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

doi:10.1016/j.bmc.2006.12.026

Bioorganic & Medicinal Chemistry

Volume 15, Issue 5, 1 March 2007, Pages 2156-2166

https://doi.org/10.1016/j.bmc.2006.12.026 Get rights and content

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 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 Å 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.

Graphical abstract

The crystal structure of the nuclear xenobiotic receptor PXR in complex with T0901317 leads to the synthesis of agonists specific to PXR relative to the nuclear receptor LXR.

Introduction

The human pregnane X receptor (PXR; alternatively SXR, PAR) responds to a variety of endogenous and exogenous compounds in liver, intestine, and other tissues, and is a key regulator of the expression of genes central to xenobiotic metabolism and excretion.1, 2, 3 PXR is also responsible for an important class of drug interactions caused by the efficient up-regulation of chemoprotective pathways that lead to the elimination of a wide range of therapeutics.4, 5, 6, 7 It has also recently been shown that T0901317 (T1317), the ligand described here in complex with PXR, protects against the development of the neurodegenerative disease Niemann-Pick C in a PXR-dependent fashion within a mouse model of this condition.⁸

Like other members of the orphan class of the nuclear receptor (NR) superfamily, PXR contains DNA-binding and ligand binding domains (DBD, LBD, respectively), acts as a heterodimer with the retinoid X receptor-α (RXRα), and binds to a range of direct- and everted-repeat elements in the regulatory region of target genes.⁹ Upon association with an activating agonist, a transcriptional coactivator protein like the steroid receptor coactivator-1 (SRC-1) is recruited to the activation function-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 Crystal structures of the human PXR LBD have been determined in complexes with a variety of small (e.g., SR12813, hyperforin) and large (e.g., rifampicin) ligands, and with fragments of the human transcriptional coactivator SRC-1.12, 13, 14, 15 These structures have revealed that PXR’s ligand binding promiscuity is a function of its large and conformable ligand binding pocket, which is framed in part by sequence elements novel to PXR relative 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 interface plays a role in receptor function and association with coactivators.¹⁶

The liver X receptor (LXR), another member of the orphan class of NRs that functions as a heterodimer with RXRα, plays an important role in monitoring the levels of oxysterols in hepatocytes and regulates the expression of genes essential for cholesterol homeostasis.17, 18, 19, 20, 21 The LBDs of the two LXR isoforms, α and β, 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 domains.22, 23, 24, 25, 26 The PXR LBD deviates from that of LXR in its ∼60-residue α1–α3 insert that adds one helix (α2) and two strands (β1, β1′) 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 β has been elucidated previously.22, 24, 26

In addition to their association with transcriptional coactivators, 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 antagonist tamoxifen. It has been shown structurally that corepressors contain an extended leucine-rich region that binds to an inactive LBD conformation distinct in structure from that of an active LBD, particularly in the position of the terminal α-helix in the LBD fold (αAF in PXR).²⁷ In ER, tamoxifen sterically blocks the active orientation of the terminal helix in that fold, producing a state that preferentially binds to transcriptional corepressors 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 protecting tissues from potentially harmful chemicals. A small number of antagonists have been described, however, including the ET-743²⁹ and the antifungal ketoconazole.30, 31 Indeed, ketoconazole has been shown recently to repress the binding of both transcriptional coactivators and corepressors to PXR.³⁰ The structural basis of the antagonism of PXR by these compounds has not been established, however.

The identification or design of new antagonists of human PXR is desirable because of the important role this receptor plays in drug metabolism, and because the current antagonists exhibit weak EC₅₀ values relative to established agonists.29, 30, 31 Repressing the ability of PXR to recognize the presence of therapeutic compounds may allow lower doses to be administered with higher efficacy and fewer side effects. Here we present the 2.8 Å 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 conformation of the receptor’s αAF. We find, however, that compounds based on this scaffold either retain their ability 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 difficult due to the promiscuous and conformable nature of the receptor’s ligand binding pocket.

Section snippets

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 Å resolution were collected, and the structure was determined and refined to R and R_free 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 orientation of bound T1317, and one fifteen-amino acid stretch of the human transcriptional coactivator steroid receptor coactivator

Discussion

T1317 is an efficacious activator of gene transcription mediated by both the nuclear receptors PXR and LXRβ.¹⁹ We show, by comparing the 2.8 Å resolution crystal structure of the PXR–T1317 complex to the LXRβ–T1317 complexes reported previously,22, 24, 25, 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 group of T1317, as well as the distinct placement of the proximal hydrogen-bonding

Protein expression and purification

Generation of human PXR LBD in complex with the 88-amino acid fragment of human SRC-1 was accomplished as described previously.12, 13, 14, 15, 16 To prevent the formation of covalent complexes with reducing agent during crystallization, as has been seen with previous structures (data not shown), Cys-284 within the ligand binding pocket of the human PXR LBD was replaced with serine.

Crystallization

The human PXR ligand binding domain/SRC-1 complex (hPXR-LBD/SRC-1) was concentrated in the presence of 10-fold

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^☆: This work was supported by National Institutes of Health Grant DK62229 (M.R.R.).

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

Abstract

Graphical abstract

Introduction

Section snippets

PXR–T1317 structure

Discussion

Protein expression and purification

Crystallization

Cell

Toxicology

Curr. Opin. Struct. Biol.

J. Mol. Biol.

Cell Metab.

Cell

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Methods Enzymol.

Proc. Natl. Acad. Sci. U.S.A.

Gene Dev.

Curr. Drug Metab.

J. Clin. Invest.

Proc. Natl. Acad. Sci. U.S.A.