activating the Nuclear Receptor
Human LRH-1 (NR5A2). PLoS ONE 11(7):
e0159316. doi:10.1371/journal.pone.0159316
Conventional efforts relying on high-throughput physical and virtual screening of large com-
pound libraries have failed to yield high-efficiency chemical probes for many of the 48
human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach
new to nuclear receptors, would provide effective lead compounds targeting human liver
receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand
binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands
are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using
disulfide-trapping, we identified a lead compound that conjugates with remarkably high-effi-
ciency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand bind-
ing domain of hLRH-1. Guided by computational modeling and cellular assays, the lead
compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no
cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing
hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of
the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3
h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1
renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are
potent activators of hLRH-1 and suggest that with further development this lead series may
yield useful chemical probes for manipulating LRH-1 activity in vivo.
Editor: Alessio Lodola, University of Parma, ITALY
Received: April 14, 2016
Accepted: June 30, 2016
Published: July 28, 2016
Copyright: © 2016 de Jesus Cortez et al. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: California Institute for Quantitative
Biosciences (http://qb3.org) PME, UCSF Pancreas
Center (http://cancer.ucsf.edu) PME, American Heart
Association (15GRNT25670067)(heart.org) PME;
National Institutes of Health (K12GM081266,
U01GM094614) (nih.gov) FdJC; National Institutes of
Health (U01GM094614) (nih.gov) RJF; American
Diabetes Association (diabetes.org) (1-15-MI-08)
HAI, UCSF NIH-funded Liver Center (P30DK026743)
(livercenter.ucsf.edu) HAI. The funders had no role in
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New Chemical Probes for Human LRH-1
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Introduction
Liver Receptor Homolog 1 (LRH-1, NR5A2) is among several nuclear receptors (NRs) that still
lack a high affinity, selective chemical probe [1]. Early crystallographic x-ray structures showed
that both the rodent and human LRH-1 ligand binding domains (LBDs) contain a large hydro-
phobic hourglass-shaped ligand binding cavity (800–1200 Å3) that can easily accommodate
ligands [2–4]. Human LRH-1 LBD structures bound to either endogenous or exogenous phos-
pholipid ligands reveal the two lipid tails buried within and occupying the entire length of the
hydrophobic pocket, and the headgroup positioned at the mouth of the pocket [2, 3, 5]. In con-
trast, mouse LRH-1 contains a salt-bridge at the mouth of the pocket that greatly diminishes
the binding of phospholipid ligands [2, 6]. Receptor-ligand interactions can greatly change the
size of the ligand binding pocket, as evidenced by the contracted binding pocket observed
when hLRH-1 LBD is bound to either the shorter-chain phospholipid ligand DLPC [5] or the
synthetic ligand GSK8470 [7], compared to the higher-affinity phosphoinositide ligands PIP2
and PIP3 [8]. Thus for hLRH-1, standard virtual screening methods that survey a static struc-
ture might fail to capture the structural dynamics of the hydrophobic ligand binding pocket.
For several nuclear receptors, co-activator peptide recruitment to the activation function 2
(AF2) in the LBD has been successfully adapted as the primary endpoint in high throughput
screening assays of compound libraries. The first reported synthetic hLRH-1 ligand, GSK8470,
emerged from a high-throughput fluorescence resonance energy transfer (FRET)-based bio-
chemical screening assay using TIF2 (NCOA2) peptide recruitment [9]. Unfortunately,
GSK8470 is both unstable and insoluble making it difficult to achieve reproducible results in
cellular assays [7]. Extensive modification of GSK8470 by Whitby and co-workers ultimately
led to RJW100 [7], which has been used with some success in specific cellular and in vivo set-
tings [10, 11]. In retrospect, newer biophysical data using hSF-1 strongly suggest that peptide
recruitment assays might fail to discriminate between low and high affinity ligands for NR5As.
Indeed, binding affinities of hSF-1 LBD for the coactivator peptide PGC-1α established that
while significant, the absolute difference in peptide affinity in the presence of a low affinity ver-
sus high affinity phospholipid ligand (or no ligand), are quite small at, 8.5 μM and 6 μM,
respectively [12]. This finding reinforces the notion that traditional screening approaches that
rely on coactivator peptide recruitment assays are less effective for NR5As than perhaps for
other NR subfamilies.
Competing Interests: MPJ is a consultant to
Schrodinger LLC, which develops and licenses some
of the software used in this work. This does not alter
the authors' adherence to all the PLOS ONE policies
on sharing data and materials.
Here, we applied a screening strategy that identifies lead compounds based on their ability
to form a disulfide bond (covalent adduct) with a naturally occurring cysteine residue that lines
the ligand binding pocket of the hLRH-1 LBD. To ensure that the formation of covalent
adducts is governed by the intrinsic affinity of the compounds for the ligand binding pocket,
rather than their reactivity with the cysteine (thiol) sidechain, the screen is carried out in the
presence of saturating concentrations of the disulfide reducing agent β-mercaptoethanol
(BME). While this technology has been successfully employed to develop ligands for a variety
of targets, it has heretofore not been applied to nuclear receptors [13, 14]. Following screening
of a library of 1280 cysteine-reactive (disulfide-linked) compounds, and computational dock-
ing to visualize hit compound orientations within the hLRH-1 LBD and guide development of
higher affinity ligands, elaborated non-cysteine reactive compounds were then tested in the
hepatocellular carcinoma HepG2 cellular model system. Our collective results show that a lead
compound identified by disulfide-trapping can be transformed into non-disulfide-linked
(unreactive) biologically active ligands, illustrating the utility of the disulfide-trapping method-
ology for developing ligands targeting nuclear receptors.
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New Chemical Probes for Human LRH-1
Results
Disulfide-Trapping Screen
A library of 1280 disulfide-linked, low molecular weight (~300 Da) compounds (in house
unpublished library) was screened against the hLRH-1 LBD. To specifically target the cysteine
residue lining the ligand binding pocket (Cys346), the two surface-exposed cysteine residues
(Cys311, Cys487) were mutated to serine. Twenty-eight compounds conjugated with high effi-
ciency (> 2.0 standard deviations above the mean) to Cys346 under stringent conditions
(500 μM BME) (S1 Fig). One compound (15.31) conjugated with remarkably high efficiency
(~85%) and was selected for further development.
Computational Modeling
Covalent docking was used to visualize conjugated compounds bound within the hLRH-1
ligand binding domain (PDB:3PLZ).The ligand binding pocket is an hourglass shaped struc-
ture with the bottom portion nearest to the solvent. Modeling of top hit 15.31 conjugated to
Cys346 indicated this compound occupies the top of the pocket, partially overlapping the bind-
ing site for GSK8470, and suggested a clear avenue for improving binding (Fig 1). The two iso-
propyl groups inadequately fill the top of the pocket, suggesting that larger substituents would
improve binding. The propyl amide bends to form the disulfide link with Cys346; absent the
disulfide the alkyl chain might be lengthened to better fill the middle of pocket. The hydroxyl
group on 15.31 is located within 2.8 Å of a backbone carbonyl (Met345), and is the only polar
contact observed in the model.
Ligand Development
Based on computational models, we prepared ten non-disulfide-linked analogs of 15.31
designed to explore the ligand-receptor pharmacophore at three positions (R1, R2, R3 in Fig 2).
Fig 1. Computational model shows orientation of and space around top hit 15.31 in the hLRH-1 pocket. A.
Computational model of top hit 15.31 covalently bound to LRH-1 through a disulfide bond to Cys346. Van der Walls
surface (blue) represents all protein within 4.5 Å of the ligand. B. Structure of 15.31 and cartoon derived from the model
illustrating the binding site for 15.31 is framed by helices 3, 5 and 11 and likely includes a polar interaction with the
backbone carbonyl of Met345
.
doi:10.1371/journal.pone.0159316.g001
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New Chemical Probes for Human LRH-1
Fig 2. Activity of PME9 Exceeds that of RJW100 in HepG2 Cells. Relative expression of CYP24A1 transcripts in HepG2 cells following 16 h
treatment with either vehicle (DMSO) or compounds (10 μM) as listed on the X-axis. For reference, activity with the existing NR5A agonist RJW100 is
shown (grey bar). For these experiments levels of hLRH-1 were low, as doxycycline (-Dox) was not added to HepG2-hLRH-1 cells (Refer to S2 Fig).
Data are representative of at least three independent experiments with error bars representing SEM, P values = **** < 0.0001.
doi:10.1371/journal.pone.0159316.g002
Specifically, the isopropyl groups were replaced with either smaller (H) or larger (tert-butyl)
groups and the propyl amide group was replaced with alkyl chains of longer lengths. Analogs
1–10 were prepared from commercially available scaffolds, by activating the carboxylic acid
and subsequently coupling to alkyl amines with the indicated chain length as described in
Materials and Methods.
Cellular Assays
Analogs 1–10 were tested in a hepatocellular carcinoma HepG2 cell line stably expressing
hLRH-1 that can be induced to express low or high levels without or with doxycycline (-Dox or
+Dox), respectively. Levels of the direct hLRH-1 target gene CYP24A1 [15] were measured by
RT-qPCR as described in Materials and Methods. A comparison of all analogs showed that
compounds 8 (PME8) and 9 (PME9) (10 μM) consistently increased endogenous CYP24A1
transcript levels 3-4-fold following a 16 h treatment time (Fig 2). The activity of PME9
exceeded that of RJW100. Importantly, when assayed in HepG2-hLRH-1 cells expressing low
levels of hLRH-1, PME8 did not change hLRH-1 transcripts (Fig 3A) or protein levels (S2 Fig).
Compound PME8 does increase CYP24A1 levels in a time-dependent (Fig 3A), and dose-
dependent manner with maximal activity observed at 16 h of treatment (Fig 3B).
To determine whether the activity of the two most potent compounds PME8 and PME9 are
specific for hLRH-1, their activities were retested after siRNA-mediated knockdown of hRH-1
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New Chemical Probes for Human LRH-1
Fig 3. PME8 induces transcription of an hLRH-1 target gene in a both time- and dose-dependent manner with activity increasing at higher hLRH-1
levels. A. Relative expression levels of the hLRH-1 target gene CYP24A1 with increasing treatment times of PME8 (10 μM) compared to the DMSO control
(black bar) ranging from 3–24 h. Corresponding expression levels of hLRH-1 transcripts in each time condition are shown in right panel without (-Dox) or with
(+Dox) induction of exogenous hLRH-1. B. Levels of CYP24A1 with DMSO or with increasing concentrations of RJW100 and PME8 treatment for 16 h
without (-Dox) or with (+Dox) induction of exogenous hLRH-1. Data are representative of at least three independent experiments with error bars representing
SEM, P values = **** < 0.0001.
doi:10.1371/journal.pone.0159316.g003
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New Chemical Probes for Human LRH-1
Fig 4. Activity of PME8 and PME9 Depends on hLRH-1. Expression of CYP24A1 transcripts was
determined in the presence of RJW100 (grey bar) or the two new compounds PME8 and PME9 (10 μM)
without (siCont) or with knockdown (siLRH-1) of hLRH-1 (left panel). SiRNA-mediated knockdown of hLRH-1
in HepG2-hLRH-1 cells expressing low levels of hLRH-1 (-Dox) was carried out as described in Materials and
Methods. Relative levels of hLRH-1 transcripts after siControl or sihLRH-1 are shown (right panel).
doi:10.1371/journal.pone.0159316.g004
in HepG2 cells. For PME8 and PME9, as well as the control RJW100 compound, a clear depen-
dence on hLRH-1 was observed as evidenced by the precipitous drop in CYP24A1 expression
following treatment with sihLRH-1, but not with siControl RNA (Fig 4). These ligands likely
bind within the ligand binding pocket as the activity of both PME8 and PME9, as well as
RJW100, decreased when tested in HepG2 cells stably expressing equivalent levels of F342W/
I416W hLRH-1, a “pocket” mutant harboring two large bulky tryptophan side chains designed
to fill the ligand binding cavity of hLRH-1 [2] (S3 Fig). Interestingly, although greatly reduced,
there are still residual levels of CYP24A1 after treatment with PME8 and PME9 in cells express-
ing this variant of hLRH-1. Consistent with these data, we observed direct binding of PME8
and PME9 to the hLRH-1 LBD using surface plasmon resonance assays (S4 Fig).
Finally, to ask how PME8 and PME9 compare to other published compounds, we directly
compared the activity of all compounds with respect to the ability to activate or repress
CYP24A1 expression in HepG2-hLRH-1 cells. For this experiment, assays were carried out
under high levels of hLRH-1 (+Dox) to maximize our ability to see any repressive effects of
compounds and the ligand concentrations used were based on published effective doses.
Results from this experiment indicate that only one of the two published agonists, RJW100,
showed the expected activity; DLPC failed to increase CYP24A1 expression (Fig 5). Of the two
published antagonist/repressors, only SR1848 decreased CYP24A1 levels. However, SR1848
also exhibited substantial repression of the housekeeping gene, TBP. Similar results were also
observed for another identified hLRH-1 target, SERPINE or PAI1 [15, 16] (data not shown).
Taken together, our findings show that PME8 and PME9 are potential new leads for creating
high affinity, efficacious synthetic ligands for hLRH-1.
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New Chemical Probes for Human LRH-1
Fig 5. Effects of other published compounds targeting hLRH-1 compared to PME8. All published
agonists and antagonists were tested in HepG2-hLRH-1 cells expressing high levels (+Dox) of hLRH-1.
Activating compounds were added for 16 h relative to the DMSO control (black bars) at the following
concentrations: RJW100 (10 μM) [7], DLPC (20 μM) [5], PME8 (10 μM) (bolded, this study) as well as the two
putative antagonist/repressors, SR1848 (10 μM) [20] and Cpd3 (10 μM) [21]. Relative levels of the hLRH-1
target, CYP24A1 (left set of bars) and the housekeeping gene, TBP (right set of bars) are shown with all
values normalized to GAPDH. Error bars represents SEM and P values = **** < 0.0001.
doi:10.1371/journal.pone.0159316.g005
Discussion
A screen of 1280 disulfide-linked compounds against hLRH-1 identified 28 hits that were
trapped within the ligand binding pocket through a disulfide exchange reaction with Cys346
.
Computational modeling of these hits conjugated to Cys346 suggested they bind within a simi-
lar region of the pocket, at the top of the hourglass with the alkyl amide pointing toward the
middle of the pocket. An advantage of covalent docking is that modeled ligand-receptor inter-
actions (poses) are expected to be particularly accurate as the possible ligand orientations
(translations and rotations) are strongly constrained by the covalent link to the protein [17,
18].
The top hit 15.31, a substituted benzamide, was selected for further study owing to its
remarkable conjugation efficiency (85%, S1 Fig) under stringent conditions (500 μM BME)
and the ease with which a small, yet informative, structure activity relationship study could be
carried out. The computationally modeled pose of 15.31 proved to be highly predictive (Fig 1).
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New Chemical Probes for Human LRH-1
Increasing the size of both the R1 and R2 aryl ring substituents, from isopropyl to tert-butyl
groups, to better fill the pocket provided compounds that were active in cells (Fig 2). The most
active compound, 9 (PME9), was even more effective than RJW100, the highest affinity agonist
described to date (Fig 2) [7]. The alkyl chain length (R3) also proved important, with N-butyl-
benzamides being more active in cells than the corresponding N-propyl- or N-pentylbenza-
mide compounds. This pattern, which is observed among compounds 1–3 and 8–10 (PME8-
10), may reflect an inability of amide-linked alkyl groups to traverse the pocket constriction.
As phospholipid tails apparently bind the length of the pocket, replacing the amide with an
alkyl or ether linkage may improve the activity of compounds with longer R3 substituents. The
hydroxyl group also contributes to efficacy, as evidenced by the difference in activity between
compounds 6 and 7 (Fig 2), the latter forming a hydrogen bond with the backbone carbonyl of
Met345 according to the computational model.
Together, these data highlight PME9 as a strong starting point for further development of
hLRH-1 ligands. Specific features of the ligand binding site yet to be exploited include polar
interactions with arginine and histidine residues positioned 4.8 and 3.7 Å of the R1 and R2 sub-
stituents. Moreover, the other 27 hits from the screen point to additional modifications of the
benzamide scaffold expected to improve ligand efficacy, such as merging structures.
Collective data obtained from numerous in vitro and in vivo studies suggest that LRH-1 is a
potential therapeutic target for several diseases including pancreatic cancer, breast cancer,
colon cancer, inflammatory bowel disease and fatty liver disease. The dearth of effective syn-
thetic probes for LRH-1 has hindered efforts to validate this nuclear receptor as a therapeutic
target. Our success in developing new ligands for LRH-1 that effectively drive target gene
expression is an important step and highlights the value of disulfide-trapping as a strategy for
discovering lead compounds targeting nuclear receptors.
Materials and Methods
Disulfide-Trapping Screen
The disulfide-trapping screen was performed according to standard procedures. Purified LRH-
1 (Cys311Ser, Cys487Ser) was diluted to 4 μM in a buffer containing 25 mM HEPES, 150 mM
NaCl and 500 μM BME. The protein was aliquoted into a 96 well-plate (25 μL per well) and
individually incubated at ambient temperature for 1 h with disulfide-linked monophores (1280
total compounds) at 150 μM. After the equilibration period, reaction mixtures were analyzed
by high-throughput mass spectrometry using Waters Acquity UPLC/ESI-TQD with a 2.1 x
50mm Acquity UPLC BEH300 C4 column. The extent of labeling was determined by compar-
ing the molecular masses of the covalently adducted protein to the molecular mass of the free
protein. Hits were defined as those compounds that conjugated with efficiencies greater than
two standard deviations (SD) above the mean.
Computational Modeling
A library of 1280 disulfide-linked compounds was covalently docked to the hLRH-1 LBD.
Library compounds were initially represented by their SMILES strings and then converted to
3D structures using Maestro’s LigPrep feature. In addition, LigPrep was used to identify and
generate tautomers of each ligand, yielding a total of 1407 compounds for covalent docking.
The library of compounds was covalently docked to Cys346 in hLRH-1 using the PDB coor-
dinate file 3PLZ, which shows the thiol sidechain oriented into the ligand binding pocket and
accessible to docked ligands. PDB 3PLZ is missing the loop near Cys346, which connects helix 2
and helix 3. To limit potential docking artifacts, the missing H2-H3 loop residues (330–337)
were manually added to the hLRH-1 sequence and the loop orientation was optimized with
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New Chemical Probes for Human LRH-1
Prime. The predicted positions of residues within the H2-H3 loop closely aligned with other
previously reported hLRH-1 crystal structures. In addition, hydrogen atoms were added and
the correct charges specified using the protein preparation wizard in Maestro. Before covalently
docking the library, leaving groups for the ligands and receptor were defined. Prime was used
to covalently dock the 1280 disulfide-linked compounds to Cys346 of hLRH1. The covalent
docking feature first docks each ligand to the receptor to generate a non-covalent pose. The
covalent bond between the compound and the Cys346 side chain is then formed and the ligand
pose is refined. The free energy of each ligand and the leaving groups were determined using
Prime. To determine the overall binding energy the following equation was used:
E ¼ Ecomplex þ ER;LG þ EL;LG ꢀ Ereceptor ꢀ Eligand
where ER,LG and EL,LG are the free energies of the receptor and ligand leaving group,
respectively.
Ligand Synthesis
All reagents were commercially available and used without further purification unless other-
wise indicated. 1H NMR spectra were recorded using a 400 MHz Varian Inova NMR spectrom-
eter. Chemical shifts (δ) are reported relative to the internal standard tetramethylsilane (TMS).
Mass spectra were obtained at the University of California at Berkeley Mass Spectrometry
Facility using electron spray ionization and an LTQ-FT instrument. Thin-layer chromatogra-
phy was performed using silica gel 60 F254-coated plates (EM Science, Gibbstown, NJ). Flash
column chromatography was carried out using silica gel 60 (EMD Chemical, Inc., Cincinnati,
OH). All reactions were carried out under an inert atmosphere of argon unless otherwise
indicated.
Compounds 1–10 were synthesized from commercially available carboxylic acids according
to the following general procedures, first activating the carboxylic acid as the para-nitrobenzyl
ester and then coupling to the alkyl amine to form the desired amide.
General procedure for preparing the activated, para-nitrobenzyl esters. To a flame-
dried round bottom flask was added commercially available carboxylic acid (1 eq) as a solution
in anhydrous THF (0.2 M), followed by para-nitrobenzyl chloroformate (1.1 eq) and NEt3 (1.2
eq) at ambient temperature. After stirring for 1 h, the mixture was poured into a separatory
funnel and extracted with EtOAc against NaHCO3. The organic layer was dried over MgSO4
and then concentrated to dryness to give the crude activated ester that was used without further
purification.
General procedure for preparing the amides. To a flame-dried round bottom flask was
added the para-nitrobenzyl ester (1 eq) as a solution in anhydrous THF (0.2 M), followed by
the appropriate amine (1.1 eq) and NEt3 (1.2 eq) at ambient temperature. After stirring over-
night, the solvent was removed via rotary evaporation and the amide was purified to homoge-
neity via flash silica gel chromatography and concentrated to dryness to give the desired final
product in an overall yield of 49%–65% over two steps.
Analytical Data for Compounds 1–10. 2-hydroxy-3,5-diisopropyl-N-propylbenzamide
(1): The title compound was synthesized in two steps from 2-hydroxy-3,5-diisopropylbenzoic
acid with an overall yield of 50%; 1H NMR (400 MHz, CDCl3) δ 7.18 (s, 1H), 6.96 (s, 1H), 6.29
(s, 1H), 3.44–3.38 (m, 2H), 3.37–3.31 (m, 1H), 2.83 (hept, J = 7.7 Hz, 1H), 1.74–1.58 (m, 2H),
1.24–1.20 (m, 12H), 0.98 (t, J = 7.4 Hz, 3H); 13C NMR (400 Mhz, CDCl3) δ 170.67, 157.42,
138.20, 137.69, 128.89, 119.85, 113.22, 41.40, 33.68, 26.68, 24.22, 22.86, 22.43, 11.42. HRMS
(ESI+) calcd for [M+H]+ = m/z 264.1958, found 264.1955.
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New Chemical Probes for Human LRH-1
N-butyl-2-hydroxy-3,5-diisopropylbenzamide (2): The title compound was synthesized
from 2-hydroxy-3,5-diisopropylbenzoic acid in two steps with an overall yield of 50%; 1H
NMR (400 MHz, CDCl3) δ 7.18 (s, 1H), 6.97 (s, 1H), 6.32 (s, 1H), 3.49–3.38 (m, 2H), 3.34
(hept, J = 7.1 Hz, 1H), 2.82 (hept, J = 7.1 Hz, 1H), 1.67–1.52 (m, 2H), 1.46–1.34 (m, 2H), 1.23–
1.22 (m, 12H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (400 Mhz, CDCl3) δ 170.86, 157.45, 138.42,
137.85, 129.06, 119.85, 113.43, 39.68, 33.89, 31.85, 26.88, 24.43, 22.65, 20.37, 13.98. HRMS
(ESI+) calcd for [M+H]+ = m/z 278.2115, found 278.2111.
2-hydroxy-3,5-diisopropyl-N-pentylbenzamide (3): The title compound was synthesized
from 2-hydroxy-3,5-diisopropylbenzoic acid in two steps with an overall yield of 49%; 1H
NMR (400 MHz, CDCl3) δ 12.50 (s, 1H), 7.19 (s, 1H), 6.97 (s, 1H), 6.32 (s, 1H), 3.45–3.39 (m,
2H), 3.35 (q, J = 6.9 Hz, 1H), 2.83 (p, J = 6.9 Hz, 1H), 1.68–1.55 (m, 2H), 1.39–1.32 (m, 4H),
1.24–1.22 (m 12 H), 0.97–0.87 (m, 3H). 13C NMR (400 Mhz, CDCl3) δ 170.61, 157.23, 138.18,
137.62, 128.83, 119.62, 113.19, 39.72, 33.66, 29.26, 29.09, 26.64, 24.19, 22.41, 22.35, 13.95.
HRMS (ESI+) calcd for [M+H]+ = m/z 292.2271, found 292.2267.
2-hydroxy-3-isopropyl-N-propylbenzamide (4): The title compound was synthesized from
2-hydroxy-3-isopropylbenzoic acid in two steps with an overall yield of 51%; 1H NMR (400
MHz, CDCl3) δ 7.31 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 6.90–6.72 (m, 1H), 6.3 (s, 1H),
3.77–3.20 (m, 3H), 1.63 (h, J = 7.1 Hz, 2H), 1.22 (d, J = 6.9 Hz, 6H), 0.97 (t, J = 7.4 Hz, 3H). 13C
NMR (400 Mhz, CDCl3) δ 170.79, 159.35, 138.12, 130.61, 122.70, 118.29, 113.84, 41.62, 26.66,
22.98, 22.60, 11.60. HRMS (ESI+) calcd for [M+H]+ = m/z 222.1489, found 222.1486.
2-hydroxy-5-isopropyl-N-propylbenzamide (5): The title compound was synthesized in
two steps from 2-hydroxy-5-isopropylbenzoic acid with an overall yield of 50%; 1H NMR (400
MHz, CDCl3) δ 12.19 (s, 1H), 7.26 (d, J = 8.6 Hz, 1H), 7.14 (s, 1H), 6.91 (d, J = 8.5 Hz, 1H),
6.40 (s, 1H), 3.46–3.33 (m, 2H), 2.96–2.70 (m, 1H), 1.85–1.56 (m, 2H), 1.22 (d, J = 7.0 Hz, 6H),
0.99 (t, J = 7.4 Hz, 3H). 13C NMR (400 Mhz, CDCl3) δ 170.33, 159.80, 139.17, 132.54, 122.85,
118.69, 114.12, 41.60, 33.66, 24.34, 23.04, 11.65. HRMS (ESI+) calcd for [M+H]+ = m/z
222.1489, found 222.1486.
3-(tert-butyl)-2-hydroxy-N-propylbenzamide (6): The title compound was synthesized in
two steps from 3-(tert-butyl)-2-hydroxybenzoic acid with an overall yield of 51%; 1H NMR
(400 MHz, CDCl3) δ 7.38 (d, J = 7.8 Hz, 1H), 7.20 (d, J = 8.1 Hz, 1H), 6.75 (t, J = 7.8 Hz, 1H),
3.48–3.32 (m, 2H), 1.65 (m, 2H), 1.42 (s, 9H), 0.99 (t, J = 7.4 Hz, 3H). 13C NMR (400 Mhz,
CDCl3) δ 170.78, 159.36, 138.52, 130.86, 122.40, 118.33, 113.85, 41.64, 30.25, 27.32, 22.62,
11.58. HRMS (ESI-) calcd for [M-H]+ = m/z 234.1500, found 234.1500.
5-(tert-butyl)-2-hydroxy-N-propylbenzamide (7): The title compound was synthesized in
two steps from 5-(tert-butyl)-2-hydroxybenzoic acid with an overall yield of 55%; 1H NMR
(400 MHz, CDCl3) δ 12.20 (s, 1H), 7.42 (dd, J = 8.7, 2.3 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 6.90
(d, J = 8.7 Hz, 1H), 6.47 (s, 1H), 3.49–3.34 (m, 2H), 1.72–1.58 (m, 2H), 1.28 (s, 9H), 0.97 (t,
J = 7.4 Hz, 3H). 13C NMR (400 Mhz, CDCl3) δ 170.48, 159.41, 141.52, 131.86, 121.40, 118.34,
113.84, 41.62, 34.32, 31.61, 23.06, 11.63. HRMS (ESI+) calcd for [M+H]+ = m/z 236.1645,
found 236.1642.
3,5-di-tert-butyl-2-hydroxy-N-propylbenzamide (8): The title compound was synthesized
in two steps from 3,5-di-tert-butyl-2-hydroxybenzoic acid with an overall yield of 56%; 1H
NMR (400 MHz, CDCl3) δ 7.45 (d, J = 1.9 Hz, 1H), 7.13 (d, J = 1.8 Hz, 1H), 6.34 (s, 1H), 3.49–
3.33 (m, 2H), 1.65 (h, J = 7.6 Hz, 2H), 1.42 (s, 9H), 1.30 (s, 9H), 0.98 (t, J = 7.4 Hz, 3H). 13C
NMR (400 Mhz, CDCl3) δ 171.46, 158.91, 139.97, 138.38, 128.87, 119.07, 113.57, 41.66, 35.41,
34.48, 31.70, 29.59, 23.08, 11.63. HRMS (ESI+) calcd for [M+H]+ = m/z 292.2271, found
292.2267.
3,5-di-tert-butyl-N-butyl-2-hydroxybenzamide (9): The title compound was synthesized
in two steps from 3,5-di-tert-butyl-2-hydroxybenzoic acid with an overall yield of 50%; 1H
PLOS ONE | DOI:10.1371/journal.pone.0159316 July 28, 2016
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New Chemical Probes for Human LRH-1
NMR (400 MHz, CDCl3) δ 12.75 (s, 1H), 7.45 (s, 1H), 7.14 (s, 1H), 6.35 (s, 1H), 3.43 (m, 2H),
1.72–1.55 (m, 2H), 1.41–1.36 (m, 2H), 1.42 (s, 9H), 1.30 (s, 8H), 0.95 (t, J = 7.3 Hz, 3H). 13
C
NMR (400 Mhz, CDCl3) δ 171.19, 158.67, 139.73, 138.12, 128.61, 118.85, 113.33, 39.51, 35.17,
34.24, 31.64, 31.46, 29.35, 20.14, 13.76. HRMS (ESI+) calcd for [M+H]+ = m/z 306.2428, found
306.2423.
3,5-di-tert-butyl-2-hydroxy-N-pentylbenzamide (10): The title compound was synthesized
from 3,5-di-tert-butyl-2-hydroxybenzoic acid in two steps with an overall yield of 65%; 1H NMR
(400 MHz, CDCl3) δ 12.76 (s, 1H), 7.45 (s, 1H), 7.14 (s, 1H), 6.36 (s, 1H), 3.90–2.71 (m, 2H),
1.77–1.50 (m, 32), 1.42 (s, 9H), 1.39–1.31 (m, 4H), 1.30 (s, 9H), 0.91 (t, J = 6.5 Hz, 3H). 13C NMR
(400 Mhz, CDCl3) δ 171.18, 158.67, 139.73, 138.12, 128.61, 118.87, 113.34, 39.79, 35.17, 34.24,
31.46, 29.36, 29.27, 29.10, 22.36, 13.96. HRMS (ESI+) calcd for [M+H]+ = m/z 320.2584, found
320.2580.
Cell lines and Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) Assay.
HepG2 3G stable cells expressing 3X-Flag-tagged WT and pocket mutant (F342W/I416W)
hLRH-1 were generated as described [15] after insertion into the doxycycline (Dox)-inducible
pTRE 3G vector (Clontech) to generate HepG2-hLRH-1 stable cell lines. The parental TET-On
3G HepG2 cell line was a generous gift from Dr. Stephen Hand. Induction of low or high levels
of hLRH-1 was achieved by addition of DMSO or 500 ng/ml Dox, respectively.
For RT-qPCR assay, cells (5 x 105) were plated on 24-well plates in 0.5 mL of media. The fol-
lowing day, fresh media was applied with compound or DMSO control. After an overnight
treatment or indicated period of time, cells were washed with ice-cold 1 X PBS and total RNA
was isolated by Trizol (Life Technologies). DNase-treated total RNA was used to generate
cDNA using High-Capacity cDNA Reverse Transcription kits (Life Technologies). RT-qPCR
was performed with SYBR (Biotool) and data analyzed essentially as described [19]. Sequences
for all primer pairs used for qPCR reactions are listed in S1 Table. For siLRH-1 knockdowns,
siLRH-1 (GS2494) and non-silencing control (SI03650318) siRNA were purchased from Qia-
gen. SiRNAs were used at a final concentration of 5 nM and reverse-transfected into
HepG2-hLRH-1 stable cells by RNAiMax (Life Technologies) for 72 h. Knock-down was con-
firmed after assaying hLRH-1 transcripts by RT-qPCR.
Surface Plasmon Resonance Assays. Data were collected on a BiaCore T100 instrument.
The biotin CAPture kit (GE Healthcare Life Science) was used as directed for chip preparation
and immobilization of protein. In brief, the CAP chip was first conditioned with 3 x 1 min injec-
tions of the provided regeneration solution (6 M Guanidine HCL, 0.25 M NaOH). CAP reagent
was then captured on flow cells 1 and 2 to a final density of ~ 2500 response units using capture
buffer (25 mM HEPES, pH 7.4, 150 mM NaCl). Avi-tagged LRH-1 was then directly injected
exclusively on to flow cell 2 achieving a final density of ~ 1000 response units (capture buffer).
Flow cell 1 served as the reference flow cell and flow cell 2 served as the active flow cell. A titration
series for each fragment was generated as a 0.6x dilution series starting from 300 μM. All samples
were made to match the running buffer used in the experiment as closely as possible (capture
buffer + 3% DMSO, + 0.05% Tween-20, 250 μM TCEP). Data was collected at 25°C with a flow
rate of 30 μL/min. Data processing included referencing to both the reference flow cell as well as
a buffer injection. Equilibrium binding constants were determined by nonlinear regression analy-
sis utilizing a simple 1:1 binding model. Steady state values used to generate dose-responses
curve correspond to the response units achieved 20 seconds after injection.
Supporting Information
S1 Fig. Disulfide-trapping screen and structure of top hit 15.31. (A) Cartoon depicting the
screening strategy. Each molecule in the library consists of two elements: a monophore (circle,
PLOS ONE | DOI:10.1371/journal.pone.0159316 July 28, 2016
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New Chemical Probes for Human LRH-1
square, oval), which is the unique chemical entity, and a common linker region (-S-S-R) that
can undergo disulfide exchange with thiols, including cysteine side chains. Library compounds
are individually incubated with the target protein (hLRH-1 LBD) under high reductant condi-
tions (500 μM BME), such that disulfide exchange is rapid. Compounds with high intrinsic
affinity for a binding site near a cysteine residue (Cys346 in hLRH-1) have superior residence
time and thus encourage the lasting formation of a disulfide bond between the protein thiol
and compound thiol. Compounds with weak inherent affinity are readily reduced off. (B)
Graph showing the conjugation efficiency for each of 1280 compounds in the library to hLRH-
1. Notably, the disulfide-trapping screen was carried out against hLRH-1 bound to a phospho-
lipid (phosphatidylethanolamine) co-expressed with the receptor in bacteria. As was observed
by Whitby and coworkers (Whitby et al., 2006), small non-polar molecules are clearly able to
displace bound bacterial phospholipid ligands from the pocket.
(EPS)
S2 Fig. Expression of hLRH-1 is unaffected by PME8. Expression of hLRH-1 detected in
Western blots using anti-Flag antibody is shown in HepG2 cells expressing low (-Dox) or
higher levels (+Dox) of hLRH-1. All three sumoylated species of hLRH-1 (SU-LRH-1) are
observed after Induction of high levels of hLRH-1, as indicated by arrows.
(EPS)
S3 Fig. Activity of RJW100, PME8 and PME9 depends on an intact hLRH-1 ligand binding
pocket. Expression of CYP24A1 transcripts in the presence of DMSO (black bar), RJW100
(grey bars), PME8 and PME9 (white bars) at 10 μM for 16 h treatment in HepG2-hLRH-1 cells
expressing low levels of wild type hLRH-1 or the hLRH-1 pocket mutant F342W/I416W vari-
ant as described in Materials and Methods.
(EPS)
S4 Fig. PME8 and PME9 bind directly to the hLRH-1 LBD. (Left) Raw surface plasmon reso-
nance sensorgrams (time vs response) for PME8 and PME9 show direct binding to biotinylated
apo hLRH-1 LBD. (Right) Dose-response curves (log ligand concentration vs response) for
PME8 and PME9 with apparent dissociation constants determined using steady state values at
20 s post injection (red dots on sensorgrams) and a 1:1 binding model.
(EPS)
S1 Table. RT-qPCR Primer Sequences.
(EPS)
Acknowledgments
PME thanks KM Shokat for helpful discussions and use of LCMS equipment, the UCSF SMDC
for access to the library of disulfide-linked compounds, and PR Griffin for generously provid-
ing a sample of SR1848.
Author Contributions
Conceived and designed the experiments: PME HAI. Performed the experiments: FdJC MS SI
JMB. Analyzed the data: PME HAI MS FdJC JMB SI. Contributed reagents/materials/analysis
tools: ES RJF MPJ. Wrote the paper: PME HAI.
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