Discovery of the first irreversible small molecule inhibitors of the interaction between the vitamin D receptor and coactivators

Article abstract of DOI:10.1021/jm300460c

The vitamin D receptor (VDR) is a nuclear hormone receptor that regulates cell proliferation, cell differentiation, and calcium homeostasis. The receptor is activated by vitamin D analogues that induce the disruption of VDR-corepressor binding and promote

Full text of DOI:10.1021/jm300460c

Article
pubs.acs.org/jmc
Discovery of the First Irreversible Small Molecule Inhibitors of the
Interaction between the Vitamin D Receptor and Coactivators
Premchendar Nandhikonda,†,∥ Wen Z. Lynt,†,∥ Megan M. McCallum,† Tahniyath Ara,†
Athena M. Baranowski,† Nina Y. Yuan,† Dana Pearson,† Daniel D. Bikle,‡ R. Kiplin Guy,§
and Leggy A. Arnold*,†
†Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, United States
‡Endocrine Research Unit, Department of Medicine, Veterans Affairs Medical Center, San Francisco, California 94121, United States
§Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, United
States
S
* Supporting Information
ABSTRACT: The vitamin D receptor (VDR) is a nuclear hormone receptor that
regulates cell proliferation, cell differentiation, and calcium homeostasis. The receptor is
activated by vitamin D analogues that induce the disruption of VDR−corepressor binding
and promote VDR−coactivator interactions. The interactions between VDR and
coregulators are essential for VDR-mediated transcription. Small molecule inhibition of
VDR−coregulator binding represents an alternative method to the traditional ligand-
based approach in order to modulate the expression of VDR target genes. A high
throughput fluorescence polarization screen that quantifies the inhibition of binding
between VDR and a fluorescently labeled steroid receptor coactivator 2 peptide was applied to discover the new small molecule
VDR−coactivator inhibitors, 3-indolylmethanamines. Structure−activity relationship studies with 3-indolylmethanamine
analogues were used to determine their mode of VDR-binding and to produce the first VDR-selective and irreversible VDR−
coactivator inhibitors with the ability to regulate the transcription of the human VDR target gene TRPV6.
and its coactivators.8 All of these antagonists are based on the
INTRODUCTION
■
secosteroid scaffold of 1,25-(OH)2D3. A different approach to
modulate gene regulation that is mediated by VDR represents
the disruption of VDR−coregulator binding in the presence of
1,25-(OH)2D3. Small molecule NR−coactivator inhibitors have
been discovered for the estrogen receptor, thyroid receptor,
androgen receptor, and pregnane X receptor using rational
design and high throughput screening (HTS).9 Mita et al.
introduced the first reversible small molecule VDR−coactivator
inhibitors.10 These compounds inhibited both VDR-mediated
and estrogen receptor β-mediated transcription. Thus, highly
potent and selective VDR−coactivator inhibitors are still
missing. It is expected that these compounds can be applied
as molecular probes to identify the biological functions of
VDR−coactivator interactions. Herein, we describe the first
HTS campaign that identified small molecule VDR−coactivator
inhibitors and their evaluation using carefully selected
secondary assays. One class of inhibitors were investigated in
regard to their mode of VDR binding, selectivity, and SARs that
resulted in the identification of the first irreversible and highly
selective VDR−coactivator inhibitor with the ability to
modulate VDR-mediated transcription.
The vitamin D receptor (VDR) is a ligand-activated tran-
scription factor that belongs to the nuclear receptor (NR)
superfamily. VDR binds to its endogenous ligand, 1,25-
dihydroxyvitamin D3 (1,25-(OH)2D3), with high affinity,1
mediating the modulation of genes responsible for cell
differentiation, proliferation, and calcium homeostasis.2 On
the basis of its biological function, the VDR has been identified
as an important pharmaceutical target for the treatment of
metabolic disorders, skin diseases, cancer, autoimmune
diseases, and cardiovascular diseases.3 The receptor contains
several functional domains, including a DNA binding domain
(VDR-DBD) and a ligand-binding domain (VDR-LBD), which
mediates ligand-dependent gene regulation.4 VDR binds DNA
as a heterodimer with the retinoid X receptor (RXR).5 In the
unliganded state, VDR is associated with corepressor proteins,
which repress transcription of VDR target genes.6 In the
presence of 1,25-(OH)2D3, the VDR-LBD undergoes a
conformational change. This conformational change prevents
corepressor binding and permits interactions with coactivator
proteins, resulting in the formation of a multiprotein complex
that activates VDR-mediated transcription.7
VDR ligand agonists have been developed to treat metabolic
bone diseases and proliferative skin disorders.3 In contrast to
the large number of reported VDR agonist, only a limited
number of VDR ligand antagonists have been described with
the ability to allosterically inhibit the interactions between VDR
Received: January 27, 2012
Published: May 7, 2012
© 2012 American Chemical Society
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Table 1. Summary of Biophysical and Biochemical Properties of Validated VDR−SRC2-3 Inhibitors
inhibition of
transcription (%)
e
g
toxicity (%)
a
b
c
d
compd % purity
solubility (μM)
permeability, log(Pe) (cm/s) VDR−SRC2-3 inhibition, IC50 (μM)
62.5 μM 20.8 μM 62.5 μM 20.8 μM
f
f
f
f
f
f
f
f
f
1
93.0
89.8
89.2
94.7
88.6
95.2
96.1
94.5
99.6
42.8
58.0
65.6
64.9
84.1
78.5
42.4
81.6
79.1
58.1
41.6
57.6
0.1
−7.86
−6.55
−8.22
−7.93
−7.70
−6.60
−6.67
−6.78
−6.36
−6.25
−6.10
−6.12
−6.11
−6.01
−6.05
−6.12
−6.01
−5.99
−8.71
−7.84
−7.83
3.3
6.0
5.5
5.7
3.1
4.7
3.6
7.6
5.5
2.4
2
273.3
0.7
4.0
3
5.9
f
4
0.2
8.6
11.5
f
5
218.7
21.9
13.8
7.1
6.8
f
7
1.6
f
f
f
8
49.9
8.1
5.3
12.5
16.7
f
9
17.6
6.9
6.2
f
f
10
11
12
13
14
15
16
17
18
19
20
21
22
34.2
22.3
9.6
2.6
7.7
342.0
422.6
403.3
132.5
84.6
100
100
100
100
100
100
90
10
0
11
12
13
14
15
16
18.0
18
19
20
21
42.8
58.0
65.6
64.9
84.1
78.5
27.7
30.3
14.1
17.3
28.9
15.4
25.1
22.2
21.6
37.7
10.5
10
90
10
468.7
201.4
277.7
427.8
466.8
453.9
462.0
f
f
9.1
100
80
0
81.6
79.1
58.1
41.6
0
100
100
10.6
60
30
f
f
13.5 (partial
50%)
23
24
25
26
27
28
29
99.2
84.1
87.1
67.6
93.2
98.9
34.4
576.0
42.1
−8.28
−6.29
−6.52
−7.48
−7.28
−6.78
−5.95
20.5
28.0
14.3
13.7
2.0
50
80
100
0
15
20
60
0
23
24
25
26
27
28
29
99.2
84.1
87.1
67.6
93.2
98.9
34.4
15.3
493.0
338.8
487.0
377.4
30
100
65
0
10.5
25.1
10
0
a
Purities were determined by high pressure liquid chromatography using a photodiode array, and identity was confirmed by mass spectrometry.
Solubilities were determined in phosphate buffered saline at pH 7.4. Permeabilities were measured using the parallel artificial membrane
b
c
permeation assay (PAMPA) at neutral pH (pH 7.4). The following permeability standards (log Pe) were used: ranitidine (−8.02 0.074 cm/s) low
permeability, carbamazepine (−6.81 0.0011 cm/s) medium permeability, and verapamil (−5.93 0.015 cm/s) high permeability. The solubility
d
and permeability assay conditions reflect conditions required for activity in cell-based assays. A fluorescence polarization competition assay was
carried out using VDR-LBD (1 μM), Alexa Fluor labeled peptide SRC2-3 (7 nM), VDR-agonist LG190178 (5 μM), and serially diluted small
molecules. IC50 values were obtained by fitting data to the following equation: Y = Bottom + (Top − Bottom)/(1 + 10(logIC −X)(HillSlope)) using three
50
e
independent experiments in quadruplet. VDR-mediated inhibition of transcription was carried out using a commercially available GeneBLAzer
f
(Invitrogen) assay. Data were normalized by signals observed for inactive and activated VDR ( 1,25(OH)2D3). IC50/LD50 values (μM) are given
instead of percentages for highly active compounds using the following nonlinear regression equation: Y = Bottom + (Top − Bottom)/(1 +
g
10(logIC −X)(HillSlope)) using three independent experiments in quadruplet. Toxicity was determined using CellTiter-Glo (Promega), and data were
50
normalized by signal observed for living and dead cells ( 100 μM 3-dibutylamino-1-(4-hexylphenyl)propan-1-one).
compound 3-dibutylamino-1-(4-hexylphenyl)propan-1-one,
which has been reported to inhibit the interaction between
coactivator SRC2 and the thyroid receptor β13 and which
inhibit the interaction between VDR and SRC2-3 (see
Supporting Information). A far-red fluorescent label (Alexa
Fluor 647, 630/685 nm) was used for SRC2-3 to minimize
fluorescent interference by fluorescence quenching and
aggregation of screening compounds. Two-hundred seventy-
five thousand compounds were tested in the primary screening
campaign at a single dosage of 30 μM. On the basis of the FP
signal, 589 compounds exhibited more than 40% inhibition of
the VDR−SRC2-3 interaction at that concentration and were
less likely to bear structure elements of promiscuous
aggregating molecules14 or electrophilic compounds.15 HTS
evaluation of the frozen stock solutions of these compounds
confirmed that 140 compounds exhibited a dose-dependent
response with IC50 values of less than 40 μM. Further analyses
RESULTS
■
The HTS was carried out using 384-well black polystyrene
plates with 20 μL of assay reagent per well. This assay reagent
included VDR-LBD (1 μM), LG190178 (5 μM), Alexa Fluor
647 labeled SRC2-3 (7.5 nM), and a selected small molecule
from the screening library (30 μM). FP was measured after 3 h
of incubation. Recently, we quantified the binding affinities
between VDR-LBD and different coregulator peptides using the
same technique and discovered that the third nuclear
interaction domain (NID) of steroid receptor coactivator 2
(SRC2), called SRC2-3, has the strongest interaction with VDR
among other coregulator peptides tested.11 The study also
showed that the binding affinities of theses peptides were
similar in the presence of either 1,25-(OH)2D3 or synthetic
agonist LG190178.12 Three-hundred different compounds were
investigated per plate, which contained also the negative
control, dimethyl sulfoxide (DMSO), and the positive control
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Figure 1. Hit structures from HTS for inhibitors of the interaction between VDR-LBD and fluorescently labeled peptide SRC2-3. Structures of
validated hits are shown, grouped by chemotype, and annotated with IC50 values that were determined using a fluorescence polarization assay that
employed VDR-LBD and fluorescently labeled peptide SRC2-3.
of these stock solutions were performed in HEK293 cells,
which express a fusion protein of VDR-LBD and GAL4-DBD.
It was determined that these cells induced the transcription of a
β-lactamase reporter gene in the presence of 1,25-(OH)2D3,
(GeneBLAzer, Invitrogen). Quantification of β-lactamase was
accomplished by detecting the decrease of fluorescence
resonance energy transfer (FRET) caused by the enzymatic
cleavage of the β-lactam containing substrate, which was added
after an incubation time of 24 h. The quantity of uncleaved
substrate, which was determined by measuring the fluorescence
emission at 447 nm, revealed that 48 of the 140 active
compounds were able to regulate the VDR-mediated tran-
scription of β-lactamase. Additionally, the abilities of these
compounds to inhibit the interaction between VDR and 1,25-
(OH)2D3 were determined to exclude allosteric VDR−
coactivator binding disruption through VDR ligand antagonism.
The application of a VDR PolarScreen (Invitrogen) confirmed
that none of the active compounds was able to replace labeled
1,25-(OH)2D3.
The 48 compounds were then purchased as solids, dissolved
in DMSO as a 10 mM solution, and analyzed by liquid
chromatography−mass spectrometry (LC−MS) to determine
purity and identity (Table 1). A subsequent dose−response
analysis using the described FP assay determined that 29 of the
48 purchased compounds exhibited IC50 values of less than 40
μM. These compounds were divided into 6 groups, based on
their scaffold similarity, and depicted in Figure 1 with their
determined FP IC50 values.
ability using PAMPA (parallel artificial membrane perme-
ability), toxicity in HEK293T cells, and their ability to inhibit
VDR-mediated transcription using the described GeneBLAzer
assay. The results are summarized in Table 1.
The 3-indolylmethanamines grouped in C showed the most
promising characteristics, which included good to excellent
solubility, high permeability, inhibition of VDR−SRC2-3
interaction at low micromolar concentrations, and the ability
to inhibit VDR-mediated transcription in the range of 62.5−
20.8 μM.
Selected analogues of 3-indolylmethanamines were regen-
erated in our laboratory in regard to the low purities and
limited commercial availability of these compounds. In order to
analyze the electronic effects of both aromatic substituents, we
substituted the pyridine ring of the compounds depicted in
Figure 1 with substituted phenyl groups as shown in Figure 2.
For synthesis details, see Supporting Information. Three series
of 3-indolylmethanamines were generated, including those
bearing electron-donating and -accepting aromatic substituents
(Figure 2).
The compounds were characterized using carefully selected
biophysical and biochemical assays that are summarized in
Table 2. The 2-chlorophenyl substitutent was chosen for series
31 because 31a exhibits a higher activity (IC50) and higher rate
constant (k) in comparison with others members of group 30.
These assays included a VDR transcription assay that
employed luminescence instead of FRET. The assay is based
on the VDR-mediated transcription of a luciferase gene under
control of the CYP24A1 gene promoter.16 The gene product of
CYP24A1 is 24(R)-hydroxylase, which is responsible for the
Further characterizations were carried out, which include the
determination of small molecule aqueous solubility, perme-
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also have lower solubility, ranging from 4.9 to 85 μM. The
permeability of all series 30−32 range from medium to high in
comparison with drug standards carbamazepine (log Pe = −6.81
cm/s, medium) and verapamil (log Pe = −5.93 cm/s, high).
Determination of the ability of 3-indolylmethanamines to
inhibit the interaction between VDR and SRC2-3 resulted in
similar IC50 values, ranging from 27 to 44 μM, for the majority
of compounds after 3 h. Significantly higher IC50 values were
observed for compound 30g, bearing a methyl group (IC50
=
104 μM) instead of an aryl group, and compounds 30e, 31e,
31f, and 31g, bearing electron-withdrawing aromatic sub-
stitutents. For the last four compounds, nonlinear fitting
resulted in IC50 values with high standard deviation caused by
lack of saturation at higher compound concentrations (Table
2). Additionally, we observed loss of activity for 3-
indolylmethanamines with the alkylation of the indole nitrogen
(32a) or nitrogen−sulfur substitution (32c). Compound 32b,
missing the 2-methylindole substituents, inhibited only 50% of
the interaction between VDR and SRC2-3. The FP analysis of
the VDR−coactivator inhibition reaction at different time
points identified significant changes of inhibition in time. This
prompted us to determine each compound’s rate constant by
fitting the data to first order dissociation kinetics (see
Supporting Information). Small standard deviations support
the application of this model and enabled us to identify large
Figure 2. Structures of synthesized 3-indolylmethanamines.
catabolism of vitamin D analogues into their 24-hydroxylated
species.17 CYP24A1 is highly and directly regulated by VDR.
The solubility of compounds from series 30 is excellent
except those bearing a 2-chloroaryl or 2-naphthalenyl
substituent (Table 2, compounds 31a and 30h). Molecules
from series 31 and 32, which bear a 2-chloroaryl substituent,
Table 2. Summary of Biophysical and Biochemical Properties of 3-Indolylmethanamines
b
solubility
a
permeability,
VDR−SRC2-3 inhibition, rate constant k for the dissociation of SRC2-3 transcription inhibition, toxicity LC50
cg dg e f
, ,
compd
(μM)
log(Pe) (cm/s)
IC50 (μM)
30.2 4.8
from VDR (10−5
)
IC50 (μM)
(μM)
30a
30b
30c
30d
30e
30f
252.9
93.9
237.5
175.1
157.7
117.6
189.0
67.3
503.4
31.6
68.0
84.2
35.9
21.0
4.9
−6.00
−6.03
−6.10
−6.34
−6.88
−6.08
−6.40
−6.30
−6.83
−6.41
−6.24
−6.30
−6.45
−7.28
−7.60
n.d.
45.1 1.6
34.2 1.0
88.0 4.7
35.7 1.4
2.0 0.16
6.7 0.12
1.8 0.15
14.2 0.28
2.3 0.21
38.6 0.89
4.5 0.59
39.1 1.3
23.7 0.35
0.87 0.13
1.2 0.16
2.5 0.07
25.2 0.36
n.o.
10.9 2.8
8.1 1.7
12.1 1.8
14.6 2.5
13.5 1.3
12.2 2.4
20.1 5.2
15.0 2.4
13.1 2.5
8.5 1.8
4.2 1.9
5.8 2.1
8.2 1.5
8.7 1.5
4.4 1.1
24.0 5.7
8.6 1.1
>70
15.3 2.9
14.6 3.4
17.2 2.5
21.7 4.2
25.8 6.3
16.2 2.5
37.4 7.7
20.8 3.5
31.4 8.2
12.6 2.2
11.6 1.7
12.1 2.2
12.7 3.0
19.4 3.8
11.0 2.1
43.1 9.1
12.1 2.7
>70
31.7 4.3
31.2 3.2
43.6 7.8
n.s.
28.5 4.5
104.8 15.2
29.6 3.1
58.6 8.1
29.8 4.5
36.7 5.1
26.5 3.4
17.7 3.2
n.s.
30g
30h
30i
31a
31b
31c
31d
31e
31f
n.s.
31g
31h
32a
32b
32c
n.d.
n.s.
59.7
15.3
52.3
11.8
−6.22
−6.58
−6.51
−6.89
24.3 4.4
n.o.
20.3 4.5 (partial)
n.o.
1.4 0.12
n.o.
21.6 3.3
>70
>70
>70
a
b
Solubilities were determined in phosphate buffered saline at pH 7.4. Permeabilities were measured using the parallel artificial membrane
permeation assay (PAMPA) at neutral pH (pH 7.4). The following permeability standards were used (log Pe): ranitidine (−8.02 0.074 cm/s) low
permeability, carbamazepine (−6.81 0.0011 cm/s) medium permeability, and verapamil (−5.93 0.015 cm/s) high permeability. The solubility
c
and permeability assay conditions reflect conditions required for activity in cell-based assays. A fluorescence polarization competition assay was
carried out using VDR-LBD (1 μM), Alexa Fluor labeled peptide SRC2-3 (7 nM), VDR-agonist LG190178 (5 μM), and serial diluted small
50−X)
molecules. IC50 values were obtained by fitting data obtained after 3 h to the following equation: Y = Bottom + (Top − Bottom)/(1 + 10(logIC
d
c
(HillSlope)) using three independent experiments in quadruplet. A fluorescence polarization assay described under footnote was monitored over
time. Dissociation rate constants were obtained by linear fitting of ln(mP) (fluorescence polarization) against time (first order kinetics). HEK293T
e
cells were transfected with CMV-VDR, a CYP24A1 promoter driven luciferase expression vector, and a Renilla luciferase control vector. The data
f
were normalized to Renilla luciferase activity and by signals observed for inactive and activated VDR ( 1,25(OH)2D3). Toxicity was determined
based on signal observed for Renilla luciferase activity and normalized to signal observed for living and dead cells ( 100 μM 3-dibutylamino-1-(4-
g
hexylphenyl)propan-1-one, CBT358);. n.d. = not determined; n.s. = no saturation of signal at higher small molecule concentration (no reliable
nonlinear fitting possible); n.o. = not observed.
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reactivity differences between the 3-indolylmethanamines
tested. As expected, we observed smaller rate constants for 3-
indolylmethanamines with higher IC50 values of more than 44
μM (30e, 30g, 30i, 31e, 31f, and 31g). Four 3-indolylmethan-
amines (30f, 30h, 31b, and 32b) exhibited IC50 values in the
range of 27−44 μM but showed relative small reaction rates.
Interestingly, compound 31d, which has the lowest IC50 values,
does not have the highest reaction rate. The ability of 3-
indolylmethanamines to displace 1,25-(OH)2D3 from VDR was
determined by a commercially available FP assay (Polarscreen,
Invitrogen) and excludes VDR ligand displacement by 3-
indolylmethanamines, which can cause allosteric disruption of
the VDR−coactivator interaction. None of the synthesized 3-
indolylmethanamines were able inhibit the interaction between
labeled 1,25-(OH)2D3 and VDR except compounds 30e and
30f, which exhibited weak inhibition at higher concentrations
(see Supporting Information). Almost all 3-indolylmethan-
amines were able to inhibit the VDR-mediated transcription at
lower micromolar concentrations except those that were not
able to inhibit the interaction between VDR and SRC2-3
(Table 2, compounds 32a and 32c). We also observed
significant cell toxicity caused by 3-indolylmethanamines at
higher concentrations. The small difference between transcrip-
tional inhibition and toxicity prompted us to use rt-PCR to
determine the modulation of gene regulation in the presence of
3-indolylmethanamines.
●
Figure 3. Identification of 31b’s reaction partner. ( ) Alexa Fluor-
labeled SRC2-3 peptide (7 nM) was incubated for 3 h with different
concentration of 31b followed by the addition of VDR-LBD (1 μM)
and LG190178 (5 μM). Fluorescence polarization was detected after 5
■
min. ( ) VDR-LBD (1 μM) and LG190178 (5 μM) were incubated
for 3 h with different concentrations of 31b followed by the addition of
Alexa Fluor labeled SRC2-3 peptide (7 nM). Fluorescence polarization
was detected after 5 min.
The azafulvenium salts are reactive electrophilies that can
undergo reactions with natural occurring nucleophilies such as
cysteine residues of proteins. The wide range of reaction rates
of the differently substituted 3-indolylmethanamines and the
fact that these compounds are likely to react with VDR
irreversibly prompted us to investigate the possibility of a linear
free energy relationship between the alkylation of VDR
measured by disruption of VDR−SRC2-3 binding and the
electronic nature of different aromatic 3-indolylmethanamine
substituents; therefore, log(kx/k0) was plotted against Hammett
σ-values for the compounds of series 30 and 31 (Figure 4).19
A strong correlation was found for both series (r2 = 0.93)
with significant negative ρ-values, supporting the proposed
mechanism that during the rate determining step, a positive
charge is building up. Additionally, we observed that this
reaction is less sensitive to substituents of compound series 30
(ρ = −1.1) than to substituents of compound series 31 (ρ =
−1.5). This supports the fact that substituents of series 30 have
a majorly inductive stabilizing effect, resulting in a smaller
absolute ρ-value than substituents of compound series 31,
which can stabilize the positive charge via resonance.
The different reaction rates of the 3-indolylmethanamines
and similar IC50 values indicate that these compounds are likely
to react with VDR or SRC2-3 in an irreversible fashion. Indeed,
it was reported that, especially under acidic conditions or
elevated temperature, 3-indolylmethanamines underwent elim-
ination reactions by breaking the carbon−nitrogen bonds and
forming the corresponding azafulvenium salts (Scheme 1).18
Scheme 1. Proposed Mechanism of Action for 3-
Indolylmethanamines
In water at a neutral pH, 3-indolylmethanamines were stable
for 24 h, but reaction occurred when high concentrations of 2-
mercaptoethanol (5 mM) were added. To determine the
selectivity of 3-indolylmethanamine (31b) toward different
nucleophiles, an FP assay of different concentrations of 2-
mercaptoethanol was carried out (Figure 5).
Two effects were observed. First, the IC50 values for 31b
under identical conditions increased with the amount of 2-
mercaptoethanol from 36.8 μM (0.01 mM 2-mercaptoethanol)
to 82.2 μM (100 mM 2-mercaptoethanol). Second, the efficacy
of each isotherm decreased with increasing amount of 2-
mercaptoethanol. These results show that VDR−SRC2-3
binding inhibition by 31b was only changed in the presence
of more than a 1000-fold excess of an alternative nucleophile,
such as 2-mercaptoethanol.
Small molecule target selectivity is very important, especially
toward different NR−coactivator interactions. To determine
the NR selectivity of 3-indolylmethanamines, five additional
NR−coactivator interactions were investigated: androgen
receptor AR−SRC2-3, thyroid receptor TRα−SRC2-2 and
TRβ−SRC2-2, estrogen receptor β (ERβ)−SRC2-2, and
peroxisome proliferator-activated receptor γ (PPARγ)−DRIP2
To discriminate which of the binding partners (VDR or
SRC2-3) is alkylated by 3-indolylmethanamines, we incubated
31b for 3 h with either VDR or SRC2-3 followed by the
addition of the other interaction partner that was SRC2-3 or
VDR, respectively (Figure 3). The binding isotherm of each
condition was different. Preincubation of 31b with SRC2-3
followed by the addition of VDR did not result in an alkylation
reaction because the FP signal did not change with higher
concentration of 31b. In contrast, preincubation of VDR with
different 31b concentrations followed by the addition of SRC2-
3 did result in a change of FP signal. The corresponding
isotherm was similar to the inhibition observed for combining
all reagents at the same time and measuring FP after 3 h.
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Figure 4. Linear free energy relationship between VDR−SRC2-3 inhibition and the electronic nature of 3-indolylmethanamines substitutents: (A)
series 30; (B) series 31. The log(kx/k0) values were calculated using rate constants given in Table 2, K0 being the nonsubstituted compounds 30a
and 31a. The σ-values were obtained from Ritchie et al. The ρ-values represent the slopes of the linear regressions with the corresponding r2 values.
Figure 6. Nuclear receptor−coactivator binding studies in the
presence of 3-indolylmethanamine 31b using fluorescence polar-
ization. FP was detected at an excitation/emission wavelength of 595/
615 nm. The conditions for different NRs are as follows: For AR,
androgen receptor LBD (5 μM), Texas Red labeled SRC2-3 (7 nM),
and dihydrotestosterone (5 μM) were incubated with small molecule
for 3 h. For TRα, thyroid receptor α LBD (2 μM), Texas Red labeled
SRC2-2 (7 nM), and triiodothyronine (1 μM) were incubated with
small molecule for 3 h. For TRβ, thyroid receptor β LBD (0.8 μM),
Texas Red labeled SRC2-2 (7 nM), and triiodothyronine (1 μM) were
incubated with small molecule for 3 h. Estrogen receptor β (3 μM),
Texas Red labeled SRC2-2 (5 nM), and estradiol (0.1 μM) were
incubated with small molecule for 3 h. Peroxisome proliferator-
activated receptor γ (5 μM), Texas Red labeled DRIP2 (7 nM), and
rosiglitazone (5 μM) were incubated with small molecule for 3 h. For
VDR, vitamin D receptor LBD (1 μM), Texas Red labeled SRC2-3 (7
nM), and LG190178 (5 μM) were incubated with small molecule for 3
h.
Figure 5. Influence of 2-mercaptoethanol for the inhibition of the
VDR−SRC2-3 binding in the presence of 3-indolylmethanamine 31b.
VDR-LBD (1 μM), LG190178 (5 μM), and Alexa Fluor labeled
SRC2-3 peptide (7 nM) were incubated in the presence of different
concentrations of 31b and in the absence and presence of different
concentrations of 2-mercaptoethanol. Interactions between VDR and
○
SRC2-3 were determined by fluorescence polarization: ( ) DMSO
□
(negative control), ( ) 3-dibutylamino-1-(4-hexylphenyl)propan-1-
one (positive control). 2-Mercaptoethanol concentrations (31b IC50
■
●
values) were as follows: ( ) 100 mM (82.2 7.3 μM), ( ) 10 mM
▲
▼
(54.5 3.1 μM), ( ) 1 mM (45.4 2.0 μM), ( ) 0.1 mM (37.5
◆
1.1 μM), ( ) 0.01 mM (36.8 0.5 μM).
(VDR-interacting protein 205). The quantification of these
NR−coactivator interactions has been reported previously.20
All 3-indolylmethanamines exclusively disrupted the VDR−
SRC2-3 interaction, as depicted for compound 31b in Figure 6.
The binding isotherms for all 3-indolylmethanamines are
presented in the Supporting Information.
We also determined the abilities of 3-indolylmethanamines
to inhibit different VDR−coregulator interactions. The
quantification of interactions between VDR and coregulator
peptides was reported recently.11 Herein, we focused on three
different coregulators, which include SRC2,21 DRIP205,22 and
Hairless [Hr].23 The results employing compound 31b are
summarized in Figure 7.
Inhibition of binding was observed in the presence of 31b for
VDR−SRC2-2, VDR−SRC2-3, and VDR−DRIP2 (Figure 7).
The efficacy of these isotherms is dissimilar because of the
different binding constants between VDR and SRC2-2 (Kd =
1.7 0.2 μM), VDR and SRC2-3 (Kd = 0.93 0.17 μM), and
VDR and DRIP-2 (Kd = 1.6 0.2 μM). In comparison, the
binding constant between VDR and SRC2-1 and VDR and Hr1
is greater than 5 μM and no inhibition by compound 31b was
observed under these conditions. Interestingly, the IC50 values
Figure 7. VDR−coactivator interactions in the presence of 31b using
FP. VDR-LBD (1 μM), LG190178 (5 μM), and different Texas Red
labeled coregulator peptides (7 nM) were incubated for 3 h in the
presence of different concentrations of compound 31b.
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of 31b and for the VDR−SRC2-2, VDR−SRC2-3, and VDR−
DRIP-2 interactions are very similar (IC50 = 25.4 6.5 μM,
IC50 = 28.5 6.9 μM, and IC50 = 33.1 3.4 μM, respectively).
The ability of 31b to partially inhibit the interaction between
VDR and SRC2, containing all three SRC2 NIDs, was tested
using a pull down assay (Figure 8). Control experiments
TRPV6 (ECaC2 or CaT1) is a membrane Ca2+ ion channel,
which is highly expressed in advanced prostate cancer24 and
was reported to be directly regulated by VDR in the presence of
1,25-(OH)2D3.25 The expression levels of TRPV6 in the
prostate cancer cell line DU145 in the presence and absence of
1,25-(OH)2D3 and compound 31b are depicted in Figure 10.
Figure 8. Western blot of in vitro binding reactions between SRC2
bearing all three NIDs and VDR-LBD in the presence of 31b. Lanes
1−3 show different concentrations of 31b in the presence of VDR,
SRC2, and 1,25(OH)2D3. Lane 4 shows VDR, SRC2, and 1,25-
(OH)2D3. Lane 5 shows no ligand (1,25(OH)2D3), no coregulator
(SRC2).
indicate that SRC2 bound to VDR in the presence of VDR
ligand 1,25(OH)2D3 (Figure 8, lane 4) but not in the absence
of 1,25(OH)2D3 (Figure 8, lane 5). The VDR−SRC2
interaction was blocked in a dose dependent manner by 31b
(Figure 8, lanes 1−3). Although significant inhibition of the
VDR−SRC2 interaction was observed at 50 and 100 μM 31b, a
residual interaction between VDR and SRC2 could still be
detected. Thus, the inhibition of the interaction between VDR
and full length SRC2 by 31b exhibits dose response
dependence, similar to the SRC2-2 peptide binding study
described above.
Figure 10. Modulation of expression of TRPV6 in the presence and
absence of 1,25-(OH)2D3 and increasing concentrations of small
molecule 31b. DU145 cells were cultured in six-well plates and treated
with 1,25-(OH)2D3 (20 nM) and/or small molecule 31b. TRPV6
expression levels were determined by semiquantitative RT-PCR and
normalized to GAPDH transcript level and to DMSO control
condition. The ΔΔCt method was used to measure the fold change
in expression of genes. Standard deviations were calculated from three
biological independent experiments performed in triplicate.
Additionally, we investigated the interaction between VDR
and SRC2 in the presence of 3-indolylmethanamine or vehicle
prior to the pull-down assay, to discriminate between 31b−
VDR and 31b−SRC2 binding, respectively (Figure 9). VDR−
In the presence of 1,25-(OH)2D3, TRPV6 was up-regulated
in DU145 cells. The single treatment of cells with 3-
indolylmethanamine 31b at 20 μM showed no regulation of
TRPV6. For 31b concentrations higher than 20 μM an
increased cytotoxicity was observed (see Supporting Informa-
tion). In contrast, in the presence of 20 nM 1,25-(OH)2D3 and
different concentrations of 31b, TRPV6 transcription was
reduced in a dose dependent manner, confirming that 31b is
modulating TRPV6 expression by interacting with VDR.
DISCUSSION
■
It was demonstrated that the application of HTS was successful
in identifying the first irreversible inhibitors of the VDR−
coactivator interactions. The screening campaign utilized a FP-
based primary assay to quantify small molecule inhibition of the
VDR−coactivator peptide binding. The original hit rate of 1.3%
was reduced to 0.32% through the application of different
scaffold filters to eliminate highly reactive molecules from the
hit compound selection. These reactive molecules included
enones, thiols, imines, and thioisocyanates. Rescreening of the
remaining 579 compounds resulted in 140 confirmed hit
compounds. Further evaluation of these molecules using
secondary cell-based assays (transcription and toxicity assays)
confirmed 48 compounds that exhibit biochemical and cellular
activity. The purchase of these compounds as solids and
screening of the freshly dissolved quality-controlled compounds
using the HTS assay confirmed only 29 compounds of the
initial 48 compounds. We hypothesize that the low hit
reproducibility is based on at least two reasons. First is the
deviation of compound activities for single point HTS assays,
Figure 9. Western Blot of in vitro binding reactions between SRC2
bearing all three NIDs and 3-indolylmethanamines. Lane 1 shows
preincubation SRC2 with 31b. Lane 2 shows preincubation with 32a,
and lane 3 shows preincubation with vehicle only. After incubation,
beads were washed and treated with VDR, and VDR−SRC2
interactions were determined by Western blot.
SRC2 interactions could be verified for all reaction conditions,
although 31b, in contrast to 32a, was able to inhibit the
interaction between VDR and SRC2 (Table 2 and Figure 8).
Thus, preincubation of 31b with SRC2 (Figure 9), in contrast
to preincubation of 31b with VDR (Figure 8), did not result in
an alkylation reaction, and therefore, VDR−SRC2 binding was
observed.
To examine the influence of VDR−coactivator inhibition by
31b with respect to VDR-mediated transcription, we
investigated the expression levels of the transient receptor
potential vanilloid type 6 gene (TRPV6). The gene product of
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and second is the limited stability of some screening
compounds in DMSO.
described transcriptions assays because of their sensitivity
toward cell toxicity.
The regulation of VDR target genes by small molecules,
which includes the inhibition of VDR−coactivator interactions,
represents a new strategy to develop new drug candidates for
diseases related to 1,25-(OH)2D3 and VDR. For prostate
cancer cells, for example, LNCaP cells, it was reported that
1,25-(OH)2D3 can induce proliferation or antiproliferation
depending on the amount of supplemental serum used.28
Additionally, it has been shown that the expression of TRPV6,
induced by 1,25-(OH)2D3, is responsible for cell proliferation
and apoptosis resistance.29 We hypothesize that especially for
prostate cancer with 1,25-(OH)2D3-independent proliferation
(DU145), VDR antagonists such as 3-indolylmethanamines
represent a new anticancer approach because of their ability to
down-regulate the transcription of TRPV6 and to induce cell
death.
From the selection of validated hit compounds, further
characterization assays of 3-indolylmethanamines were chosen
based on their promising characteristics, which included good
to excellent solubility, high permeability, inhibition of VDR−
SRC2-3 interaction at low micromolar concentrations, and the
ability to inhibit VDR-mediated transcription in cells. In order
to identify SARs for 3-indolylmethanamines, we synthesized
structural analogues with diverse substituents at different
positions of the scaffold. These compounds were evaluated
with assays mentioned above for the initial HTS hit
compounds. In general, 3-indolylmethanamines with electron
donating aryl substituents inhibited the interaction between
VDR and SRC2-3 coactivator peptide more quickly than 3-
indolylmethanamines with electron withdrawing ones. The
significance of this correlation was demonstrated with the
application of a linear free energy relationship (LFER)
equation, which indicated a strong and different relationship
for series 30 and 31. The negative ρ values support the
hypothesized mode of binding of 3-indolylmethanamines,
which includes the formation of an azafulvenium salt. This
salt is then expected to react with solvent-exposed nucleophilic
residue of VDR inhibiting the interaction with SRC2. The
reactivity of 3-indolylmethanamines toward thiols was demon-
strated by conducting FP assays in the presence of 2-
mercaptoethanol, which diminished binding between VDR
and compound 31b at higher concentrations.
Many irreversible antagonists are among FDA-approved
drugs, such as dibenzyline, which is an α-adrenoceptor blocker
used for hypertension. Most of these drugs are reactive toward
biological nucleophilies, but they usually exhibit high
selectivities among them. An exclusive selectivity could be
demonstrated for 3-indolylmethanamines in regard to their
ability to disrupt the VDR−coactivator interaction in
comparison with five other NR−coactivator interactions. This
is remarkable because different electrophilic inhibitors (β-
aminoketones and methylsulfonylnitrobenzoates) have been
developed for three of these interactions (TRα−SRC2, TRβ−
SRC2, and PPARγ−DRIP2).26 The micromolar activity of 3-
indolylmethanamine is comparable to the reported activity of
direct irreversible and reversible inhibitors of other NR−
coactivator interactions9,26 as well as to the binding affinities
reported for native coactivator peptides.11 Additionally, a
selectivity of 3-indolylmethanamines toward the interactions
between VDR−SRC2-3, VDR−SRC2-2, and VDR−DIP2 was
observed among other NR−coregulator interactions tested.
This is consistent with the fact that coactivator SRCs and DRIP
are binding VDR at the same interaction site.27
CONCLUSION
■
This study reports a successful HTS strategy to identify small
molecule inhibitors of VDR−coregulator interactions. To date,
these are the first irreversible inhibitors of VDR−coactivator
interactions that have been reported. This new class of VDR
antagonists exhibits excellent selectivity among different NR−
coregulator interactions and inhibits the interaction between
VDR and different coactivators. This inhibition regulated the
expression of VDR target genes such as TRPV6, which has been
shown to be up-regulated in last stage prostate cancers. The
application of 3-indolylmethanamines such as 31b is expected
to be part of new therapy to reduce prostate cancer cell growth
and enables investigations toward the function of VDR−
coactivator interactions during gene regulation. 3-Indolylme-
thanamines also have a great potential as novel drug candidates
for human diseases associated with the overproduction of 1,25-
(OH)2D3, such as sarcoidosis30 or Crohn’s disease.31 We are
currently developing more selective and more potent 3-
indolylmethanamines in order to study their modulation of
VDR-mediated transcription and their biological activity in
vivo. These future studies will include prostate cancer
xenographs that enable the determination of efficacy of 3-
indolylmethanamines by monitoring the expression of VDR
target genes in tumors as well as the expected tumor regression.
Simultaneously, any signs of acute and chronic toxicity will be
determined by observation and necropsy.
EXPERIMENTAL SECTION
■
Chemistry. All materials were obtained from commercial suppliers
and used without further purification. All solvents used were dried
using an aluminum oxide column. Thin-layer chromatography was
performed on precoated silica gel 60 F254 plates. Purification of
compounds was carried out by normal phase column chromatography
(SP1 [Biotage], silica gel 230−400 mesh) followed by evaporation.
Purity determinations were performed by element analysis (EA1110,
CarloErba) or using a LC−MS (Surveyor&MSQ) with a C18 column.
The total flow rate was 1.0 mL/min, and the gradient program started
at 90% A (0.1% formic acid in H2O), changed to 95% B (0.1% formic
acid in methanol) and then to 90% A. The mass spectrometer was
operated in positive-ion mode with electrospray ionization. All
compounds presented were confirmed at 95% purity or better using
either method. NMR spectra are recorded on a Bruker 400 MHz and
referenced internally to the residual resonance in CDCl3 (δ 7.26 ppm
for hydrogen and δ 77 ppm for carbon atoms).
Problematic is the quantification of transcriptional inhibition
for 3-indolylmethanamines because of their ability to induce
cell death at a similar concentration. This resulting narrow
therapeutic window is of concern for the development of
nontoxic VDR−coactivator inhibitor. We hypothesize that the
inhibition of transcription by itself has the potential to induce
cell death including apoptosis, which has been demonstrated
for TRPV6 expression by using siRNA-TRPV6.28 Similar, 3-
indolylmenthanamine 31b reduced the expression of TRPV6 in
a dose-depended manner as determined by rt-PCR and induced
cell death at similar concentrations. Unfortunately, this
antiproliferative behavior of 3-indolylmenthanamines compli-
cates the identification of transcriptional inhibition using the
General Procedure for the Aza Friedel−Crafts Reaction. In a
dry flask, aniline (2 mmol) and aldehyde (2 mmol) were dissolved in
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toluene (2 mL) and stirred for 1 h. Then indole (2 mmol) and
decanoic acid (0.2 mmol, 10 mol %)32 were added slowly as a solution
in toluene (2 mL). The reaction mixture was stirred at room
temperature and monitored by TLC. After the reaction was
completed, saturated aqueous NaHCO3 (6 mL) was added. The
mixture was extracted with dichloromethane (3 × 10 mL). The
organic layer was combined, washed with brine (10 mL), and dried
over anhydrous Na2SO4. The solvents were removed under reduced
pressure, and the residue was purified by recrystallization or
chromatography through Biotage SP1 flash system.
(OH)2D3 and LG190178 (positives) and DMSO (negative). Toxicity
was determined by luminescence using Cell-Titer Glo (Promega),
which was added to the plates after recording the FRET signal.
Controls for cell viability were 3-dibutylamino-1-(4-hexylphenyl)-
propan-1-one (100 μM in DMSO) (positive) and DMSO (negative).
Two independent experiments were conducted in triplicate.
CYP24A1 Promoter Transcription Assay. This assay was used
to determine the regulation of transcription of the VDR-target gene,
CYP24A1, in the presence of small molecules. Briefly, HEK 293T cells
(ATTC) were cultured in 75 cm2 flasks using MEM/EBSS (Hyclone)
with L-glutamine (2 mM), glucose (1 mM), nonessential amino acids,
sodium pyruvate (1 mM), penicillin and streptomycin, and 10% heat
inactivated FBS (Hyclone). At 50−70% confluency, cell medium was
changed to phenol red free MEM/EBSS with L-glutamine (2 mM),
glucose (1 mM), nonessential amino acids, sodium pyruvate (1 mM),
penicillin and streptomycin, and 10% dialyzed and heat inactivated
FBS (Invitrogen), followed by the addition of 2 mL of untreated
MEM/EBSS containing 1.56 μg of a VDR-pRc/CMV plasmid, 16 μg
of a luciferase reporter gene plasmid containing a rat 24-hydroxylase
gene promoter (−1399 to +76), 17.4 μg of a Renilla luciferase control
vector (Promega), Lipofectamine LTX (75 μL), and PLUS reagent
(25 μL). After 16 h, the cells were harvested and plated in sterile cell
culture treated black 384-well plates with optical bottom (Nunc
142761) at 15 000 cells per well. After 2 h, plated cells were treated
with small molecules in vehicle DMSO, followed by a 16 h incubation
time. Transcription was determined using a Dual-Luciferase reporter
assay (Promega). Cell viability was determined using the Renilla
luciferase signal. IC50 values and standard errors were calculated based
on two independent experiments performed in quadruplicate.
Controls for this assay were 1,25-(OH)2D3 and LG190178 (positives)
and DMSO (negative). Controls for cell viability were 3-dibutylamino-
1-(4-hexylphenyl)propan-1-one (100 μM in DMSO) (positive) and
DMSO (negative). Two independent experiments were conducted in
quadruplicate.
Example: N-((2-Methyl-1H-indol-3-yl)(phenyl)methyl)aniline
(30a). Rf = 0.3 (EtOAc/hexanes = 1/4). 230 mg white solid, 37%
1
yield. H NMR (300 MHz, CDCl3, TMS): δ 2.34 (s, 3H), 4.34 (s,
1H), 5.76 (s, 1H), 6.59 (dd, J = 1.8, 4.8 Hz, 2H), 6.69−6.73 (m, 1H),
6.69−7.05 (m, 1H), 7.08−7.18 (m, 4H), 7.26−7.29 (m, 3H), 7.38 (d, J
= 7.5 Hz, 2H), 7.49 (d, J = 7.8 Hz, 1H), 7.82 (s, 1H). 13C NMR (300
MHz, CDCl3, TMS): 12.35, 54.40, 110.8, 112.45, 113.28, 116.12,
118.76, 119.10, 120.43, 126.79, 128.58, 143.9, 144.1, 148.88, 148.96.
Anal. Calcd for C22H20N2: C 84.58, H 6.45, N 8.97. Found: C 84.2027,
H 6.72, N 8.66.
Reagents. 1,25-(OH)2D3 (calcitriol) was purchased from
Endotherm, Germany. LG190178 was synthesized using a published
procedure.12
Labeled Coregulator Peptides. Peptides, such as SRC2-3
(CLQEKHRILHKLLQNGNSPA),11 were purchased and labeled
with cysteine-reactive fluorophores, such as Texas-Red maleimides
and Alexa Fluor 647 maleimides, in DMF/PBS, 50:50. After
purification by HPLC, the corresponding labeled peptides were
dissolved in DMSO and stored at −20 °C.
Protein Expression and Purification. The VDR-LBDmt DNA
was kindly provided by D. Moras33 and cloned into pMAL-c2X vector
(New England Biolabs). For a detailed expression and purification
protocol, see ref 11. For detailed expression and purification of
protocols of PPARγ-LBD, TRα-LBD, TRβ-LBD, and AR-LBD, see ref
26b.
High Throughput FP Assay. The HTS was carried out at St. Jude
Children’s Research Hospital. The small molecule collection consisted
of 275 000 unique compounds from commercial sources (ChemDiv,
ChemBridge, and Life Chemicals). The FP assay was conducted in
384-well black polystyrene microplates (Corning, no. 3573). The assay
solution contained buffer (25 mM PIPES (pH 6.75), 50 mM NaCl,
0.01% NP-40, 2% DMSO, VDR-LBD protein (1 μM), LG190178 (5
μM), and Alexa Fluor 647-labeled SRC2-3 (7.5 nM). Small molecule
transfer into 20 μL of assay solution was accomplished using a 50H
hydrophobic coated pin tool (V&P Scientific), delivering 60 nL of a 10
mM compound solution, which resulted in a final concentration of 30
μM. Inhibition of binding was detected using FP performed on an
EnVision multilabel plate reader (GE) with a 620 nm excitation filter, a
688 nm S polarized emission filter, a 688 nm R polarized emission
filter, and a Cy5 FP dichroic mirror. Automation was realized using a
VDR Ligand Competition Assay. Ligand antagonism was
determined by using a FP assay (PolarScreen, Invitrogen), which
employs a fluorescently labeled 1,25-(OH)2D3 analogue. Two
independent experiments were conducted in quadruplicate, and data
were analyzed using nonlinear regression with variable slope
(GraphPrism).
Solubility Assay. In a 384 UV plate (Corning no. 3675), 16
compounds were serially diluted in quadruplicate starting from a 10
mM compound stock solution in DMSO. Therefore, buffer (90 mM
ethanolamine, 90 mM KH2PO4, 90 mM potassium acetate, and 30
mM KCl (pH 7.4)) containing 20% acetonitrile was used. The plate
was sealed (Corning no. 6570), sonicated for 1 min, and agitated for
an additional 5 min before scanning from 230 to 800 nm at 5 min
increments. A calibration plot was prepared for each compound for the
maximal absorbance using background-subtracted values. A 384-well
filter plate (Pall no. 5037) was prewetted with 20% acetonitrile/buffer
and filled with buffer (47.5 μL) and 10 mM compound in DMSO (2.5
μL). The final DMSO concentration was 5%. After sonication (1 min)
and agitation (12 h), the mixtures were filtered and 30 μL of each well
was transferred into a 384-well UV plate, together with the addition of
20 μL of acetonitrile. The plate was agitated for 5 min and scanned
from 230 to 800 nm at 5 min increments. The solubility was
determined using background-subtracted values and the following
equation: sol = (absorbance at λmax)/[(slope)(5/3)]. Each plate had
the following solubility standards: 4,5-diphenylimidazole (67.3 3.7
μM), β-estradiol (43.0 2.3 μM), diethylstilbestrol (108.3 5.4 μM),
ketoconazole (134.5 2.4 μM), and 3-phenylazo-2,6-diaminopyridine
(357.7 7.0 μM). All experiments were conducted in quadruplicate.
Permeability Assay. This assay was carried out using Millipore’s
Multiscreen protocol AN1725EN00. Each plate had the following
standards with the following permeability values (log Pe): Ranitidine
system developed by high resolution engineering, which uses a Staubli
̈
T60 robot arm to transfer plates from instrument to instrument. The
assay solution was dispensed in bulk into empty plates using Matrix
Wellmates (Matrix Technologies), followed by compound addition,
centrifugation using a Vspin plate centrifuge (Velocity 11), and
incubation for 3 h at room temperature. The positive control (3-
dibutylamino-1-(4-hexylphenyl)propan-1-one26a) and negative control
(DMSO) were measured within each plate to determine the assay
plate quality and to enable data normalization.
Fluorescence Resonance Energy Transfer (FRET) Tran-
scription Assay. A GeneBLAzer (Invitrogen) assay was used to
identify VDR−coactivator inhibitors that regulate VDR-mediated
transcription. The provided HEK293 cells of this assay expressed a
fusion protein of VDR-LBD and the GAL4-DBD, which was activated
by 1,25-(OH)2D3, and induced transcription of a β-lactamase reporter
gene. Quantification of β-lactamase was accomplished by detecting the
decrease in FRET caused by the enzymatic cleavage of the β-lactam-
containing substrate, which was added after an incubation time of 24 h.
The cleaved substrate concentration was quantified by measuring the
fluorescence emission at 447 nm. Controls for this assay were 1,25-
(−8.02
0.074 cm/s) represents low permeability. Carbamazepine
0.0011 cm/s) represents medium permeability, and
(−6.81
verapamil (−5.93
experiments were conducted in triplet.
0.015 cm/s) represents high permeability. All
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NR−Coactivator Binding Studies in the Presence of 3-
Indolylmethamines. These assays were conducted in 384-well black
polystyrene microplates (Corning) using a buffer (20 mM Tris (pH
7.50), 100 mM NaCl, 0.01% NP-40, 2% DMSO) and analyzed with a
M1000 reader (Tecan) to detect FP at an excitation/emission
wavelength of 595/615 nm. For the androgen receptor, AR-LBD (5
μM), Texas Red labeled SRC2-3 (7 nM) and dihydrotestosterone (5
μM) were incubated in buffer with small molecule for 3 h. For the
thyroid receptor α, TRα-LBD (2 μM), Texas Red labeled SRC2-2 (7
nM) and triiodothyronine (1 μM) were incubated with small molecule
for 3 h. For the thyroid receptor β, TRβ-LBD (0.8 μM), Texas Red
labeled SRC2-2 (7 nM) and triiodothyronine (1 μM) were incubated
with small molecule for 3 h. For the peroxisome proliferator-activated
receptor γ, PPARγ-LBD (5 μM), Texas Red labeled DRIP2 (7 nM)
and rosiglitazone (5 μM) were incubated with small molecule for 3 h.
For the VDR, VDR-LBD (1 μM), Texas Red labeled SRC2-3 (7 nM)
and LG190178 (5 μM) were incubated with small molecule for 3 h.
Two independent experiments were carried out in quadruplicate, and
data were analyzed using nonlinear regression with variable slope
(GraphPrism).
were calculated from three biological independent experiments
performed in triplicate.
ASSOCIATED CONTENT
* Supporting Information
■
S
Inhibition of VDR−SRC2-3 binding in the presence of 3-
dibutylamino-1-(4-hexylphenyl)propan-1-one, synthesis and
characterization of molecules, graphical representation of rate
constants, nuclear receptor−coactivator binding isotherms, and
VDR−ligand competition isotherms. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: (414) 229 2612. Fax: (414) 229 5530. E-mail:
arnold2@uwm.edu.
Author Contributions
∥Both authors contributed equally to this work.
VDR−Coactivator Binding Studies in the Presence of 3-
Indolylmethamine 31b. These assays were conducted in 384-well
black polystyrene microplates (Corning) using a buffer (25 mM PIPES
(pH 6.75), 50 mM NaCl, 0.01% NP-40, 2% DMSO) and analyzed
with a M1000 reader (Tecan) to detect FP at an excitation/emission
wavelength of 595/615 nm. VDR-LBD protein (1 μM), LG190178 (5
μM), and 7.5 nM Texas Red labeled SRC1-3 [CESKDHQLL-
RYLLDKDEKDL], Texas Red labeled SRC2-3 [CLQEKH-
RILHKLLQNGNSPA], Texas Red labeled SRC3-3 [CKKENNALL-
R Y L L D R D D P S D ] , o r T e x a s R e d l a b e l e d D R I P 2
[CNTKNHPMLMNLLKDNPAQD] were incubated with different
concentration of 31b. Two independent experiments were carried out
in quadruplicate, and data were analyzed using nonlinear regression
with variable slope (GraphPrism).
Western Blot of in Vitro Binding Reactions between SRC2
Bearing All Three NIDs and VDR-LBD in the Presence of 31b.
GST fusion to the SRC2 bearing all three NIDs was expressed in
Escherichia coli BL21. Cultures were grown to OD600 = 0.5−0.6 at 22
°C and induced with 0.5 mM isopropyl-D-thiogalactoside for 12 h. The
cultures were centrifuged (1000g), and bacterial pellets were
resuspended in 20 mM Tris, pH 7.4, 200 mM NaCl, 1 mM NaN3,
0.5 M EDTA, 1 mM DTT, protein inhibitors cocktail (Roche) and
sonicated. Debris was pelleted by centrifugation (100000g). The
supernatant was incubated with glutathione-Sepharose 4B beads
(Amersham Biosciences) and washed. Protein on bead was stored
with 10% glycerol at −20 °C. Each pull-down reaction was carried out
in 100 μL of buffer (25 mM PIPES (pH 6.75), 50 mM NaCl, 0.01%
NP-40, 2% DMSO) using 100 nM calcitriol, 10 μM VDR-LBD-MPB,
and 31b. After 2 h at rt, 15 μL of SRC2-beads was added to each
reaction followed by 30 min of incubation. The mixture was filtered,
washed with buffer (100 μL), and eluted from the bead using a buffer
and 10 mM reduced glutathione. Separation was carried out using
SDS−PAGE followed by Western blotting using standard procedures
with anti-MBP (E8032S, New England BioLabs) and anti-mouse IgG-
Tr (sc-2781, Santa Cruz, CA).
Semiquantitative Real Time PCR. DU145 cells were incubated
at 37 °C with 31b (20 μM) in the presence or absence of 20 nM
calcitriol for 18 h. Total RNA was isolated from cells using an
RNAeasy kit (Qiagen). Genomic DNA was removed, and cDNA was
generated using equal amounts of RNA (QuantiTect reverse
transcription kit, Qiagen). The cDNA mixture was then diluted 5-
fold, and the QuantiFast SYBR Green PCR kit (Qiagen) was used for
the real time PCR following manufacturer’s recommendations. Primers
used in these studies are as follows: GAPDH forward primer 5-
accacagtccatgccatcac-3, reverse primer 5-tccaccaccctgttgctgta-3;
TRPV6 FP 5-ACTGTCATTGGGGCTATCATC-3, RP 5- CAGCA-
GAATCGCATCAGGTC-3. rReal-time rt-PCR was carried out on a
Mastercycler (Eppendorf). We used the ΔΔCt method to measure the
fold change in gene expression of target genes. Standard deviations
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Taosheng Chen, Fu-Yue Zeng, Wenwei Lin, and
Jimmy Cui for their assistance with the HTS at the St. Jude
High Throughput Screening Center, as well as Aaron Kosinski
for his assistance with protein preparation. We also thank Paul
Webb and Phuong Nguyen for providing us with the SRC2
plasmid. This work was supported by the University of
Wisconsin-Milwaukee (L.A.A.), NIH Grants DA031090
(L.A.A.) and DK58080 (R.K.G.), the American Lebanese
Syrian Associated Charities (ALSAC) (R.K.G.), and St. Jude
Children’s Research Hospital (R.K.G.).
ABBREVIATIONS USED
■
VDR, vitamin D receptor; NR, nuclear hormone receptor; FP,
fluorescence polarization; SRC2, steroid receptor coactivator 2;
SRC2-3, labeled steroid receptor coactivator 2 peptide; SAR,
structure−activity relationship; TRPV6, transient receptor
potential vanilloid type 6 gene; 1,25-(OH)2D3, 1,25-dihydrox-
yvitamin D3; DBD, DNA binding domain; LBD, ligand-binding
domain; RXR, retinoid X receptor; HTS, high throughput
screening; DMSO, dimethyl sulfoxide; HEK293, human
embryonic kidney cell; FRET, fluorescence resonance energy
transfer; LC−MS, liquid chromatography−mass spectrometry;
PAMPA, parallel artificial membrane permeability; AR,
androgen receptor; ERβ, estrogen receptor β; TRα, thyroid
receptor α; TRβ, thyroid receptor β; PPARγ, peroxisome
proliferator-activated receptor γ; DRIP205, vitamin D receptor
interacting protein 205; GST, glutathione S-transferase;
DU145, human prostate cancer cell; GAPDH, glyceraldehyde
3-phosphate dehydrogenase; LFER, linear free energy relation-
ship
REFERENCES
■
(1) Brumbaugh, P. F.; Haussler, M. R. 1 Alpha,25-dihydroxychole-
calciferol receptors in intestine. I. Association of 1 alpha,25-
dihydroxycholecalciferol with intestinal mucosa chromatin. J. Biol.
Chem. 1974, 249, 1251−1257.
(2) Jurutka, P. W.; Whitfield, G. K.; Hsieh, J. C.; Thompson, P. D.;
Haussler, C. A.; Haussler, M. R. Molecular nature of the vitamin D
receptor and its role in regulation of gene expression. Rev. Endocr.
Metab. Disord. 2001, 2, 203−216.
4649
dx.doi.org/10.1021/jm300460c | J. Med. Chem. 2012, 55, 4640−4651

Journal of Medicinal Chemistry
Article
(3) Feldman, D.; Pike, J. W.; Glorieux, F. H. Vitamin D, 2nd ed.;
Elsevier: Burlington, VT, 2005; Vol. 1−2.
(14) McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. A
common mechanism underlying promiscuous inhibitors from virtual
and high-throughput screening. J. Med. Chem. 2002, 45, 1712−1722.
(15) Huth, J. R.; Song, D.; Mendoza, R. R.; Black-Schaefer, C. L.;
Mack, J. C.; Dorwin, S. A.; Ladror, U. S.; Severin, J. M.; Walter, K. A.;
Bartley, D. M.; Hajduk, P. J. Toxicological evaluation of thiol-reactive
compounds identified using a la assay to detect reactive molecules by
nuclear magnetic resonance. Chem. Res. Toxicol. 2007, 20, 1752−1759.
(16) Chen, K. S.; DeLuca, H. F. Cloning of the human 1 alpha,25-
dihydroxyvitamin D-3 24-hydroxylase gene promoter and identifica-
tion of two vitamin D-responsive elements. Biochim. Biophys. Acta
1995, 1263, 1−9.
(17) Holick, M. F.; Kleiner-Bossaller, A.; Schnoes, H. K.; Kasten, P.
M.; Boyle, I. T.; DeLuca, H. F. 1,24,25-Trihydroxyvitamin D3. A
metabolite of vitamin D3 effective on intestine. J. Biol. Chem. 1973,
248, 6691−6696.
(18) Xie, W. H.; Bloomfield, K. M.; Jin, Y. F.; Dolney, N. Y.; Wang,
P. G. Lanthanide triflates catalyzed reactions of imines with indole in
protic media. Synlett 1999, 498−500.
(19) Ritchie, C. D.; Sager, W. F. An examination of structure−activity
relationships. Prog. Phys. Org. Chem. 1964, 2, 323−400.
(20) (a) Estebanez-Perpina, E.; Moore, J. M.; Mar, E.; Delgado-
Rodrigues, E.; Nguyen, P.; Baxter, J. D.; Buehrer, B. M.; Webb, P.;
Fletterick, R. J.; Guy, R. K. The molecular mechanisms of coactivator
utilization in ligand-dependent transactivation by the androgen
receptor. J. Biol. Chem. 2005, 280, 8060−8068. (b) Moore, J. M.;
Galicia, S. J.; McReynolds, A. C.; Nguyen, N. H.; Scanlan, T. S.; Guy,
R. K. Quantitative proteomics of the thyroid hormone receptor−
coregulator interactions. J. Biol. Chem. 2004, 279, 27584−27590.
(21) (a) Masuyama, H.; Brownfield, C. M.; St-Arnaud, R.;
MacDonald, P. N. Evidence for ligand-dependent intramolecular
folding of the AF-2 domain in vitamin D receptor-activated
transcription and coactivator interaction. Mol. Endocrinol. 1997, 11,
1507−1517. (b) Hong, H.; Kohli, K.; Garabedian, M. J.; Stallcup, M.
R. GRIP1, a transcriptional coactivator for the AF-2 transactivation
domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol. Cell.
Biol. 1997, 17, 2735−2744. (c) Li, H.; Gomes, P. J.; Chen, J. D. RAC3,
a steroid/nuclear receptor-associated coactivator that is related to
SRC-1 and TIF2. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8479−8484.
(22) Rachez, C.; Suldan, Z.; Ward, J.; Chang, C. P.; Burakov, D.;
Erdjument-Bromage, H.; Tempst, P.; Freedman, L. P. A novel protein
complex that interacts with the vitamin D3 receptor in a ligand-
dependent manner and enhances VDR transactivation in a cell-free
system. Genes Dev. 1998, 12, 1787−1800.
(23) Hsieh, J. C.; Sisk, J. M.; Jurutka, P. W.; Haussler, C. A.; Slater, S.
A.; Haussler, M. R.; Thompson, C. C. Physical and functional
interaction between the vitamin D receptor and hairless corepressor,
two proteins required for hair cycling. J. Biol. Chem. 2003, 278,
38665−38674.
(24) (a) Wissenbach, U.; Niemeyer, B.; Himmerkus, N.; Fixemer, T.;
Bonkhoff, H.; Flockerzi, V. TRPV6 and prostate cancer: cancer growth
beyond the prostate correlates with increased TRPV6 Ca2+ channel
expression. Biochem. Biophys. Res. Commun. 2004, 322, 1359−1363.
(b) Nijenhuis, T.; Hoenderop, J. G.; van der Kemp, A. W.; Bindels, R.
J. Localization and regulation of the epithelial Ca2+ channel TRPV6 in
the kidney. J. Am. Soc. Nephrol. 2003, 14, 2731−2740.
(4) Yamada, S.; Shimizu, M.; Yamamoto, K. Vitamin D receptor.
Endocr. Dev. 2003, 6, 50−63.
(5) Toell, A.; Polly, P.; Carlberg, C. All natural DR3-type vitamin D
response elements show a similar functionality in vitro. Biochem. J.
2000, 352 (Part2), 301−309.
(6) Kim, J. Y.; Son, Y. L.; Lee, Y. C. Involvement of SMRT
corepressor in transcriptional repression by the vitamin D receptor.
Mol. Endocrinol. 2009, 23, 251−264.
(7) Rachez, C.; Freedman, L. P. Mechanisms of gene regulation by
vitamin D(3) receptor: a network of coactivator interactions. Gene
2000, 246, 9−21.
(8) (a) Lamblin, M.; Spingarn, R.; Wang, T. T.; Burger, M. C.;
Dabbas, B.; Moitessier, N.; White, J. H.; Gleason, J. L. An o-
aminoanilide analogue of 1alpha,25-dihydroxyvitamin D(3) functions
as a strong vitamin D receptor antagonist. J. Med. Chem. 2010, 53,
7461−7465. (b) Kato, Y.; Nakano, Y.; Sano, H.; Tanatani, A.;
Kobayashi, H.; Shimazawa, R.; Koshino, H.; Hashimoto, Y.; Nagasawa,
K. Synthesis of 1alpha,25-dihydroxyvitamin D3-26,23-lactams
(DLAMs), a novel series of 1 alpha,25-dihydroxyvitamin D3
antagonist. Bioorg. Med. Chem. Lett. 2004, 14, 2579−2583. (c) Inaba,
Y.; Yoshimoto, N.; Sakamaki, Y.; Nakabayashi, M.; Ikura, T.;
Tamamura, H.; Ito, N.; Shimizu, M.; Yamamoto, K. A new class of
vitamin D analogues that induce structural rearrangement of the
ligand-binding pocket of the receptor. J. Med. Chem. 2009, 52, 1438−
1449. (d) Nakabayashi, M.; Yamada, S.; Yoshimoto, N.; Tanaka, T.;
Igarashi, M.; Ikura, T.; Ito, N.; Makishima, M.; Tokiwa, H.; DeLuca, H.
F.; Shimizu, M. Crystal structures of rat vitamin D receptor bound to
adamantyl vitamin D analogs: structural basis for vitamin D receptor
antagonism and partial agonism. J. Med. Chem. 2008, 51, 5320−5329.
(e) Saito, N.; Kittaka, A. Highly potent vitamin D receptor
antagonists: design, synthesis, and biological evaluation. ChemBioChem
2006, 7, 1479−1490. (f) Cho, K.; Uneuchi, F.; Kato-Nakamura, Y.;
Namekawa, J.; Ishizuka, S.; Takenouchi, K.; Nagasawa, K. Structure−
activity relationship studies on vitamin D lactam derivatives as vitamin
D receptor antagonist. Bioorg. Med. Chem. Lett. 2008, 18, 4287−4290.
(g) Herdick, M.; Steinmeyer, A.; Carlberg, C. Antagonistic action of a
25-carboxylic ester analogue of 1alpha, 25-dihydroxyvitamin D3 is
mediated by a lack of ligand-induced vitamin D receptor interaction
with coactivators. J. Biol. Chem. 2000, 275, 16506−16512. (h) Ishizuka,
S.; Kurihara, N.; Miura, D.; Takenouchi, K.; Cornish, J.; Cundy, T.;
Reddy, S. V.; Roodman, G. D. Vitamin D antagonist, TEI-9647,
inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin
D3 from pagetic bone marrow cells. J. Steroid Biochem. Mol. Biol. 2004,
89−90, 331−334.
(9) Moore, T. W.; Katzenellenbogen, J. A. Inhibitors of nuclear
hormone receptor/coactivator interactions. Annu. Rep. Med. Chem.
2009, 44, 443−457.
(10) Mita, Y.; Dodo, K.; Noguchi-Yachide, T.; Miyachi, H.;
Makishima, M.; Hashimoto, Y.; Ishikawa, M. LXXLL peptide mimetics
as inhibitors of the interaction of vitamin D receptor with coactivators.
Bioorg. Med. Chem. Lett. 2010, 20, 1712−1717.
(11) Teichert, A.; Arnold, L. A.; Otieno, S.; Oda, Y.; Augustinaite, I.;
Geistlinger, T. R.; Kriwacki, R. W.; Guy, R. K.; Bikle, D. D.
Quantification of the vitamin D receptor−coregulator interaction.
Biochemistry 2009, 48, 1454−1461.
(12) Boehm, M. F.; Fitzgerald, P.; Zou, A.; Elgort, M. G.; Bischoff, E.
D.; Mere, L.; Mais, D. E.; Bissonnette, R. P.; Heyman, R. A.; Nadzan,
A. M.; Reichman, M.; Allegretto, E. A. Novel nonsecosteroidal vitamin
D mimics exert VDR-modulating activities with less calcium
mobilization than 1,25-dihydroxyvitamin D3. Chem. Biol. 1999, 6,
265−275.
(25) Meyer, M. B.; Watanuki, M.; Kim, S.; Shevde, N. K.; Pike, J. W.
The human transient receptor potential vanilloid type 6 distal
promoter contains multiple vitamin D receptor binding sites that
mediate activation by 1,25-dihydroxyvitamin D3 in intestinal cells. Mol.
Endocrinol. 2006, 20, 1447−1461.
(26) (a) Arnold, L. A.; Estebanez-Perpina, E.; Togashi, M.; Jouravel,
N.; Shelat, A.; McReynolds, A. C.; Mar, E.; Nguyen, P.; Baxter, J. D.;
Fletterick, R. J.; Webb, P.; Guy, R. K. Discovery of small molecule
inhibitors of the interaction of the thyroid hormone receptor with
transcriptional coregulators. J. Biol. Chem. 2005, 280, 43048−43055.
(b) Hwang, J. Y.; Huang, W.; Arnold, L. A.; Huang, R.; Attia, R. R.;
Connelly, M.; Wichterman, J.; Zhu, F.; Augustinaite, I.; Austin, C. P.;
Inglese, J.; Johnson, R. L.; Guy, R. K. Methylsulfonylnitrobenzoates, a
(13) Arnold, L. A.; Kosinski, A.; Estebanez-Perpina, E.; Fletterick, R.
J.; Guy, R. K. Inhibitors of the interaction of a thyroid hormone
receptor and coactivators: preliminary structure−activity relationships.
J. Med. Chem. 2007, 50, 5269−5280.
4650
dx.doi.org/10.1021/jm300460c | J. Med. Chem. 2012, 55, 4640−4651

Journal of Medicinal Chemistry
Article
new class of irreversible inhibitors of the interaction of the thyroid
hormone receptor and its obligate coactivators that functionally
antagonizes thyroid hormone. J. Biol. Chem. 2011, 286, 11895−11908.
(27) (a) Ciesielski, F.; Rochel, N.; Moras, D. Adaptability of the
Vitamin D nuclear receptor to the synthetic ligand Gemini:
remodelling the LBP with one side chain rotation. J. Steroid Biochem.
Mol. Biol. 2007, 103, 235−242. (b) Vanhooke, J. L.; Benning, M. M.;
Bauer, C. B.; Pike, J. W.; DeLuca, H. F. Molecular structure of the rat
vitamin D receptor ligand binding domain complexed with 2-carbon-
substituted vitamin D3 hormone analogues and a LXXLL-containing
coactivator peptide. Biochemistry 2004, 43, 4101−4110.
(28) Lehen’kyi, V.; Raphael, M.; Oulidi, A.; Flourakis, M.;
Khalimonchyk, S.; Kondratskyi, A.; Gordienko, D. V.; Mauroy, B.;
Bonnal, J. L.; Skryma, R.; Prevarskaya, N. TRPV6 determines the effect
of vitamin D3 on prostate cancer cell growth. PLoS One 2011, 6,
e16856.
(29) Lehen’kyi, V.; Flourakis, M.; Skryma, R.; Prevarskaya, N.
TRPV6 channel controls prostate cancer cell proliferation via Ca(2+)/
NFAT-dependent pathways. Oncogene 2007, 26, 7380−7385.
(30) Stern, P. H.; De Olazabal, J.; Bell, N. H. Evidence for abnormal
regulation of circulating 1 alpha,25-dihydroxyvitamin D in patients
with sarcoidosis and normal calcium metabolism. J. Clin. Invest. 1980,
66, 852−855.
(31) Bosch, X. Hypercalcemia due to endogenous overproduction of
1,25-dihydroxyvitamin D in Crohn’s disease. Gastroenterology 1998,
114, 1061−1065.
(32) Shirakawa, S.; Kobayashi, S. Carboxylic acid catalyzed three-
component aza-Friedel−Crafts reactions in water for the synthesis of
3-substituted indoles. Org. Lett. 2006, 8, 4939−4942.
(33) Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.; Moras, D.
The crystal structure of the nuclear receptor for vitamin D bound to its
natural ligand. Mol. Cell 2000, 5, 173−179.
4651
dx.doi.org/10.1021/jm300460c | J. Med. Chem. 2012, 55, 4640−4651