Overcoming the Pregnane X Receptor Liability: Rational Design to Eliminate PXR-Mediated CYP Induction

Article abstract of DOI:10.1021/acsmedchemlett.1c00187

The pregnane X receptor (PXR) regulates expression of proteins responsible for all three phases required for the detoxification mechanism, which include CYP450 enzymes, phase II enzymes, and multidrug efflux pumps. Therefore, PXR is a prominent receptor t

Full text of DOI:10.1021/acsmedchemlett.1c00187

pubs.acs.org/acsmedchemlett
Letter
Overcoming the Pregnane X Receptor Liability: Rational Design to
Eliminate PXR-Mediated CYP Induction
Joshi M. Ramanjulu,* Shawn P. Williams, Ami S. Lakdawala, Michael P. DeMartino, Yunfeng Lan,
and Robert W. Marquis
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ABSTRACT: The pregnane X receptor (PXR) regulates
expression of proteins responsible for all three phases required
for the detoxification mechanism, which include CYP450 enzymes,
phase II enzymes, and multidrug efflux pumps. Therefore, PXR is a
prominent receptor that is responsible for xenobiotic excretion and
drug−drug interactions. Pyrimidinone 1 is an antagonist of the
calcium sensing receptor (CaSR) and a strong activator of PXR.
Repeat oral administration revealed diminished exposures over
time, which prohibited further progression. A medicinal chemistry
campaign was initiated to understand and abolish activation of
PXR in order to increase systemic exposures. Rational structure−activity relationship investigations utilizing cocrystal structures and
a de novo pharmacophore model resulted in compounds devoid of PXR activation. These studies culminated in the first orally active
CaSR antagonist 8 suitable for progression. Cocrystallography, the pharmacophore model employed, and additional observations
reported herein supported rational elimination of PXR activation and have applicability across diverse chemical classes to help erase
PXR-driven drug−drug interactions.
KEYWORDS: Pregnane X receptor, drug−drug interactions, CYP induction, calcium sensing receptor, parathyroid hormone, pyrimidinone,
pharmacophore model
is highly evolved to regulate an entire network of genes that are
he family of cytochrome P450 (CYP) enzymes play an
Tintegral role in drug−drug interactions because of their involved in the metabolism and excretion of endo- and
xenobiotics from the body (Figure 1).10,11
efficient yet promiscuous metabolizing capabilities. Among
Crystal structures of the human PXR LBD apo- and ligand-
bound complexes were first reported in 2001.12 Subsequently,
several other cocrystal structures have been reported.13 The
PXR LBD binding pocket is extremely large, flexible, and
hydrophobic in nature. The cavity is lined with 28−30 amino
acid residues, among which only six are polar residues, and
these are evenly distributed throughout the surface of the
largely hydrophobic cavity. The volume of the apo binding
pocket is ∼1250 Å3 and has been shown to expand to ∼1544
Å3 to accommodate molecules of various sizes (≤230 to >800
Da). Despite the presence of hydrophobic residues, this pocket
is capable of binding polar molecules (cLogP > 1) and yet
flexible enough to bind to hydrophobic molecules (cLogP =
8). The initial crystal structures reported by Watkins et al.12
demonstrated a single ligand existing in multiple binding
CYP isoforms, CYP3A4 is known to metabolize greater than
50% of marketed drugs. The induction of CYP3A has been
heavily investigated in preclinical drug discovery and human
trials.1 Pregnane xenobiotic receptor (PXR) has garnered
much attention because it plays a major role in the induction of
CYP3A, leading to drug−drug interactions (DDIs).2 PXR
belongs to the nuclear receptor (NR) superfamily and is
mainly expressed in the liver, intestine, and colon, where most
drug-metabolizing enzymes are also expressed and regulated.
PXR is a multidomain protein with two key domains, a highly
conserved DNA-binding domain (DBD) and the largest,
moderately conserved ligand-binding domain (LBD).3 Sub-
stantial divergence in recognition of small molecules is noted
among species as a result of the low sequence conservation of
the LBD. For example, the LBD shares amino acid identities of
only 76% between human and rodent species and significantly
less for lower-order species (∼50%).4−7 Upon activation, PXR
transcriptionally induces genes that express proteins known to
play key roles in all three phases of metabolism−excretion
mechanisms: (a) CYP enzymes, including CYP3A4; (b)
conjugation enzymes, including carboxylesterases; and (c)
transporters such as MDR1.8,9 Thus, PXR-mediated clearance
Received: April 2, 2021
Accepted: August 2, 2021
https://doi.org/10.1021/acsmedchemlett.1c00187
© XXXX American Chemical Society
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Figure 1. Role of PXR in the regulation of xenobiotic metabolism.
Table 1. Mouse Toxicokinetic Parameters (Composite Sampling) for Compound 1
AUC0−t (μg h/mL)
Cmax (μg/mL)
dose (mg kg−1 day−1
)
day 1
day 10
fold diff.
day 1
day 10
fold diff.
300
1000
2000
2.88
10.8
15.1
1.26
2.85
1.58
−2.28
−3.79
−9.56
0.98
2.96
3.60
0.82
0.68
0.48
−1.2
−4.4
−7.5
orientations. Inspection of subsequent cocrystal structures
revealed different amino acid residues engaging ligands from
diverse structural classes, increasing the difficulty of predicting
the key interactions required for affinity and activation.14 In
silico models developed previously to guide structure−activity
relationship (SAR) investigations were deemed unreliable
given the promiscuous nature of the binding cavity coupled
with the possibility of multiple binding orientations.15,16 In
balancing the learnings from these articles with advantages and
disadvantages of 3D−5D QSAR and docking models, we have
pursued a combination of methods relying on internal and
external crystal structures to develop a pharmacophore model
to guide docking algorithms.
The calcium sensing receptor (CaSR) located on the
parathyroid gland functions as the principal regulator of
parathyroid hormone (PTH) secretion.17 PTH(1−84) has
been clinically validated as a bone-forming agent and used to
treat osteoporosis in both its truncated (Forteo) and full-
length (Preos) forms.17 Both forms of approved PTH therapies
require daily injections. An alternative therapeutic approach
would be to use orally bioavailable small-molecule antagonists
of the CaSR to stimulate secretion of endogenous PTH, thus
making them potential treatments for osteoporosis. One such
CaSR antagonist, compound 1, was identified as suitable for
progression into rodent toxicology studies. Oral administration
of compound 1 in mice at 300, 1000, and 2000 mg kg−1 day−1
for 10 days resulted in significantly diminished exposures (both
area under the curve (AUC) and maximum concentration
(Cmax)) on day 10 compared with day 1. The reduction in
exposure was dose-dependent, with about 2-, 4-, and 10-fold
drops at 300, 1000, and 2000 mg/kg, respectively (Table 1).
Compound 1 is a substrate for CYP3A4 and was therefore
presumed to be eliminated via phase I metabolism.18 As this
mechanism was unable to explain the larger drop at higher
doses, it was hypothesized that the compound may induce
higher levels of the CYP3A4 enzyme upon repeated treatment,
thus leading to diminishing exposure over the course of the
study. With this in mind, compound 1 was evaluated in a PXR
functional assay, which confirmed the induction potential for
CYP3A4. This result was further validated in a human
hepatocyte induction assay that has a strong clinical correlation
with drugs that elicit PXR-mediated CYP3A4 induction.19,20
Further evaluation of compound 1 in a binding assay
confirmed its affinity toward human PXR (hPXR) and
demonstrated greater induction potential in mouse and
human compared with rat PXR assays (pp S7, S17 and S18
in the Supporting Information (SI)). Finally, other members
from this class of pyrimidinones also exhibited high affinity for
hPXR, thus confirming the observations from the toxicology
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Scheme 1. Synthesis of 2-(3-Fluoro-2-hydroxyphenyl)-6-methyl-5-(5-methylthien-2-yl)-3-(2-phenylethyl)-4(3H)-
a
pyrimidinone (1)
a
Reagents and conditions: (i) benzyl bromide, K2CO3, DMF; (ii) NaOH, MeOH, H2O; (iii) ethyl chloroformate, TEA, THF, NH4OH; (iv) 10%
Pd/C, 40 psi, EtOH; (v) phenethylamine, Et2O, reflux; (vi) Ti(iPrO)4, e, reflux; (vii) benzyl bromide, K2CO3, DMF; (viii) Br2, AcOH; (ix)
Pd(PPh3)4, EtOH, H2O, Na2CO3, 5-methylthiophene-2-boronic acid, microwave.
Figure 2. (a) Structure of compound 2. (b, c) Cocrystal structures showing pocket residues in contact with and in proximity to the bound ligand.
study and the induction potential of the pyrimidinone class of
compounds.
conditions with 5-methylthiophene-2-boronic acid furnished
compound 1.
To explore and expedite the SAR to overcome PXR
induction, several synthetic methodologies were applied
(Scheme 1).21−24 The synthesis of compound 1 began with
3-fluoro-2-hydroxybenzoic acid, which was reacted with benzyl
bromide followed by hydrolysis to provide carboxylic acid c.
Carboxylic acid c was then converted to the amide using the
mixed anhydride, and treatment with ammonium hydroxide
followed by benzyl deprotection provided the intermediate 3-
fluoro-2-hydroxybenzamide (e). The second key intermediate,
3-oxo-N-(2-phenylethyl)butanamide (f), was prepared by
refluxing diketene in anhydrous ether with phenethylamine.
Next, the reaction of intermediates e and f in refluxing titanium
isopropoxide, a novel synthetic methodology developed at
GSK,23 furnished the pyrimidinone scaffold in one step, and
subsequent benzyl protection produced pyrimidinone g.
Selective bromination at C5 under acidic conditions followed
by Pd(PPh3)4-catalyzed Suzuki reaction under microwave
As discussed earlier, published pharmacophore models
generated from computational methods proved to be
unreliable to identify residues critical for PXR induction.14−16
Additionally, the possible existence of multiple binding modes
also complicates the development of in silico models to aid
SAR investigations. To date, no pharmacophore model has
been successfully applied to eliminate PXR-mediated induction
of CYP3A4. With this in mind, we initiated cocrystallization
trials for pyrimidinone-based agonists of PXR that were highly
active in the functional assay (% maximum response > 70%
and EC50 < 3 μM). Several pyrimidinone-based compounds
resulted in cocrystal structures at a resolution of 2.25−3.00 Å.
To our delight, all of the crystal structures unambiguously
revealed a single binding mode (Figure 2). The C2 aromatic
moiety (2-fluorophenol) is twisted out of the plane to engage
with His407 via a hydrogen bond (H-bond) interaction.
Gln285 is involved in a second H-bond interaction with the
C
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carbonyl group of the pyrimidinone scaffold. Both His407 and
Gln285 are involved in H-bond interactions, as previously
reported by several groups.14 Taken together, these results
indicate that these two residues play a prominent role via
“anchoring” and “aligning” small molecules, thereby facilitating
subsequent high-affinity hydrophobic interactions. The anch-
ored pyrimidinone projects the C5 benzyl group into the
highly hydrophobic bottom pocket that is shaped by Phe281,
Phe288, Trp299, and Tyr306. In this orientation, the C5
benzyl group engages in a face-to-face π−π stacking interaction
with Trp299 and edge-to-face interactions with Phe288 and
Tyr306 at distances ranging from 3.67 to 4.40 Å. The N3
phenethyl substituent nestles into the hydrophobic back
pocket that is encapsulated by Leu240, Ile414, and Phe420.
The extended phenyl group is engaged in edge-to-face π−π
stacking with Phe420. The contribution of hydrophobicity to
the drug−receptor complex is more energetically favorable
than polar interactions. Thus, the collective contributions of
the above hydrophobic interactions lead to the tight binding
nature of pyrimidinone-based CaSR analogues to the LBD of
PXR.
The ligand-binding pocket located in the LBD is
predominately hydrophobic in nature and is lined by 28
amino acid residues that are desolvated when bound to
compound 2 (Figure 2). Comparison of the apo and ligand-
bound structures revealed only minor changes with a root-
mean-square deviation of 0.242 Å over all atoms in the 28
residues. The cavity volume is 1289 Å3, which is 29 Å3 larger
than that of the apo structure but only 5 Å3 smaller than the
pocket in the SR12813 complex25,26 and many of the reported
structures.12,13 Four polar residues (Gln285, His407, Arg410,
and Ser208) are situated at the peripheral portion of the cavity,
while hydrophobic groups are lined up in the inside. The two
critical polar groups (Gln285 and His407) extend out into the
middle of this pocket to anchor small molecules that can
engage such interactions.
Our data demonstrating a single binding mode are at odds
with an earlier hypothesis that cocrystallization methods in the
absence of the coactivator SRC-1 likely result in multiple
binding modes.13 SRC-1 binds to the activation factor 2 region
within the LBD and is thought to be involved in rigidifying the
binding pocket, thus limiting multiple orientations. To gather
further evidence to support the observed single binding mode,
cocrystallization of additional analogues was pursued using
identical crystallization methods. The results indicated that,
similar to the structures with compound 2, a single binding
mode was present for each analogue examined (compounds 9
and 10; SI p S10), and the orientations and conformations of
all three compounds within the pocket are similar.
Furthermore, all of the residues surrounding the binding
cavity remain fixed except for Leu209 (Figure 2). The key H-
bonds with His407 and Gln285 are maintained with the C5
moiety projected into the hydrophobic cavity at the bottom of
the pocket (Figure 3). A possible explanation for the existence
of only one conformation is that the contribution from the H-
bond interactions and hydrophobic interactions is significantly
higher for pyrimidinones relative to other binders, thus limiting
the motion within the binding pocket. In contrast, the H-bond
donor (phenol moiety) and corresponding polar interaction
for SR12813 (Table S1) are significantly weakened by the
presence of two ortho tert-butyl groups, leading to existence of
multiple binding conformations.
Figure 3. (a) Structure of compound 2 anchored via the two polar
residues His407 and Gln285, with the benzyl group sandwiched by
Phe288 and Thr299. (b) Structure of T0901317 displaying H-bond
interactions of the sulfonyl group with Gln285 and His407 and
interactions of the phenyl group with Phe281 and Leu209.
Armed with robust structural biology and institutional
knowledge, we built a pharmacophore model in order to
rationally design out PXR affinity associated with the
pyrimidinone class of CaSR antagonists and for wider
applicability. Upon examination of several cocrystal structures,
both in-house and published, we selected 10 structures based
on molecular properties and bound conformations (Table S1).
In particular, both crystal structures of SR12813 were included
to dissect the multiconformational binding potential of the
LDB.12 Striking properties of the LBD include the large and
flexible binding site, movement of a flexible loop containing
residues 309−321 (which allows expansion of the cavity), and
binding of compounds with diverse molecular properties. The
binding site cavity is composed of 20 hydrophobic, four polar,
and four charged residues. We have identified five key polar
residues, namely, Ser208, Ser247, Gln285, His407, and
Arg410, that are spread across the top of the binding site to
form significant interactions with bound ligands. The crystal
structures were critically analyzed for ligand binding modes
and H-bond and hydrophobic interactions. H-bond inter-
actions were further scrutinized for donor and acceptor
characteristics as well as bond strength related to distance
and angle. Hydrophobic interactions were inspected similarly
and classified as aliphatic versus aromatic, with aromatic
contacts further distinguished into face-to-face and edge-to-
face interactions. All compounds evaluated had at least two
hydrophobic interactions and one H-bond interaction. The
pharmacophoric features of each ligand were calculated using
Molecular Operating Environment (MOE) and compared to
those of other molecules generated with SRC-1 cofactor (SI pp
S4 and S11). The resulting 3D pharmacophore features were
overlaid, and an overall “average” model was identified (Figure
4). In general, the pharmacophore model has four residues
dispersed in the cavity to anchor molecules in the center via H-
bond and/or charged interactions. Among the four residues,
Leu209 always engaged the molecules via the carbonyl group
and contributed to a wider array of molecules compared with
Ser247. Gln285 and His407 are the key polar residues, and one
of them engaged in H-bonds in all of the cocrystal structures
evaluated to date. Hydrophobic residues form the overall
“triangular” shape of the cavity, with seven residues
concentrated at the three corners. The top left corner is
occupied by Met243, while the top right corner contains
Leu207 and Leu411, which are hydrophobic in nature but not
optimal for engaging aryl groups. The lower portion of the
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assessed simultaneously for their CaSR antagonist activity (SI
p S5) and in the PXR gene reporter assay (Figure 7and SI pp
S7, S8, S17, and S18). The first set of analogues probed the
back pocket (N3 substituents); however, these displayed
reduced affinity toward PXR and suffered from poor activity
against CaSR. The 2-position of the pyrimidinone is the most
sensitive, and structural changes are not well-tolerated with
respect to CaSR activity. Incorporation of polar heterocycles
(compound 5) dramatically lowered the PXR affinity despite
maintaining the key H-bond to His407, suggesting that
hydrophobic interactions significantly boost the affinity toward
PXR. We then examined the contributions of the H-bond
interactions between Gln285 and the carbonyl of the
pyrimidinone template. Replacement of the pyrimidinone
with pyrazine 3, which is not capable of engaging in the H-
bond interaction with Gln285, demonstrated high levels of
PXR affinity, perhaps due to the anchoring H-bond interaction
with His407 via 2-hydroxy-3-fluorophenyl moiety at C2. This
corroborates our hypothesis that one H-bond interaction is
sufficient to anchor small molecules to facilitate secondary
hydrophobic interactions as suggested by our analysis.
As detailed earlier, the crystal structure and the docking
confirmed that the substituent at C5 of the pyrimidinone
template projects out into the bottom “rigid” hydrophobic
cavity. To ensure the predominately hydrophobic nature of
this pocket area, hydroxymethylthiophene analogue 4 and
thiazole analogue 5 were evaluated and, as anticipated,
produced dramatic reductions in PXR affinity (Figure 7).
Incorporation of a rigid 5-phenylthien-2-yl moiety at C5 of the
pyrimidinone resulted in loss of affinity, suggesting a severe
steric clash with Trp299. Even though compound 6 is capable
of π−π stacking and/or edge-to-face interactions with either
Phe288 or Trp299, this portion of the binding pocket is not
flexible enough to accommodate larger groups. This agrees
with an earlier report that flexibility was seen only within the
three regions that are located at the top of the cavity (200−
209, 229−235, and 310−317) but not at the bottom portion of
the cavity.27 On this basis, incorporation of rigid and polar
groups would likely produce the optimal profile. In fact,
incorporation of polarity through a 5-(pyridin-2-yl)thien-2-yl
(7) and 4-(pyridin-2-yl)-5-methylthien-2-yl group (8) at C5
resulted in dramatic losses in PXR affinity while retaining
activity against the CaSR receptor (Figure 7 and SI p S18).
Docking clearly indicated that stereoelectronic repulsive forces
of the C5 substituent overcame several detrimental interactions
(Figure 6). Compound 8 demonstrated a clean profile in the
gene reporter assay as well as in the industry-standard human
hepatocyte assay. Gratifyingly, assessment of compound 8 in a
10 day repeat dose rat toxicology study demonstrated no PXR-
mediated reduction in exposures from day 1 to day 10 at 10-
fold higher systemic exposure (Table 2). In comparison,
compound 1 had decreased exposure (0.72× in AUC) at day
10 while the compound 8 exposure increased (29.5 vs 41.1 μg
h/mL) over the course of the study. As observed in the mouse
toxicology study (Table 1), compound 1 induced rodent PXR
activation at much lower concentrations, leading to diminished
exposure, while with compound 8, even at >20-fold higher
levels (Cmax), AUC exposure increased 1.4-fold on day 10.
In order to understand whether any general trends related to
physicochemical properties were present, we evaluated a much
broader data set from the GSK compound collection.
Evaluation of PXR activity data from >15 000 compounds
provided a few general trends. Molecules that are hydrophobic,
Figure 4. (a) Pharmacophore model displaying key residues engaged
binding to a diverse group of compounds. Purple and green spheres
represent H-bond and hydrophobic interactions, respectively, while
the size of each sphere reflects number of compounds. Distances
captured between residues/spaces are depicted. (b) Average distances
between the three key groups of hydrophobic residues.
pocket has significant hydrophobic character, is rigid, and is
surrounded by three aromatic residues (Phe288, Trp299, and
Tyr306). This area accommodates aliphatic and aryl groups
and is capable of interacting with aryls in both a face-to-face
and edge-to-face manner.
The pharmacophore model triggered a structure-guided
investigation to overcome the PXR affinity associated with the
pyrimidinone class of CaSR antagonists. As described above,
all of the crystal structures of the pyrimidinone analogues
demonstrated a single bound conformation. Docking of
compound 1 resulted in a similar conformation with the C5
substituent extended into the bottom hydrophobic cavity while
anchored to Gln285 via the carbonyl group. The second H-
bond is observed to involve the hydroxy group of the C2 aryl
group, while the N3 phenethyl group projects out to the back
in the pocket without any significant interactions. Overlay of
the docked and crystal structures of compound 2 (Figure 5)
Figure 5. (a) Cocrystal structure of compound 2 depicting key
interactions. (b) Docked structure of compound 2. Both the docked
and crystal structures show the critical H-bonds between compound 2
and residues Gln285 and His407.
enhances our confidence that like other cocrystallized
pyrimidinones, it exists in the conformation observed
experimentally and validates our use of the docking model to
drive SAR investigations.
Using information derived from the cocrystal structures and
subsequent docking studies, an SAR strategy was employed
with the intention of examining the five key interactions
observed (Figure 6). All of the new analogues prepared were
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Figure 6. (a) Pharmacophore-model-informed SAR strategy. (b) Docked structure of compound 8 (magenta) supporting elimination of PXR
affinity (overlaid with the docked structure of compound 1 in yellow).
Figure 7. Structures and in vitro data of pyrimidinone-based CaSR antagonists 1−8.
Table 2. Rat Toxicokinetic Parameters of Compounds 1 and 8 (Results Are Reported as Mean (n = 3) and [Range])
AUC0−t (μg h/mL)
Cmax (μg/mL)
compound
dose (mg kg−1 day−1
)
day 1
day 10
fold diff.
day 1
day 10
fold diff.
1
8
300
100
2.5 [1.3−4.7]
29.5 [22.6−34.8]
1.8 [1.0−2.5]
41.1 [20.4−77.0]
−1.4
+1.4
0.33
8.9
0.36
12
+1.1
+1.3
neutral, and flexible demonstrated a high propensity to activate
PXR. Contrarily, no single chemical property (molecular
weight (MW), number of H-bond donors, number of H-bond
acceptors, cLogD, number of ionizable groups) that abolishes
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Figure 8. (a) Plots of MW and cLogD (pH 7.4) for compounds from the GSK compound collection. (b) Binning of MW and cLogD (pH 7.4) for
the GSK compounds. Color coding reflects PXR activity (% maximum response), with red, yellow, and green corresponding to >70% (high), 30−
70% (medium), and <30% (low) induction relative to 10 μM rifampicin, respectively (SI pp S8 and S9). (c) Exemplary functional groups observed
as PXR activators.
PXR activity emerged. However, lower lipophilicity (cLogD <
2.5) and low MW displayed the strongest correlations (Figure
8). Additionally, not all CYP3A4 substrates are PXR inducers
(data not shown). Since no clear trends to abolish PXR activity
are available, the pharmacophore model that allows a priori
prediction of PXR activation should greatly aid in compound
design.
safety data will be the subject of a future publication. In this
paper, we have highlighted the ambiguity associated with PXR
activation in pharmacophore models and SARs, thus fulfilling
the need for the development of pharmacophore model with
wider applicability.
ASSOCIATED CONTENT
■
PXR activation is a primary reason for CYP3A4-induced
drug−drug interactions and leads to attrition of preclinical and
clinical assets.27 Because of its indiscriminatory binding pocket,
PXR accommodates a wide range of chemical classes
commonly encountered in lead optimization. High levels of
PXR induction by the pyrimidinone class of CaSR antagonists
prompted us to investigate a rational way to abolish PXR-
mediated CYP induction. Multiple conformations observed
with SR12813 have injected uncertainty into cocrystal
structure interpretations and subsequent pharmacophore
models derived from them. The pharmacophore models
reported to date were insufficient to rationalize the SAR
trends for the PXR activity observed for the pyrimidi-
nones.28−30 Furthermore, in silico models reported to date
were unable to emphasize the critical nature of specific
interactions observed.14−16,31 For instance, H-bonding to
Gln285 was reported to be indispensable, and hydrophobic
contacts are not essential for receptor activation. The above
annotations have led to the development of a comprehensive
model derived from diverse chemical classes. Our PXR model,
unlike earlier models, emphasizes one key binding model for
focused SAR investigations to overcome PXR affinity:
incorporation of polar and/or rigid hydrophobic moieties at
the bottom pocket featuring Phe288 and Trp299. A recent
report provides evidence that Trp299 is a critical residue for
binding and transactivation.32 Elimination of H-bond-acceptor
properties to remove the molecule anchoring ability may
diminish binding but are not enough as claimed by the
previous reports. Utilization of our pharmacophore model has
led to a focused yet rapid SAR campaign on the pyrimidinone
scaffold that produced several analogues devoid of PXR
affinity. A full report detailing SAR investigations as well as
drug metabolism, pharmacokinetic, pharmacodynamic, and
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00187.
Experimental procedures; molecular modeling protocols;
conditions and protocols for biological assays; inter-
pretation of PXR activation; crystallography experimen-
tal methods, data, and structures; development of the
pharmacophore model; and profile of compounds
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
Joshi M. Ramanjulu − GlaxoSmithKline, Collegeville,
Pennsylvania 19426, United States; orcid.org/0000-
0003-3471-651X; Phone: (484) 574-2304;
Email: Joshi.M.Ramanjulu@gsk.com
Authors
Shawn P. Williams − GlaxoSmithKline, Collegeville,
Pennsylvania 19426, United States
Ami S. Lakdawala − GlaxoSmithKline, Collegeville,
Pennsylvania 19426, United States
Michael P. DeMartino − GlaxoSmithKline, Collegeville,
Pennsylvania 19426, United States
Yunfeng Lan − GlaxoSmithKline, Collegeville, Pennsylvania
19426, United States
Robert W. Marquis − GlaxoSmithKline, Collegeville,
Pennsylvania 19426, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00187
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(11) Pavek, P. Pregnane X Receptor (PXR)-Mediated Gene
Repression and Crosstalk of PXR with Other Nuclear Receptors via
Coactivator Interactions. Front. Pharmacol. 2016, 7, 456.
(12) Watkins, R. E.; Wisely, G. B.; Moore, L. B.; Collins, J. L.;
Lambert, M. H.; Williams, S. P.; Willson, T. M.; Kliewer, S. A.;
Redinbo, M. R. The human nuclear xenobiotic receptor PXR:
structural determinants of directed promiscuity. Science 2001, 292,
2329−2333.
(13) Xue, Y.; Chao, E.; Zuercher, W. J.; Willson, T. W.; Collins, J. L.;
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Author Contributions
A.S.L. conducted computational modeling, generation of the
pharmacophore model, and molecular docking. S.P.W.
performed X-ray analysis of the crystals. Y.L. and M.P.D.
conducted the synthesis of pyrimidinone derivatives and
interpreted the data for characterization of the compounds.
J.M.R. oversaw PXR screening, supervised SAR studies,
developed the concept, and wrote the manuscript. R.W.M.
contributed to the program strategy. All of the authors
approved the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge Dr. Iain McLay, Group Leader
(retired from GSK), for performing initial computational
analysis of in-house crystal structures; Yingtao Lu, Xiaoyang
Dong, and Robert Trout for preparation of compounds 2, 3,
and 4, respectively; Andrew Brown and Metul Patel for PXR
assay writeup and data generation; Mark Mellinger for
generation of analytical data; Tim Hart for providing safety
assessment data; and Grazyna-Graczyk Millbrandt for the
interpretation of NMR spectra.
(15) Ekins, S.; Kortagere, S.; Iyer, M.; Reschly, E.; Lill, M. A.;
Redinbo, M. R.; Krasowski, M. D. Challenges Predicting Ligand−
Receptor Interactions of Promiscuous Proteins: The Nuclear
Receptor PXR. PLoS Comput. Biol. 2009, 5, e1000594.
(16) Kortagere, S.; Krasowski, M. D.; Ekins, S. Ligand- and
structure-based pregnane X receptor models. Methods Mol. Biol. 2012,
929, 359−375.
ABBREVIATIONS
(17) Hannan, E. M.; Kallay, E.; Chang, W.; Brandi, M. L.; Thakker,
R. V. The calcium-sensing receptor in physiology and in calcitropic
and noncalcitropic diseases. Nat. Rev. Endocrinol. 2019, 15, 33−52.
(18) After administration of ketoconazole, the dose-normalized
AUC0−∞ of compound 1 increased an average of 3.9-fold, and the
dose-normalized Cmax increased an average of 2.1-fold. Compound 1
is a substrate of 3A4. It is not a P-glycoprotein (P-gp) substrate.
Ketoconazole is a dual inhibitor/modulator of human CYP3A/P-gp
that is expressed extensively in the gut and liver. Ketoconazole had
been shown to increase absorption and decrease first-pass metabolism
of some CYP3A/P-gp substrates (e.g., erythromycin) in the monkey.
The data from this study confirmed that 1 is a substrate of CYP3A.
(19) Luo, G.; Cunningham, M.; Kim, S.; Burn, T.; Lin, J.; Sinz, M.;
Hamilton, G.; Rizzo, C.; Jolley, S.; Gilbert, D.; Downey, A.; Mudra,
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D.; Selling, B.; LeCluyse, E.; Gan, L. S. CYP3A4 Induction by Drugs:
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(21) Casillas, L. N.; Charnley, A. K.; Harris, P. A.; Jeong, J. U.;
Marquis, R. W.; Ramanjulu, J. M.; Trout, R. Preparation of 5-thienyl-
4(3H) pyrimidinone derivatives as calcium receptor antagonists. WO
2010065383 A1, 2010.
■
PXR, pregnane X receptor; DBD, DNA-binding domain; LBD,
ligand-binding domain; NR, nuclear receptor; M.R, maximum
response; CaSR, calcium sensing receptor; SAR, structure−
activity relationship; PTH, parathyroid hormone; DDI, drug−
drug interaction
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