Affinity labeling of the nuclear vitamin D receptor with nonsteroidal alkylating agents.

Article abstract of DOI:10.1016/S0960-894X(02)00878-8

Synthesis of an affinity alkylating non steroidal mimic of 1alpha,25-dihydroxyvitamin D(3) and its radiolabeled counterpart is presented. We also report the affinity labeling of the VDR-ligand binding domain (VDR-LBD) with this analogue.

Full text of DOI:10.1016/S0960-894X(02)00878-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 213–216
Affinity Labeling of the Nuclear Vitamin D Receptor with
Nonsteroidal Alkylating Agents
Ana Fernandez-Gacio,y Carlos Fernandez-Marcos,{ Narasimha Swamyx and Rahul Ray*
Bioorganic Chemistry and Structural Biology, Section in Endocrinology, Diabetes and Metabolism, Department of Medicine, Boston
University School of Medicine, 85 East Newton Street, Boston, MA 02118, USA
Received 14 June 2002; accepted 10 October 2002
Abstract—Synthesis of an affinity alkylating non steroidal mimic of 1a,25-dihydroxyvitamin D3 and its radiolabeled counterpart is
presented. We also report the affinity labeling of the VDR-ligand binding domain (VDR-LBD) with this analogue.
# 2002 Elsevier Science Ltd. All rights reserved.
1a,25-Dihydroxyvitamin D3 (1,25(OH)2 D3) is a multi-
functional seco-steroid. Its biological properties include
calcium and phosphorus homeostasis, growth and
maturation control of a broad range of normal and
malignant cells, and immune-regulation. Therapeutic
value of 1,25(OH)2 D3 has been amply demonstrated in
many diseases including cancers of various organs, leu-
kemia, psoriasis, osteoporosis, autoimmune diabetes,
etc.1 However, 1,25(OH)2 D3 causes hypercalcemia in
rodents leading to soft-tissue calcification, loss of body-
weight, and death. In humans 1,25(OH)2 D3 induces
hypercalcemia, which is manifested in kidney stones,
hypercalciuria, and related diseases.2
(i) have enhanced antiproliferation and decreased calce-
mic properties, (ii) show tissue-specific effects, and (iii)
display antihormone activities.
Mechanism of 1,25(OH)2 D3-action closely parallels
estrogen, another steroid hormone. Thus, strong and
specific interaction between 1,25(OH)2 D3 and its
nuclear receptor, vitamin D receptor (VDR) promotes a
conformational change in the latter and allosteric bind-
ing to retinoid X receptor (RXR), and binding of the
VDR-1,25(OH)2 D3-RXR to several co-activators and
to the upstream promoter region of the vitamin
D-regulated genes, initiating gene expression.4 The key
to such highly specific molecular interactions, leading to
observed biological activities, lies in the three-dimensional
architectures of the interacting molecules.5
Adverse side effects of 1,25(OH)2 D3 have garnered a
strong interest in developing analogues of 1,25(OH)2 D3
with intact beneficial effects and reduced toxicity. Dur-
ing the past several years numerous vitamin D analo-
gues have been synthesized and their biological activity
evaluated.3 Although a number of such analogues, with
significant separation between antiproliferative effects
and systemic toxicity, have been developed, no infor-
mation is available to date that demonstrates pre-
dictable trends in modification of analogues that will
In the case of estrogen receptor (ER), a nuclear receptor
of the same family as VDR, several artificial non-
steroidal ligands, having drastically different structures
than the parent hormone, have been developed. Some of
these nonsteroidal mimics have shown tissue-specific
activity and antihormonal effects with high therapeutic
values.6 It has been demonstrated that some of these
compounds, for example, tamoxifen and raloxifen sta-
bilized the nuclear estrogen receptor by causing drasti-
cally different conformational changes in the estrogen
receptor (ER) than the parent hormone, particularly in
the relative positioning of the C-terminal helix-12 (H-
12); and such changes are reflected in their unique bio-
logical properties.7 On the other hand, nonsteroidal
anticancer agents flutamide and biculatamide are
known to act as androgen receptor (AR) ligands, and
*Corresponding author. Tel.: +1-617-638-8199; fax: +1-617-638-
8194; e-mail: bapi@bu.edu
yFulbright Fellow from Departamento Quımica Organica, Facultad de
Quımica, Universidad de Santiago de Compostela, Spain.
{On leave from Departamento Quımica Organica, Facultad de Veter-
inaria, Universidad de Extremadura, Caceres, Spain.
xCurrent address: Women’s and Infants Hospital, Providence, RI,
USA.
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00878-8

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A. Fernandez-Gacio et al. / Bioorg. Med. Chem. Lett. 13 (2003) 213–216
214
stabilize AR differently than the parent hormone.8
Similarly a novel vitamin D analogue has been shown to
act as a VDR antagonist.9 Recently Moras’s group
carried out X-ray diffraction analysis of VDR, com-
plexed with the hormone and two noncalcemic side-
chain analogues, and showed that these compounds
stabilized VDR stronger than the hormone without
causing drastic conformational changes in VDR.10
as well as hydrolysis of the ester to produce the triol (B)
(Scheme 1).
We decided to carry out the protection of the
hydroxyl groups of the ketone (A) as TBDMS ethers.
The mono- (C) and di-protected (D) derivatives were
obtained by standard procedure. Esterification of
the mono-TBDMS ether (C) with bromoacetic acid
afforded the bromoacetate. But attempted deprotection
of the silyl group largely produced the diol (E). Alter-
natively, borohydride reduction, bromoacetic acid
esterification and TBAF deprotection of the primary
hydroxyl group produced the triol (B) (Scheme 2).
Boehm et al. recently reported that a class of biphenyl
compounds (Fig. 1) mimicked the actions of 1,25(OH)2
D3, such as anti-proliferation/pro-differentiation of sev-
eral normal and malignant cells and regulation of vita-
min D-controlled genes, without eliciting hypercalcemia
in mice.11 Furthermore, one compound in this group
(LG190178) showed strong activity in reducing skin
xenograft human prostate tumor in nude mice.12 This
provided a unique opportunity to study the interaction
between this class of non-vitamin D compounds with
VDR.
The di-protected ketone (D) was quantitatively reduced
with NaBH4 in methanol, and the resulting alcohol (F)
was esterified with bromoacetic acid to give the mono-
ester (G). Deprotection of (G) with TBAF resulted in
the hydrolysis of the ester exclusively producing the triol
(B). However, deprotection of the hydroxyl groups with
10% HF in dichloromethane efficiently produced the
desired bromoacetate (H) (Scheme 3).
We initiated the present study to develop potential affi-
nity alkylating derivatives of LG190178 (Fig. 1) to
determine the spatial orientation of these compounds
inside the vitamin D-binding pocket of VDR by identi-
fying the contact points between these compounds and
peptide backbone of VDR. In this communication we
report the synthesis of an affinity alkylating derivative
(H) and its radiolabeled counterpart. We also report
affinity labeling of VDR-ligand binding domain (VDR-
LBD) with this analogue.
Radiochemical synthesis of (H) was accomplished by
replacing bromoacetic acid in Scheme
3
with
[1-14C]bromoacetic acid (Sigma Chemical Co., Milwau-
kee, WI, USA, sp. ac. 10.8 mCi/mmol). A parallel reac-
tion with unlabeled bromoacetic acid, spiked with
[1-14C]bromoacetic acid, was carried out to purify (by
TLC), characterize and determine radiochemical purity
of the labeled products (G) and (H).
Our original synthetic strategy included the esterifica-
tion of the ketone (A) to obtain the corresponding bro-
moesters. The addition of one equivalent of
bromoacetic acid to the ketone (A) produced a separ-
able mixture of two bromoesters. However, sodium
borohydride resulted in the reduction of the carbonyl,
Incubation of two samples of VDR-LBD (103–427,
M
rapp 39,000)13 (10 mg, 0.26 nmol, in a buffer containing
50 mM Tris–HCl, 150 mM NaCl, 5 mM DTT, 0.1%
Triton X, pH 8.3) at 0 ꢀC for 2 h with [14C]-H (Sp.
activity 8.4 mCi/mmol, 5000 cpm, 0.6 nmol in EtOH)
covalently labeled the protein, as evidenced by the SDS-
PAGE analysis and radioactive scanning (Fig. 2, Lane
1). On the other hand, when the incubation was carried
out in the presence of an excess of 1,25(OH)2 D3 (1 mg,
2.4 nmol), labeling was significantly reduced (Fig. 2,
Lane 2). These results strongly suggested that [14C]-H
specifically labeled the 1,25(OH)2 D3-binding pocket of
VDR-LBD. It should be noted that labeling was not
completely obliterated by co-incubation with 1,25(OH)2
Figure 1. Structures of 1,25(OH)2 D3, its non-steroidal mimics and
their affinity analogues.
Scheme 1.

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A. Fernandez-Gacio et al. / Bioorg. Med. Chem. Lett. 13 (2003) 213–216
215
Scheme 2.
Scheme 3.
D3. This is to be expected because we could not employ
a large excess of 1,25(OH)2 D3 due to insolubility prob-
lem. In addition, the labeling process is rapid, so that
the unlabeled VDR-LBD is always expected to be con-
taminated with a finite amount of the labeled protein.
According to the published report all the nonsteroidal
1,25(OH)2 D3-mimics (Fig. 1)11 and certain A-ring
modified steroidal 1,25(OH)2 D3-mimics14 with drasti-
cally reduced VDR-binding affinities (than the parent
hormone) displayed significant 1,25(OH)2 D3-like activ-
ities in vitro. Results of our studies described in this
communication strongly suggested the mediation of
VDR in the 1,25(OH)2 D3-like activities of LG190178,
one of the nonsteroidal mimics. Currently we are in the
process of identifying the contact point/points between
LG190178 and VDR-LBD.
It was mentioned earlier that nonsteroidal 1,25(OH)2
D3-mimics showed poor VDR-binding activity, which
might compromise their bioavailability in vivo. We
Figure 2. Affinity labeling f VDR-LBD (105-427) with 14C-H.

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A. Fernandez-Gacio et al. / Bioorg. Med. Chem. Lett. 13 (2003) 213–216
216
hypothesize that (H), the affinity labeling derivative of
LG190178 and similar reagents might cross-link to
endogenous VDR in an in vivo system, and make this
analogue more bioavailable for desired biological activ-
ities. Therefore these affinity analogues could poten-
tially have better therapeutic index than the parent
compounds.
5. (a) Carlberg, C.; Quack, M.; Herdick, M.; Bury, Y.; Polly, P.;
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Acknowledgements
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J.-C.; Moras, D.; Sultan, C. J. Biol. Chem. 2000, 275, 24022.
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This work was supported in part by a grant from the
National Institutes of Health (DK 44337 to RR). Fer-
nandez-Gacio would like to thank the Commission for
Educational Exchange between the USA and Spain for a
Fulbright-Lee Kennedy fellowship.
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