Evolution of pharmacologic specificity in the pregnane X receptor

1471-2148-8-103 1471-2148 Research article Evolution of pharmacologic specificity in the pregnane X receptor Ekins Sean ekinssean@yahoo.com Reschly J Erica ejr15@pitt.edu Hagey R Lee lhagey@ucsd.edu Krasowski D Matthew mdk24@pitt.edu

Collaborations in Chemistry, Inc., Jenkintown, PA, USA

Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD, USA

Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ, USA

Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA

Department of Medicine, University of California at San Diego, San Diego, CA, USA

BMC Evolutionary Biology 1471-2148 2008 8 1 103 http://www.biomedcentral.com/1471-2148/8/103 18384689 10.1186/1471-2148-8-103 28 8 2007 02 4 2008 02 4 2008 2008 Ekins et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The pregnane X receptor (PXR) shows the highest degree of cross-species sequence diversity of any of the vertebrate nuclear hormone receptors. In this study, we determined the pharmacophores for activation of human, mouse, rat, rabbit, chicken, and zebrafish PXRs, using a common set of sixteen ligands. In addition, we compared in detail the selectivity of human and zebrafish PXRs for steroidal compounds and xenobiotics. The ligand activation properties of the Western clawed frog (Xenopus tropicalis) PXR and that of a putative vitamin D receptor (VDR)/PXR cloned in this study from the chordate invertebrate sea squirt (Ciona intestinalis) were also investigated.

Results

Using a common set of ligands, human, mouse, and rat PXRs share structurally similar pharmacophores consisting of hydrophobic features and widely spaced excluded volumes indicative of large binding pockets. Zebrafish PXR has the most sterically constrained pharmacophore of the PXRs analyzed, suggesting a smaller ligand-binding pocket than the other PXRs. Chicken PXR possesses a symmetrical pharmacophore with four hydrophobes, a hydrogen bond acceptor, as well as excluded volumes. Comparison of human and zebrafish PXRs for a wide range of possible activators revealed that zebrafish PXR is activated by a subset of human PXR agonists. The Ciona VDR/PXR showed low sequence identity to vertebrate VDRs and PXRs in the ligand-binding domain and was preferentially activated by planar xenobiotics including 6-formylindolo-[3,2-b]carbazole. Lastly, the Western clawed frog (Xenopus tropicalis) PXR was insensitive to vitamins and steroidal compounds and was activated only by benzoates.

Conclusion

In contrast to other nuclear hormone receptors, PXRs show significant differences in ligand specificity across species. By pharmacophore analysis, certain PXRs share similar features such as human, mouse, and rat PXRs, suggesting overlap of function and perhaps common evolutionary forces. The Western clawed frog PXR, like that described for African clawed frog PXRs, has diverged considerably in ligand selectivity from fish, bird, and mammalian PXRs.

Background

The pregnane X receptor (PXR; NR1I2; also known as steroid and xenobiotic receptor) is a member of the nuclear hormone receptor (NR) superfamily 12. PXR functions as a ligand-activated transcription factor and regulates the metabolism, transport, and excretion of exogenous compounds, steroid hormones, vitamins, bile salts, and xenobiotics. A remarkably diverse array of compounds activate human PXR, although generally only at micromolar concentrations (less commonly at high nanomolar concentrations), consistent with a hypothesized function of PXR as a toxic compound sensor 34 (see Figure 1 for chemical structures of some PXR activators).

Figure 1

Chemical structures of PXR activators

Chemical structures of PXR activators. Chemical structures of the PXR activators 5β-pregnane-3,20-dione, 5α-androstan-3α-ol, 5β-lithocholic acid, 5α-cyprinol 27-sulfate, 3-aminoethylbenzoate, and 6-formylindolo-[3,2-b]-carbozole. The key bond positions are numbered for the steroids and bile salts, and the lettering of the steroidal rings is indicated for pregnanedione and lithocholic acid. The structure to the right of lithocholic acid illustrates the most stable orientation of the A, B, and C steroid rings for 5β-bile salts (like lithocholic acid) with the A/B cis configuration (referring to the relative orientation of the hydrogen atom substituents on carbon atoms 5 and 10). The structure to the right of 5α-cyprinol sulfate shows the most stable orientation of 5α-bile salts (like 5α-cyprinol sulfate) that prefentially adopt the A/B trans configuration.

PXR genes have been cloned and functionally characterized from a variety of vertebrate species, including human, rhesus monkey, mouse, rat, rabbit, dog, pig, chicken, frog, and zebrafish 1456789101112. Like other NRs, PXRs have a modular structure with two major domains: an N-terminal DNA-binding domain (DBD) and a larger C-terminal ligand-binding domain (LBD). The PXR LBD is unusually divergent across species, compared to other NRs, with only 50% sequence identity between mammalian and non-mammalian PXR sequences; other NRs tend to have corresponding sequence identities at least 10–20% higher 1213. Even the PXR DBD, which is more highly conserved across species than the LBD, shows more cross-species sequence diversity than other NRs 1213141516. A detailed phylogenetic analysis of the entire vertebrate NR superfamily demonstrated evidence of positive evolutionary selection for the LBD of PXRs 17.

In this study, we compare in detail the selectivity of human and zebrafish PXRs for steroid hormones and related compounds. We also compare human, mouse, rat, rabbit, chicken, frog, and zebrafish PXRs with a set of common compounds that activate most PXRs. These in vitro data are used to develop pharmacophore models to capture the essential structural and chemical features of activators of these PXRs (pharmacophore models summarize the key features important for biological activity). Commonly used features in pharmacophore models include hydrophobic, hydrogen bond acceptor, hydrogen bond donor, and excluded volumes (areas where atoms are not allowed, e.g., due to steric overlap with receptor amino acid residues).

We sought to probe the distant evolutionary history of PXR and the related vitamin D receptor (VDR; NR1I1) by studying an invertebrate NR1I-like receptor. The draft genome of the chordate invertebrate Ciona intestinalis (sea squirt; a urochordate) revealed a single gene [GenBank: BR000137] with close sequence similarity to the vertebrate VDRs, PXRs, and constitutive androstane receptors (CARs, NR1I3) 1819 (see Additional file 1 for sequence alignment). NR1I-like genes were also detected in the genomes of the fruitfly (Drosophila melanogaster) and the nematode Caenorhabditis elegans 20, although these genes have yet to be functionally characterized. The draft genome of the purple sea urchin (Strongylocentrotus purpuratus) revealed several putative NR1H-like genes but no NR1I-like genes 21. The early evolutionary history of the NR1I subfamily (VDR, PXR, CAR) in vertebrates is not completely clear, but one hypothesis is that a single ancestral 'VDR/PXR' gene duplicated, with the two genes then diverging into distinct VDRs and PXRs, both of which are currently found in both mammalian and non-mammalian species 22. We follow the convention of referring to the non-mammalian PXR/CAR-like genes as PXRs 12, although it is not clear whether the function of the single gene in fishes and chicken is more similar to mammalian CAR or PXR 910. The duplication of a single VDR/PXR gene into two different genes may have occurred during a complex series of gene duplications that are thought to have occurred in early vertebrate evolution, based on analysis of lamprey and hagfish genes 23. Later in vertebrate evolution (probably early on in or before the evolution of mammals), a single PXR-like ancestral gene then duplicated with subsequent divergence into the separate PXR and CAR genes found in all mammals sequenced thus far 9. For simplicity, the single Ciona intestinalis NR1I-like gene will be referred to as 'Ciona VDR/PXR'. One advantage of studying Ciona intestinalis, in addition to the genome project data available, is that this animal is a member of Urochordata, a subphylum now thought to contain the closest extant relatives of modern vertebrates 24.

Additional file 1

Sequence alignment of nine PXRs and the Ciona VDR/PXR. Sequence alignment of the DNA-binding and ligand-binding domains of PXRs and the Ciona intestinalis VDR/PXR

Click here for file

From the Ghost database of Ciona intestinalis Genomic and cDNA Resources 25, cDNA clone IDs ciem829d05 and cilv048e18 correspond to the Ciona VDR/PXR. Based on the expressed sequence tag counts, these cDNAs show highest expression in the larvae and juvenile life stages and lower expression in eggs, cleaving embryos, young adults, and mature adults. For adult animals, expression was seen in gonadal tissue and blood cells. Although invertebrates are not known to produce and utilize vitamin D pathways, we speculated that the Ciona VDR/PXR may bind ligands structurally similar to vitamin D, based on the subsequent evolutionary development and ligand preferences of vertebrate VDRs. Alternatively, the Ciona VDR/PXR may function more like vertebrate PXRs, and assist in protection from toxic levels of endogenous and/or exogenous compounds, in which case it might bind a diverse array of ligands. We therefore cloned and expressed the Ciona VDR/PXR to determine how similar this receptor is to vertebrate NR1I receptors in terms of activation by ligands.

Results

Selectivity of human PXR

We first assessed the ability of a diverse set of compounds to activate human PXR by determining detailed concentration-response curves for activation of human PXR for 25 androstane steroids (Table 1), 11 estrane steroids (Table 1), 29 pregnane steroids (Table 2), 50 bile salts (Additional file 2; some bile salts were previously published 15), and 50 additional diverse compounds that included xenobiotics and vitamins (Table 3) (see Figure 1 for selected chemical structures of an androstane steroid, a pregnane steroid, two bile salts, and two additional compounds). These activation data further confirm the broad ligand specificity of human PXR, with most compounds only activating at micromolar concentrations.

Additional file 2

Table of activation data for human and zebrafish PXRs by bile salts. Summary of concentration-response data for activation of human and zebrafish PXRs by bile salts

Click here for file

Table 1

Activation of human and zebrafish PXRs by androstane and estrane steroids

Cmp. #

Compound

hPXR Activity

hPXR Efficacy

zfPXR Activity

zfPXR Efficacy

Toxicity

ANDROSTANES

AN1

5α-Androstan-3α,17β-diol

5.38

0.68

5.19

0.84

None

AN2

5α-Androstan-3,17-dione (androstanedione)

4.90

0.87

5.50

0.86

None

AN3

5α-Androstan-3α-ol (androstanol)

5.20

0.5

5.34

1.00

None

AN4

5α-Androstan-3α-ol-17-one (androsterone)

4.73

0.93

5.60

0.87

None

AN5

5α-Androstan-17β-ol-3-one (dihydrotestosterone)

4.94

0.39

5.21

0.59

None

AN6

5β-Androstan-3α-ol-17-one (etiocholanolone)

5.24

0.54

5.47

0.88

200

AN7

4-Androsten-3,17-dione (androstenedione)

4.69

0.59

5.44

0.14

None

AN8

4-Androsten-17β-ol-3-one (testosterone)

4.14

0.22

5.61

0.12

None

AN9

5-Androsten-3β-ol-17-one (DHEA)

4.49

0.52

4.89

0.35

None

AN10

5α-Androst-16-en-3α-ol (androstenol)

5.26

0.7

5.44

1.02

None

AN11

5β-Androstan-3α,11β-diol-17-one

4.72

0.51

4.52

1.04

None

AN12

5-Androsten-3β-sulfate-17-one (DHEA sulfate)

4.32

0.22

None

AN13

5β-Androstan-3α-ol-17-one (epiandrosterone)

5.31

0.7

5.02

0.43

None

AN14

5β-Androstan-3α-ol-11,17-dione

4.39

0.15

5.01

0.49

None

AN15

4-Androsten-17α-ol-3-one (epitestosterone)

4.17

0.9

None

AN16

4-Androsten-17α-glucosiduronate-3-one (epitestosterone glucuronide)

4.86

0.69

None

AN17

4-Androsten-17α-sulfate-3-one (epitestosterone sulfate)

5.47

0.67

None

AN18

5β-Androstan-3α-glucosiduronate-17-one (etiocholanolone glucuronide)

None

AN19

5α-Androstane

None

100

AN20

5α-Androstan-3β-ol

6.10

0.43

5.57

1.66

AN21

5α-Androst-16-en-3β-ol

5.32

1.01

5.48

2.11

AN22

5α-Androst-16-en-3-one

5.52

0.96

5.58

0.68

100

AN23

5β-Androstan-3α-ol

5.85

1.12

5.59

0.33

None

AN24

Androst-4,16-dien-3-one

5.15

0.64

5.96

0.17

100

AN25

Androst-5,16-dien-3β-ol

None

5.60

1.50

100

ESTRANES

ES1

1,3,5(10)-Estratrien-3,17β-diol (estradiol)

4.80

0.34

None

200

ES2

1,3,5(10)-Estratrien-3-ol-17-one (estrone)

4.42

0.47

None

200

ES3

1,3,5(10)-Estratrien-3,16α,17β-triol (estriol)

None

200

ES4

1,3,5(10)-Estratrien-3,16α-diol-17-one (16α-hydroxyestrone)

5.60

0.42

5.70

0.17

None

ES5

1,3,5(10)-Estratrien-3-ol-4-methoxy-17-one (4-methoxyestrone)

5.40

0.93

5.62

0.19

None

ES6

1,3,5(10)-Estratrien-3,15α,16α,17β-tetrol (estetrol)

5.67

0.29

None

200

ES7

1,3,5(10)-Estratrien-2,3-diol-17-one (2-hydroxyestrone)

5.44

0.93

5.74

0.19

None

ES8

1,3,5(10)-Estratrien-17-one-3-sulfate (estrone sulfate)

5.47

0.43

None

ES9

1,3,5(10)-Estratrien-17β-ol-3-glucosiduronate (estradiol glucuronide)

None

ES10

1,3,5(10)-Estratrien-17β-ol-3-sulfate (estradiol sulfate)

6.05

0.6

None

200

ES11

1,3,5(10)-Estratrien-17α-ethinyl-3,17β-diol (ethinyl estradiol)

5.72

0.68

None

200

Activities are in -log(EC₅₀), with EC₅₀in molar units for the activation of human or zebrafish PXR. Efficacy is relative to 10 μM rifampicin (human PXR) or 20 μM 5α-androstan-3α-ol (zebrafish PXR) which are assigned an efficacy of 1.0. Toxicity is the lowest concentration in micromolar that produced significant toxicity in the HepG2 cells.

Table 2

Activation of human and zebrafish PXRs by pregnane steroids and related compounds

Cmp. #

Compound

hPXR Activity

hPXR Efficacy

zfPXR Activity

zfPXR Efficacy

Toxicity

PR1

5β-Pregnan-3α,20α-diol (5β-pregnanediol)

5.29

0.34

None

100

PR2

5β-Pregnan-3,20-dione (5β-pregnanedione)

5.59

0.97

6.08

0.85

None

PR3

4-Pregnen-11β,21-diol-3,20-dione (corticosterone)

5.00

0.54

None

PR4

4-Pregnen-17,21-diol-3,20-dione (cortexolone)

4.64

0.49

None

PR5

4-Pregnen-11β,21-diol-3,18,20-trione (aldosterone)

4.26

0.21

None

PR6

4-Pregnen-17,21-diol-3,11,20-trione (cortisone)

4.16

0.28

None

200

PR7

4-Pregnen-3,20-dione (progesterone)

4.83

0.57

None

200

PR8

4-Pregnen-17-ol-3,20-dione

4.75

0.7

None

PR9

4-Pregnen-21-ol-3,20-dione (cortexone)

5.61

0.3

None

200

PR10

4-Pregnen-3β,17,21-triol-3,20-dione (cortisol)

4.32

0.66

None

PR11

5-Pregnen-3β,17-diol-20-one

4.47

0.36

None

PR12

5-Pregnen-3β-diol-20-one (pregnenolone)

5.64

1.26

6.32

2.05

None

PR13

5-Pregnen-16α-cyano-3β-ol-20-one

None

PR14

5α-Pregnan-3α-ol-20-one (allopregnanolone)

5.38

0.46

5.40

0.30

None

PR15

5α-Pregnan-3α,20α-diol (allopregnanediol)

4.28

0.16

4.58

0.29

None

PR16

5β-Pregnan-3α,20α-diol-3-glucosiduronate (pregnanediol glucuconide)

4.26

0.17

4.78

1.42

None

PR17

5β-Pregnan-3α,11β,17,20α-21-pentol (cortol)

4.33

0.83

3.95

0.98

200

PR18

5β-Pregnan-3α,17,20α-21-tetrol-11-one (cortolone)

4.35

0.72

4.34

0.25

200

PR19

5β-Pregnan-3α,17,21-triol-11,20-dione

4.28

0.8

4.43

0.33

None

PR20

5α-Pregnan-3α,11β,21-triol-20-one

4.90

0.26

None

PR21

4-Pregnen-17α,20β-diol-3,20-dione

None

200

PR22

4-Pregnen-20β-ol-3,20-dione-17α-sulfate

5.73

0.66

None

PR23

5β-Pregnan-3α,20β-diol

5.42

0.49

5.95

0.25

None

PR24

5β-Pregnan-3α,11β,17,21-tetrol-20-one

4.33

0.73

3.94

0.11

None

PR25

5β-Pregnan-3α-ol-20-one

4.98

0.55

6.64

1.22

100

PR26

5-Pregnen-20-one-3β-sulfate

None

4.95

0.23

None

PR27

4-Estren-17α-ethynyl-18-homo-17β-ol-3-one (levonorgestrel)

5.37

0.35

6.00

0.86

None

PR28

4-Estren-17α-ethynyl-17β-ol-3-one (norethindrone)

4.59

0.32

5.85

0.40

None

PR29

1,4-Pregnadien-9α-fluoro-16α-methyl-11β,17,21-triol-3,20-dione (dexamethasone)

4.39

0.83

None

Table 3

Activation of human and zebrafish PXRs by xenobiotics and vitamins

Cmp. #

Compound

hPXR Activity

hPXR Efficacy

zfPXR Activity

zfPXR Efficacy

Toxicity

MI1

Acetaminophen

None

MI2

3-Aminobenzoic acid

None

MI3

Benzo [a]pyren

4.75

0.55

4.00

0.06

100

MI4

n-Butyl-4-aminobenzoate

4.88

1.35

4.86

0.69

100

MI5

Butylbenzoate

None

MI6

Caffeine

None

MI7

Carbamazepine

4.20

0.37

None

200

MI8

Carbamazepine epoxide

4.09

0.57

None

200

MI9

β-Carotene

5.46

0.67

None

100

MI10

Chlorpyrifos

4.59

2.05

5.44

0.88

None

MI11

Chlorzoxazone

None

500

MI12

Cyclosporine

None

MI13

Ecdysone

None

MI14

Ethyl-2-aminobenzoate

None

MI15

Flurbiprofen

4.10

1.59

4.10

0.53

None

MI16

Folic acid

None

MI17

Guggulsterone

None

MI18

GW3965

None

MI19

Hyperforin

7.22

1.29

None

MI20

Mevastatin

5.23

0.51

None

MI21

Mycophenolic acid

None

MI22

Nifedipine

5.33

0.41

4.91

0.99

MI23

Oxcarbazepine

4.74

0.35

~4.70

~0.30

200

MI24

Paclitaxel

4.92

0.13

None

100

MI25

Phenobarbital

3.43

1.19

3.49

0.10

None

MI26

Phenytoin

4.26

0.52

None

200

MI27

n-Propyl-4-hydroxybenzoate

4.51

0.32

4.28

0.31

100

MI28

Provitamin D₃

None

MI29

Provitamin D₂

None

MI30

Reserpine

4.91

0.72

None

MI31

Retinol

5.80

0.20

None

MI32

Rifampicin

7.00

1.00

None

200

MI33

SR12813

6.41

0.90

None

MI34

TCDD

7.17

1.78

6.32

6.17

MI35

TCPOBOP

5.25

0.66

None

200

MI36

T-0901317

7.66

1.24

None

100

MI37

α-Tocopherol

~4.30

~0.25

None

100

MI38

β-Tocopherol

4.85

0.33

None

100

MI39

δ-Tocopherol

5.14

0.64

None

100

MI40

γ-Tocopherol

None

100

MI41

1α,25-Dihydroxyvitamin D₃

None

MI42

1α-Hydroxyvitamin D₂

None

MI43

1α-Hydroxyvitamin D₃

None

MI44

Vitamin K₁

4.99

0.13

None

100

MI45

Vitamin K₂

5.04

0.80

None

100

MI46

Vitamin K₃

~4.30

~0.15

None

100

Comparison of human and zebrafish PXRs

In two prior studies that compared PXRs from different species, human and zebrafish PXRs were found to share some activating ligands, including pregnanes, androstanes, and a few xenobiotics such as nifedipine and phenobarbital 1215. Activation of zebrafish PXR by the much larger set of 165 compounds tested on human PXR was determined in this study, and these two species showed considerable overlap in their ligand specificity (Tables 1, 2, 3, Additional file 2). Human PXR has very broad specificity for steroid hormones and their synthetic intermediates (Figure 2A) albeit mostly at micromolar concentrations likely to exceed typical physiologic concentrations 28.

Figure 2

PXR activation and steroid pathways

PXR activation and steroid pathways. Steroid pathways typical of vertebrates are indicated. (A) Human PXR is activated by a large number of steroid hormones, although typically at micromolar concentrations. The coloring indicates at which concentrations the various steroids activate human PXR (see key in bottom right of panel). (B) Zebrafish PXR is activated by a smaller number of steroid hormones than human PXR, although there is much overlap between the selectivity of the two PXRs. Zebrafish PXR tends to be more sensitive to steroid hormone activation, at least for the functional assay used in this study. The coloring indicates at which concentrations the various steroids activate zebrafish PXR using the same key as in (A). Abbreviations: dehydroepiandrosterone, DHEA; DHEA sulfate; DHEA SO₄; dihydrotesterone, DHT.

Zebrafish PXR was activated by far fewer steroid compounds which were essentially a subset of those that activate human PXR (Figure 2B). For both human and zebrafish PXRs, pregnane steroids showed the highest activity (Figure 2, Table 2). Human and zebrafish PXRs showed more differences in regard to bile salt activators with zebrafish PXR being activated by very few of the bile salts tested (Additional file 2). In terms of the evolution of bile salts, human PXR is activated by both evolutionary 'early' bile salts 262728 (e.g., 27-carbon bile alcohol sulfates such as 5α-cholestan-3α,7α,12α,26,27-pentol [cyprinol] 27-sulfate) and 'recent' bile salts (e.g., cholic acid) (Additional files 2 and 3). Zebrafish PXR is activated only by early bile salts, including 5α-cyprinol sulfate and 5β-scymnol (5β-cholestan-3α,7α,12α,24,26,27-hexol) 27-sulfate (Additional files 2 and 3). The results are consistent with crystallographic studies of human PXR that show a large, flexible ligand-binding pocket 293031323334. This pocket can accommodate bile salts of both 5α (A/B trans) and 5β (A/B cis) orientation (Figure 1), as well as those with differing side-chain lengths and conjugation. This is in contrast to studies of farnesoid X receptors (FXRs; NR1H4) and VDRs, two other NRs that are activated by bile acids 35363738. In particular, FXRs are antagonized by 5α-bile alcohol sulfates 39 while VDRs are essentially only activated by the smallest bile salt, lithocholic acid (5β-cholan-3α-ol-24-oic acid), and its metabolites 384041.

Additional file 3

Comparison of bile salt activation of human and zebrafish PXRs. Comparison of bile salt synthetic pathways for humans and zebrafish, indicating which bile salts and intermediates activate human and zebrafish PXRs.

Click here for file

Pharmacophore models for six PXRs

In a comparative study, we determined concentration-response curves for a common set of 16 compounds (steroids, bile salts, xenobiotics) in a set of PXRs from six species (human, zebrafish, mouse, rat, rabbit, and chicken; Tables 1, 2, 3, 4, Additional file 2). The pharmacophores generated are shown mapped to two of the generally more active ligands, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 5β-pregnane-3,20-dione (Figure 3). Human, rat, and mouse PXRs showed very similar pharmacophores with 4–5 hydrophobic features and multiple excluded volumes (Figure 3A,C,D). The pharmacophores for these three PXRs all suggest generally large ligand-binding pockets with differences only in positions of the features. It is interesting that compared with previous pharmacophores for human PXR 424344 which contained 4–5 hydrophobic features and at least 1–2 hydrogen bonding moieties, there are no hydrogen bonding features in the current human PXR pharmacophore. This could be due to the molecules used in the current training set being mostly bile salts and having active and inactive compounds with similar features. As the Catalyst pharmacophore generation method looks for differences between the extremes of activity to describe the features contributing to the pharmacophore, this may represent a limitation of the method. While a single universal pharmacophore for human PXR (and perhaps PXRs from other species) may be impossible due to the size and flexibility of the binding site, it is likely in the current study that the 16 selected molecules may just be a sub-section of the binding pocket. For example, this may be where steroidal compounds fit 33 as modelled previously with a pharmacophore 45. Therefore, the pharmacophores serve as a novel qualitative method for analysis of PXR ligand specificity across the species.

Table 4

Activation of mouse, rat, rabbit, and chicken PXRs

Cmp #

Compound

Mouse PXR Activity (efficacy, ε, in parentheses)

Rat PXR Activity (efficacy, ε, in parentheses)

Rabbit PXR Activity (efficacy, ε, in parentheses)

Chicken PXR Activity (efficacy, ε, in parentheses)

BI004

Murideoxycholic acid

5.09 (ε = 0.76)

4.80 (ε = 0.22)

4.52 (ε = 1.86)

No effect

BI005

Chenodeoxycholic acid

No effect

4.70 (ε = 0.42)

4.70 (ε = 0.36)

BI008

Deoxycholic acid

No effect

5.00 (ε = 0.32)

4.44 (ε = 0.37)

No effect

BI011

Lithocholic acid

4.86 (ε = 0.48)

4.78 (ε = 0.42)

4.80 (ε = 0.70)

5.09 (ε = 0.17)

BI020

Cholic acid

No effect

4.82 (ε = 0.42)

4.02 (ε = 0.67)

4.47 (ε = 0.36)

BI023

5β-Cholestan-3α,7α,12α-triol

5.85 (ε = 1.23)

5.65 (ε = 0.72)

5.41 (ε = 0.37)

5.89 (ε = 0.27)

BI034

5β-Scymnol sulfate

4.44 (ε = 0.85)

4.40 (ε = 0.85)

4.09 (ε = 1.93)

4.37 (ε = 0.88)

BI036

5α-Cyprinol sulfate

4.78 (ε = 0.29)

4.50 (ε = 0.28)

4.09 (ε = 0.43)

4.51 (ε = 0.61)

BI038

3α,7α,12αtTrihydroxy-5β-cholestan-27-oic acid, taurine conjugated

No effect

BI046

Tauro-β-muricholic acid

No effect

BI047

7α-Hydroxycholesterol

No effect

PR2

5β-Pregnane-3,20-dione

5.36 (ε = 0.84)

5.24 (ε = 1.01)

4.90 (ε = 1.0)

5.59 (ε = 0.81)

MI3

Benzo [a]pyren

4.86 (ε = 0.94)