Crystal structures of rat vitamin D receptor bound to adamantyl vitamin D analogs: Structural basis for vitamin D receptor antagonism and partial agonism

Article abstract of DOI:10.1021/jm8004477

The X-ray crystal structures of the rat VDR ligand-binding domain complexed with 19-norvitamin D compounds that contain an adamantyl substituent at the side-chain terminus, 2a (ADTT), 2b (ADNY), and 2c (ADMI4) and a coactivator peptide derived from DRIP205 are reported. These compounds show a series of partial agonistic ( 10-75% efficacy)/antagonistic activities. All of these complexed receptors are crystallized in the canonical active conformation, regardless of their activity profiles. The bulky adamantyl side chain does not crowd helix 12 but protrudes into the gap formed by helix 11, loop 11-12, helix 3, and loop 6-7, thereby widening the ligand binding pocket. We suggest that these structural changes destabilize the active protein conformation and reduce its contribution to equilibrium among the active and inactive conformations. The coactivator peptide traps the minor active conformation, and the equilibrium shifts to the active conformation. As a result, these ligands show partial agonistic activities.

Full text of DOI:10.1021/jm8004477

5320
J. Med. Chem. 2008, 51, 5320–5329
Crystal Structures of Rat Vitamin D Receptor Bound to Adamantyl Vitamin D Analogs:
Structural Basis for Vitamin D Receptor Antagonism and Partial Agonism
Makoto Nakabayashi,† Sachiko Yamada,*,†,‡ Nobuko Yoshimoto,† Takashi Tanaka,† Miharu Igarashi,† Teikichi Ikura,†
Nobutoshi Ito,† Makoto Makishima,‡ Hiroaki Tokiwa,§ Hector F. DeLuca,| and Masato Shimizu†
School of Biomedical Science and Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental UniVersity,
2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, School of Medicine, Nihon UniVersity, 30-1 Ohyaguchikami-machi,
Itabashi-ku, Tokyo 173-0086, Department of Chemistry, Faculty of Science, Rikkyo UniVersity, 3-34-1 Nishi-Ikebukuro,
Toshima-ku, Tokyo 171-8501, and Department of Biochemistry, UniVersity of Wisconsin-Madison, Madison, Wisconsin 53706-1544
ReceiVed April 19, 2008
The X-ray crystal structures of the rat VDR ligand-binding domain complexed with 19-norvitamin D
compounds that contain an adamantyl substituent at the side-chain terminus, 2a (ADTT), 2b (ADNY), and
2c (ADMI4) and a coactivator peptide derived from DRIP205 are reported. These compounds show a series
of partial agonistic (10-75% efficacy)/antagonistic activities. All of these complexed receptors are crystallized
in the canonical active conformation, regardless of their activity profiles. The bulky adamantyl side chain
does not crowd helix 12 but protrudes into the gap formed by helix 11, loop 11-12, helix 3, and loop 6-7,
thereby widening the ligand binding pocket. We suggest that these structural changes destabilize the active
protein conformation and reduce its contribution to equilibrium among the active and inactive conformations.
The coactivator peptide traps the minor active conformation, and the equilibrium shifts to the active
conformation. As a result, these ligands show partial agonistic activities.
Introduction
to which coactivators16 are recruited, and chromatin remodel-
ing17 and transactivation follow. When accommodating an
antagonist, the receptors preferentially adopt inactive conforma-
tions that interact with corepressors,16 and transactivation is kept
silent. When binding a SRM, NRs adopt both active and inactive
conformations and therefore have the potential to interact with
either coactivators or corepressors and exert partial activity.
Therefore, the activity of SRM-bound receptors depends on the
relative expression of coactivators and corepressors in a given
cell environment.
In the NR field, many antagonists and partial agonists have
been developed as clinical medicines. For example, hydroxyta-
moxifen (OHT)18 and raloxifene (RAL)19 have been developed
as selective estrogen receptor (ER) modulators (SERMs),15 the
mineralocorticoid receptor (MR) antagonist spironolactone20 has
been developed as an antihypertensive agent, and androgen
receptor (AR) antagonists such as flutamide21 and bicalutamide22
have been developed for the treatment of prostate cancer. The
X-ray crystal structures of these steroid hormone receptors
complexed with their antagonistic and partially agonistic ligands
have been determined.23-30 These crystallographic studies have
revealed that the conformations of antagonist- and partial-
agonist-bound NRs are varied whereas agonist-bound NRs adopt
the same canonical active conformation.
The hormonally active vitamin D metabolite 1,25(OH)2D3
expresses its function by binding to the vitamin D receptor
(VDRa, NR1I1),1-7 which is a ligand-regulated transcription
factor and a member of the nuclear receptor (NR) superfamily.8,9
The VDR plays physiological roles in the regulation of the
mineral homeostasis and proliferation, the differentiation, and
the immune response of cells.10,11 Active vitamin D3 and some
potent synthetic analogs have been used clinically to treat
calcium and bone disorders such as osteoporosis and the skin
disorder psoriasis.10,11 However, despite some degree of target
selectivity, the further development of vitamin D analogs as
medicines for the treatment of malignant tumors and autoim-
mune disorders and for selective bone formation has been
limited because of the adverse effect of hypercalcemia.12-14 The
development of VDR modulators with selectivity at the tissue
level (i.e., SVDRM) to overcome this limitation has been
awaited, and for this purpose, a search for VDR partial agonists/
antagonists is of great interest.59
Selective receptor modulators (SRM) exhibit agonistic or
antagonistic activity in a cell and tissue context-dependent
manner.15 In NRs without ligands (apo), both the active and
the inactive conformations are in equilibrium. With a bound
agonist (holo), NRs predominantly adopt the active conformation
In the VDR field, a few vitamin D analogs that have
antagonistic activity, partial agonistic activity, or both have been
reported, including compounds that contain a bulky alkoxycar-
bonyl group at the side-chain terminus, such as 5,31-34
compounds that have a methylene lactone structure in the side
chain, such as 6,34-36 and their analogs,37-39 and nonsteroidal
VDR modulators (Figure 1).40 However, the X-ray crystal
structure of VDR complexed with an antagonistic ligand has
never been reported, although a number of agonist complexes
have been determined.41-49 We have designed and synthesized
vitamin D derivatives that have the bulky adamantyl (AD)
substituent at the terminus of side chains of various lengths50-53
(Figure 1). These compounds have partial agonistic activity
* To whom correspondence should be addressed. Tel: +81 42 664 1629.
Fax: +81 42 664 1629. E-mail: yamada.vd@image.ocn.ne.jp.
†
Tokyo Medical and Dental University.
‡
Nihon University.
Rikkyo University.
University of Wisconsin-Madison.
§
|
a Abbreviations: VDR, vitamin D receptor; NR, nuclear receptor;
SVDRM, selective vitamin D receptor modulator; ER, estrogen receptor;
MR, mineralocorticoid receptor; AR, androgen receptor; SRM, selective
receptor modulator; OHT, hydroxytamoxifen; RAL, raloxifene; SERM,
selective estrogen receptor modulator; AD, adamantane; LBD, ligand
binding domain; LBP, ligand binding pocket; GEN, genistein; THC,
tetrahydrochrysene; 2D ASMA, two-dimensional alanine scanning muta-
tional analysis.
10.1021/jm8004477 CCC: $40.75
2008 American Chemical Society
Published on Web 08/19/2008

Crystal Structures of VDR/Antagonists Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5321
Figure 1. Structures of vitamin D analogs discussed in this article.
(10-75% efficacy), antagonistic activity, or both. In the present
study, we determined the X-ray crystal structures of the rat VDR
ligand-binding domain (LBD) complexed with AD 19-norvi-
tamin D compounds with various side-chain lengths and various
activity profiles (25-AD-25-hydroxyl (2a), 24-AD-24-hydroxyl
(2b), and 26-AD-25-hydroxyl (2c) (Figure 1)) and an LXXLL-
containing coactivator peptide. In all of these ternary complexes,
the VDR was found to adopt the canonical active conformation
of NR, regardless of their activity profiles. These results provide
a structural basis for VDR antagonism and partial agonism for
the first time as well as supporting evidence of the general
molecular mechanism of SRM that was previously suggested.15
chain aldehyde (7) was employed. The reductive elimination
of the hydroxysulfone group via the acetate, followed by the
deprotection of the hydroxyl groups, afforded the desired
compounds.
Crystal Structures. The crystals of rVDR LBD (116-423,
∆165-211) were grown in the presence of a ligand (AD
compounds 2a-2c or 1) and a peptide containing the LXXLL
motif that was derived from coactivator DRIP 205. The
complexes of rVDR LBD with AD compounds 2a and 2b were
readily crystallized in space group C2 and were refined to
resolutions of 1.70 and 2.10 Å, respectively. In contrast, the
complex of 2c was crystallized rather slowly (resolution 2.35
Å), which suggests that the ligand was accommodated in the
LBP with its very minor conformation. The crystal data are
given in the Supporting Information.
The overall folds of rVDR LBD bound to 2a, 2b, and 2c
(Figures 2A-C) are identical to each other and to those of the
VDR complexes of the natural hormone (1)41,43 and its agonistic
analogs.42,44-49 Contrary to our expectation, all three of these
complexes adopted the canonical active conformation of the NR
LBD, and the coactivator peptide bound to the coactivator
recognition surface (AF-2 surface). The VDR complexes of AD
compounds (2a, 2b, and 2c) are aligned at CR atoms with the
complex of 1, which was crystallized in this study from the
same recombinant rVDR LBD, with average root-mean-square
(rms) deviations of 0.344, 0.262, and 0.385 Å, respectively.
The ligands are accommodated in the ligand binding pockets
(LBP), which are significantly widened at the tip of the side
chains compared with the complex with the natural hormone
(1). The volumes of the LBP with 1, 2a, 2b, and 2c are 942,
1040, 1092, and 1102 Å3, respectively. The LBP of the rVDR
Results
Synthesis. We designed AD 19-nor-2-methylenevitamin D
compounds as candidates for potential VDR partial agonists and
antagonists because (1) the rigid, bulky side chain with the AD
moiety together with the 22-double would interfere with H12
in the active conformation and (2) the 19-nor-2-methylene
A-ring motif that was introduced by DeLuca’s group54,55
promises a high VDR affinity that is important for antagonistic
activity. It is also advantageous that the 19-norvitamin D moiety
is chemically stable and easy to synthesize.
New AD compounds, 2a and 2b, were synthesized by the
methods shown in Scheme 1, which are essentially the same as
the method that was previously reported for the synthesis of
26-AD-19-nor-vitamin D compounds including 2c.51 The C22
secosteroid unit and side-chain fragment were combined by the
Julia coupling reaction. In the synthesis of 2a, the C22 aldehyde
(8) was combined with side-chain sulfone 5d, whereas in the
synthesis of 2b, the reversed pair of C22 sulfone (13b) and side-

5322 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Nakabayashi et al.
Scheme 1a
a Reagents and conditions: (a) Zn, BrCH2COOEt, benzene, reflux (72.9%); Et3SiCl, imidazole, DMF (94.2%). (b) DIBAL-H, toluene, -78 °C (98.3%).
(c) TsCl, pyridine, 0 °C (91%); PhSH, t-BuOK, DMF, 0 °C (83%); m-CPBA, CH2Cl2, 0 °C (94%). (d) TMSCN, ZnI2, CH2Cl2, 0 °C (75.3%). (e) DIBAL-H,
toluene/CH2Cl2, 0 °C (68.7%). (f) LDA, THF, -20 °C. (g) Ac2O, DMAP, pyridine; Na-Hg, Na2HPO4 (37.4% from 8). (h) CSA, MeOH (80.5%). (i) PhSH,
t-BuOK, DMF, 0 °C (99.8%); Na2WO4 ·2H2O, 30% H2O2, CH2Cl2/MeOH (3:5), 0 °C ∼room temperature (65%). (j) AcOH/THF/H2O; Ac2O, DMAP,
pyridine; Na-Hg, Na2HPO4 (34% from 13b). (k) n-Bu4NF, DMF/THF (98.8%).
LBD complexed with 2a is shown in Figure 2D in comparison
with that of the 1,25(OH)2D3 complex. The ligand 2a (space-
filling model) is accommodated in its LBP but protrudes from
that of the 1,25(OH)2D3 complex.
Complex with 2a. 25-Adamantyl-25-hydroxyl compound 2a
shows very potent partial agonistic activity, antagonistic activity,
deviations are found in the loop parts. Specifically, CR rms
deviations of loop 11-12 are most significant. The CR rms
deviations are also significant at Ala148-Pro155 (the loop
following H1), His367-Gln374 (loop 10-11), and L303-Lys311
(H7). However, the rms deviations of functionally important
residues such as those on H12 and the signature region (highly
conserved sequences among the NR family consisting of the
C-terminus of H3, loop 3-4, and H4) are not noticeable. As
described above, the LBP is widened around the part that is
surrounded by the C-terminus of H11, loop 11-12, and the
N-terminus of H3 (Figures 2D). Here, the side-chain conforma-
tions of three Leu residues, 223 (H3), 400 (H11), and 410 (loop
11-12), differ significantly from those of the 1,25(OH)2D3
complex (Figure 3D): rms deviations, Leu 223 Cδ1 and Cδ2
(2.79 and 2.53 Å, respectively); Leu400 Cγ, Cδ1, and Cδ2 (2.12,
3.11, and 3.21 Å, respectively); Leu410 Cδ1 and Cδ2 (2.52 and
2.67 Å, respectively). We have shown by alanine scanning
mutational analysis (ASMA)50 that these residues are important
in the folding of the active conformation of the hVDR LBD.
The widening of the LBP here may explain why compound 2a
shows antagonistic activity. This weakens the interactions among
the secondary structures here and destabilizes the active
conformation. It has been reported that the volume of the hVDR
51
or both:
VDR affinity, ∼75% that of natural hormone 1;
transactivation, EC50 ) 10-9 M, 15% efficacy compared to that
of 1; and inhibition of the action of 1, IC50 ) 3 × 10-9 M.
Regardless of 2a’s potent antagonistic activity, the VDR
complex of 2a adopts the transcriptionally active conformation.
This active conformation complex is conceivably a minor
component in the equilibrium with major inactive conformations
and was trapped by the LXXLL peptide and was crystallized
out. In accord with the high VDR affinity, ligand 2a is anchored
strongly by three pairs of hydrogen bonds at its three hydroxyl
groups as natural hormone 1: 1R-OH, Ser233 and Arg270
(2.81 and 2.98 Å, respectively); 3ꢀ-OH, Tyr143 and Ser274
(2.80 and 2.86 Å, respectively); 25-OH, His301 and His 393
(2.82 and 2.71 Å, respectively) (Figure 3A). The CR rms
deviations of individual residues are shown in Figure 2E.
(e0.24, 0.25-0.44, 0.45-0.74, 0.75-1.04, and g1.05 Å are
yellow, orange, red, purple, and black, respectively.) Large rms

Crystal Structures of VDR/Antagonists Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5323
Figure 2. Crystal structures of the ternary complexes of the rVDR LBD (116-423, ∆165-211) with AD vitamin D compounds and a coactivator
peptide derived from DRIP 205. The overall folds of the rVDR LBD bound to (A) 2a, (B) 2b, and (C) 2c with ribbon-loop rendering and secondary-
structure colors with the coactivator peptide in green and the ligands in stick representation with atom-type colors. (D) LBP of the rVDR LBD
complexed with 2a (transparent yellow) in comparison with that of the 1,25(OH)2D3 complex (transparent blue) (stereoview, relaxed eyes). Individual
CR rms deviations of the rVDR complexes with (E) 2a, (F) 2b, and (G) 2c shown on the LBD CR structures. (e0.24, 0.25-0.44, 0.45-0.74,
0.75-1.04, and g1.05 Å are yellow, orange, red, purple, and black, respectively.) (stereoview, relaxed eyes).

5324 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Nakabayashi et al.
Figure 3. Overlay of ligands in the rVDR LBD complexes. The complexes were aligned at CR atoms of the protein, and the ligands and some
interacting residues are overlaid. (A) 2a (green stick with red ball of oxygen), (B) 2b (cyan stick with red ball of oxygen), and (C) 2c (magenta stick
with red ball of oxygen) are overlaid with 1 (atom-type stick with red ball of oxygen) accompanied by six residues that hydrogen bond with its
three hydroxyl groups (shown in the same topology). (D) Ligand 2a and three Leu residues (green stick with red oxygen) interacting with its AD
moiety are overlaid with 1 and the same Leu residues (atom-type) in its complex.
LBP is not significantly changed when it binds superagonists
with long, bulky side chains such as 342 and 4 (Figure 1).44
Complex with 2b. The 24-adamantyl-24-hydroxyl compound
2b, which has the shortest side chain of the three AD
compounds, is a potential partial agonist of the VDR (VDR
affinity is about 20% of that of 1; transactivation, EC50 ) 2 ×
10-11 M; efficacy is 74% of that of 1).52 It forms three pairs of
hydrogen bonds at its three hydroxyl groups (Figure 3B): 1R-
OH, Ser233 and Arg270 (2.76 and 2.91 Å, respectively); 3ꢀ-
OH, Tyr143 and Ser274 (2.96 and 3.00 Å, respectively); and
24-OH, His301 and His393 (2.93 and 2.55 Å, respectively).
Compared with the 1,25(OH)2D3 complex, significant CR rms
deviations are found in the residues that interact with the
adamantane moiety and the 24-hydroxyl group, H3 N-terminal
(Leu226∼Asp228), H6 to loop 6-7 (Val296∼Leu303), H11
(Glu391∼Lys395), and loop 11-12 (Leu400-Pro404 and
Ser407-Thr411) (Figure 2F). Again, the rms deviations of the
residues that form the AF-2 surface are minimal.
Complex with 2c. 26-Adamantyl-25-hydroxyl compound 2c
has the longest side chain of the three compounds and shows
potent antagonistic activity (compared with 1: VDR affinity,
1/20; transactivation, EC50 ) 2.4 × 10-8 M; efficacy, 10%;
antagonistic activity, IC50 ) 3 × 10-8 M).51 Compound 2c is
anchored in the LBP by two pairs of hydrogen bonds at the
A-ring hydroxyl groups (Figure 3C): (1) 1R-OH, Ser233 and
Arg270 (2.57 and 3.26 Å, respectively) and (2) 3ꢀ-OH, Tyr143

Crystal Structures of VDR/Antagonists Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5325
and Ser274 (2.67 and 2.90 Å, respectively). However, the side-
chain hydroxyl group is not within hydrogen-bond distances
from the two His residues (His301 and His393 (4.41 and 7.20
Å, respectively)). For this reason, 2c has a lower VDR affinity
than the other two compounds.
Because the 25-hydroxyl group is not fixed at H11 (His 393),
the long and bulky side chain of 2c could be packed in the
LBP and could adopt a significantly different conformation
compared with 2a and 2b (Figure 3A-C). Compared with those
of the 1,25(OH)2D3 complex, the CR rms deviations are
significant at a part of loop 1-3 that faces the 3ꢀ-hydroxyl group
(Thr146-Asp152), at H6 to loop 6-7 (Tyr291-Leu303), and
at the H11 N-terminal to loop 11-12 (Ser394-Leu410) (Fig-
ure 2G).
5) or not (type II, 7). Two groups of compounds showed distinct
behaviors in the 2D ASMA.
The rVDR complexed with AD 19-norvitamin D compounds
(2a-c), which show a series of partial agonistic (10-75%
efficacy) to antagonistic activities, adopts the canonical active
conformation; H12 is placed on the opening of the LBP, which
forms the AF-2 surface to which the coactivator-peptide binds
(Figure 2A-C). The bulky AD moiety does not crowd H12, as
we proposed in our previous docking study,50-53 but protrudes
into the gap that is surrounded by the H11 C-terminus, loop
11-12, the H3 N-terminus, and loop 6-7. As a result, in all of
these complexes, the LBPs are expanded (Figure 2D). These
results contrast those that were obtained from VDR superago-
nists with long and bulky side chains, such as 342 and 4 (Figure
1).44 These superagonists reportedly do not widen the receptor
LBP. Because of its bulkiness and rigidity, the AD side chain
forces a change in the conformations of the VDR at N-terminal
H3, loop 6-7, and H11 to loop 11-12, and weakens the
interaction among these secondary structures. This structural
change would destabilize the active conformation and reduce
its contribution in the equilibrium among various conformations
of the complexes (Figure 4).
Three AD 19-norvitamin D compounds (2a-2c) can be
categorized as type I antagonists by our definition, but they are
difficult to classify as either active- or passive-type antagonists.
The schematic molecular mechanism in Figure 4 explains the
activity profiles of the AD 19-norvitamin D series. The VDR
LBD complexed with AD vitamin D ligands (2a-2c) adopts
multiple relatively stabilized conformations (D, E, F, etc.).
Whether the ligand exhibits agonist or antagonistic activity
depends on the population of these conformations and the
concentrations of coactivator (CoA) and corepressor (CoR)
complexes under given cellular conditions. The population of
the active conformation (F) would decrease in the order 2b >
2a . 2c; accordingly, the transcriptional activities would
decrease in the same order. However, the transcriptional
activities of these compounds (2a-2c) would be changed in a
cell and tissue context-dependent manner following the equi-
librium shown in Figure 4. If the CoA complex level is high,
then the equilibrium between complexes F and H shifts to H,
and agonistic activities would become predominant. If the CoA
complex level is low, the CoR complex level is high, or both,
then the whole equilibrium among complexes D to H would
converge to G, and transactivation would be suppressed.15
Complexes of coactivators and corepressors possess sensing
activities for multiple signaling pathways16 and thus the activities
of VDR ligands are affected by cell signaling via the regulation
of these coregulator levels.
Discussion
A number of crystal structures of NRs that are complexed
with antagonists, partial agonists, or both have been reported.23-30
Whereas NRs that are bound to agonist ligands invariably adopt
the same canonical active conformation, those that are bound
to antagonists/partial agonists exhibit various conformations.
When ERR binds RAL23 and OHT,24 the bulky side chains with
the tertiary amine group protrude from the LBP opening and
are fixed on H3 (Asp351), forming a salt bridge via the amine
group. In this way, the bulky ligands directly inhibit H12 from
adopting the active conformation. Instead, H12 binds to the
coactivator recognition surface (AF-2 surface) that mimicks the
LXXLL motif of the coactivator NR box by its LLEML
sequence. ERꢀ adopts a slightly different conformation when
it binds the partial agonist genistein (GEN)25 and the pure
antagonist tetrahydrochrysene (THC) compound.26 These ligands
do not have a bulky substituent and are easily accommodated
in the LBP. Therefore, these ligands do not directly interfere
with H12, but H12 does not adopt the active conformation. It
is placed over the LBP in a position such that it occludes the
AF-2 surface only partially, and the transactivation is suppressed.
This type of antagonism has been termed “passive antagonism”
by Shiau et al.26 as opposed to the active antagonism that was
exhibited by RAL23 and OHT.24 The same ligands (GEN and
THC) are similarly accommodated in the ERR, but here the
H12 is in the active conformation, and the compounds act as
agonists for ERR. It is reported that in the ERꢀ/THC complex,
small differences (1.6-2.3 Å) in the positioning of residues on
H11 cause this conformational and activity difference. So-called
passive antagonists are rather common in other NRs such as
progesterone and spironolactone for MR27,28 and flutamide and
bicalutamide for AR.29,30 In the case of AR, it was possible to
crystallize the complexes with antagonists only by mutating the
residues that cause antagonism, and the mutant receptors were
crystallized in the canonical active conformation. It was
suggested that in the passive-type antagonist complexes the LBD
body that accepts H12 is not properly folded so that H12 is not
placed in the correct position for the active conformation. For
the proper folding of the LBD body, the tight packing of H3
and H4/5 with the assistance of ligands and the correct
positioning of H11 are believed to be important.26-30
Conclusions
The crystal structures of the rVDR LBD complexed with a
series of AD 19-norvitamin D compounds (2a-2c) and a
coactivator peptide derived from DRIP205 have been solved.
These complexes adopt the canonical active conformation, and
the coactivator peptide binds to the AF-2 surface, regardless of
the partial agonistic/antagonistic activities of the ligands. We
suggest that the rigid and bulky adamantyl side chain that
protrudes into a gap formed by the H11 C-terminus, loop 11-12,
the H3 N-terminus, and loop 6-7 weakens the interactions
among these secondary structures and destabilizes the active
conformation. As a result, the contribution of the active
conformation in the equilibrium among multiple protein con-
formations is significantly reduced, and the transcriptional
potencies of these compounds (2a-2c) are decreased. The
We have investigated the structure-activity relationship of
VDR ligands on the basis of their activity profiles in the 2D
alanine scanning mutational analysis (2D ASMA) of all 34
VDR-LBP residues.50 In this study, we also analyzed the
behaviors of the two VDR antagonists/partial agonists, 7 and 8
(Figure 1),50 compared with that of the full antagonist, 5, and
we classified these antagonists/partial agonists into two groups
simply by whether they have bulky substituent (type I, 8 and

5326 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Nakabayashi et al.
Figure 4. Molecular mechanism of the transactivation regulated by the VDR.
(1-Adamantan-1-yl-3-benzenesulfonyl-propoxy)-triethylsilane (5d).
A toluene solution of DIBAL-H (1.02 M, 12.8 mL, 13.1 mmol)
was added to a solution of 4b (1.6 g, 4.37 mmol) in toluene (10
mL) at -78 °C, and the mixture was stirred for 1.5 h at the same
temperature. The reaction was quenched with 10% potassium
sodium tartrate solution and was then worked up to yield alcohol
derivative 5a (1.4 g, 98.3%). This alcohol was dissolved in dry
pyridine (6 mL). TsCl (1.43 mL, 7.5 mmol) was added to this
solution at 0 °C, and the whole solution was stirred for 4 h at that
temperature. The reaction was quenched with water at 0 °C for 1 h
and was worked up to yield tosylate 5b (1.87 g, 91%). Thiophenol
(445 mL, 4.33 mmol) and t-BuOK (526.8 mg, 4.69 mmol) were
added to a solution of 5b (1.73 g, 3.61 mmol) in DMF (16 mL),
and the mixture was stirred at 0 °C for 40 min. The reaction was
quenched with water and was then worked up to yield sulfide 5c
(1.24 g, 83%). M-CPBA (65%, 1.82 mg, 6.89 mmol) was added to
a solution of sulfide 5c (1.31 g, 3.13 mmol) in CH2Cl2 (26 mL) at
0 °C, and the mixture was stirred for 30 min at 0 °C and for 2 h at
room temperature. Water was added to the reaction, and the mixture
was worked up to produce sulfone 5d (1.32 g, 94%). 1H NMR
(5d, δ): 0.536, 0.541 (6H, q, J ) 7.9 Hz), 0.91 (9H, t, J ) 7.9 Hz),
1.3-1.8 (12H, m), 1.95 (3H, m), 3.05 (1H, m), 3.16 (1H, dd, J )
7.6 and 3.5 Hz), 3.25 (1H, m), 7.58 (2H, m), 7.67 (1H, m), 7.91
(2H, m).
results presented a structural basis for VDR antagonism and
partial agonism for the first time.
Experimental Section
General. All nonaqueous reactions were carried out under argon
or nitrogen in freshly distilled anhydrous solvents. In all chemical
reactions, the products underwent a general workup procedure that
included extraction with organic solvents, solvent evaporation after
drying the extracts over anhydrous MgSO4, and purification by
column chromatography on silica gel. We conducted high-pressure
liquid chromatography (HPLC) by using Jasco 880-PU pumps
equipped with an 801-SC solvent programmer and a Uvidec-100V
variable-length UV-vis detector. Nuclear magnetic resonance (1H
and 13C) spectra were recorded in CDCl3 solution on a Bruker ARX
400 MHz spectrometer. Low- (MS) and high-resolution mass
spectra (HRMS) were obtained by electronic ionization (70 eV)
on a JEOL JMS-AX505HA spectrometer. Ultraviolet spectra were
recorded on a Hitachi U-3200 spectrophotometer.
Chemicals. 26-Adamantyl-1R,25R-dihydroxy-2-methylene-22,23-
didehydro-19,27-dinor-20-epivitamin D3 (2c) was synthesized in
our laboratory as reported.51 All commercial reagents were used
without further purification.
3-Adamantan-1-yl-3-hydroxy-propionic Acid Ethyl Ester (4a).
To a refluxing suspension of aldehyde 3 (1.11 g, 6.75 mmol) and
Zn powder (573 mg, 8.75 mmol) in benzene (13 mL) was added
ethyl bromoacetate (895 µL, 8.10 mmol) in one portion, and the
mixture was refluxed for 40 min. After the reaction was cooled to
0 °C, 3% HCl was added to the reaction, and the whole mixture
was extracted with CH2Cl2. After a general workup, ethyl ester 4a
25-Adamantyl-1r,25-dihydroxy-2-methylene-22,23-didehydro-
19,26,27-trinor-20-epivitamin D3 1,3-Bis-(tert-butyl-trimethysilyl)
25-triethylsilyl Ether (11). Diisopropylamine (28 µL, 0.2 mmol),
followed by n-BuLi (1.54 M hexane solution, 107 µL, 0.16 mmol),
was added to a solution of sulfone 5d (74 mg, 0.17 mmol) in THF
(500 µL) at -20 °C, and the mixture was stirred for 10 min at that
temperature. To this mixture, a solution of aldehyde 851 (62.8 mg,
0.11 mmol) in THF (750 µL) was added, and the reaction continued
to be stirred at -20 °C for 20 min. The reaction was quenched
with water and was worked up to yield 9 (146 mg) as a mixture of
diastereomers. Hydroxyl sulfone 9 (124 mg) was dissolved in
pyridine (1 mL) and was treated with acetic anhydride (115 µL,
1.22 mmol) and dimethylaminopyridine (3.2 mg, 0.026 mmol) at
room temperature for 4 h. Acetate 10 (123 mg) was obtained after
a general workup. Na-Hg (10%, 403 mg, 1.8 mmol) and Na2HPO4
(254 mg, 1.8 mmol) were added to a solution of acetate 10 in THF
(1 mL) at 0 °C, and then the mixture was stirred at room temperature
for 1 h. The reaction was quenched with 1 N HCl and was worked
up to yield bis-TBS ether 11 as a 1:1 mixture of C25 epimers (38.1
mg, 37.4% from 8). 1H NMR (11, δ): 0.03, 0.05, 0.07, 0.08 (each
3H, s), 0.51 (3H, s), 0.61 (6H, m), 0.86, 0.89 (each 9H, s), 0.95
1
(1.24 g, 72.9%) was obtained as a colorless oil. H NMR (4a, δ):
1.28 (3H, t, J ) 7.2 Hz), 1.4-1.8 (12H, m), 2.0 (3H, m), 2.36
(1H, dd, J ) 16.1 and 10.5 Hz), 2.52 (1H, dd, J ) 16.1 and 2.3
Hz), 2.74 (1H, d, J ) 3.8 Hz), 3.54 (1H, m), 4.17 (2H, q, J ) 7.2
Hz).
3-Adamantan-1-yl-3-triethylsilanyloxy-propionic Acid Ethyl Es-
ter (4b). Imidazole (3.4 g, 50 mmol) and Et3SiCl (4.2 mL, 25.0
mmol) were added to a solution of 4a (2.10 g, 8.32 mmol) in DMF
(20 mL), and the mixture was stirred at room temperature for 3 h.
The reaction was cooled (0 °C), quenched with water, and worked
up to yield triethylsilyl ether 4b (2.87 g, 94.2%). 1H NMR (4b, δ):
0.60 (6H, q, J ) 7.9 Hz), 0.95 (9H, t, J ) 7.9 Hz), 1.27 (3H, t, J
) 7.2 Hz), 1.4-1.8 (12H, m), 1.97 (3H, m), 2.29 (1H, dd, J )
15.8 and 7.8 Hz), 2.56 (1H, dd, J ) 15.8 and 3.8 Hz), 3.74 (1H,
dd, J ) 7.8 Hz), 4.14 (2H, m).

Crystal Structures of VDR/Antagonists Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5327
(9H, m), 2.81 (1H, m), 3.13 (1H, m), 4.42 (2H, m), 4.92 (1H, s),
4.97 (1H, s), 5.2-5.5 (2H, m), 5.83 (1H, d, J ) 11.3 Hz), 6.21
(1H, d, J ) 11.3 Hz).
0.04, 0.06, 0.07 (each 3H, s), 0.52 (3H, s), 0.86, 0.89 (each 9H, s),
1.20 (3H, d, J ) 6.5 Hz), 2.79 (1H, m), 2.87 (1H, dd, J ) 14.2 and
9.6 Hz), 3.16 (1H, m), 4.41 (2H, m), 4.92 (1H, s), 4.97 (1H, s),
5.80 (1H, d, J ) 11.2 Hz), 6.19 (1H, d, J ) 11.2 Hz), 7.57 (2H,
m), 7.63 (1H, m), 7.91 (2H, m). 13C NMR (13b, δ): -4.9, -4.7,
12.1, 18.4, 18.5, 20.5, 22.3, 23.4, 26.0, 26.3, 27.7, 28.8, 32.8, 38.8,
40.5, 45.9, 47.8, 56.0, 56.4, 62.3, 71.8, 72.7, 106.6, 116.8, 122.4,
128.1, 129.5, 133.5, 133.7, 140.4, 140.6, 153.1. IR (neat, cm-1):
2952, 2928, 2855, 1471, 1306, 1252, 1147, 1086. MS m/z (%):
698 (M+, 15), 683 (3), 641 (18), 566 (100), 509 (30), 424 (10),
366 (35), 351 (5), 293 (8), 257 (10), 197 (8), 147 (12), 135 (10),
73 (60). HRMS: calcd for C40H66O4Si2S, 698.4220; found, 698.4235.
24-Adamantyl-1r,24-dihydroxy-2-methylene-22,23-didehydro-
19,25,26,27-tetranorvitamin D3 1,3-Bis-(tert-butyl-dimethylsilyl)
Ether (17a and 17b). To a solution of sulfone 13b (34.2 mg, 0.049
mmol) in THF (500 µL) at -20 °C was added diisopropylamine
(21 µL, 0.15 mmol), followed by n-BuLi (1.54 M hexane solution,
80 µL, 0.12 mmol), and the resulting yellow solution was stirred
for 10 min at that temperature. To this solution was added a solution
of aldehyde 7 (25.4 mg, 0.096 mmol) in THF (500 µL), and the
mixture was stirred for 30 min at the same temperature. The reaction
was quenched with water, and it then underwent a general workup
procedure to yield a complex mixture of diastereomers of coupling
product 14 (49 mg). The TMS ether was deprotected by treating
14 (49 mg) with AcOH/THF/H2O (8/8/1, 1 mL) at room temperature
for 20 h. A 5% NaHCO3 solution was added to the reaction at 0
°C, and the mixture was worked up to yield 15 (29.5 mg). Diol 15
was acetylated by treatment with Ac2O (150 µL) and DMAP (3.9
mg, 0.032 mmol) in pyridine (500 µL) at room temperature for
6 h. The reaction was quenched with water at 0 °C for 1 h and was
then worked up to produce acetate 16 (29.5 mg). This acetate was
dissolved in THF (400 mL) and MeOH (600 mL), was cooled to
0 °C, was treated with Na2HPO4 (190 mg, 1.3 mmol) and 10%
Na-Hg (306 mg, 1.3 mmol), and was then stirred at room
temperature for 3 h. The reaction was quenched with 10% HCl at
0 °C and was worked up to produce less-polar 17a (4.6 mg, 17.4%
(25R)-25-Adamantyl-1r,25-dihydroxy-2-methylene-22,23-dide-
hydro-19,26,27-trinor-20-epivitamin D3 (2a). Camphor sulfonic acid
(50.1 mg, 0.22 mmol) was added to a solution of silyl ether 11
(46.4 mg, 0.054 mmol) in MeOH (1 mL) at room temperature, and
the mixture was stirred for 2.5 h. The reaction was quenched with
5% NaHCO3 and was worked up to yield a 1:1 mixture of 2a and
its C25 epimer 2a′ (22.5 mg, 80.5%), which were separated by
HPLC (YMC-Pack ODS-AM SH-342-5; 150 × 20 mm2; H2O/
MeOH, 1/9; 8 mL/min) to give 2a (11 mg) and 2a′ (10.5 mg),
1
which eluted in this order. H NMR (2a (25R epimer), δ): 0.52
(3H, s), 0.94 (3H, d, J ) 6.6 Hz), 3.01 (1H, dd, J ) 10.7 and 1.8
Hz), 4.49 (2H, m), 5.09 (1H, s), 5.11 (1H, s), 5.33 (1H, ddd, J )
15.2, 8.6, and 5.0 Hz), 5.44 (1H, dd, J ) 15.2 and 9.2 Hz), 5.88
(1H, d, J ) 11.0 Hz), 6.36 (1H, d, J ) 11.0 Hz). MS m/z (%): 520
(M+, 28), 520 (20), 484 (10), 251 (35), 223 (15), 165 (60), 135
(100), 105 (45). HRMS: calcd for C35H52O3, 520.3916; found,
520.3925. UV (95% EtOH) λmax, nm (log ε): 246 (4.48), 254 (4.54),
263 (4.37). 1H NMR (2a′ (25S epimer), δ): 0.53 (3H, s), 0.93 (3H,
d, J ) 6.6 Hz), 3.07 (1H, m), 4.49 (2H, m), 5.09 (1H, s), 5.11 (1H,
s), 5.41 (2H, m), 5.87 (1H, d, J ) 11.0 Hz), 6.35 (1H, d, J ) 11.0
Hz). MS m/z (%): 520 (M+, 8), 385 (65), 251 (10), 165 (30), 133
(100), 79 (25). HRMS: calcd for C35H52O3, 520.3916; found,
520.3918. UV (95% EtOH) λmax, nm: 246, 254, 263.
Adamantan-1-yl-trimethylsilanyloxy-acetonitrile (6). TMSCN
(346 µL, 2.6 mmol) and ZnI2 (24.6 mg, 0.077 mmol) were added
to a solution of aldehyde 3 (213 mg, 1.3 mmol) in CH2Cl2 (5 mL)
at 0 °C, and the mixture was stirred for 1 h at that temperature.
The reaction was quenched with 5% Na2S2O3 solution at 0 °C and
1
was then worked up to produce cyanide 6 (257 mg, 75.3%). H
NMR (6, δ): 0.19 (9H, s), 1.5-1.8 (12H, m), 1.9-2.2 (3H, m),
3.86 (1H, s). 13C NMR (6, δ): -0.3, 28.1, 36.9, 37.3, 37.6, 71.3,
119.0. MS m/z (%): 263 (M+, 8), 248 (10), 135 (100), 107 (5), 93
(8), 79 (89, 73 (5).
1
Adamantan-1-yl-trimethylsilanyloxy-acetaldehyde (7). A toluene
solution of DIBAL-H (1.01 M, 135 µL, 0.14 mmol) was added to
a solution of cyanide 6 (24 mg, 0.091 mmol) in CH2Cl2 (500 µL)
at 0 °C, and the mixture was stirred for 1 h at that temperature.
The reaction was quenched with 10% potassium sodium tartrate
solution and was worked up to yield aldehyde 7 (166 mg, 68.7%).
1H NMR (7, δ): 0.10 (9H, s), 1.5-1.8 (12H, m), 1.9-2.2 (3H, m),
3.33 (1H, d, J ) 2.9 Hz), 9.65 (1H, d, J ) 2.9 Hz). 13C NMR (7,
δ): 0.2, 28.4, 37.1, 38.0, 38.3, 85.0, 205.6. MS m/z (%): 237 (M+
- C2H5, 87), 135 (100), 107 (5), 93 (12), 73 (42).
1r-Hydroxy-2-methylene-22-phenylsulfanyl-19,23,24,25,26,27-
hexanorvitamin D3 1,3-Bis-(tert-butyl-dimethylsilyl) Ether (13a).
Tosylate 12 was treated with thiophenol under conditions similar
to those described in the synthesis of 5c to produce 13a (99.8%).
1H NMR (13a, δ): 0.02, 0.05, 0.06, 0.08 (each 3H, s), 0.54 (3H,
s), 0.86, 0.89 (each 9H, s), 1.13 (3H, d, J ) 6.5 Hz), 2.68 (1H, dd,
J ) 12.1 and 8.8 Hz), 2.82 (1H, m), 3.15 (1H, dd, J ) 12.1 and
2.7 Hz), 4.42 (2H, m), 4.92 (1H, s), 4.97 (1H, s), 5.84 (1H, d, J )
11.2 Hz), 6.21 (1H, d, J ) 11.2 Hz), 7.15 (1H, m), 7.26 (2H, m),
7.33 (2H, m). 13C NMR (13a, δ): -4.9, -4.7, -4.6, 12.3, 18.4,
18.5, 19.2, 22.4, 23.6, 26.0, 26.1, 27.8, 28.9, 37.0, 38.8, 40.6, 41.4,
46.1, 47.8, 56.0, 56.3, 71.9, 72.7, 106.5, 116.5, 122.5, 125.7, 129.0,
129.1, 133.2, 138.0, 141.0, 153.1. IR (neat, cm-1): 2927, 2855,
1253, 1101, 836. MS m/z (%): 666 (Μ+, 18), 609 (10), 534 (100),
477 (5), 402 (20), 366 (25), 309 (5), 279 (8), 251 (10), 197 (12),
147 (10), 123 (12), 75 (55), 73 (40).
from 12) and more-polar 17b (4.4 mg, 16.6% from 12). H NMR
(17a, δ): 0.03, 0.05, 0.07, 0.08 (each 3H, s), 0.57 (3H, s), 0.86,
0.89 (each 9H, s), 1.05 (3H, d, J ) 6.6 Hz), 2.83 (1H, m), 3.47
(1H, m), 4.42 (2H, m), 4.92 (1H, s), 4.97 (1H, s), 5.45 (2H, m),
5.83 (1H, d, J ) 11.2 Hz), 6.22 (1H, d, J ) 11.2 Hz). IR (neat,
cm-1): 3749, 2927, 1716, 1540, 1507. MS m/z (%): 716 (M+
-
H2O, 22), 659 (3), 602 (50), 584 (70), 513 (5), 467 (7), 449 (6),
366 (45), 351 (8), 234 (10), 135 (100), 73 (50). HRMS: calcd for
C46H78O3Si2, 734.5489; found, 734.5468. 1H NMR (17b, δ): 0.02,
0.05, 0.06, 0.08 (each 3H, s), 0.57 (3H, s), 0.86, 0.89 (each 9H, s),
1.05 (3H, d, J ) 6.5 Hz), 2.83 (1H, m), 3.49 (1H, m), 4.43 (2H,
m), 4.92 (1H, s), 4.97 (1H, s), 5.47 (2H, m), 5.84 (1H, d, J ) 11.1
Hz), 6.22 (1H, d, J ) 11.1 Hz). IR (neat, cm-1): 3749, 2927, 2902,
2853, 1732, 1541, 1256. MS m/z (%): 716 (M+ - H2O, 10), 659
(2), 602 (38), 584 (36), 513 (2), 467 (5), 449 (3), 366 (32), 351
(5), 234 (10), 197 (12), 135 (100), 93 (15), 73 (65).
(24R)-24-Adamantyl-1r,24-dihydroxy-2-methylene-22,23-dide-
hydro-19,25,26,27-tetranorvitamin D3 (2b). A solution of n-Bu4NF
(1 M THF solution, 48 µL, 0.048 mmol) was added to a solution
of 17a (3.5 mg, 4.8 µmol) in THF (200 µL) at room temperature,
and the mixture was stirred for 6.5 h. The reaction was quenched
with water and was then worked up to yield 2b (2.4 mg, 98.8%).
1H NMR (2b, δ): 0.58 (3H, s), 1.06 (3H, d, J ) 6.6 Hz), 3.48 (1H,
m), 4.47 (2H, m), 5.09 (1H, s), 5.11 (1H, s), 5.45 (2H, m), 5.89
(1H, d, J ) 11.2 Hz), 6.36 (1H, d, J ) 11.2 Hz). MS m/z (%): 506
(M+, 15), 452 (20), 437 (5), 251 (12), 202 (10), 135 (100), 93
(20), 79 (18). HRMS: calcd for C34H50O3, 506.3760; found,
506.3755. UV (95% EtOH) λmax, nm: 245, 254, 263.
Protein Expression and Purification. The rat VDR LBD (residues
116-423, ∆165-211) was cloned as an N-terminal His6-tagged
fusion protein into the pET14b expression vector and was over-
produced in Escherichia coli C41. The cells were grown at 37 °C
in LB medium (including 100 mg/L ampicilin) and were subse-
quently induced for 6 h with 15 µM isopropyl-ꢀ-D-thiogalactopy-
ranoside (IPTG) at 23 °C. The purification procedure included
1r-Hydroxy-2-methylene-22-phenylsulfonyl-19,23,24,25,26,27-
hexanorvitamin D3 1,3-Bis-(tert-butyl-dimethylsilyl) Ether (13b).
To a solution of 13a (19 mg, 0.028 mmol) in CH2Cl2/MeOH (3/5,
800 µL) was added sodium tungstate(VI) dihydrate (14.7 mg, 0.045
mmol) at 0 °C. After 5 min, 30% H2O2 (50 µL) was added, and
the whole mixture was stirred for 7.5 h at room temperature. The
reaction was quenched with 2 N Na2S2O3 at 0 °C, and it underwent
a general workup to produce sulfone 13b (12.8 mg, 65%) and the
corresponding sulfoxide (5.1 mg, 26.3%). 1H NMR (13b, δ): 0.02,

5328 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Nakabayashi et al.
(9) Tsai, M. J.; O’Malley, B. W. Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu. ReV. Biochem.
1994, 63, 451486.
(10) DeLuca, H. F. Overview of general physiologic features and functions
of vitamin D. Am. J. Clin. Nutr. 2004, 80, 1689S–1696S.
(11) Emerging Therapeutic Uses. In Vitamin D; Feldman, D., Glorieux,
F. H., Pike, J. W., Eds.; Academic Press: New York, 1997, pp 1089-
1227.
(12) Yamada, S.; Shimizu, M.; Yamamoto, K. Structure-function relation-
ships of vitamin D including ligand recognition by the vitamin D
receptor. Med. Res. ReV. 2003, 23, 89–115.
(13) Yamada, S.; Yamamoto, K. Ligand recognition by vitamin D receptor:
total alanine scanning mutational analysis of the residues lining the
ligand binding pocket of vitamin D receptor. Curr. Top. Med. Chem.
2006, 6, 1255–1265.
(14) Makishima, M.; Yamada, S. Targeting the vitamin D receptor:
advances in drug discovery. Expert Opin. Ther. Pat. 2005, 15, 1133–
1145.
(15) Smith, C. L.; O’Malley, B. W. Coregulator function: a key to
understanding tissue specificity of selective receptor modulators.
Endocr. ReV. 2004, 25, 45–71.
(16) Rosenfeld, M. G.; Lunyak, V. V.; Glass, C. K. Sensors and signals:
a coactivator/corepressor/epigenetic code for integrating signal-de-
pendent programs of transcriptional response. Genes DeV. 2006, 20,
1405–1428.
(17) Aoyagi, S.; Archer, T. K. Dynamics of coactivator recruitment and
chromatin modifications during nuclear receptor mediated transcription.
Mol. Cell. Endocrinol. 2008, 280, 1–5.
(18) Jordan, V. C. The strategic use of antiestrogens to control the
development and growth of breast cancer. Cancer 1992, 70, 977–
982.
(19) Delmas, P. D.; Bjarnason, N. H.; Mitlak, B. H.; Ravoux, A. C.; Shah,
A. S.; Huster, W. J.; Draper, M.; Christiansen, C. Effects of raloxifene
on bone mineral density, serum cholesterol concentrations, and uterine
endometrium in postmenopausal women. N. Engl. J. Med. 1997, 337,
1641–1647.
(20) Sutanto, W.; de Kloet, E. R. Mineralocorticoid receptor ligands:
biochemical, pharmacological, and clinical aspects. Med. Res. ReV.
1991, 11, 617–639.
(21) Kolvenbag, G. J.; Furr, B. J. Relative potency of bicalutamide (casodex)
and flutamide (eulexin). Urology 1999, 54, 194–197.
(22) Fradet, Y. Bicalutamide (casodex) in the treatment of prostate cancer.
Expert ReV. Anticancer Ther. 2004, 4, 37–48.
(23) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.; Bonn,
T.; Engstrom, O.; Ohman, L.; Greene, G. L.; Gustafsson, J. A.;
Carlquist, M. Molecular basis of agonism and antagonism in the
oestrogen receptor. Nature 1997, 389, 753–758.
(24) Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P. J.;
Agard, D. A.; Greene, G. L. The structural basis of estrogen receptor/
coactivator recognition and the antagonism of this interaction by
tamoxifen. Cell 1998, 95, 927–937.
(25) Pike, A. C.; Brzozowski, A. M.; Hubbard, R. E.; Bonn, T.; Thorsell,
A. G.; Engstro¨m, O.; Ljunggren, J.; Gustafsson, J. A.; Carlquist, M.
Structure of the ligand-binding domain of oestrogen receptor beta in
the presence of a partial agonist and a full antagonist. EMBO J. 1999,
18, 4608–4618.
(26) Shiau, A. K.; Barstad, D.; Radek, J. T.; Meyers, M. J.; Nettles, K. W.;
Katzenellenbogen, B. S.; Katzenellenbogen, J. A.; Agard, D. A.;
Greene, G. L. Structural characterization of a subtype-selective ligand
reveals a novel mode of estrogen receptor antagonism. Nat. Struct.
Biol. 2002, 9, 359–364.
affinity chromatography on a Ni-NTA column, followed by dialysis
and ion-exchange chromatography (SP-Sepharose). After tag
removal by thrombin digestion, the protease was removed by
filtration through a HiTrap benzamidine column, and the protein
was further purified by gel filtration on a Superdex200 column.
The purity and homogeneity of the rVDR LBP were assessed by
SDS-PAGE.
Crystallization. Purified rVDR LBD solution was concentrated
to about 0.75 mg/mL by ultrafiltration. To an aliquot (800 µL) of
the protein solution was added a ligand (ca. 10 equiv); the solution
was further concentrated to about 1/8, and then a solution (25 mM
Tris-HCl, pH 8.0; 50 mM NaCl; 10 mM DTT; 0.02% NaN3) of
coactivator peptide (H2N-KNHPMLMNLLKDN-CONH2) derived
from DRIP205 was added. This solution of VDR/ligand/peptide
was allowed to crystallize by the vapor-diffusion method that used
a series of precipitant solutions containing 0.1 M MOPS-NaOH
(pH 7.0), 0.1-0.4 M sodium formate, 12-22% (w/v) PEG4000,
and 5% (v/v) ethylene glycol. Droplets for crystallization were
prepared by mixing 2 µL of complex solution and 1 µL of
precipitant solution, and droplets were equilibrated against 500 µL
of precipitant solution at 20 °C. It took 1 to 2 days to obtain crystals
of X-ray diffraction quality for VDR complexes with 1,25(OH)2D3,
2a, or 2b as a ligand.
Diffraction Experiment and Structure Analysis. Prior to the
diffraction data collection, crystals were soaked in a cryoprotectant
solution that contained 0.1 M MOPS-NaOH (pH 7.0), 0.1-0.4 M
sodium formate, 15-20% PEG4000, and 17-20% ethylene glycol.
Diffraction data sets were collected at 100 K in a stream of nitrogen
gas at beamlines BL-6A of KEK-PF and NW12A of PF-AR
(Tsukuba, Japan). Reflections were recorded with an oscillation
range per image of 1.0°. Diffraction data were indexed, integrated,
and scaled using the program HKL2000.56 The structures of the
complex were solved by molecular replacement with the program
CNS,57 and finalized sets of atomic coordinates were obtained after
iterative rounds of model modification with the program XtalView58
and after refinement with CNS by rigid body refinement, simulated
annealing, positional minimization, water molecule identification,
and individual isotropic B-value refinement. We calculated the LBP
volumes of the VDR/adamantyl vitamin D and 1,25(OH)2D3
complexes by using the fast Connolly channel surface program of
the SYBYL molecular modeling software (Tripos, St. Louis).
Supporting Information Available: Data collection and refine-
ment statistics of rVDR complexes. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Yamada, S.; Shimizu, M.; Yamamoto, K. Vitamin D Receptor. In
Vitamin D and Rickets; Hochberg, Z., Ed.; Karger: New York, 2003;
pp 50-68.
(2) McDonnell, D. P.; Mangelsdorf, D. J.; Pike, J. W.; Haussler, M. R.;
O’Malley, B. W. Molecular cloning of complementary DNA encoding
the avian receptor for vitamin D. Science 1987, 235, 1214–1217.
(3) Baker, A. R.; McDonnell, D. P.; Hughes, M. R.; Crisp, T. M.;
Mangelsdorf, D. J.; Haussler, M. R.; Pike, J. W.; Shine, J. Cloning
and expression of full-length cDNA encoding human vitamin D
receptor. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 3294–3298.
(4) Burmester, J. K.; Maeda, N.; DeLuca, H. F. Isolation and expression
of rat 1,25-dihydroxyvitamin D receptor cDNA. Proc. Natl. Acad. Sci.
U.S.A. 1988, 85, 1005–1009.
(5) Kamei, Y.; Kawada, T.; Fukuwatari, T.; Ono, T.; Kato, S.; Sugimoto,
E. Cloning and sequencing of the gene encoding the mouse vitamin
D receptor. Gene 1995, 152, 281–282.
(6) Li, Y. C.; Bergwitz, C.; Juppner, H.; Demay, M. B. Cloning and
characterization of the vitamin D receptor from Xenopus laeVis.
Endocrinology 1997, 138, 2347–2353.
(27) Bledsoe, R. K.; Madauss, K. P.; Holt, J. A.; Apolito, C. J.; Lambert,
M. H.; Pearce, K. H.; Stanley, T. B.; Stewart, E. L.; Trump, R. P.;
Willson, T. M.; Williams, S. P. A ligand-mediated hydrogen bond
network required for the activation of the mineralocorticoid receptor.
J. Biol. Chem. 2005, 280, 31283–31293.
(28) Fagart, J.; Huyet, J.; Pinon, G. M.; Rochel, M.; Mayer, C.; Rafestin-
Oblin, M. E. Crystal structure of a mutant mineralocorticoid receptor
responsible for hypertension. Nat. Struct. Mol. Biol. 2005, 12, 554–
555.
(29) Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T. Structural
basis for antagonism and resistance of bicalutamide in prostate cancer.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6201–6206.
(30) Bohl, C. E.; Miller, D. D.; Chen, J.; Bell, C. E.; Dalton, J. T. Structural
basis for accommodation of nonsteroidal ligands in the androgen
receptor. J. Biol. Chem. 2005, 280, 37747–37754.
(31) Bury, Y.; Steinmeyer, A.; Carlberg, C. Structure activity relationship
of carboxylic ester antagonists of the vitamin D3 receptor. Mol.
Pharmacol. 2000, 58, 1067–1074.
(7) Chun, R. F.; Chen, H.; Boldrick, L.; Sweet, C.; Adams, J. S. Cloning,
sequencing, and functional characterization of the vitamin D receptor
in vitamin D-resistant New World primates. Am. J. Primatol. 2001,
54, 107–118.
(8) Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schutz,
G.; Umesono, K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon,
P.; Evans, R. M. The nuclear receptor superfamily: the second decade.
Cell 1995, 83, 835–839.
(32) Va¨isa¨nen, S.; Pera¨kyla¨, M.; Ka¨rkka¨inen, J. I.; Steinmeyer, A.; Carlberg,
C. Critical role of helix 12 of the vitamin D3 receptor for the partial
agonism of carboxylic ester antagonists. J. Mol. Biol. 2002, 315, 229–
238.

Crystal Structures of VDR/Antagonists Complexes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5329
(33) Lempia¨inen, H.; Molna´r, F.; Macias Gonzalez, M.; Pera¨kyla¨, M.;
Carlberg, C. Antagonist- and inverse agonist-driven interactions of
the vitamin D receptor and the constitutive androstane receptor with
corepressor protein. Mol. Endocrinol. 2005, 19, 2258–2272.
(34) Toell, A.; Gonzalez, M. M.; Ruf, D.; Steinmeyer, A.; Ishizuka, S.;
Carlberg, C. Different molecular mechanisms of vitamin D(3) receptor
antagonists. Mol. Pharmacol. 2001, 59, 1478–1485.
(35) Ishizuka, S.; Kurihara, N.; Reddy, S. V.; Cornish, J.; Cundy, T.;
Roodman, G. D. (23S)-25-Dehydro-1R-hydroxyvitamin D3-26,23-
lactone, a vitamin D receptor antagonist that inhibits osteoclast
formation and bone resorption in bone marrow cultures from patients
with Paget’s disease. Endocrinology 2005, 146, 20232030.
(36) Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.; Norman, A. W.;
Ishizuka, S. Antagonistic action of novel 1R,25-dihydroxyvitamin D3-
26, 23-lactone analogs on differentiation of human leukemia cells (HL-
60) induced by 1R,25-dihydroxyvitamin D3. J. Biol. Chem. 1999, 274,
16392–16399.
(37) Fujishima, T.; Kojima, Y.; Azumaya, I.; Kittaka, A.; Takayama, H.
Design and synthesis of potent vitamin D receptor antagonists with
A-ring modifications: remarkable effects of 2R-methyl introduction
on antagonistic activity. Bioorg. Med. Chem. 2003, 11, 3621–3631.
(38) Kato, Y.; Nakano, Y.; Sano, H.; Tanatani, A.; Kobayashi, H.;
Shimazawa, R.; Koshino, H.; Hashimoto, Y.; Nagasawa, K. Synthesis
of 1R,25-dihydroxyvitamin D3-26,23-lactams (DLAMs), a novel
series of 1R,25-dihydroxyvitamin D3 antagonist. Bioorg. Med. Chem.
Lett. 2004, 14, 2579–2583.
(39) Yoshimoto, N.; Inaba, Y.; Yamada, S.; Makishima, M.; Shimizu, M.;
Yamamoto, K. 2-Methylene 19-nor-25-dehydro-1R-hydroxyvitamin D3
26,23-lactones: synthesis, biological activities, and molecular basis
of passive antagonism. Bioorg. Med. Chem. 2008, 16, 457–473.
(40) Ma, Y.; Khalifa, B.; Yee, Y. K.; Lu, J.; Memezawa, A.; Savkur, R. S.;
Yamamoto, Y.; Chintalacharuvu, S. R.; Yamaoka, K.; Stayrook, K. R.;
Bramlett, K. S.; Zeng, Q. Q.; Chandrasekhar, S.; Yu, X.-P.; Linebarger,
J. H.; Iturria, S. J.; Burris, T. P.; Kato, S.; Chin, W. W.; Nagpal1, S.
Identification and characterization of noncalcemic, tissue-selective,
nonsecosteroidal vitamin D receptor modulators. J. Clin. InVest. 2006,
116, 892–904.
(41) 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.
(42) Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. M.; Mitschler, A.; Moras,
D. Crystal structures of the vitamin D receptor complexed to
superagonist 20-epi ligands. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
5491–5496.
(43) 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 D(3) hormone
analogues and a LXXLL-containing coactivator peptide. Biochemistry
2004, 43, 4101–4110.
(44) Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. M.; Moras, D. Crystal
structures of the vitamin D nuclear receptor liganded with the vitamin
D side chain analogues calcipotriol and seocalcitol, receptor agonists
of clinical importance. Insights into a structural basis for the switching
of calcipotriol to a receptor antagonist by further side chain modifica-
tion. J. Med. Chem. 2004, 47, 1956–1961.
(46) Hourai, S.; Fujishima, T.; Kittaka, A.; Suhara, Y.; Takayama, H.;
Rochel, N.; Moras, D. Probing a water channel near the A-ring of
receptor-bound 1R,25-dihydroxyvitamin D3 with selected 2R-
substituted analogues. J. Med. Chem. 2006, 49, 5199–5205.
(47) Ciesielski, F.; Rochel, N.; Moras, D. Adaptability of the vitamin D
nuclear receptor to the synthetic ligand gemini: remodeling the LBP
with one side chain rotation. J. Steroid Biochem. Mol. Biol. 2007,
103, 235–242.
(48) Vanhooke, J.; Tadi, B. P.; Benning, M. M.; Plum, L. A.; DeLuca,
H. F. Crystal structure of the vitamin D nuclear receptor ligand binding
domain in complex with a locked side chain analog of calcitriol. Arch.
Biochem. Biophys. 2007, 460, 161–165.
(49) Rochel, N.; Hourai, S.; Rerez-Garcia, X.; Rumbo, A.; Mourino, A.;
Moras, D. Crystal structure of the vitamin D nuclear receptor ligand
binding domain in complex with a locked side chain analog of
calcitriol. Arch. Biochem. Biophys. 2007, 460, 172–176.
(50) Yamamoto, K.; Abe, D.; Yoshimoto, N.; Choi, M.; Yamagishi, K.;
Tokiwa, H.; Shimizu, M.; Makishima, M.; Yamada, S. Vitamin D
receptor: ligand recognition and allosteric network. J. Med. Chem.
2006, 49, 1313–1324.
(51) Igarashi, M.; Yoshimoto, N.; Yamamoto, K.; Shimizu, M.; Ishizawa,
M.; Makishima, M.; DeLuca, H. F.; Yamada, S. Identification of a
highly potent vitamin D receptor antagonist: (25S)-26-adamantyl-25-
hydroxy-2-methylene-22,23-didehydro-19,27-dinor-20-epi-vitamin D3
(ADMI3). Arch. Biochem. Biophys. 2007, 460, 240–253.
(52) Yoshimoto, N. Vitamin D Receptor Antagonists: Design, Synthesis,
Biological Activities, and Molecular Basis of Antagonism. Thesis,
Tokyo Medical and Dental University, 2008.
(53) Inaba, Y.; Yamamoto, K.; Yoshimoto, N.; Matsunawa, M.; Uno, S.;
Yamada, S.; Makishima, M. Vitamin D3 derivatives with adamantane
or lactone ring side chains are cell type-selective vitamin D receptor
modulators. Mol. Pharmacol. 2007, 71, 1298–1311.
(54) Sicinski, R. R.; Prahl, J. M.; Smith, C. M.; DeLuca, H. F. New 1R,25-
dihydroxy-19-norvitamin D3 compounds of high biological activity:
synthesis and biological evaluation of 2-hydroxymethyl, 2-methyl, and
2-methylene analogues. J. Med. Chem. 1998, 41, 4662–4674.
(55) Shevde, N. K.; Plum, L. A.; Clagett-Dame, M.; Yamamoto, H.; Pike,
J. W.; DeLuca, H. F. A potent analog of 1R,25-dihydroxyvitamin D3
selectively induces bone formation. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 13487–13491.
(56) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 1997, 276, 307–326.
(57) Bru¨nger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J.-S.; Kuszewski, J.; Nilges, M.;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Crystallography & NMR system: a new software suite for macromo-
lecular structure determination. Acta Crystallogr. 1998, D54, 905–
921.
(58) McRee, D. E. XtalView/Xfitsa versatile program for manipulating
atomic coordinates and electron density. J. Struct. Biol. 1999, 125,
156–165.
(59) The Protein Data Bank accession numbers for the coordinates of the
structures of the VDR complexes with (25R)-25-adamantyl-1R,25-
hydroxy-2-methylene-22,23-didehydro-19,26,27-trinor-20-epivita-
min D3 (2a), (24R)-24-adamantyl-1R,24-hydroxy-2-methylene-22,23-
didehydro-19,25,26,27-tetranor-vitamin D3 (2b), (25R)-26-adamantyl-
1R,25-hydroxy-2-methylene-22,23-didehydro-19,27-dinor-20-
epivitamin D3 (2c), and 1,25(OH)2D3 (1) reported in this article are
2ZMI, 2ZMH, 2ZMJ, and 2ZLC, respectively.
(45) Eelen, G.; Verlinden, L.; Rochel, N.; Claessens, F.; De Clercq, P.;
Vandewalle, M.; Tocchini-Valentini, G.; Moras, D.; Bouillon, R.;
Verstuyf, A. Superagonistic action of 14-epi-analogs of 1,25-dihy-
droxyvitamin D explained by vitamin D receptor-coactivator interac-
tion. Mol. Pharmacol. 2005, 67, 1566–1573.
JM8004477