Crystal structure of the PXR-T1317 complex provides a scaffold to examine the potential for receptor antagonism

Article abstract of DOI:10.1016/j.bmc.2006.12.026

The human pregnane X receptor (PXR) recognizes a range of structurally and chemically distinct ligands and plays a key role in regulating the expression of protective gene products involved in the metabolism and excretion of potentially harmful compounds. The identification and development of PXR antagonists is desirable as a potential way to control the up-regulation of drug metabolism pathways during the therapeutic treatment of disease. We present the 2.8 A resolution crystal structure of the PXR ligand binding domain (LBD) in complex with T0901317 (T1317), which is also an agonist of another member of the orphan class of the nuclear receptor superfamily, the liver X receptor (LXR). In spite of differences in the size and shape of the receptors' ligand binding pockets, key interactions with this ligand are conserved between human PXR and human LXR. Based on the PXR-T1317 structure, analogues of T1317 were generated with the goal of designing an PXR antagonist effective via the receptor's ligand binding pocket. We find that selectivity in activating PXR versus LXR was achieved; such compounds may be useful in addressing neurodegenerative diseases like Niemann-Pick C. We were not successful, however, in producing a PXR antagonist. Based on these observations, we conclude that the generation of PXR antagonists targeted to the ligand binding pocket may be difficult due to the promiscuity and structural conformability of this xenobiotic sensor.

Full text of DOI:10.1016/j.bmc.2006.12.026

Bioorganic & Medicinal Chemistry 15 (2007) 2156–2166
Crystal structure of the PXR–T1317 complex provides a scaffold
to examine the potential for receptor antagonismq
Yu Xue,a Esther Chao,b William J. Zuercher,b Timothy M. Willson,b
Jon L. Collinsb and Matthew R. Redinboa,c,
*
aDepartment of Chemistry, CB#3290, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, USA
bNuclear Receptor Discovery Research, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
cDepartment of Biochemistry and Biophysics, and the Lineberger Comprehensive Cancer Center, School of Medicine,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Received 3 October 2006; revised 6 December 2006; accepted 11 December 2006
Available online 20 December 2006
Abstract—The human pregnane X receptor (PXR) recognizes a range of structurally and chemically distinct ligands and plays a key
role in regulating the expression of protective gene products involved in the metabolism and excretion of potentially harmful com-
pounds. The identification and development of PXR antagonists is desirable as a potential way to control the up-regulation of drug
˚
metabolism pathways during the therapeutic treatment of disease. We present the 2.8 A resolution crystal structure of the PXR
ligand binding domain (LBD) in complex with T0901317 (T1317), which is also an agonist of another member of the orphan class
of the nuclear receptor superfamily, the liver X receptor (LXR). In spite of differences in the size and shape of the receptors’ ligand
binding pockets, key interactions with this ligand are conserved between human PXR and human LXR. Based on the PXR–T1317
structure, analogues of T1317 were generated with the goal of designing an PXR antagonist effective via the receptor’s ligand bind-
ing pocket. We find that selectivity in activating PXR versus LXR was achieved; such compounds may be useful in addressing neu-
rodegenerative diseases like Niemann-Pick C. We were not successful, however, in producing a PXR antagonist. Based on these
observations, we conclude that the generation of PXR antagonists targeted to the ligand binding pocket may be difficult due to
the promiscuity and structural conformability of this xenobiotic sensor.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Like other members of the orphan class of the nuclear
receptor (NR) superfamily, PXR contains DNA-bind-
ing and ligand binding domains (DBD, LBD, respective-
ly), acts as a heterodimer with the retinoid X receptor-a
(RXRa), and binds to a range of direct- and everted-re-
peat elements in the regulatory region of target genes.9
Upon association with an activating agonist, a tran-
scriptional coactivator protein like the steroid receptor
coactivator-1 (SRC-1) is recruited to the activation func-
tion-2 (AF-2) region of the PXR LBD, which facilitates
changes in chromatin structure and activation of the
basal transcriptional machinery. A leucine-rich LxxLL
motif in transcriptional coactivators (where x is any
amino acid) has been shown to interact with a groove
present in the active orientation of NR LBDs.10,11 Crys-
tal structures of the human PXR LBD have been deter-
mined in complexes with a variety of small (e.g.,
SR12813, hyperforin) and large (e.g., rifampicin) li-
gands, and with fragments of the human transcriptional
coactivator SRC-1.12–15 These structures have revealed
that PXR’s ligand binding promiscuity is a function of
its large and conformable ligand binding pocket, which
The human pregnane X receptor (PXR; alternatively
SXR, PAR) responds to a variety of endogenous and
exogenous compounds in liver, intestine, and other tis-
sues, and is a key regulator of the expression of genes cen-
tral to xenobiotic metabolism and excretion.1–3 PXR is
also responsible for an important class of drug interac-
tions caused by the efficient up-regulation of chemopro-
tective pathways that lead to the elimination of a wide
range of therapeutics.4–7 It has also recently been shown
that T0901317 (T1317), the ligand described here in com-
plex with PXR, protects against the development of the
neurodegenerative disease Niemann-Pick C in a PXR-de-
pendent fashion within a mouse model of this condition.8
Keywords: Nuclear receptor; Xenobiotics; LXR; Antagonism; Struc-
ture–activity relationships; Structure-based design.
q
This work was supported by National Institutes of Health Grant
DK62229 (M.R.R.).
*
Corresponding author. Tel.: +1 919 843 8910; fax: +1 919 966
3675; e-mail: redinbo@unc.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.026

Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
2157
˚
is framed in part by sequence elements novel to PXR rel-
ative to other NR LBDs. In addition, the PXR LBD
forms a unique homodimer mediated by a tryptophan
zipper-like motif, and it has been shown that this inter-
face plays a role in receptor function and association
with coactivators.16
the 2.8 A resolution crystal structure of the PXR LBD
in complex with T1317. Using this structure as a guide,
and inspired by selective ER modulators produced from
ER agonists, we generate T1317 analogues designed to
act as PXR antagonists by disrupting the active confor-
mation of the receptor’s aAF. We find, however, that
compounds based on this scaffold either retain their abil-
ity to bind to PXR, and thus serve as agonists, or simply
do not bind to the receptor. We conclude that the design
of effective pocket-targeted PXR antagonists may be dif-
ficult due to the promiscuous and conformable nature of
the receptor’s ligand binding pocket.
The liver X receptor (LXR), another member of the or-
phan class of NRs that functions as a heterodimer with
RXRa, plays an important role in monitoring the levels
of oxysterols in hepatocytes and regulates the expression
of genes essential for cholesterol homeostasis.17–21 The
LBDs of the two LXR isoforms, a and b, both share
31% sequence identity with the human PXR LBD (and
77% with one another), as well as the conserved overall
structural fold common to NR ligand binding do-
mains.22–26 The PXR LBD deviates from that of LXR
in its ꢀ60-residue a1–a3 insert that adds one helix (a2)
and two strands (b1,b10) and frame a significantly larger
ligand binding pocket relative to LXR.10 The synthetic
NR ligand T0901317 (T1317) is an established agonist
for LXR, exhibiting robust up-regulation of target gene
expression.19 The structural basis of T1317 binding to
LXR isoform b has been elucidated previously.22,24,26
2. Results
2.1. PXR–T1317 structure
Crystals of the human PXR LBD were grown in the
presence of 10-fold molar excess T0901317 (T1317),
˚
X-ray diffraction data to 2.8 A resolution were collected,
and the structure was determined and refined to R and
Rfree values of 0.228 and 0.240, respectively (Table 1).
Two ternary complexes were observed in the asymmetric
unit, each containing one human PXR LBD, one orien-
tation of bound T1317, and one fifteen-amino acid
stretch of the human transcriptional coactivator steroid
In addition to their association with transcriptional coac-
tivators, NR LBDs bind to transcriptional corepressors
(e.g., NCoR, SMRT) that exert opposite effects on gene
transcription by mechanisms that include enhancing the
condensed structure of chromatin.4 Interactions between
NRs and corepressors can occur in the absence of ligand,
but are enhanced in the presence of antagonizing ligands.
A paradigm for this effect is provided by efficient down-
regulation of transcription caused by the association of
the estrogen receptor (ER) with the established antago-
nist tamoxifen. It has been shown structurally that core-
pressors contain an extended leucine-rich region that
binds to an inactive LBD conformation distinct in struc-
ture from that of an active LBD, particularly in the posi-
tion of the terminal a-helix in the LBD fold (aAF in
PXR).27 In ER, tamoxifen sterically blocks the active ori-
entation of the terminal helix in that fold, producing a
state that preferentially binds to transcriptional core-
pressors and down-regulates gene expression.28 In the
case of PXR, most ligands are found to act as agonists
of this receptor, in line with its established role in protect-
ing tissues from potentially harmful chemicals. A small
number of antagonists have been described, however,
including the ET-74329 and the antifungal ketocona-
zole.30,31 Indeed, ketoconazole has been shown recently
to repress the binding of both transcriptional coactiva-
tors and corepressors to PXR.30 The structural basis of
the antagonism of PXR by these compounds has not
been established, however.
receptor coactivator
1 (SRC-1, residues 682–696)
(Fig. 1). The PXR LBD is expressed with an 88-amino
acid fragment of SRC-1 (residues 623–710) to improve
protein stability. This is the first structure of PXR in
which the coactivator fragment remains attached during
crystallization. The retention of this fragment is likely
due to the relatively high affinity and potent agonist
activity of T1317. The 88-amino acid region of human
SRC-1 employed contains two leucine-rich NR box mo-
tifs (at 633–637, LVQLL and 690–694, LHRLL). Only
the second NR box was observed associated with the
Table 1. Crystallographic statistics for the PXR-T0901317 complex
˚
Resolution (A; highest shell)
˚
50–2.8 A (2.9–2.8)
Space group
Asymmetric unit
P212121
Two molecules
a = 83.9 b = 90.6
c = 105.7
a = b = c = 90
SER-CAT (APS)
119,110
˚
Cell constants (A, ꢁ)
Data collection facility
Total reflections
Unique reflections
20,332
5.8 (5.4)
Mean redundancy (highest shell)
a
Rsym (%; highest shell)
9.4 (28.1)
97.6 (91.1)
25.9 (5.1)
22.8
Completeness (%; highest shell)
Mean I/r (highest shell)
Rcryst
b
c
Rfree
24.0
2O9I
The identification or design of new antagonists of hu-
man PXR is desirable because of the important role this
receptor plays in drug metabolism, and because the cur-
rent antagonists exhibit weak EC50 values relative to
established agonists.29–31 Repressing the ability of
PXR to recognize the presence of therapeutic com-
pounds may allow lower doses to be administered with
higher efficacy and fewer side effects. Here we present
RCSB Accession Code
P
P
a Rsym
=
|I-hIi|/ I, where I is the observed intensity and hIi is the
average intensity of multiple symmetry-related observations of that
reflection.
P
P
b Rcryst
observed and calculated structure factors, respectively.
=
kFobs| ꢁ |Fcalck/ |Fobs|, where Fobs and Fcalc are the
P
P
c Rfree
= kFobs| ꢁ |Fcalck/ |Fobs| for 10% of the data not used at any
stage of structural refinement.

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Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
The PXR LBD in this T1317 complex retains the same
overall structure observed in previous PXR structures,
˚
sharing, for example, 1 A root-mean-square deviation
(rmsd) over Ca positions with the structure of the apo
(unliganded) PXR LBD.15 The LBDs also form a
homodimer consistently observed either via crystallo-
graphic or, as in this case, non-crystallographic symme-
try. This homodimer interaction is mediated largely by
interdigitating aromatic residues from b10 in each mono-
mer, and its formation has been shown to be central to
transcriptional activity and coactivator recruitment by
PXR.16 Only a small number of shifts in the positions
of amino acids that line the ligand binding pocket of
PXR were observed between the T1317 and apo struc-
tures. For example, Leu-209 and Met-323 undergo rot-
˚
amer changes and shifts in position of 6.0 A. It was
also noted that the side chains of both His-407 and
˚
Phe-429 shift 1.0 A toward the bound ligand relative
˚
to the apo structure, and in doing so form an 3.8 A aro-
matic edge-to-edge van der Waals contact (compared to
˚
5.3 A for the same atoms in the apo structure). Because
Phe-429 is located on the aAF of the PXR’s AF-2 re-
gion, this interaction likely stabilizes the active form of
the receptor during the up-regulation of gene
transcription.
2.2. PXR–T1317 interactions
T1317 forms three polar and 12 van der Waals contacts
with amino acid side chains that line the PXR ligand
binding pocket (Fig. 2 Table 2). His-407 is positioned
˚
2.4 A from the ligand’s hydroxyl group, while the polar
Figure 1. Crystal structure of the homodimer in the asymmetric unit of
the ligand binding domain of human PXR (PXR LBD) in complex
with T0901317 (T1317). The PXR LBD in one monomer is rendered in
red, yellow, and green, and in the other monomer in cyan, magenta,
and pink. The fragments of the steroid receptor coactivator 1 (SRC-1)
are shown in aqua and orange, and the T1317 ligands in magenta and
green.
˚
groups of the Gln-285 side chain are 2.9 A and 3.2 A
˚
from one sulfoxy oxygen and His-327 is 3.3 A from the
˚
other. This is the first time His-327 has been observed
within hydrogen bonding distance (generously defined)
of a bound ligand in any of the PXR complexes deter-
mined to date. Similarly, Tyr-306, which forms an
edge-to-face with the free benzyl ring of T1317, has also
not been observed to contact ligand in previous struc-
tures. The same T1317 benzyl ring forms parallel and
edge-to-face aromatic stacking interactions with Phe-
288 and Trp-299, respectively. The two CF3 groups of
the ligand form van der Waals contacts with five resi-
AF-2 region of the PXR LBD in both molecules in the
crystallographic asymmetric unit. This implies that the
sequence around or including LHRLL may have higher
affinity for PXR relative to the region around the
LVQLL motif.
˚
dues, including a 3.5 A interaction with Met-425 located
Figure 2. Stereoview of the binding of T0901317 within the ligand binding pocket of the human PXR LBD. Residues making polar contacts are
rendered in green, those making hydrophobic interactions are in red, with Met-425 from aAF highlighted in yellow.

Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
2159
Table 2. Comparison of residues contacting T0901317 in PXR and
LXR
PXR
LXR
Gln-285
His-327
His-407
Leu-209
Val-211
Leu-240
Met-243
Met-246
Phe-288
Trp-299
Tyr-306
Leu-411
Ile-413
Leu-313
(Ser-357)
His-435
a
—
—
a
Phe-268
Phe-271
Leu-274
Thr-316
Ile-327
(Tyr-335)
Val-439
Leu-442
Leu-449
Phe-420
Met-425
Leu-453
Met-312
Phe-329
Leu-345
Ile-353
(Cys-284-Ser)b
(Cys-301)c
(Leu-206)
(Met-323)
(Phe-429)
Trp-457
Amino acids forming hydrogen bonding interactions with ligand are
listed in bold, those on the terminal aAF helices of the receptors are
underlined, and those not contacting ligand are in italics and
parentheses.
Figure 3. Superposition of the human PXR LBD monomer (red) on
that of the human LXRb LBD (gold), with the T0901317 ligands
present in both structures shown in magenta for PXR and blue for
LXR.
a Residues 209 and 211 are on a sequence insert novel to PXR; thus, no
equivalent side chains exist in LXR.
b Cys-284 was mutated to serine to improve the crystallization
behavior of PXR; see Materials and methods.
c While Cys-301 corresponds in sequence to Phe-329, the PXR side
chain Phe-288 overlaps structurally with this LXR phenylalanine; see
Figure 4.
hydrogen bonds with the ligand (His-435 and Thr-
316). His-435 in LXR corresponds in both sequence
and structure to His-407 in PXR; however, Gln-285,
which forms two polar contacts with T1317 in the
PXR complex, is replaced by a leucine in LXR
(Table 2, Fig. 4). Thr-316, which forms the second
polar interaction in LXR, is related in sequence to
Phe-288 in PXR, but corresponds in structure to
on the receptor’s aAF that likely helps to stabilize the ac-
tive conformation of the AF-2 surface. The volume of
the PXR ligand binding pocket in this structure was mea-
˚
His-327, albeit shifted in position by ꢀ5.5 A. The ben-
3
˚
zyl ring of T1317 forms aromatic contacts with phenyl-
alanines 271 and 329 in LXR, which helps to position
the ligand distinctly within the pocket when compared
to PXR. It is also noted that distinct histidine-aromatic
interactions are observed in PXR relative to LXR. An
edge-to-face aromatic interaction between His-435 and
Trp-457 had been noted previously for LXR; this con-
sured to be 1334 A and it was further found that the
3
T1317 ligand occupied all but 442 A of that space. In to-
˚
tal, the 15 residues contacted by T1317 are the largest ob-
served for a small ligand in PXR’s pocket, but do not
exceed the 18 residues contacted by the large macrolide
antibiotic rifampicin.12
˚
tact is replaced in PXR by a 3.8 A edge-to-edge inter-
action between His-407 and Phe-429. A second
histidine aromatic contact is observed in PXR: a
2.3. T1317 Binding by LXR versus PXR
We next compared the structure of the human PXR
LBD-T1317 complex to that of the human LXRb
LBD complexed to the same ligand.22,24–26 The LBDs
˚
3.3 A face-to-edge interaction between Trp-299 and
His-327 (Fig. 4). In total, PXR and LXR share one
conserved polar contact and eight conserved hydropho-
bic interactions in their respective complexes with
T1317 (Table 2). In addition, both receptors directly
contact corresponding residues located on the aAF
helices of their AF-2 surfaces (Met-425 in PXR, Leu-
453 in LXR). LXR apparently further stabilizes its
AF-2 region by the formation of the aromatic contact
between His-435 and Trp-457, which is located on
aAF. Taken together, these observations show that,
in spite of differences in pocket shape and ligand orien-
tation, numerous key contacts are conserved between
these related receptors, leading to the efficient up-regu-
lation of target genes by T1317.
˚
exhibited the same overall fold, sharing 2.2 A rmsd
over Ca positions (and 27% sequence identity),
although the secondary structural elements present on
the a1–a3 insert novel to PXR, including b1, b10,
and a2, are not present in the LXR LBD (Fig. 3).
LXR’s ligand binding pocket is roughly half the size
3
˚
of PXR’s (650 A ) and the observed binding of
T1317 within the pocket uses essentially all the avail-
able space. The distinct and limited shape of the
LXR pocket causes the T1317 ligand to bind in a posi-
˚
tion rotated by ꢀ30ꢁ and shifted by up to 5.3 A relative
to the position observed in the PXR complex. Sixteen
LXR side chains contact T1317, two of which form

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Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
Figure 4. Stereoview comparing the binding of T0901317 to the LBDs of human PXR and LXRb. Human PXR residues and T1317 ligand are
colored as in Figure 2, except Phe-429 and Met-243, which are shown in white. In LXR, residues forming polar ligand contacts are shown in cyan,
while those forming hydrophobic interactions are in gold or, for Leu-452 and Tyr-335, in pink.
2.4. Design and examination of putative antagonists
modating larger adducts than was previously appreciat-
ed. The structural basis of these effects may be based on
the mobile a2 region of PXR, which is present on a se-
quence insert unique to this receptor relative to other
members of the nuclear receptor superfamily. This
stretch of the PXR LBD structure has been observed
Because T1317 appears to bind particularly strongly to
PXR, we next sought to design a PXR antagonist using
the T1317 structure as a scaffold. It is known that antag-
onists of other nuclear receptors (e.g., tamoxifen for the
estrogen receptor) appear to function by sterically
blocking the active position of aAF28. Thus, we chose
to change the structure of the CF3-containing moiety
in T1317, which binds adjacent to aAF in PXR
(Fig. 2). Eleven analogues were synthesized that includ-
ed both smaller and larger groups at this position
(Fig. 5; Scheme 1). These compounds, along with
T1317, were tested for their ability to bind to PXR in
an in vitro ligand-competition assay, and to activate
PXR-mediated transcription in transient transfection as-
says in cultured cells (Table 3). Compounds 2–5, which
retained the hydroxyl group and one CF3 group, all
exhibited good binding to and activation of PXR. In
contrast, compounds 6 and 7, which retain the hydroxyl
group but not a proximal CF3, are poor binders and
activators of PXR. Similarly, compound 8, which con-
tains a acetyl group, was the least efficacious compound
examined. These data support the importance of the
hydrogen bond donated by the T1317 hydroxyl group
to the His-407 side chain in PXR, in particular the elec-
tron withdrawing character of the CF3 groups that
polarize the hydroxyl group and improve its ability to
share its hydrogen atom.25
˚
to shift in position by several A between different li-
gand-bound complexes, and, in the case of the PXR
structure bound to the large macrolide antibiotic rifam-
picin, to become completely disordered.12–15 The large
adducts placed in the T1317 scaffold in compounds
9–12 may be directed toward this region of PXR and
may create space for themselves by disrupting the posi-
tion of a2 rather than aAF. In addition, they could ex-
tend from the receptor’s pocket into solvent by creating
a pore adjacent to Leu-240 (Fig. 2). In either case, these
observed effects underscore the fundamentally promis-
cuous nature of PXR in terms of ligand binding—the
receptor contains a polypeptide insert that allows its
binding pocket to expand and contract, facilitating the
productive binding of a wide range of chemical
structures.
The activation of gene expression by T1317 analogues
was also examined via LXRb by transient transfection
assays in cultured cells (Table 3). Similar to PXR, com-
pounds 6–8, which do not retain the hydroxyl group
proximal to a CF3 moiety, were poor ligands for
LXR, highlighting the importance of the His-435 hydro-
gen received from the polarized ligand hydroxyl group.
In contrast to PXR, however, compounds 9 and 11 were
poor agonists of LXR, while compounds 10 and 12 were
relatively effective. Considering the structure of the
LXRb–T1317 complex, the benzyl group in 10 may
stack upon the aromatic Phe-268 side chain, an interac-
tion not possible with compound 9 or 11. For 12, the
flexible nature of its CF3-rich extension may facilitate
productive binding by protruding past Phe-268 into sol-
vent. Taken together, these observations regarding the
impact of T1317 analogues on LXR-mediated gene
expression reveal that this receptor, with its smaller li-
gand binding pocket and more narrow agonist profile,
is more accommodating to changes in chemical structure
Compounds 9–12 were designed to include large adducts
adjacent to the hydroxyl and CF3 groups, with the goal
of sterically disrupting aAF position. We were surprised
to find, however, that each compound bound well to
PXR and served as an effective agonist rather than an
antagonist (Table 3). For example, cyclohexyl or benzyl
rings (compounds 9 and 10) were apparently accommo-
dated by the receptor’s ligand binding pocket. Indeed,
even a benzyl group contained on an extended and rigid
two-methylene linker (compound 11) did not disrupt
either ligand binding or receptor activation. These
observations suggest that the aAF and AF-2 region of
PXR is reasonably conformable and capable of accom-

Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
2161
Figure 5. Structures of analogues of T0901317 (compound 1) examined for their impact on human PXR and human LXRb.
than was expected. This highlights the plasticity present
in the conserved nuclear receptor LBD fold, particularly
for members of the former orphan class.
group of T1317, as well as the distinct placement of
the proximal hydrogen-bonding residue Thr-316, caus-
es the ligand to adopt a position in LXR rotated ꢀ30ꢁ
˚
and shifted by ꢀ5 A relative to that observed in PXR.
Still, nearly half the contacts between protein and li-
gand are conserved in both complexes, including the
close hydrogen bond between a histidine side chain
and a polarized hydroxyl group of the ligand. Both
LBDs also utilize an interaction between the same his-
tidine side chain and an aromatic residue located on
AF-helices of the receptors’ AF-2 regions: Trp-457 in
LXR and Phe-429 in PXR. Indeed, it was noted in this
PXR complex that the presence of the ligand appears
3. Discussion
T1317 is an efficacious activator of gene transcription
mediated by both the nuclear receptors PXR and
LXRb.19 We show, by comparing the 2.8 A resolution
˚
crystal structure of the PXR–T1317 complex to the
LXRb–T1317 complexes reported previously,22,24–26
that these two LBDs share some analogous contacts
to the ligand, but exhibit key differences as well. The
packing of aromatic side chains against the benzyl
˚
to mediate the formation of a direct 3.8 A aromatic
contact between these side chains caused by their shift

2162
Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
OH
HO
O
O
a,b
HO
F3C
O
N
O
c, d
S
S
N
NH2
F3C
OH
F3C
6
2
R
4: R= Et
5: R= n-Pr
O
O
1) e
F3C
S
9: R= cyclohexyl
10: R= phenyl
N
2) f, g, or d
11: R= benzeneethynyl
12: R= heptafluoropropyl
F3C
O
O
O
N
O
S
a,b
H3C
NH2
F3C
8
OH
R
O
O
d or h
H3C
S
N
F3C
3: R = CF3
7: R = H
Scheme 1. Reactions and conditions: (a) benzenesulfonyl chloride, pyridine, 60 ꢁC; (b) 2,2,2-trifluoroethyl trifluoromethylsulfonate, K2CO3, MeCN,
80 ꢁC; (c) oxalyl chloride, DMSO, Et3N, rt; (d) trifluoromethyltrimethylsilane, TBAF, THF, 50 ꢁC; (e) Dess-Martin periodinane, pyridine, DCM, rt;
(f) RMgBr, THF, ꢁ78 ꢁC to rt; (g) phenylacetylene, n-BuLi, THF, ꢁ78 ꢁC to rt; (h) vinylmagnesiun bromide, THF, ꢁ78 ꢁC to rt.
Table 3. T0901317 analogues and their impact on human PXR and
LXRb
in the control of gene expression by both nuclear
receptors.
Compound
PXR
pIC50
PXR
pEC50
PXR
%max
LXR
pEC50
LXR
%max
We were surprised both by the difficulty we experienced
in designing an antagonist to either receptor, and by the
plasticity the LXR receptor exhibits upon derivatization
of the efficacious ligand T1317. Given the numerous
interactions observed between the T1317 and the ligand
binding pockets of both receptors, it was expected that
adding bulky groups to the region adjacent to the
hydroxyl and dual CF3 moieties on the ligand would pro-
duce compounds that sterically block the active orienta-
tion of the AF-helix (as seen in the estrogen receptor with
tamoxifen, for example).28 Instead, we found for PXR
that compounds either bound and were agonists, or ap-
peared incapable of binding to the receptor. In addition,
in the cases where reasonably large groups were added to
the T1317 scaffold, the region of PXR’s ligand binding
pocket adjacent to aAF was remarkably amenable to
accommodating bulky additional atoms. This is perhaps
not surprising for PXR, given its well-established
1
2
7.4
5.0
5.4
6.6
6.5
5.1
5.6
5.8
7.6
7.2
6.2
7.7
7.9
6.0
6.3
6.9
6.8
5.0
5.0
5.0
8.5
7.8
6.0
8.0
93
90
7.0
<4.5
5.8
100
19
50
73
27
3
3
104
101
96
4
5.4
5.6
5
6
23
45
<4.5
<4.5
<4.5
<4.5
5.5
7
3
8
11
3
9
103
99
9
10
11
12
30
5
139
98
<4.5
6.2
58
See Figure 5 for analogue structures. Compound 1 is T0901317 present
in the PXR complex structure presented here.
˚
by 1 A in position toward the bound T1317. It is likely
that the additional stabilization of the active conforma-
tion of aAF by this interaction plays an important role

Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
2163
promiscuous ligand binding character and the structural
flexibility of unique regions (such as a2) in its ligand
binding pocket. It would appear that for PXR, in con-
trast to more rigid and specific steroid receptors like
the estrogen receptor, ligands can bridge between rigid
portions of the pocket, while more flexible regions of
the protein can shift to accommodate a variety of distinct
chemical structures.
binding pocket of the human PXR LBD was replaced
with serine.
4.2. Crystallization
The human PXR ligand binding domain/SRC-1 com-
plex (hPXR-LBD/SRC-1) was concentrated in the pres-
ence of 10-fold molar excesses of T0901317 (T1317;
synthesized in-house) to a final concentration of 5 mg/
mL. Crystallization was achieved by hanging-drop
vapor diffusion against the following conditions at 22
ꢁC: 50 mM imidazole at pH 7.1, 10% 2-propanol, v/v.
While the promiscuity of PXR is well known, we were
not expecting to find that LXRb would also show evi-
dence for conformability in accommodating larger
T1317 analogues. For example, compounds 10 and 12
were relatively effective agonists for LXR, in spite of
the presence of large groups attached adjacent to
T1317’s hydroxyl moiety (Fig. 5). The structural basis
of this is likely centered on the region around Phe-268
in the LXR pocket, which may stack with the aromatic
group in 10 and provide a pore for the conformable
group in 12. LXR did exhibit more specificity for the
types of groups it can accommodate in this position rel-
ative to PXR, however, as non-aromatic or longer rigid
adducts in 9 and 11, respectively, that still activated
PXR were clearly not agonists for LXRb. Thus, the li-
gand binding domains of numerous members of the
nuclear receptor superfamily, particularly the former or-
phan receptors, are likely to possess some degree of
structural flexibility to assist in the accommodation of
chemically distinct ligands. It would appear that PXR,
though, with its large and mobile a1–a3 insert, is at
the extreme of this flexibility continuum with these un-
ique features that are central to its significant
promiscuity.
4.3. Data collection and structure determination
The structure of the T1317-bound form of the ligand
binding domain of human PXR was determined by
molecular replacement using the crystal structure of
the apo (unliganded) PXR as a search model.15 Rotation
and translation function searches were performed using
AMoRe;32 clear solutions for each monomer of the di-
mer in the asymmetric unit were obtained in the proper
space group, P212121. The structure was refined using
CNS with the maximum likelihood function as a target,
and included an overall anisotropic B-factor and a bulk
solvent correction.33 Ten percent of the observed data
were set aside for cross-validation using the free-R sta-
tistic prior to any structural refinement.34 Manual
adjustments and building of the model (including the
placement of the T0901317 ligands and SRC-1 coactiva-
tor fragments) were performed using O35 and rA-
weighted electron density maps.36 The structure exhibits
good geometry (Table 1) with no Ramachandran
outliers.
We conclude that the unique aspects of the PXR ligand
binding pocket may make antagonist design particularly
difficult for this member of the nuclear receptor super-
family. It is noted that compounds 9 and 11 appear to
be selective for PXR relative to LXRb; as such, they
may be useful as leads to address neurodegenerative dis-
eases like Niemann-Pick C.8 However, it may be neces-
sary to look outside the ligand binding pocket of PXR
to find sites effective at antagonizing the receptor using
small molecules. Candidate sites on the LBD include
the AF-2 surface30 and the PXR homodimer interface,
the disruption of which has been shown to impact pro-
ductive coactivator binding by the receptor.16 An effec-
tive PXR antagonist, if identified, may be of
significant clinical use to reduce the activation of xeno-
biotic metabolism pathways during the therapeutic
treatment of disease.
4.4. Compound syntheses
See Scheme 1 for steps involved in compound genera-
tion. 1H NMR spectra were recorded on a Varian Gem-
ini 400 MHz NMR spectrometer. H NMR spectra are
1
reported as chemical shift d, number of protons, multi-
plicity (s, singlet; d, doublet; t, triplet; m, multiplet; br
s, broad singlet), and coupling constant (J) in Hertz.
Electron Spray (ES) or Chemical Ionization (CI) was
recorded on a Hewlett Packard 5989A mass spectrome-
ter. Mass spectrometry results are reported as the mass
over charge. Purity by HPLC [Luna 20 · 4 mm 3.0 mi-
cron C18(2) column, water (+0.1% v/v formic acid)/
MeOH (+0.075% v/v formic acid) gradient: 50% MeOH
to 100% MeOH for 5 min, holding at 100% MeOH for
final 1 min, flow rate = 2.0 mL/min]. Starting materials
are either available from commercial sources or via liter-
ature procedures. Abbreviations used in the examples
below have their accepted meanings in the chemical lit-
erature. For example, DCM (dichloromethane), THF
(tetrahydrofuran), MeCN (acetonitrile), DMSO
(dimethylsulfoxide), and TBAF (tetrabutylammonium
fluoride).
4. Materials and methods
4.1. Protein expression and purification
Generation of human PXR LBD in complex with the
88-amino acid fragment of human SRC-1 was accom-
plished as described previously.12–16 To prevent the for-
mation of covalent complexes with reducing agent
during crystallization, as has been seen with previous
structures (data not shown), Cys-284 within the ligand
4.4.1. Compound 6. 4-Aminobenzyl alcohol (10 g,
80 mmol) and benzenesulfonyl chloride (11.5 mL,
89 mmol) in pyridine were heated at 60 ꢁC overnight.
Water was added and extracted with DCM. The organic

2164
Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
layer was separated and evaporated to dryness. The res-
idue was purified by silica gel chromatography using
ethyl acetate to give 17 g (80%) of intermediate sulfon-
4.4.5. Compound 3. Compound 3 was prepared in an
analogous fashion from compound 8 to given 30 mg
(25%) as a racemic mixture: H NMR (MeOH-d4) d:
7.49–7.65 (m, 7H), 7.10 (d, J = 8.6 Hz, 2H), 4.40 (m,
2H), 1.70 (s, 3H), LC/MS (ES+) m/e 428 (M+H), 98%
purity.
1
1
amide: H NMR (DMSO-d6) d: 7.8 (s, 1H), 7.65 (m,
3H), 7.10 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H),
5.04 (br s, 1H), 4.45 (s, 2H). The intermediate sulfon-
amide (1.2 g, 4.6 mmol), 2,2,2-trifluoroethyltrifluoro-
methane sulfonate (1.97 g, 9.1 mmol), and K2CO3
(1.25 g, 9.1 mmol) in MeCN (20 mL) were heated at
80 ꢁC overnight. The solvents were removed under re-
duced pressure. The residue was purified by silica gel
chromatography using 50% ethyl acetate in hexanes to
give 6 (1.0 g, 80%): 1H NMR (DMSO-d6) d: 7.5–7.8
(m, 5H), 7.26 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 8.5 Hz,
2H), 5.30 (br s, 1H), 4.62 (m, 2H), 4.43 (s, 2H); LC/
MS (ES+): m/e 346 (M+H), 100% purity.
4.4.6. Compound 4. Compound 2 (2.5 g, 6.0 mmol),
Dess-Martin periodinane (5.2 g, 12.0 mmol) and pyri-
dine (4 mL, 48 mmol) in DCM (60 mL) were stirred at
room temperature overnight. The reaction mixture was
treated with 60 mL of an aqueous solution of NaH-
CO3/Na2S2O3 (5:1) and stirred at room temperature
for 2 h. The organic layer was separated and evaporated
to dryness. The residue was purified by silica gel chro-
matography using 50% ethyl acetate in hexanes to give
an intermediate trifluoromethyl ketone (2.2 g, 90%):
1H NMR (MeOH-d4) d: 7.46–7.62 (m, 7H), 7.21 (d,
J = 8.6 Hz, 2H), 4.45 (m, 2H). One molar ethyl magne-
sium bromide in THF (0.5 mL) was slowly added to a
solution of the above ketone (100 mg, 0.24 mmol) in
THF (3 mL) at ꢁ78 ꢁC. The reaction mixture was grad-
ually warmed to room temperature overnight. Saturated
aqueous NH4Cl was added, and the aqueous layer was
extracted with ethyl acetate. The organic layer was sep-
arated and evaporated to dryness. The residue was puri-
fied by reverse phase chromatography on RP-C18 using
50–100% MeCN in water to give 4 (5 mg, 10%) as a
racemic mixture: 1H NMR (MeOH-d4) d: 7.54–7.71
(m, 7H), 7.11 (d, J = 8.6 Hz, 2H), 4.45 (m, 2H), 2.21
(m, 1H), 1.98 (m, 1H), 0.87 (m, 3H); LC/MS (ES+)
m/e 445 (M+H), 98% purity.
4.4.2. Compound 8. Prepared in a similar fashion from 4-
aminoacetophenone (5.0 g, 37.0 mmol): 1H NMR
(DMSO-d6) d: 7.90 (d, J = 8.4 Hz, 2H), 7.56–7.71 (m,
5H), 7.27(d, J = 8.4 Hz, 2H), 4.64 (m, 2H), 2.48 (s,
3H), LC/MS (ES+) m/e 358 (M+H), 95% purity.
4.4.3. Compound 2. DMSO (5.5 mL, 28 mmol) was add-
ed dropwise to 2 M oxalyl chloride in DCM (19 mL)at
ꢁ78 ꢁC under nitrogen followed by sequential addition
of compound 6 (10 g, 30 mmol) in DMSO/DCM (1:1)
(20 mL) and neat triethylamine (75 mL). The reaction
mixture was gradually warmed to room temperature
overnight whereupon the solvents were removed under
reduced pressure. The residue was purified by silica gel
chromatography with DCM to give an intermediate
1
aldehyde (8.5 g, 85%): H NMR (DMSO-d6) d: 9.8 (s,
1H), 7.8 (m, 2 H), 7.52–7.81 (m, 5H), 7.38 (m, 2H).
The following compounds were prepared in an analo-
gous fashion from the above intermediate trifluorometh-
yl ketone:
The intermediate aldehyde (5.0 g, 14.6 mmol) and triflu-
oromethyltrimethylsilane (2.5 g, 17.5 mmol) in anhy-
drous THF (15 mL) were treated with a catalytic
amount of TBAF at 0 ꢁC. The reaction mixture was
gradually warmed to room temperature overnight. The
reaction mixture was treated with 1 N hydrochloric acid
at room temperature for 1 h. Water was added and
extracted with DCM. The organic layer was separated
and evaporated to dryness. The residue was purified
by silica gel chromatography using 20% ethyl acetate
4.4.7. Compound 5. Using 2 M n-propyl magnesium bro-
mide in diethyl ether followed by purification by reverse
phase chromatography on RP-C18 with 50–100%
MeCN in water gave rise to compound 5 (7 mg, 10%)
as a racemic mixture: 1H NMR (MeOH-d4) d: 7.47–
7.65 (m, 7H), 7.11 (d, J = 8.6 Hz, 2H), 4.41 (m, 2H),
2.22 (m, 1H), 1.99 (m, 1H), 1.38 (m, 2H), 0.91 (m,
3H); LC/MS (ES+) m/e 456 (M+H), 95% purity.
1
in hexanes to give 2 (5.0 g, 85%): H NMR (MeOH-
d4) d: 7.65 (m, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.52 (m,
2H), 7.45 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H),
5.03 (m, 1H), 4.43 (m, 2H); LC/MS (ES+) m/e 414
(M+H), 96% purity.
4.4.8. Compound 9. Using 2 M cyclohexyl magnesium
bromide in diethyl ether (0.13 mL) followed by purifica-
tion by reverse phase chromatography on RP-C18 with
50–100% MeCN in water provided compound 9 (20 mg,
20%) as a racemic mixture: 1H NMR (MeOH-d4) d:
7.43–7.89 (m, 7H), 7.11 (d, J = 8.6 Hz, 2H), 4.41 (m,
2H), 2.61–2.05 (4m, 5H), 1.03 (m, 3H), 0.93 (m, 3H);
LC/MS (ES+) m/e 496 (M+H), 94% purity.
The following compounds were prepared in an analo-
gous fashion:
4.4.4. Compound 12. Treatment of intermediate alde-
hyde with heptafluoropropyl-trimethylsilane (0.06 mL,
0.29 mmol) and purification by reverse phase chroma-
tography on RP-C18 with 50–100% MeCN in water
provided compound 12 (20 mg, 15%) as a racemic mix-
ture: 1H NMR (MeOH-d4) d: 7.72 (m, 3H), 7.52 (m, 4H)
7.17 (d, J = 8.6 Hz, 2H), 4.41 (m, 2H), 2.22 (m, 1H), 1.99
(m, 1H), 1.38 (m, 2H), 0.91 (m, 3H); LC/MS (ES+) m/e
456 (M+H), 98% purity.
4.4.9. Compound 10. Using 3 M phenyl magnesium
bromide in diethyl ether (0.09 mL) followed by purifi-
cation via reverse phase chromatography on RP-C18
with 50–100% MeCN in water gave rise to compound
10 (70 mg, 70%) as a racemic mixture: 1H NMR
(MeOH-d4) d: 7.33–7.69 (m, 12H), 7.11 (d,
J = 8.5 Hz, 2H), 4.42 (m, 2H), LC/MS (ES+) m/e
490 (M+H), 100% purity.

Y. Xue et al. / Bioorg. Med. Chem. 15 (2007) 2156–2166
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1
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