Development of novel vitamin D receptor-coactivator inhibitors

Article abstract of DOI:10.1021/ml400462j

Nuclear receptor coregulators are master regulators of transcription and selectively interact with the vitamin D receptor (VDR) to modulate cell differentiation, cell proliferation, and calcium homeostasis. Herein, we report the syntheses and evaluation of highly potent and selective VDR-coactivator inhibitors based on a recently identified 3-indolylmethanamine scaffold. The most active compound, PS121912, selectively inhibited VDR-mediated transcription among eight other nuclear receptors tested. PS121912 is also selectively disrupting the binding between VDR and the third nuclear receptor interaction domain of the coactivator SRC2. Genetic studies revealed that PS121912 behaves like a VDR antagonist by repressing 1,25-(OH)₂D₃ activated gene transcription. In addition, PS121912 induced apoptosis in HL-60.

Full text of DOI:10.1021/ml400462j

Letter
pubs.acs.org/acsmedchemlett
Development of Novel Vitamin D Receptor−Coactivator Inhibitors
Preetpal S. Sidhu,† Nicholas Nassif,† Megan M. McCallum,† Kelly Teske,† Belaynesh Feleke,†
Nina Y. Yuan,† Premchendar Nandhikonda,† James M. Cook,† Rakesh K. Singh,‡ Daniel D. Bikle,§
and Leggy A. Arnold*,†
†Department of Chemistry and Biochemistry, University of Wisconsin−Milwaukee, Milwaukee, Wisconsin 53211, United States
‡Molecular Therapeutics Laboratory, Program in Women’s Oncology, Department of Obstetrics and Gynecology, Woman and
Infant’s Hospital of Rhode Island, Alpert Medical School of Brown University, Providence, Rhode Island 02903, United States
§Endocrine Research Unit, Department of Medicine, Veterans Affairs Medical Center, San Francisco, California 94121, United States
S
* Supporting Information
ABSTRACT: Nuclear receptor coregulators are master regulators of
transcription and selectively interact with the vitamin D receptor (VDR)
to modulate cell differentiation, cell proliferation, and calcium homeo-
stasis. Herein, we report the syntheses and evaluation of highly potent
and selective VDR−coactivator inhibitors based on a recently identified
3-indolylmethanamine scaffold. The most active compound, PS121912,
selectively inhibited VDR-mediated transcription among eight other
nuclear receptors tested. PS121912 is also selectively disrupting the
binding between VDR and the third nuclear receptor interaction domain
of the coactivator SRC2. Genetic studies revealed that PS121912
behaves like a VDR antagonist by repressing 1,25-(OH)2D3 activated
gene transcription. In addition, PS121912 induced apoptosis in HL-60.
KEYWORDS: Vitamin D receptor, VDR, steroid receptor coactivator, fluorescence polarization, high throughput screening,
3-indolyl-methanamines, CYP24A1, CAMP, apoptosis, HL60
VDR.11 Interestingly, none of these antagonists have been
he vitamin D receptor (VDR) is a ligand-inducible
Ttranscription factor that belongs to the superfamily of
further developed as therapeutics.
nuclear receptors (NR).1 Like most NRs, VDR has a modular
structure consisting of a DNA binding domain and a ligand
Recently, coregulator proteins have been identified as
transcriptional master regulators.12 In its unligated state, VDR
is bound with corepressors. Upon binding with 1,25-(OH)2D3,
VDR undergoes a conformational change that disrupts
corepressor binding and enables the interaction between
VDR and coactivators. The activated form of VDR interacts
with retinoid X receptor (RxR) to form a heterodimer that
binds to vitamin D response elements on the DNA leading to
transcription of associated genes. VDR is known to interact
with 2700 human DNA sites and effects the expression of 229
gene products.13
A new approach to modulate VDR function without causing
hypercalcemia but to induce cell proliferation and differ-
entiation is the design of small molecules that target the
interactions between VDR and coregulators. In general, the
approach of inhibiting protein−protein interactions is challeng-
ing in terms of potency and specificity. Further adding to the
complexity is the fact that the family of more than 300
coregulators interact with different NR.14 Thus, selectivity with
respect to different NRs and inhibition of specific NR−
binding domain (LBD) connected by a flexible hinge region.
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), a hormonally
active form of vitamin D, is the endogenous high affinity
ligand for VDR.2 Traditionally, 1,25-(OH)2D3 is known for its
role in cell differentiation and calcium homeostasis, but lately it
has been reported to be involved in inflammation, immune
response, and cell proliferation.3
Human clinical studies with 1,25(OH)2D3 are dose-limited
because of the reported induction of hypercalcemia and
hypercalciurea.4,5 This dysregulation can cause psychosis,
bone pain, calcification of soft tissue, coronary artery disease,
and, in severe cases, coma and cardiac arrest. During the last
decades, hundreds of VDR agonists have been synthesized to
develop VDR ligands with lower calcemic activity. However,
two synthetic VDR ligands that were developed to systemically
treat cancer were not active or induced hypercalcemia in clinical
trials.6,7 In addition, a smaller number of VDR antagonist were
developed, which include the irreversible antagonist TEI-96478
and those bearing bulky side chains such as 25-carboxylic esters
(ZK168218 and ZK159222),9 26-adamantly substituted antag-
onists (ADTT and analogues),10 and 22-butyl-branched
compounds that ultimately destabilized the active form of
Received: November 13, 2013
Accepted: January 8, 2014
Published: January 8, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
inhibitor. Importantly, we demonstrated that these unique
compounds modulated gene transcription and cell proliferation.
A diversified library of 3-indolylmethanamines was generated
by using one-pot modified microwave-based Aza−Friedel−
Craft reaction (Scheme 1).
Scheme 1. Improved Synthesis of 3-Indolylmethanamines
The first step involves the combination of an amine and an
aromatic aldehyde to form an imine intermediate at 100 °C
within 10 to 30 min using microwave irradiation. To that
solution, indole and a catalytic amount of decanoic acid was
added to afford the desired products at room temperature,
which are summarized in Figure 1. The purified compounds
were analyzed using a battery of biophysical and biochemical
assays (Table 1).
The biophysical properties determined include small
molecule solubility and permeability. The solubility of
synthesized 3-indolylmethanamines in PBS buffer (pH 7.4)
with 5% DMSO ranged between 150 and 3 μM. The
compounds substituted with polar heterocyclic side chains
showed excellent solubility (>100 μM). The small molecule
permeability was determined using a parallel artificial
membrane permeation assay (PAMPA) employing a hexade-
cane membrane. In comparison to the used standards
coregulator interactions is essential for novel promising NR−
coregulator inhibitors.
Various small molecule NR−coactivator inhibitors have been
discovered for thyroid receptor (TR), pregane X receptor
(PxR), estrogen receptor (ER), and the androgen receptor
(AR).15 Recently, the first reversible VDR−coactivator
inhibitors were developed to disrupt the VDR-mediated gene
transcription, but these compounds lack the important
selectivity among other NRs.16,17 However, drawing from a
pool of hundreds of thousands of molecules, high throughput
screening (HTS) is a superior method to identify ideal
candidates that not only possess selectivity for VDR but also
for the various VDR−coregulator interactions.
The first HTS method to quantify the interaction between
VDR and coregulator peptides was introduced by Arai et al.
using fluorescence intensity.18 Six compounds were investigated
in this study. Recently, we described a HTS campaign in
collaboration with NIH investigating 390 000 molecules that
led to the identification of GW0742 as a competitive VDR
antagonist.19 Furthermore, we reported a second HTS
campaign with 275 000 molecules that resulted in the
development of an irreversible VDR−coactivator binding
inhibitor.20 Herein, we report our recent successes to improve
the potency and selectivity of these VDR−coactivator binding
(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
majority of 3-indolylmethanamines exhibited medium to high
permeability (Table 1).
A fluorescence polarization (FP) assay was used to determine
the ability of synthesized molecules to inhibit the interactions
between VDR-LBD and Alexa Fluor 647 labeled coactivator
peptide SRC2−3. The compounds were analyzed in a dose-
dependent manner, and potencies are reported as IC50 values.
In order to assess the ability of 3-indolylmethanamines to
Figure 1. Structures of synthesized 3-indolylmethanamines.
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ACS Medicinal Chemistry Letters
Letter
Table 1. Summary of Biophysical and Biochemical Properties of 3-Indolylmethanamines
a
b
c
d
e
compd
solubility (μM)
permeability log (Pe) (cm/s) VDR-SRC2−3 IC50 (μM)
VDR-mediated transcription IC50 (μM)
toxicity LC50 (μM)
1
2
3
4
5
6
7
8
9
3.6
7.36 0.10
7.03 0.07
6.26 0.02
6.64 0.02
6.16 0.02
5.86 0.03
6.26 0.01
7.42 0.52
7.68 0.31
n.d.
12.8 0.8
7.2 0.4
22.8 2.2
21.8 1.7
11.3 0.5
n.o.
n.o.
36.4 7.4
>50
57.9
96.5
91.5
94.0
48.1
25.2
8.5
3.0 0.7
2.2 0.5
4.4 2.1
3.2 0.7
9.3 3.1
5.0 3.4
5.2 2.7
7.3 2.9
5.8 3.1
0.59 0.1
5.4 3.5
5.8 2.1
9.1 2.2
3.4 0.6
3.2 1.4
3.7 1.8
14.1 6.6
n.o.
15.3 1.7
14.1 1.6
41.7 13.3
>100
17.2 4.8
15.6 1.4
63.1 13.6
n.o.
>75
>50
19.8
29.4
68.9
60.4
45.5
18.2
20.1
123.3
128.3
52.8
3.6
>100
10
>75
f
PS121912
12
6.36 0.02
6.21 0.01
5.60 0.19
6.67 0.04
6.41 0.01
6.47 0.11
6.50 0.14
7.57 0.32
n.d.
12.4 0.7 (8.1 2.3)
7.2 0.4
59.9 4.5
35.2 12.5
9.5 0.4
>75
27.3 2.7
42.7 2.7
>75
13
14
53.7 16.8
10.8 1.6
>100
15
16
17
51.5 10.4
14.2 1.4
66.8 10.3
16.7 0.8
11.9 0.7
n.o.
>100
18
>100
19
>100
20
176.7
97.9
99.3
114.4
3.0
6.71 0.01
6.14 0.13
6.92 0.16
n.d.
6.1 2.5
3.8 2.1
n.o.
>75
21
>75
22
>100
23
>75
n.o.
>75
24
n.d.
n.o.
>25
>100
25
95.3
130.1
120.3
101.8
150.2
144.8
6.68 0.11
5.87 0.01
6.09 0.01
6.14 0.04
5.78 0.01
5.88 0.02
15.8 1.2
101.4 15.2
29.3 5.7
17.3 0.7
32.3 6.4
>25
>75
26
12.2 3.3
6.7 2.5
8.5 2.7
5.4 2.1
n.d.
>75
27
>100
28
>50
29
18.6 2.5
>75
30
n.o.
a
b
Solubility was determined in phosphate-buffered saline at pH 7.4. Permeability was measured using the parallel artificial membrane permeation
c
assay (PAMPA) at neutral pH (pH = 7.4). 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 molecules. IC50 values were obtained by fitting data
obtained after 2 h to the following equation: Y = bottom + (top − bottom)/(1 + 10((log IC − X)hillslope)) using three independent experiments in
50
d
quadruplet. Transcription assay: HEK293T cells were transfected with CMV-VDR and a CYP24A1 promoter driven luciferase expression vector in
e
f
the presence of 1,25(OH)2D3. Toxicity was determined under the conditions of the transcription assay using CellTiter-Glo. Two-hydrid assay:
HEK293T cells were transfected with a VP16-VDR-LBD, SRC1-GAL4, and luciferase reporter plasmid vector in the presence of 1,25(OH)2D3.21
n.d. = not determined; n.o. = not observed.
inhibit the VDR−coactivator interaction in cells, a VDR two-
hybrid assay and a VDR-mediated transcription assay was used.
The toxicity of compounds under the conditions of the
transcription assay was determined with CellTiter-Glo
(Promega).
All 3-indolylmethanamines in group A (Table 1, compounds
1−10 and PS121912) exhibited cellular activities in the low
micromolar to nanomolar range. The compound activities
measured with the biochemical FP assay are generally higher
probably due to compound off-targets effects. The compound
toxicities are ranging between 14.1 and >100 μM. The
compound PS121912 exhibited the highest activity in the
VDR-mediated transcription assay (IC50 = 590 100 nM) and
largest therapeutic index.
For compounds in group B, bearing benzylamine sub-
stitutents, low micromolar activities were determined for the
transcriptional inhibition of VDR. The activities for the FP
assay ranged between 7.2 to 59.9 μM. Importantly, 3-
indolylmethanamines are irreversible inhibitors acting through
the formation of an azafulvenium salt that react with
nucleophilies like mercaptoethanol (see Supporting Informa-
tion). Thus the activity of these inhibitors depends on the
incubation time, the environment, and the electronic
substituent effects.20 Compound 15 was the most toxic
compound within the library of 3-indolylmethanamines with
a LD50 value of 10.8 1.6 μM.
For compounds in group C, various heterocyclic substituents
were introduced. Interestingly, the majority of these 3-
indolylmethanamine were not toxic but very active inhibitors
of VDR-mediated transcription. Compound 16 exhibited the
largest therapeutic index of more than 31 in group C, but it was
still inferior to compound PS121912 with a therapeutic index of
46. The substitution of the secondary nitrogen by oxygen or
carbon prevented the generation of a reactive electrophilic
compound and thus resulted in inactive compounds 22 and 23.
The NR-selectivity of the most potent compound, PS121912,
was determined by measuring the inhibition of transcription for
a panel of nine different NRs. These include the peroxisome
proliferator-activated receptors α, γ, and δ, the retinoic acid
receptor α, the thyroid receptors α and β, and the estrogen
receptors α and β. The results are summarized in Table 2.
The 3-indolylmethanamine PS121912 selectively inhibited
the transcription of VDR at nanomolar concentrations, whereas
for all other NRs we determined IC50 values ranging between
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ACS Medicinal Chemistry Letters
Letter
Table 2. Inhibition of NR-Mediated Transcription in the
b
Presence of Compound PS121912
a
compd
nuclear receptor
IC50 (μM)
b
1
2
3
4
5
6
7
8
9
VDR
0.59 0.1
20.8 1.1
c
PPAR-α
PPAR-γ
PPAR-δ
RxR-α
TR-α
d
>25
e
>30
f
>25
g
>30
g
TR-β
24.1 1.1
26.2 3.3
Figure 3. Gene regulation by PS121912 (7.5 μM) in HL-60 cells after
18 h in the presence and absence of 1,25-(OH)2D3 (20 nM). Standard
errors of mean were calculated from two biological independent
experiments performed in triplicate. Stars represent P < 0.001 (***)
(Student’s t test).
h
ER-α
h
ER-β
>25
a
Three independent experiments were conducted in quadruplicate,
and data was analyzed using nonlinear regression with variable slope
b
c
(GraphPrism). 1,25(OH)2D3 (10 nM). GW7647 (30 nM).
d
e
f
Rosiglitazone (300 nM). GW0742 (50 nM). Bexarotene (200
g
h
nM). T3 (10 nM). Estradiol (10 nM).
Figure 4. HL-60 viability, toxicity, and apoptosis assay after 18 h in the
presence of PS121912.
Figure 2. Selectivity studies for VDR−coactivator interactions
inhibition in the presence of PS121912 using FP assay. VDR-LBD
(1−10 μM), VDR agonist LG190178 (5 μM), and different
coactivator peptides (7 nM) were incubated for 3 h in the presence
of different concentrations of compound PS121912.
compound PS121912 exhibited a loss of induction of
transcription, whereas cells treated with only PS121912 showed
comparable expression to that of vehicle treated HL-60 cells.
The transcriptional inhibition of the CYP24A1 gene product
24-hydroxylase by PS121912 is important because it has been
shown that deactivation of 24-hydroxylase, using vitamin D
analogues29 or general P450 enzyme inhibitors30 promotes
antiproliferation and differentiation of cancer cells.
20 and 26 μM. It is important to point out that the LD50 value
of PS121912 in HEK293T cells was 27.3 μM; thus, the reduced
luciferase activity measured at concentrations above 20 μM is
probably due to cell death and not caused by inhibition of NR-
mediated transcription. Therefore, the NR selectivity of
PS121912 is at least 35-fold.
The selectivity of PS121912 to inhibit the interaction
between VDR and coactivator SRC1,22 SRC2,23 SRC3,24 or
DRIP20525 was assessed using FP assays employing different
NR interaction domains (NIDs) of these coactivators (Figure
2).
The quantification of interactions between VDR and
different coactivator peptides was reported recently.26 Among
the interactions tested, only the interactions between VDR and
coactivator peptide SRC2−3 was fully inhibited by compound
PS121912 with an IC50 value of 12.4 0.7 μM (Table 1). The
NIDs of SRC1, SRC3, and DRIP2, which exhibit a similarly
strong interaction with VDR, were not inhibited by PS121912.
A very weak partial inhibition for the VDR−SRC2−2
interaction was observed in the presence of PS121912 leaving
88% of the VDR−coactivator binding intact.
Therefore, viable, apoptotic, and dead HL-60 cells were
determined in the presence of different concentrations of
PS121912 as depicted in Figure 4.
Three different assays were used: (a) CellTiter-Glo that
quantifies the amount of ATP (toxicity), (b) CellTiter-Fluor
that quantifies the amount of active cellular proteases
(viability), and (c) Caspase-Glo 3/7 that quantifies the amount
of active cellular caspase 3 and 7 (apoptosis). As expected, the
amount of intact and dead HL-60 cells at the same
concentration of PS121912 was inversely proportional with
EC50 values of around 20 μM. Importantly, a dose-dependent
increase of caspase 3 and 7 activity was observed in the
presence of PS121912. The increase of the apoptotic enzymes
was proportional to the amount of dead cells, confirming that
programmed cell death and not necrosis was the mode of
action of PS121912 at higher concentrations.
Furthermore, the expression levels of VDR target genes
CYP24A127 and CAMP28 were determined in HL-60 cells
treated with 7.5 μM of compound PS121912 in the presence
and absence of 20 nM 1,25-(OH)2D3 for 18 h. The results are
summarized in Figure 3.
A strong induction of CYP24A1 and CAMP by 1,25-
(OH)2D3 was observed. Cells treated 1,25-(OH)2D3 and
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed procedures and characterization of all compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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ACS Medicinal Chemistry Letters
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AUTHOR INFORMATION
Corresponding Author
*(L.A.A.) E-mail: arnold2@uwm.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
Funding
This work was supported by the University of Wisconsin−
Milwaukee [to L.A.A.], the UWM Research Growth Initiative
(RGI grant 2012) [to L.A.A.], the NIH R03DA031090 [to
L.A.A.], the UWM Research Foundation (Catalyst grant), the
Lynde and Harry Bradley Foundation [to L.A.A.], the Richard
and Ethel Herzfeld Foundation [to L.A.A.], and RO1
AR050023 [to D.D.B.].
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
We thank Stephen D. Ayers for providing us with the nuclear
receptor plasmids.
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ABBREVIATIONS
■
NR, nuclear receptors; VDR, vitamin D receptor; LBD, ligand
binding domain; SRC, steroid receptor coactivator; DMSO,
dimethylsulfoxide; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3;
RxR, retinoid X receptor; TR, thyroid receptor; PxR, pregame
X receptor; ER, estrogen receptor; AR, androgen receptor;
HTS, high throughput screening; PAMPA parallel artificial
membrane permeation assay; CYP24A1, cytochrome
P450_24A1; CAMP, cathelicidin antimicrobial peptide gene
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