LXXLL peptide mimetics as inhibitors of the interaction of vitamin D receptor with coactivators

Article abstract of DOI:10.1016/j.bmcl.2010.01.079

Suppression of vitamin D receptor (VDR)-mediated transcription is expected be of therapeutic value in Paget's disease. Once an agonist activates VDR, recruitment of additional coactivator proteins is essential for transcription. Neither non-secosteroidal VDR antagonists nor non-peptide coactivator binding inhibitors for VDR have been reported so far. Based on the X-ray structure of VDR and an LXXLL-containing peptide fragment of the coactivator (where L is leucine and X is any amino acid), which adopts a partially α-helical conformation, benzodiazepine molecules were rationally designed as non-peptide coactivator mimetics. TR-FRET assay showed that the synthesized compounds inhibited the interaction between VDR and a coactivator peptide fragment. Compound 2 showed an IC₅₀ of 20 μM. Compound 2 also inhibited VDR-mediated transcription, and this activity was independent of the concentration of co-existing agonist. Furthermore, compound 2 did not inhibit estrogen receptor α-mediated transcription, indicating that it is not a non-selective inhibitor of other nuclear receptors.

Full text of DOI:10.1016/j.bmcl.2010.01.079

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1712–1717
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
LXXLL peptide mimetics as inhibitors of the interaction of vitamin D
receptor with coactivators
Yusuke Mita a, Kosuke Dodo b, Tomomi Noguchi-Yachide a, Hiroyuki Miyachi c, Makoto Makishima d,
a,
Yuichi Hashimoto a, , Minoru Ishikawa
*
*
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b Synthetic Organic Chemistry Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
c Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 1-1-1, Tsushima-Naka, Kita-ku, Okayama
700-8530, Japan
d Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Suppression of vitamin D receptor (VDR)-mediated transcription is expected be of therapeutic value in
Paget’s disease. Once an agonist activates VDR, recruitment of additional coactivator proteins is essential
for transcription. Neither non-secosteroidal VDR antagonists nor non-peptide coactivator binding inhib-
itors for VDR have been reported so far. Based on the X-ray structure of VDR and an LXXLL-containing
peptide fragment of the coactivator (where L is leucine and X is any amino acid), which adopts a partially
Received 3 December 2009
Revised 7 January 2010
Accepted 8 January 2010
Available online 21 January 2010
a
-helical conformation, benzodiazepine molecules were rationally designed as non-peptide coactivator
mimetics. TR-FRET assay showed that the synthesized compounds inhibited the interaction between
VDR and a coactivator peptide fragment. Compound 2 showed an IC50 of 20 M. Compound 2 also inhib-
ited VDR-mediated transcription, and this activity was independent of the concentration of co-existing
agonist. Furthermore, compound 2 did not inhibit estrogen receptor a-mediated transcription, indicating
that it is not a non-selective inhibitor of other nuclear receptors.
Keywords:
Vitamin D
Antagonist
Co-factor
Nuclear receptor
Non-peptide
l
Molecular design
Protein interaction inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
The active form of vitamin D, 1,25-dihydroxyvitamin D3
(1,25(OH)2D3), is associated with regulation of calcium homeosta-
sis, bone mineralization, proliferation and differentiation of various
types of cells, and immune modulation.1–3 These biological effects
of 1,25(OH)2D3 are known to be elicited by binding of 1,25(OH)2D3
to the ligand binding domain (LBD) of vitamin D receptor (VDR), a
member of the nuclear receptor (NR) superfamily. This binding re-
sults in heterodimer formation with retinoid X receptor (RXR),
which enables high-affinity binding to the vitamin D-responsive
element (VDRE) sequence within, and subsequent transcription
of vitamin D target genes, including the genes encoding bone pro-
teins osteocalcin and osteopontin, and a metabolic enzyme, 25-
hydroxyvitamin D3-24-hydroxylase.4,5 Chemical modification of
1,25(OH)2D3 has yielded a number of secosteroidal agonists that
bind to the LBD of VDR. Some of them have been reported to elicit
a higher ratio of desirable effects to unwanted calcium mobiliza-
tion effects.1 One of these analogs, MC903 is currently in use as a
topical treatment for mild to moderate psoriasis.6 In addition,
non-secosteroidal VDR agonists7–10 with greater stability, easier
synthesis, and reduced calcium-raising effects have been devel-
oped. Some of them do not bind to serum vitamin D binding pro-
tein,7 a property that has been correlated with lower calcemic
potential in vivo.11
Paget’s disease of bone (PD) is characterized by an increased
number of osteoclasts and excessive bone resorption in focal
areas.12 The excessive breakdown and formation of bone tissue
that occur with PD cause weakened bones, resulting in bone pain,
arthritis, deformities and fractures. Osteoclast precursors from pa-
tients with PD are hyperresponsive to 1,25(OH)2D3.13 This hyperre-
sponsiveness may promote osteoclast formation and play a role in
the pathogenesis of PD. Therefore, VDR antagonists are expected to
be of therapeutic value. In fact, VDR antagonists were reported to
suppress excessive bone resorption and osteoclast formation.13,14
Many vitamin D analogs have been synthesized so far, but almost
all of these compounds are VDR agonists. Only a few families of
secosteroidal VDR antagonists that bind to LBD have been re-
ported,15–21 and no non-secosteroidal VDR antagonists are known.
As an alternative approach to block the VDR signal, we focused
on the interaction between 1,25(OH)2D3-activated VDR and coacti-
vator proteins. Once the ligand-activated VDR/RXR heterodimer
binds to VDRE on a target gene, the complex recruits additional
coactivators, including vitamin D receptor interacting proteins
* Corresponding authors. Tel.: +81 3 5841 7849; fax: +81 3 5841 8495.
E-mail addresses: hashimot@iam.u-tokyo.ac.jp (Y. Hashimoto), m-ishikawa@
iam.u-tokyo.ac.jp (M. Ishikawa).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.079

Y. Mita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1712–1717
1713
(DRIP) and the steroid receptor coactivator (SRC) family of pro-
teins, which are essential for transcriptional activation of the
responsive genes.22,23
groups in 1 were designed to mimic the leucine side chains in posi-
tions i, i+3 and i+4 of the -helix. The carbonyl group in the diaze-
a
pine moiety was anticipated to act as a part of the charge clamp by
forming a hydrogen bond with the amino group of Lys 242. As the
other part of the charge clamp, interacting with the carboxyl group
of Glu 416, an anilino group was introduced to mimic the main
chain of Leu 630. In addition, several side chains were introduced
as candidate substitutes for the main chain of Met 629 that would
interact with Glu 416.
Our plan for the synthesis of benzodiazepine 1 is outlined in
Scheme 1. We envisioned a late-stage convergent installation of
the charge clamp. The benzodiazepine 2, in turn, was expected to
be obtained by cyclization of 3 via Buchwald–Hartwig cross cou-
pling reaction. Introduction of the i-butyl group was to be accom-
plished by Suzuki coupling reaction.
Nucleophilic substitution of commercially available 4 with 4-
methoxybenzylamine followed by removal of the PMB group gave
compound 6 (Scheme 2). Successive iodination39 of 6 and reduction
of the nitrile group afforded tetrasubstituted benzene 8. After reduc-
tive amination of the primary amino group of 8, amidation with N-
Boc leucine furnished 10. The regioselectivity of the reductive ami-
nation was confirmed with 7, having diBoc protection (Scheme 3).
DiBoc-protected 12 was converted to triBoc-protected 13 by the
same method used to prepare 10 from 7. Removal of the triBoc group
of 13 followed by regioselective introduction of a Boc group at the
primary amino group led to 10. Next, two kinds of coupling reactions
were investigated (Scheme 2). Suzuki coupling reaction40 of 10 with
boronateafforded3in64%yield. Stillecouplingreactionof10didnot
give a better result. After removal of the Boc group, cyclization by
means of the intramolecular Buchwald–Hartwig coupling reaction41
was studied (Table 1). When the reaction was carried at 85 °C
according to the literature, the reaction proceeded in only 14% yield
(entry 1). The highest yield (39%) of 2 was obtained when the reac-
tion was carried at 110 °C (entry 2). Microwave-promoted reaction
at 130 °C gave a complex mixture (entry 3). Hydrogenation of 2 gave
1a possessing the saturated alkyl chain.
The coactivator proteins of NRs, including VDR, possess multi-
ple copies of a general amino acid sequence motif, that is, a con-
served LXXLL motif (where L is leucine and X is any amino acid).
Extensive studies have shown that this short LXXLL sequence is
necessary and sufficient for the binding of these proteins to NRs
and for enhancing transcriptional activity.24 In the search for inhib-
itors of this interaction, various short peptide derivatives based on
the LXXLL sequence have been shown to disrupt the interactions of
coactivators with NRs,25–27 including VDR.28,29 However, there
have been only a few reports of non-peptide inhibitors designed
to bind to this surface region of NR and block the binding of coac-
tivators (Fig. 1).30–36 In particular, no non-peptide inhibitor of VDR-
coactivator binding has been reported so far. In this report, we
present the design, synthesis and evaluation of non-peptide inhib-
itors of the interaction between activated VDR and a coactivator
peptide fragment.
The X-ray structure of the complex consisting of 1,25(OH)2D3-
bound VDR and
KNHPMLMNLLKDN-NH2) of the coactivator DRIP 205 has been re-
ported (Fig. 2a).37 The LXXLL peptide adopts a short
-helical con-
a
peptide fragment (residues 625–637:
a
formation, and binds in a surface groove formed by helices 3, 4, and
12 of VDR. The side chains of leucines 630, 633 and 634 of the
LXXLL motif are buried within the pocket and surrounded by
hydrophobic amino acids of VDR. In addition, the carboxylate oxy-
gens of Glu 416 in VDR accept hydrogens from the main chain
amides of Met 629 and Leu 630, and the primary amino group of
Lys 242 in VDR donates a hydrogen to the main-chain carbonyl
group of Leu 633. These hydrogen bonds form a ‘charge clamp’,
as previously described for the interaction between other NRs
and coactivators.38
To generate a non-peptide compound that inhibits the interac-
tion between VDR and its coactivator, we employed structure-
based drug design. We focused on five atoms in the pharmaco-
phore of the peptide fragment as shown in Figure 2b because these
Introduction of the charge clamp moieties was achieved as
shown in Scheme 4. Neither reductive amination of 1a nor depro-
tection of the nosyl group introduced at the non-substituted anili-
no group of 1a proceeded. Instead, alkylation of 1a with alkyl
bromide gave 1b in 33% yield. After removal of the Boc group, acet-
ylation of 1c afforded 1d. Guanidino derivative 1e was synthesized
from 1a by the use of a pyrazole reagent.
atoms are expected to be spatially rigidly fixed due to
a-helical
conformation. We designed non-peptide templates that mimic
the shape of the above-mentioned pharmacophore in the VDR-
bound conformation of the coactivator. The benzodiazepine deriv-
ative 1 (Fig. 2c) was designed as a possible structure to match the
pharmacophore, as shown in Figure 2d. Three branched alkyl
Figure 1. Reported inhibitors of binding between estrogen receptor (ER) or androgen receptor (AR) and their coactivators.

1714
Y. Mita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1712–1717
Figure 2. (a) Interaction between coactivator peptide fragment and VDR (PDB ID 1RK3). The VDR surface is shown in gray. The peptide is shown in green or magenta. Key
leucines in the peptide are shown in magenta. The image was drawn with PYMOL. (b) Key coactivator peptide fragment (Met 629-Leu 634) and important atoms for the
interaction. (c) Designed benzodiazepine 1. (d) Superposition of (b) and (c).
Scheme 1. Retrosynthetic analysis of benzodiazepine 1.
We used a time-resolved fluorescence resonance energy trans-
fer (TR-FRET) assay kit (Invitrogen) to evaluate the activity of syn-
thesized compounds. Glutathione-S-transferase (GST)-tagged VDR-
LBD (0.5 nM), terbium-labeled anti-GST antibody, a fluorescein-la-
beled fragment of coactivator peptide (100 nM), and 1,25(OH)2D3
(10 nM) were mixed and incubated. The binding affinity between
VDR and coactivator peptide is evaluated in terms of the TR-FRET
ratio (520 nm:495 nm). In the presence of agonist (1,25(OH)2D3),
the labeled coactivator peptide binds to VDR-LBD, allowing trans-
fer of fluorescence resonance energy from Tb donor to the fluores-
cence acceptor. The binding affinity was maximum when the
concentration of 1,25(OH)2D3 was higher than 10 nM. Inhibition
of binding between VDR and coactivator by synthesized com-
pounds was assayed in terms of decrease of this FRET signal. The
assay was performed in quintuplicate. After eliminating the high-
est and the lowest points, the average value of the remaining trip-
licate points was calculated. This assay was highly reproducible. A
secosteroidal VDR antagonist DLAM-2P17 was used as a positive
control. Under the assay conditions used, DLAM-2P (10 M) inhib-
ited the binding by 87%. Synthesized compounds 1a and 2 inhib-
ited the FRET signal at 30
M (Table 2). Among them, 242
inhibited coactivator binding with an IC50 value of 20 M. Com-
pound 2, possessing an isocrotyl group, showed stronger activity
than saturated 1a, which may indicate that the isocrotyl group fits
better inside the hydrophobic pocket than does the isobutyl group.
To investigate the cell-level VDR-inhibitory activity of the ben-
zodiazepine series, we utilized a VDR-responsive reporter gene as-
say with CMX-GAL4N-hVDR LBD as the recombinant receptor
gene, TK-MH100x4-LUC as the reporter gene, and the CMX b-galac-
tosidase gene for normalization, as previously reported.43 The cells
were incubated with 1,25(OH)2D3 (3 nM) in the presence or ab-
sence of a benzodiazepine or DLAM-2P. After incubation, cells were
assayed for luciferase reporter gene and b-galactosidase activities.
None of the compounds evaluated in our experiments reduced
l
l
l

Y. Mita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1712–1717
1715
Scheme 2. Reagents and conditions: (a) 4-methoxybenzylamine, 140 °C, 91%; (b) DDQ, DCM, H2O, rt, 85%; (c) NIS, AcOH, rt, 82%; (d) BH3, THF, 85 °C, 69%; (e) 3-
methylbutanal, NaBH(OAc)3, AcOH, DCM, 0 °C, 59%; (f) N-Boc-leucine, EDC, i-Pr2EtN, HOBt, DCM, rt, 82%; (g) 2-methyl-1-propylboronic acid pinacol ester, PdCl2(dppf), K3PO4,
DMF, 80 °C, 64%; (h) TFA, DCM, rt, 98%; (i) Pd2(dba)3, (R)-BINAP, Cs2CO3, toluene, 110 °C, 39%; (j) 10% Pd/C, H2 (0.3 MPa), AcOEt, 50 °C, 100%.
Scheme 3. Reagents and conditions: (a) (Boc)2O, DMAP, i-Pr2EtN, THF, rt, 89%; (b)
BH3, THF, 100 °C, 55%; (c) 3-methylbutanal, NaBH(OAc)3, AcOH, DCM, 0 °C, 76%; (d)
N-Boc-leucine, EDC, i-Pr2EtN, HOBt, DCM, rt, 71%; (e) TFA, DCM, rt, 24%; (f) (Boc)2O,
THF, 0 °C, 100%.
Scheme 4. Reagents and conditions: (a) BocNHCH2CH2Br, n-BuLi, THF, ꢀ78 °C, 33%;
Table 1
(b) HCl, 1,4-dioxane, rt, 42%; (c) AcOH, EDC, i-Pr2EtN, HOBt, DCM, rt, 100%; (d) N,N0-
Preparation of 2 by Buchwald–Hartwig coupling of 11
diBoc-1H-pyrazole-1-carboxyamidine, i-Pr2EtN, DMF, 65 °C, 29%; (e) HCl, 1,4-
dioxane, 0 °C.
Entry
Temperature (°C)
Time (h)
Yield (%)
1
2
3
85
8.5
11.5
2
14
39
28
110
pete with 1,25(OH)2D3 for the vitamin D binding site. Addition of
one hundred times higher concentration of 1,25(OH)2D3
(300 nM) did not affect the observed reduction by 1a and 2 of
the 1,25(OH)2D3-induced reporter gene luciferase activity (Fig. 3).
On the other hand, the addition of a high concentration of
1,25(OH)2D3 resulted in a dramatic decrease of the VDR-antagonis-
tic efficacy of DLAM-2P, that is, the IC50 value of DLAM-2P was fif-
130a
a
A microwave reactor was used.
b-galactosidase activity in the concentration range investigated.
Under the assay conditions used, the positive control DLAM-2P
antagonized the 1,25(OH)2D3-induced reporter gene luciferase
teen times higher (3.3
compared to that observed in the presence of 3 nM 1,25(OH)2D3
(0.22 M). These facts support the view that these benzodiazepine
lM) in the presence of 300 nM 1,25(OH)2D3
activity with an IC50 value of 0.22 lM (Table 2). Compounds 1a
and 2 also caused a reduction in the 1,25(OH)2D3-induced reporter
gene luciferase activity. This result indicates that 1a and 2 inhibit
VDR-mediated transcriptional activation of responsive genes. The
l
compounds are not competing with the ligand 1,25(OH)2D3 as a
conventional antagonist would, but rather they inhibit the binding
between VDR and coactivator. Furthermore, selectivity with re-
IC50 value of 2 in our assay system was calculated to be 17
lM.
Next, we investigated whether benzodiazepine compounds com-
spect to other NRs was also investigated. ERa and ERb reporter

1716
Y. Mita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1712–1717
Table 2
Inhibitory activity towards interaction between VDR and coactivator peptide fragment, and selectivity for other nuclear receptors
Compound
VDR
ER
a
ERb
TR-FRET IC50
(
l
M)
Reporter gene IC50
(
lM)
Reporter gene IC50
(
l
M)
Reporter gene IC50
(lM)
Reporter gene IC50
Estradiol (0.3 nM)
(lM)
Agonist
1,25(OH)2D3 (10 nM)
1,25(OH)2D3 (3 nM)
1,25(OH)2D3 (300 nM)
Estradiol (0.3 nM)
1a
2
DLAM-2P
>30 (48%)a
20 (76%)a
nt (87%)b
>30 (47%)a
17 (68%)a
30 (53%)a
23 (59%)a
3.3 (66%)b
>30 (8%)a
>30 (8%)a
nt
>30 (18%)a
22 (56%)a
nt
0.22 (98%)b
a
Inhibition ratio at 30
Inhibition ratio at 10
l
l
M.
M.
b
Table 3
140
120
100
80
Inhibitory activity of 1b–e on the interaction between VDR and coactivator peptide
fragment
Compound
VDR TR-FRET IC50 (lM)
1b
1c
1d
1e
13
28
>30 (43%)a
>30 (46%)a
60
a
Inhibition ratio at 30 lM.
40
20
Because proof of principle for inhibition of the interaction of VDR
with coactivators had been achieved, we next introduced side chains
in an attempt to improve the activity. All the synthesized benzodiaz-
0
0.01
0.1
1
10
100
concentration (mm)
epine molecules inhibited the FRET signal at 30
them, 1b and 1c inhibited coactivator binding with IC50 values of 13
and 28 M, respectively. Compounds 1c–e showed weaker activity,
lM (Table 3). Among
Figure 3. Dose–response of DLAM-2P (square), 1a (triangle) and 2a (circle) on VDR-
responsive reporter gene activity. Closed symbols represent lower concentration of
1,25(OH)2D3 (3 nM). Open symbols represent higher concentration of 1,25(OH)2D3
(300 nM).
l
indicating that the introduced side chains were unable to interact
with Glu 416. On the other hand, 1b46 possessing a bulky side chain
exhibited the strongest activity, implying that the side chain of 1b
might mimic the side chain of Met 629, thereby affording an addi-
tional hydrophobic interaction with VDR.
gene assay with CMX-GAL4N-hERa LBD or CMX-GAL4N-hERb LBD
as reporter genes, and estradiol (0.3 nM) as an ER agonist were
used instead of CMX-GAL4N-hVDR LBD and 1,25(OH)2D3. Com-
pound 2 also inhibited ERb reporter gene activity with an IC50 value
In conclusion, benzodiazepine molecules were rationally de-
signed as non-peptide LXXLL mimetics based on the VDR-coactiva-
of 22
reporter gene activity (Table 2). On the other hand, 1a showed only
slight inhibitory effects on ER and ERb reporter gene activities.
lM, whereas 2 showed only a slight inhibitory effect on ERa
tor interaction. TR-FRET assay showed that
interaction between VDR and a coactivator peptide fragment with
an IC50 value of 20 M. Compound 2 also inhibited VDR-mediated
transcription with almost the same IC50 value (17 M) in reporter
gene assay. Inhibition of VDR-mediated transcription by 2 was
independent of the concentration of co-existing agonist. Further-
more, VDR-coactivator interaction inhibitor 2 did not inhibit
2 inhibits the
a
l
Thus, VDR-coactivator binding inhibitors 1a and 2 can be judged
l
to be VDR-selective. Superposition of peptide fragments in coacti-
vators of VDR, ER
shown in Figure 4. Peptide fragments of VDR and ERb showed good
overlap. On the other hand, the coactivator fragment of ER did not
a
44 and ERb45 in the NR-bound conformation are
a
ERa-mediated transcription. Although these inhibitory activities
match those of VDR and ERb, especially at Leu 630 and Leu 634.
This conformational difference is consistent with the selectivity
of 1a and 2.
were not highly potent, this is the first report of non-peptide inhib-
itors of the binding between VDR and its coactivators, and of non-
secosteroidal inhibitors of VDR-mediated transcription to our
knowledge. Structural development of the benzodiazepine series
and further studies on selectivity with respect to other nuclear
receptors are in progress.
Acknowledgments
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan, and the Japan
Society for the Promotion of Science. We thank Dr. Motomu Kanai
for carrying out the microwave-promoted reaction.
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