WO2002095652A1 - Crystallized human xenobiotic nuclear receptor pxr/sxr ligand binding domain polypeptide and screening methods employing same - Google Patents

Crystallized human xenobiotic nuclear receptor pxr/sxr ligand binding domain polypeptide and screening methods employing same Download PDF

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Publication number
WO2002095652A1
WO2002095652A1 PCT/US2002/015701 US0215701W WO02095652A1 WO 2002095652 A1 WO2002095652 A1 WO 2002095652A1 US 0215701 W US0215701 W US 0215701W WO 02095652 A1 WO02095652 A1 WO 02095652A1
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pxr
polypeptide
ligand
ligand binding
binding domain
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PCT/US2002/015701
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French (fr)
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Steven Kliewer
Matthew R Redinbo
Ryan E. Watkins
George Bruce Wisely
Shawn P. Williams
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Smithkline Beecham Corporation
University Of North Carolina
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Priority to EP02726884A priority Critical patent/EP1393235A1/en
Publication of WO2002095652A1 publication Critical patent/WO2002095652A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates generally to the structure of the ligand binding domain of PXR, and more particularly to the crystalline structure of the ligand binding domain of PXR.
  • the invention further relates to methods by which modulators and ligands of PXR can be identified.
  • HRE hormone response element kDa kilodalton(s)
  • Nuclear receptors represent a superfamily of proteins that specifically bind a physiologically relevant small molecule, such as a hormone or vitamin. As a result of a molecule binding to a nuclear receptor, the nuclear receptor changes the ability of a cell to transcribe DNA, i.e. nuclear receptors modulate the transcription of DNA. However they can also have transcription independent actions. Unlike integral membrane receptors and membrane-associated receptors, nuclear receptors reside in either the cytoplasm or nucleus of eukaryotic cells. Thus nuclear receptors comprise a class of intracellular, soluble ligand-regulated transcription factors.
  • Nuclear receptors include but are not limited to receptors for giucocorticoids, androgens, mineralcorticoids, progestins, estrogens, thyroid hormones, vitamin D retinoids, and icosanoids. Many nuclear receptors, identified by either sequence homology to known receptors (See, Drewes et al., (1996) Mol. Cell. Biol. 16:925-31) or based on their affinity for specific DNA binding sites in gene promoters (See, Sladek et a , Genes Dev. 4:2353-65), have unascertained ligands and are therefore termed "orphan receptors".
  • nuclear receptors are generally characterized by two distinct structural elements.
  • nuclear receptors comprise a central DNA binding domain that targets the receptor to specific DNA sequences, which are known as hormone response elements (HREs).
  • HREs hormone response elements
  • the DNA binding domains of these receptors are related in structure and sequence, and are located within the middle of the receptor.
  • the C-terminal region of nuclear receptors encompasses the ligand binding domain (LBD). Upon binding a ligand, the receptor shifts to a transcriptionally active state.
  • LBD ligand binding domain
  • cytochrome P450 family of heme-containing proteins play critical roles in the oxidative metabolism of drugs and other xenobiotics in the liver and small intestine.
  • the CYP3A gene products bind and hydroxylate a wide variety of chemical structures, including >50% of all drugs. Maurel, (1996) in Cytochrome P450: Metabolic and Toxicological Aspects (lonnides, ed.) CRC Press, Inc., Boca Raton, Florida, 241-70.
  • Expression of CYP3A is induced at the level of transcription by a variety of xenobiotics, including many that are metabolized by CYP3A.
  • the pregnane X receptor (PXR; NR112), a member of the nuclear receptor family of ligand-activated transcription factors, is a key regulator of CYP3A gene expression in mammalian liver and small intestine. Kliewer et al., (1998) Cell 92: 73-82; Lehmann et al., (1998) J. Clin. Invest. 102: 1016- 23; Bertilsson et al., (1998) Proc. Nat. Acad. Sci. U.S.A. 95: 12208-13; Blumberg et al., (1998) Gene Dev. 12:3195-205; Xie et al., (2000) Nature 406: 435-39.
  • PXR pregnane activated receptor
  • SXR steroid and xenobiotic receptor
  • PXR contains both a DNA binding domain and a ligand binding domain (LBD).
  • PXR binds to the xenobiotic response elements in the regulatory regions of CYP3A genes as a heterodimer with the 9-cis retinoic acid receptor (RXR). Kliewer et al., (1998) Cell 92: 730-82; Lehmann et al., (1998) J. Clin. Invest. 102: 1016-23; Blumberg et al., (1998) Gene Dev. 12: 3195-205.
  • RXR 9-cis retinoic acid receptor
  • PXR mediates potentially dangerous drug-drug interactions by upregulating CYP3A expression in response to one compound, which in turn can then lead to the metabolism of other drugs vital to survival.
  • patients taking the herbal anti-depressant St. John's wort have exhibited a dramatic drop in serum levels of other critical drugs, including the antiretroviral drug Indinavir (also known as CRIXIVAN®) and the immunosuppressant compound cyclosporin.
  • CRIXIVAN® antiretroviral drug Indinavir
  • human PXR is activated efficiently by rifampicin and the cholesterol-lowering drug SR12813 (Berkhout et al friendship (1996) J. Biol. Chem. 271 : 14376-82; Berkhout et al., (1997) Atherosclerosis 133: 203-21 ), whereas mouse PXR is not (Jones et al., (2000) Mol. Endocrinol. 14: 27-39).
  • the mouse version of this receptor is activated by the synthetic steroid pregnenolone 16 ⁇ -carbonitrile (PCN), whereas the human receptor is not.
  • PCN synthetic steroid pregnenolone 16 ⁇ -carbonitrile
  • Polypeptides including the ligand binding domain of PXR, have a three-dimensional structure determined by the primary amino acid sequence and the environment surrounding the polypeptide. This three-dimensional structure establishes the polypeptide's activity, stability, binding affinity, binding specificity, and other biochemical attributes. Thus, knowledge of a protein's three-dimensional structure can provide much guidance in designing agents that mimic, inhibit, or improve its biological activity in soluble or membrane bound forms.
  • the three-dimensional structure of a polypeptide can be determined in a number of ways. Many of the most precise methods employ X-ray crystallography (See, e.g., Van Holde, (1971) Physical Biochemistry, Prentice- Hall, N. J., 221-39).
  • PXR shows structural homology with the three-dimensional fold of other proteins.
  • a solved PXR-ligand crystal structure would provide structural details and insights necessary to design a modulator of PXR that maximizes preferred requirements for any modulator, i.e. potency and specificity.
  • By exploiting the structural details obtained from a PXR-ligand crystal structure it would be possible to design a PXR modulator that, despite PXR's similarity with other proteins, exploits the unique structural features of PXR.
  • a PXR modulator developed using structure-assisted design would take advantage of heretofore unknown PXR structural considerations and thus be more effective than a modulator developed using homology-based design. Potential or existent homology models cannot provide the necessary degree of specificity.
  • a PXR modulator designed using the structural coordinates of a crystalline form of PXR would also provide a starting point for the development of modulators of other structurally similar proteins.
  • a substantially pure PXR ligand binding domain polypeptide in crystalline form is disclosed.
  • the crystalline form is a tetragonal crystalline form.
  • the crystalline form has a space group of P4 3 2 ⁇ 2.
  • the PXR ligand binding domain polypeptide has the amino acid sequence shown in SEQ ID NO: 4.
  • the PXR ligand binding domain polypeptide is in complex with a ligand. More preferably, the ligand is a hypocholesterolemic drug. Even more preferably, the hypocholesterolemic drug is SR12813.
  • a method for determining the three-dimensional structure of a crystallized PXR ligand binding domain polypeptide to a resolution of about 3.0 A or better comprises (a) crystallizing a PXR ligand binding domain polypeptide; and (b) analyzing the PXR ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized PXR ligand binding domain polypeptide, whereby the three- dimensional structure of a crystallized PXR ligand binding domain polypeptide is determined to a resolution of about 3.0 A or better.
  • a method of designing a modulator of a PXR polypeptide comprises (a) designing a potential modulator of a PXR polypeptide that will form bonds with amino acids in a ligand binding site based upon a crystalline structure of a PXR ligand binding domain polypeptide; (b) synthesizing the modulator; and (c) determining whether the potential modulator modulates the activity of the PXR polypeptide, whereby a modulator of a PXR polypeptide is designed.
  • a method of designing a modulator that selectively modulates the activity of a PXR polypeptide in accordance with the present invention comprises: (a) obtaining a crystalline form of a PXR ligand binding domain polypeptide; (b) evaluating the three-dimensional structure of the crystallized PXR ligand binding domain polypeptide; and (c) synthesizing a potential modulator based on the three-dimensional crystal structure of the crystallized PXR ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of a PXR polypeptide is designed.
  • the method further comprises contacting a PXR ligand binding domain polypeptide with the potential modulator; and assaying the PXR ligand binding domain polypeptide for binding of the potential modulator, for a change in activity of the PXR ligand binding domain polypeptide, or both.
  • the crystalline form is such that the three-dimensional structure of the crystallized PXR ligand binding domain polypeptide can be determined to a resolution of about 3.0 A or better.
  • a method of designing a modulator of a PXR polypeptide in accordance with the present invention comprises: (a) selecting a candidate PXR ligand; (b) determining which amino acid or amino acids of a PXR polypeptide interact with the ligand using a three-dimensional model of a crystallized protein comprising a PXR LBD; (c) identifying in a biological assay for PXR activity a degree to which the ligand modulates the activity of the PXR polypeptide; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the PXR polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) performing the chemical modification on the ligand to form a modified ligand; (f) contacting the modified ligand with the PXR polypeptide; (g) identifying in a biological assay for PXR activity a degree to which the modified ligand modulates the biological activity of the PXR
  • the PXR polypeptide is a PXR polypeptide. More preferably, the three-dimensional model of a crystallized protein is a PXR LBD polypeptide with a bound ligand. Even more preferably, the method further comprises repeating steps (a) through (f), if the biological activity of the PXR polypeptide in the presence of the modified ligand varies from the biological activity of the PXR polypeptide in the presence of the unmodified ligand.
  • a method for identifying a PXR modulator comprises (a) providing atomic coordinates of a PXR ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits spatially into a binding cavity or on the surface of the PXR ligand binding domain to thereby identify a PXR modulator.
  • the method further comprises identifying in an assay for PXR-mediated activity a modeled ligand that increases or decreases the activity of the PXR.
  • a method of identifying a PXR modulator that selectively modulates the activity of a PXR polypeptide compared to other polypeptides comprises (a) providing atomic coordinates of a PXR ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits spatially into a binding cavity or on the surface of a PXR ligand binding domain and that interacts with conformationally constrained residues of a PXR that are conserved among PXR isoforms to thereby identify a PXR modulator.
  • the method further comprises identifying in a biological assay for PXR-mediated activity a modeled ligand that selectively binds to the PXR ligand binding domain and increases or decreases the activity of the PXR.
  • the assay method comprises: (a) designing a test inhibitor compound capable of modulating PXR activity, based on the atomic coordinates of a PXR ligand binding domain; (b) synthesizing the test inhibitor compound; (c) incubating a PXR polypeptide with a ligand in the presence of a test inhibitor compound; (d) determining an amount of ligand that is bound to the PXR polypeptide, wherein decreased binding of ligand to the PXR protein in the presence of the test inhibitor compound relative to binding of ligand in the absence of the test inhibitor compound is indicative of inhibition; and (e) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed, whereby a compound that inhibits binding of a ligand to a PXR polypeptide is identified.
  • the ligand is a hypocholesterolemic drug
  • a method of evaluating a candidate therapeutic agent in humans using mouse model system comprises: (a) providing atomic coordinates of a human PXR ligand binding domain to a computerized modeling system; (b) modeling a candidate therapeutic agent that fits spatially into a binding cavity or on the surface of a human PXR ligand binding domain; (c) providing a mouse PXR polypeptide; (d) selecting one or more mutations to be introduced into an amino acid sequence of the mouse PXR polypeptide, the mutations being selected so as to alter the mouse PXR polypeptide to be similar to a human PXR polypeptide; (e) providing a mutant mouse PXR polypeptide comprising the one or more mutations selected in step (d); (f) contacting the candidate therapeutic agent modeled in step (b) with the mutant mouse PXR polypeptide; (g) determining an effect of the candidate therapeutic agent on the mutant mouse PXR polypeptide; and (h) evaluating the potential of the candidate therapeutic agent
  • Figure 1 is a ribbon diagram depicting the structure of the ligand binding domain of the human xenobiotic receptor PXR.
  • Figure 2 is a cut-away view of the ligand binding cavity of the human xenobiotic PXR from a first perspective.
  • Figure 3 is a cut-away view of the ligand binding cavity of the human xenobiotic PXR from a second perspective.
  • Figure 4 is an experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown directly. Amino acid side chains are shown in blue.
  • SR12813 is depicted as a wireframe model and interacting atoms are presented as a spacefilling model comprising light gray spheres.
  • Figure 5 is an experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown schematically, van der Waals contacts are indicated by solid arrows and hydrogen bonds with dashed arrows.
  • Figure 6 is a second experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown directly. Amino acid side chains are shown in blue.
  • SR12813 is depicted as a wireframe model and interacting atoms are presented as a spacefilling model comprising light gray spheres.
  • Figure 7 is a second experimentally-observed position of SR12813 in the ligand binding cavity of human PXR.
  • intermolecular interactions are shown schematically, van der Waals contacts are indicated by solid arrows and hydrogen bonds with dashed arrows.
  • Figure 8 is a third experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown directly. Amino acid side chains are shown in blue. SR12813 is depicted as a wireframe model and interacting atoms are presented as a spacefilling model comprising light gray spheres.
  • Figure 9 is a third experimentally-observed position of SR12813 in the ligand binding cavity of human PXR.
  • intermolecular interactions are shown schematically, van der Waals contacts are indicated by solid arrows and hydrogen bonds with dashed arrows.
  • Figure 10 is a ribbon diagram depicting two salt bridges (Glu-321 to Arg-410 and Asp-205 to Arg-413) adjacent to the ligand binding cavity of human PXR.
  • Figures 11A-11 D are a series of plots representing the luciferase (normalized luciferase activity counts per second (CPS) x 1000, y axis) and alkaline phosphatase activity of several PXR mutants in the presence of rifampicin or SR12813 (concentration in M, x axis).
  • CV- 1 cells were transfected with expression plasmids for hPXR, D205A, R413A, E321A, or R410A.
  • Figure 12 is a computer-generated model depicting the homodimerization interaction observed in human PXR. The various distances between interacting residues are presented in the figure. The individual monomers are denoted by blue and green coloring.
  • Figure 13 is a ribbon diagram depicting an overview of the crystallographically-observed homodimer of human PXR. Individual monomers are denoted by red and gold coloring, with green and blue coloring representing interacting structure.
  • Figure 14 is a plot representing the luciferase and alkaline phosphatase activity (normalized luciferase activity counts per second (CPS) x 1000, y axis) of PXR mutants in the presence of rifampicin(RIF) or SR12813(SR) (concentration in M, x axis).
  • CV-1 cells were transfected with expression plasmids encoding wildtype hPXR or mutant hPXR comprising the mutations W223A, Y225A and the XREM-CYP3A4- luciferase reporter.
  • 0 WT-SR
  • D W223A, Y225A-RIF
  • • WT-SR
  • ⁇ W223A, Y225A-RIF.
  • Figures 15A-15C are a series of bar graphs demonstrating that four point mutants "humanize” mouse PXR's sensitivity to ligands.
  • CV-1 cells were transfected with expression plasmids for mouse PXR (Fig. 15A), human PXR (Fig. 15B), or R203L, P205S, Q404H, Q407R (mouse ⁇ human) mouse PXR (Fig. 15C) and the XREM-CYP3A4-luciferase reporter.
  • bars from left to right represent vehicle, PCN and SR12813.
  • SEQ ID NO: 1 is a DNA sequence encoding a full-length human PXR polypeptide (Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
  • SEQ ID NO: 2 is an amino acid sequence of a full-length human PXR polypeptide and is derived from the DNA sequence of SEQ ID NO: 1 (Swiss- Prot Accession No. 075469; GenBank Accession No. AF061056).
  • SEQ ID NO: 3 is a DNA sequence encoding the ligand binding domain of human PXR. The sequence codes for residues 130-434, which corresponds to the ligand binding domain of PXR (Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
  • SEQ ID NO: 4 is an amino acid sequence of the PXR ligand binding domain and is derived from the DNA sequence of SEQ ID NO: 3 (Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
  • SEQ ID NO: 5 is an amino acid sequence of the loop involving residues 309-321 in hPXR (Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
  • SEQ ID NO: 6 is a DNA sequence encoding a full-length human VDR polypeptide (Swiss-Prot Accession No. P11473; GenBank Accession No. J03258).
  • SEQ ID NO: 7 is an amino acid sequence of a full-lenth human VDR polypeptide and is derived from the DNA sequence of SEQ ID NO: 6 (Swiss- Prot Accession No. P11473; GenBank Accession No. J03258).
  • SEQ ID NO: 8 is a DNA sequence encoding the ligand binding domain of human VDR. The sequence codes for residues 192-427, which corresponds to the ligand binding domain of PXR (Swiss-Prot Accession No. P11473; GenBank Accession No. J03258).
  • SEQ ID NO: 9 is an amino acid sequence of the VDR ligand binding domain and is derived from the DNA seqence of SEQ ID NO: 8 (Swiss-Prot Accession No. P11473; GenBank Accession No. J03258).
  • SEQ ID NO: 10 is a DNA sequence encoding an N-terminal polyhistidine tagged PXR ligand binding domain fusion protein comprising residues 130-434 from human PXR (fragment from Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
  • SEQ ID NO: 11 is an amino acid sequence of the N-terminal polyhistidine tagged fusion protein comprising residues 130-434 from human PXR and is derived from the DNA seqence of SEQ ID NO: 10 (fragment from Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
  • SEQ ID NO: 12 is a DNA sequence encoding residues 623-710 of the human SRC-1 gene (fragment from GenBank Accession No. U59302).
  • SEQ ID NO: 13 is an amino acid sequence of residues 623-710 of the human SRC-1 gene and is derived from the DNA sequence of SEQ ID NO: 12 (fragment from GenBank Accession No. U59302).
  • crystalline polypeptides provide other advantages. For example, the crystallization process itself further purifies the polypeptide, and satisfies one of the classical criteria for homogeneity. In fact, crystallization frequently provides unparalleled purification quality, removing impurities that are not removed by other purification methods such as HPLC, dialysis, conventional column chromatography, etc. Moreover, crystalline polypeptides are often stable at ambient temperatures and free of protease contamination and other degradation associated with solution storage.
  • Crystalline polypeptides can also be useful as pharmaceutical preparations.
  • crystallization techniques in general are largely free of problems such as denaturation associated with other stabilization methods (e.g., lyophilization).
  • crystallographic data provides useful structural information that can assist the design of compounds that can serve as agonists or antagonists, as described herein below.
  • the crystal structure provides information useful to map a receptor-binding domain, which could then be mimicked by a small non-peptide molecule that would serve as an antagonist or agonist.
  • mutation carries its traditional connotation and means a change, inherited, naturally occurring or introduced, in a nucleic acid or polypeptide sequence, and is used in its sense as generally known to those of skill in the art.
  • labeled means the attachment of a moiety, capable of detection by spectroscopic, radiologic or other methods, to a probe molecule.
  • target cell refers to a cell, into which it is desired to insert a nucleic acid sequence or polypeptide, or to otherwise effect a modification from conditions known to be standard in the unmodified cell.
  • a nucleic acid sequence introduced into a target cell can be of variable length.
  • a nucleic acid sequence can enter a target cell as a component of a plasmid or other vector or as a naked sequence.
  • transcription means a cellular process involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene.
  • the process includes, but is not limited to the following steps: (a) the transcription initiation, (b) transcript elongation, (c) transcript splicing, (d) transcript capping, (e) transcript termination, (f) transcript polyadenylation, (g) nuclear export of the transcript, (h) transcript editing, and (i) stabilizing the transcript.
  • the term “expression” generally refers to the cellular processes by which a polypeptide is produced from RNA.
  • transcription factor means a cytoplasmic or nuclear protein which binds to a gene, or binds to an RNA transcript of a gene, or binds to another protein which binds to a gene or an RNA transcript or another protein which in turn binds to a gene or an RNA transcript, so as to thereby modulate expression of the gene. Such modulation can additionally be achieved by other mechanisms; the essence of a "transcription factor for a gene” pertains to a factor that alters the level of transcription of the gene in some way.
  • hybridization means the binding of a probe molecule, a molecule to which a detectable moiety has been bound, to a target sample.
  • detecting means confirming the presence of a target entity by observing the occurrence of a detectable signal, such as a radiologic or spectroscopic signal that will appear exclusively in the presence of the target entity.
  • sequencing means determining the ordered linear sequence of nucleic acids or amino acids of a DNA or protein target sample, using conventional manual or automated laboratory techniques.
  • the term “isolated” means oligonucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which they can be associated, such association being either in cellular material or in a synthesis medium.
  • the term can also be applied to polypeptides, in which case the polypeptide will be substantially free of nucleic acids, carbohydrates, lipids and other undesired polypeptides.
  • substantially pure means that the polynucleotide or polypeptide is substantially free of the sequences and molecules with which it is associated in its natural state, and those molecules used in the isolation procedure.
  • substantially free means that the sample is at least 50%, preferably at least 70%, more preferably 80% and most preferably 90% free of the materials and compounds with which is it associated in nature.
  • the term “primer” means a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and more preferably more than eight and most preferably at least about 20 nucleotides of an exonic or intronic region. Such oligonucleotides are preferably between ten and thirty bases in length.
  • DNA segment means a DNA molecule that has been isolated free of total genomic DNA of a particular species.
  • a DNA segment encoding a PXR polypeptide refers to a DNA segment that contains SEQ ID NO: 1 , but can optionally comprise fewer or additional nucleic acids, yet is isolated away from, or purified free from, total genomic DNA of a source species, such as Homo sapiens.
  • DNA segment includes DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like.
  • the phrase "enhancer-promoter” means a composite unit that contains both enhancer and promoter elements. An enhancer- promoter is operatively linked to a coding sequence that encodes at least one gene product.
  • operatively linked means that an enhancer-promoter is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter.
  • Techniques for operatively linking an enhancer-promoter to a coding sequence are well known in the art; the precise orientation and location relative to a coding sequence of interest is dependent, inter alia, upon the specific nature of the enhancer-promoter.
  • candidate substance and “candidate compound” are used interchangeably and refer to a substance that is believed to interact with another moiety, for example a given ligand that is believed to interact with a complete, or a fragment of, a PXR polypeptide, and which can be subsequently evaluated for such an interaction.
  • candidate substances or compounds include "xenobiotics”, such as drugs and other therapeutic agents, carcinogens and environmental pollutants, natural products and extracts, as well as “endobiotics”, such as steroids, fatty acids and prostaglandins.
  • hormones e.g., opioid peptides, steroids, etc.
  • biological activity means any observable effect flowing from interaction between a PXR polypeptide and a ligand.
  • Representative, but non-limiting, examples of biological activity in the context of the present invention include dimerization of a PXR and association of a PXR with DNA.
  • modified means an alteration from an entity's normally occurring state.
  • An entity can be modified by removing discrete chemical units or by adding discrete chemical units.
  • modified encompasses detectable labels as well as those entities added as aids in purification.
  • structure coordinates and "structural coordinates” mean mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a molecule in crystal form.
  • the diffraction data are used to calculate an electron density map of the repeating unit of the crystal.
  • the electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal.
  • any set of structure coordinates determined by X-ray crystallography is not without standard error.
  • any set of structure coordinates for PXR or a PXR mutant that have a root mean square deviation (RMSD) from ideal preferably no more than 1.5 A, more preferably no more than 1.0 A, and most preferably no more than 0.5 A when superimposed, using the polypeptide backbone atoms, on the structure coordinates listed in Table 4 shall be considered identical.
  • RMSD root mean square deviation
  • space group means the arrangement of symmetry elements of a crystal.
  • the term "molecular replacement” means a method that involves generating a preliminary model of the wild-type PXR ligand binding domain, or a PXR mutant crystal whose structure coordinates are unknown, by orienting and positioning a molecule whose structure coordinates are known within the unit cell of the unknown crystal so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal.
  • molecular replacement can be used to determine the structure coordinates of a crystalline mutant or homologue of the hPXR ligand binding domain, or of a different crystal form of the hPXR ligand binding domain.
  • isomorphous replacement means a method of using heavy atom derivative crystals to obtain the phase information necessary to elucidate the three-dimensional structure of a native crystal (Blundell et al., (1976) Protein Crystallography, Academic Press; Otwinowski, (1991), in Isomorphous Replacement and Anomalous Scattering, (Evans & Leslie, eds.), 80-86, Daresbury Laboratory, Daresbury, United Kingdom).
  • the phrase “heavy-atom derivatization” is synonymous with the term “isomorphous replacement”.
  • ⁇ -sheet and "beta-sheet” mean the conformation of a polypeptide chain stretched into an extended zig-zig conformation. Portions of polypeptide chains that run “parallel” all run in the same direction. Polypeptide chains that are "antiparallel” run in the opposite direction from the parallel chains.
  • -helix and alpha-helix mean the conformation of a polypeptide chain wherein the polypeptide backbone is wound around the long axis of the molecule in a left-handed or right-handed direction, and the R groups of the amino acids protrude outward from the helical backbone, wherein the repeating unit of the structure is a single turnoff the helix, which extends about 0.56 nm along the long axis.
  • unit cell means a basic parallelepiped shaped block. The entire volume of a crystal can be constructed by regular assembly of such blocks. Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal. Thus, the term “unit cell” means the fundamental portion of a crystal structure that is repeated infinitely by translation in three dimensions. A unit cell is characterized by three vectors a, b, and c, not located in one plane, which form the edges of a parallelepiped.
  • Angles ⁇ , ⁇ and ⁇ define the angles between the vectors: angle ⁇ is the angle between vectors b and c; angle ⁇ is the angle between vectors a and c; and angle ⁇ is the angle between vectors a and b.
  • the entire volume of a crystal can be constructed by regular assembly of unit cells; each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal.
  • the vectors a, b and c describe the unit cell edges and the angles ⁇ , ⁇ , and ⁇ describe the unit cell angles.
  • crystal lattice means the array of points defined by the vertices of packed unit cells.
  • ligand binding site and "ligand binding domain” are used interchangeably and mean that site in a polypeptide where substrate binding occurs.
  • the ligand binding domain comprises the residues 130-434 of the full-length human PXR protein.
  • PXR means nucleic acids encoding a pregnane X receptor (PXR) nuclear receptor polypeptide that can bind DNA and/or one or more ligands, and/or has the ability to form multimers.
  • PXR includes invertebrate homologs; however, preferably, PXR nucleic acids and polypeptides are isolated from vertebrate sources.
  • PXR further includes vertebrate homologs of PXR family members, including, but not limited to, mammalian and avian homologs. Representative mammalian homologs of PXR family members include, but are not limited to, murine and human homologs.
  • PXR gene product PXR protein
  • PXR polypeptide PXR polypeptide
  • PXR peptide PXR peptides having amino acid sequences which are substantially identical to native amino acid sequences from an organism of interest and which are biologically active in that they comprise all or a part of the amino acid sequence of a PXR polypeptide, or cross-react with antibodies raised against a PXR polypeptide, or retain all or some of the biological activity (e.g., DNA or ligand binding ability and/or dimerization ability) of the native amino acid sequence or protein.
  • biological activity can include immunogenicity.
  • PXR gene product PXR protein
  • PXR polypeptide PXR polypeptide
  • PXR peptide also include analogs of a PXR polypeptide.
  • analog is intended that a DNA or peptide sequence can contain alterations relative to the sequences disclosed herein, yet retain all or some of the biological activity of those sequences. Analogs can be derived from genomic nucleotide sequences as are disclosed herein or from other organisms, or can be created synthetically. Those skilled in the art will appreciate that other analogs, as yet undisclosed or undiscovered, can be used to design and/or construct PXR analogs.
  • PXR gene product "PXR protein”, “PXR polypeptide”, or “PXR peptide” to comprise all or substantially all of the amino acid sequence of a PXR polypeptide gene product. Shorter or longer sequences are anticipated to be of use in the invention; shorter sequences are herein referred to as “segments”. Thus, the terms "PXR gene product”, “PXR protein”, “PXR polypeptide”, and “PXR peptide” also include fusion, chimeric or recombinant PXR polypeptides and proteins comprising sequences of the present invention. Methods of preparing such proteins are disclosed herein and are known in the art.
  • polypeptide means any polymer comprising any of the 20 protein amino acids, regardless of its size.
  • protein is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies.
  • polypeptide refers to peptides, polypeptides and proteins, unless otherwise noted.
  • protein polypeptide
  • polypeptide and “peptide” are used interchangeably herein when referring to a gene product.
  • modulate means an increase, decrease, or other alteration of any, or all, chemical and biological activities or properties of a wild-type or mutant PXR polypeptide.
  • modulation refers to both upregulation (i.e., activation or stimulation) and downregulation (i.e. inhibition or suppression) of a response.
  • PXR gene and “recombinant PXR gene” mean a nucleic acid molecule comprising an open reading frame encoding a PXR polypeptide of the present invention, including both exon and (optionally) intron sequences.
  • gene is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences. Preferred embodiments of genomic and cDNA sequences are disclosed herein.
  • DNA sequence encoding a PXR polypeptide can refer to one or more coding sequences within a particular individual.
  • genes for a particular polypeptide can exist in single or multiple copies within the genome of an individual. Such duplicate genes can be identical or can have certain modifications, including nucleotide substitutions, additions or deletions, all of which still code for polypeptides having substantially the same activity.
  • intron means a DNA sequence present in a given gene that is not-translated into protein.
  • interact means detectable interactions between molecules, such as can be detected using, for example, a yeast two hybrid assay.
  • the term “interact” is also meant to include “binding" interactions between molecules. Interactions can, for example, be protein- protein or protein-nucleic acid in nature.
  • the terms "cells,” “host cells” or “recombinant host cells” are used interchangeably and mean not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny might not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • agonist means an agent that supplements or potentiates the bioactivity of a functional PXR gene or protein, of a polypeptide encoded by a gene that is up- or down-regulated by a PXR polypeptide, and/or a polypeptide encoded by a gene that contains a PXR binding site in its promoter region.
  • an antagonist means an agent that decreases or inhibits the bioactivity of a functional PXR gene or protein, or that supplements or potentiates the bioactivity of a naturally occurring or engineered non-functional PXR gene or protein.
  • an antagonist can decrease or inhibit the bioactivity of a functional gene or polypeptide encoded by a gene that is up- or down-regulated by a PXR polypeptide and/or contains a PXR binding site in its promoter region.
  • An antagonist can also supplement or potentiate the bioactivity of a naturally occurring or engineered non-functional gene or polypeptide encoded by a gene that is up- or down- deregulated by a PXR polypeptide, and/or contains a PXR binding site in its promoter region.
  • chimeric protein or "fusion protein” are used interchangeably and mean a fusion of a first amino acid sequence encoding a PXR polypeptide with a second amino acid sequence defining a polypeptide domain foreign to, and not homologous with, any domain of one of a PXR polypeptide.
  • a chimeric protein can present a foreign domain that is found in an organism that also expresses the first protein, or it can be an "interspecies” or “intergenic” fusion of protein structures expressed by different kinds of organisms.
  • a fusion protein can be represented by the general formula X — PXR — Y, wherein PXR represents a portion of the protein which is derived from a PXR polypeptide, and X and Y are independently absent or represent amino acid sequences which are not related to a PXR sequence in an organism, which includes naturally occurring mutants.
  • the term "chimeric gene” refers to a nucleic acid construct that encodes a "chimeric protein" or "fusion protein” as defined herein.
  • the term "therapeutic agent” is a chemical entity intended to effectuate a change in an organism.
  • the organism is a human being. It is not necessary that a therapeutic agent be known to effectuate a change in an organism; chemical entities that are suspected, predicted or designed to effectuate a change in an organism are therefore encompassed by the term "therapeutic agent.”
  • the effectuated change can be of any kind, observable or unobservable, and can include, for example, a change in the biological activity of a protein.
  • Representative therapeutic compounds include small molecules, proteins and peptides, oligonucleotides of any length, "xenobiotics”, such as drugs and other therapeutic agents, carcinogens and environmental pollutants, natural products and extracts, as well as “endobiotics”, such as steroids, fatty acids and prostaglandins.
  • xenobiotics such as drugs and other therapeutic agents, carcinogens and environmental pollutants, natural products and extracts, as well as “endobiotics”, such as steroids, fatty acids and prostaglandins.
  • therapeutic agents can include, but are not restricted to, agonists and antagonists of a PXR polypeptide, toxins and venoms, viral epitopes, hormones (e.g., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, co-factors, lectins, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins, small molecules and monoclonal antibodies.
  • hormones e.g., opioid peptides, steroids, etc.
  • Table 1 is a table summarizing the crystal and data statistics obtained from the crystallized ligand binding domain of human PXR. Data on the unit cell are presented, including data on the crystal space group, unit cell dimensions, molecules per asymmetric cell and crystal resolution.
  • Table 2 is a table comparing human PXR-LBD with nuclear receptor LBDs of known structure.
  • Table 3 is a table summarizing the calculated buried solvent-accessible surface area of amino acid residues that line the ligand binding cavity of human PXR.
  • Table 4 is a table of the atomic structure coordinate data obtained from X-ray diffraction from the ligand binding domain of human PXR in complex with a ligand.
  • Table 5 is a table of the atomic structure coordinate data obtained from X-ray diffraction from human vitamin D receptor that was used in the molecular replacement solution of the human PXR ligand binding domain.
  • CYP3A genes are induced at the level of transcription by a variety of xenobiotics, including many that are metabolized by CYP3A.
  • This transcriptional regulation of CYP3A expression provides a mechanism for amplifying the physiologic response during periods of prolonged xenobiotic challenge.
  • the induction of CYP3A expression also represents the basis for an important class of drug-drug interactions.
  • PXR pregnane X receptor
  • PXR The human ortholog of PXR is alternately referred to as the pregnane-activated receptor (PAR) or the steroid and xenobiotic receptor (SXR). Like other nuclear receptors PXR contains a DNA binding domain (DBD) and a ligand binding domain (LBD). PXR binds to xenobiotic response elements in the regulatory regions of CYP3A genes as a heterodimer with the 9-cis retinoic receptor (RXR). Notably, PXR is activated by most of the xenobiotics that are known to induce CYP3A gene expression, including commonly used drugs such as the antibiotic rifampicin and the glucocorticoid dexamethasone.
  • DBD DNA binding domain
  • LBD ligand binding domain
  • RXR 9-cis retinoic receptor
  • PXR Xenoic acid
  • C21 steroids C21 steroids
  • PXR has evolved in order to detect structurally diverse substrates.
  • human PXR is activated efficiently by the rifampicin and the hypocholesterolemic drug SR12813, whereas mouse PXR is not.
  • mouse PXR is activated by the synthetic steroid pregnenolone 16a- carbonitrile (PCN), whereas the human receptor is not.
  • PCN synthetic steroid pregnenolone 16a- carbonitrile
  • the present invention will usually be applicable mutatis mutandis to all PXR polypeptides, as discussed herein based, in part, on the patterns of PXR structure and modulation that have emerged as a consequence of determining the three dimensional structure of human PXR in complex with a ligand.
  • PXR homologs and orthologs display substantial regions of amino acid homology.
  • the PXRs display an overall structural motif comprising three modular domains: 1 ) a variable amino-terminal domain;
  • DBD DNA-binding domain
  • each domain can usually be separately expressed with its original function intact or, as discussed herein below, chimeric proteins comprising two different proteins can be constructed, wherein the chimeric proteins retain the properties of the individual functional domains of the respective polypeptides from which the chimeric proteins were generated.
  • the amino terminal domain of PXR is the least conserved of the three domains. This domain is involved in transcriptional activation and, in some cases, its uniqueness can dictate selective receptor-DNA binding and activation of target genes by PXR.
  • the DBD is the most conserved structure in PXR. It typically contains about 70 amino acids that fold into two zinc finger motifs, wherein a zinc ion coordinates four cysteines.
  • the DBD generally contains two perpendicularly oriented ⁇ -helices that extend from the base of the first and second zinc fingers. The two zinc fingers function in concert along with non-zinc finger residues to direct the PXRs to specific target sites on DNA.
  • Various amino acids in the DBD influence spacing between two half-sites (which usually comprises six nucleotides) for receptor homodimerization. The optimal spacings facilitate cooperative interactions between DBDs, and D box residues are part of the dimerization interface. Other regions of the DBD facilitate DNA-protein and protein-protein interactions required for PXR-RXR heterodimerization.
  • the LBD is the second most highly conserved domain in these receptors. Whereas the integrity of several different LBD sub-domains is important for ligand binding, truncated molecules containing only the LBD can retain normal ligand binding activity. This domain also participates in other functions, including dimerization, nuclear translocation and transcriptional regulation activities. Importantly, this domain can bind a ligand and can undergo ligand-induced conformational changes. Ligand binding allows the activation domain to serve as an interaction site for essential co-activator proteins that function to stimulate or inhibit transcription.
  • the carboxy-terminal activation subdomain is in close three- dimensional proximity in the LBD to the ligand, so as to allow for ligands bound to the LBD to coordinate (or interact) with amino acid(s) in the activation subdomain.
  • the LBD of PXR is expressed, crystallized and its three dimensional structure determined. Computational and other methods for the design of ligands to the LBD are also disclosed.
  • the native and mutated PXR polypeptides, and fragments thereof, of the present invention can be chemically synthesized in whole or part using techniques that are well-known in the art (See, e.g., Creighton, (1983) Proteins: Structures and Molecular Principles, W.H. Freeman & Co., New York, incorporated herein in its entirety).
  • methods that are well known to those skilled in the art can be used to construct expression vectors containing a partial or the entire native or mutated PXR polypeptide coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination.
  • a variety of host-expression vector systems can be utilized to express a PXR coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a PXR coding sequence; yeast transformed with recombinant yeast expression vectors containing a PXR coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a PXR coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a PXR coding sequence; or animal cell systems.
  • the expression elements of these systems vary in their strength and specificities.
  • any of a number of suitable transcription and translation elements can be used in the expression vector.
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.
  • promoters such as the baculovirus polyhedrin promoter can be used.
  • promoters derived from the genome of plant cells such as heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) can be used.
  • promoters derived from the genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • SV40-, BPV- and EBV-based vectors can be used with an appropriate selectable marker.
  • the present invention provides crystals of PXR.
  • the crystals were obtained using the methodology disclosed in the Laboratory Examples.
  • the structures of the apo and SR12813-bound forms of PXR were solved by molecular replacement using the structure of the human vitamin D receptor as a search model (Rochel et al., (2000) Mol. Cell 5: 173-79, PDB ID: 1DB1 ; GenBank Accession No. XM007046; available online at http://www.rcsb.org/pdb/).
  • the apo form was refined to a resolution of about 2.5 A.
  • the ligand-bound form was refined to a resolution of about 2.75 A.
  • the heavy atom derivatized form of the PXR LBD-ligand structure can be solved using single isomorphous replacement anomalous scattering (SIRAS) techniques and/or multiwavelength anomalous diffraction (MAD) techniques.
  • SIRAS single isomorphous replacement anomalous scattering
  • MAD multiwavelength anomalous diffraction
  • a derivative crystal is prepared that contains an atom that is heavier than the other atoms of the sample.
  • Heavy atom derivative crystals are commonly prepared by soaking a crystal in a solution containing a selected heavy atom salt.
  • some heavy atom derivative crystals have been prepared by soaking a crystalline form of the protein of interest in a solution of methyl mercury chloride (MeHgCI).
  • MeHgCI methyl mercury chloride
  • Another representative heavy atom that can be incorporated into a derivative crystal is iodine.
  • Heavy atoms can associate with the protein of interest, or can be localized in a ligand that associates with a protein of interest. In the present invention, the latter approach was taken. Specifically, an iodine-containing form of the ligand SR12813 was co-crystallized with the PXR LBD and was used to assist in the positioning of the ligand relative to the PXR LBD.
  • Analysis of derivative crystals takes advantage of differences in the reflections from the derivative crystal as compared to the underivatized crystal. Symmetry-related reflections in the X-ray diffraction pattern, which are usually identical, are altered by the anomalous scattering contribution of the heavy atoms. The measured differences in symmetry-related reflections are used to determine the position of the heavy atoms, leading to an initial estimation of the diffraction phases, and subsequently, an electron density map is prepared. The prepared electron density map is then used to identify the position of the other atoms in the sample.
  • VA Preparation of PXR Crystals
  • the native and derivative co-crystals, and fragments thereof, disclosed in the present invention can be obtained by a variety of techniques, including batch, liquid bridge, dialysis, vapor diffusion and hanging drop methods (See, e.g., McPherson, (1982) Preparation and Analysis of Protein Crystals, John Wiley, New York.; McPherson, (1990) Eur. J. Biochem. 189:1-23.; Weber, (1991 ) Adv. Protein Chem. 41 :1-36).
  • the vapor diffusion and hanging drop methods are used for the crystallization of PXR polypeptides and fragments thereof.
  • native crystals of the present invention are grown by dissolving substantially pure PXR LBD polypeptide or a fragment thereof in an aqueous buffer containing a precipitant at a concentration just below that necessary to precipitate the protein. Water is removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases.
  • native crystals are grown by vapor diffusion (See, e.g., McPherson, (1982) Preparation and Analysis of Protein Crystals, John Wiley, New York; McPherson, (1990) Eur. J. Biochem. 189:1-23).
  • the polypeptide/precipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir having a precipitant concentration optimal for producing crystals.
  • a precipitant concentration optimal for producing crystals.
  • less than about 25 ⁇ L of PXR LBD polypeptide solution is mixed with an equal volume of reservoir solution, giving a precipitant concentration about half that required for crystallization.
  • This solution is suspended as a droplet underneath a coverslip, which is sealed onto the top of the reservoir. The sealed container is allowed to stand, until crystals grow. Crystals generally form within two to six weeks, and are suitable for data collection within approximately seven to ten weeks.
  • those of skill in the art will recognize that the above- described crystallization procedures and conditions can be varied.
  • Derivative crystals of the present invention can be obtained by soaking native crystals in mother liquor containing salts of heavy metal atoms.
  • a ligand comprising a heavy atom can be associated with a protein, and subsequently co- crystallized.
  • Such derivative crystals are useful for phase analysis in the solution of crystals of the present invention.
  • This mechanism provides derivative crystals suitable for use as isomorphous replacements in determining the X-ray crystal structure of a PXR polypeptide. Additional reagents useful for the preparation of the derivative crystals of the present invention will be apparent to those of skill in the art after review of the disclosure of the present invention presented herein.
  • Co-crystals of the present invention can be obtained by soaking a native crystal in mother liquor containing compounds known or predicted to bind the LBD of a PXR, or a fragment thereof.
  • co-crystals can be obtained by co-crystallizing a PXR LBD polypeptide or a fragment thereof in the presence of one or more compounds known or predicted to bind the polypeptide.
  • the ligand SR12831 or an iodinated form of the ligand SR12831 , is co- crystallized with a PXR-SRC-1 complex.
  • Crystal structures of the present invention can be solved using a variety of techniques including, but not limited to, isomorphous replacement anomalous scattering or molecular replacement methods.
  • Computer software packages will also be helpful in solving a crystal structure of the present invention.
  • Applicable software packages include but are not limited to X- PLORTM program (Br ⁇ nger, (1992) X-PLOR, Version 3.1. A System for X-ray Crystallography and NMR, Yale University Press, New Haven, Connecticut; X-PLOR is available from Molecular Simulations, Inc., San Diego, California), Xtal View (McRee, (1992) J. Mol.
  • Two structures of the LBD of human PXR are disclosed in the present invention.
  • a 2.5 A apo structure and second, a 2.75 A structure in complex with the high affinity ligand SR12813.
  • a 1 :1 complex comprising residues 130-434 of human PXR and residues 623-710 of the human transcriptional co-activator protein SRC-1 , was incubated in the crystallization mixture. The mixture was crystallized. The 88-amino acid co- activator peptide SRC-1 was not observed in the crystal structure, and the examination of extensively washed and dissolved crystals by SDS-PAGE revealed that this co-activator fragment was not present in the crystals of the PXR LBD.
  • Residues 142-177 and 198-431 of human PXR LBD were traced in the structures reported in an aspect of the present invention, while ordered electron density of the remaining amino acids, including the 20-residue stretch from 178-197, was not observed at any time during refinement.
  • the hydrophobic ligand binding cavity of human PXR contains a small number of polar residues, permitting the ligand SR12813 to bind in three distinct orientations.
  • human and mouse PXR are activated by different collections of xenobiotics.
  • Critical residues responsible for this directed promiscuity are disclosed in the present invention.
  • the mutation of only four residues "humanizes" murine PXR. That is, the mutation of four residues is observed to change the binding profile of murine PXR from a murinic profile to that of the human PXR.
  • the crystal structures of the present invention disclose important insights into how human PXR detects xenobiotics and thus, can be useful in predicting and avoiding drug-drug interactions.
  • the apo structure was refined to final R and Rf ree values of 0.209 and 0.284 respectively.
  • the ligand-bound structure (PXR- SR12813) was refined to R and R fre e values of 0.213 and 0.274, respectively.
  • Table 1 Residues 142-177 and 198-431 of the human PXR LBD were traced in the structures reported in the present invention.
  • the human PXR LBD is an " ⁇ -helical sandwich" comprising three layers: ⁇ 1/ ⁇ 3, ⁇ 7/ ⁇ 10, and a middle layer comprising ⁇ -helices 4, 5 and 8. See Figures 1 and 13.
  • This region of the molecule is similar to nuclear receptor ligand binding domains having known structure (Moras & Gronenmeyer, (1998) Curr. Opin. Cell Biol. 10: 384-91 ; Weatherman et al friction (1999) Annu. Rev. Biochem. 68: 559-81 ).
  • the standard three-stranded ⁇ - sheet is expanded to a five-stranded antiparallel ⁇ -sheet in the human PXR LBD, comprising ⁇ -strands 1 , 1', 2, 3 and 4, and the ligand binding cavity is localized at the bottom of the molecule. This structural feature is shown in Figure 1.
  • the structures of the apo and ligand-bound forms of the hPXR LBD are essentially identical, exhibiting a root-mean-square deviation (RMSD) of 0.68 A over the C ⁇ positions, and 0.89 A over all atoms.
  • RMSD root-mean-square deviation
  • the activation function 2 helix ( ⁇ -AF2) which plays a critical role in transcriptional activation by nuclear receptors, is packed against the body of the receptor in a position that appears permissive for coactivator interactions.
  • the human PXR (SEQ ID NOs: 1 and 2) LBD (SEQ ID NOs: 3 and 4) is most closely related in structure to the vitamin D receptor (VDR) (Rochel et aL, (2000) Mol. Cell 5: 173-79), with which it shares 45% sequence identity (SEQ ID NOs: 8 and 9) and exhibits a 1.8 A RMSD over 225 equivalent C ⁇ positions. Table 2 illustrates this similarity.
  • helix ⁇ 6 which is common to many nuclear receptors, is replaced in hPXR by a conserved, flexible loop composed of residues 309- 321 (See Figures 1 and 13). This region lies adjacent in space to the ligand binding cavity of hPXR. This feature might be related to the ability of the hPXR receptor to accommodate both small and large ligands in the binding cavity.
  • hPXR has two additional ⁇ -strands that have not previously been observed in a nuclear receptor ligand binding domain. This feature is depicted in Figure 1. Weatherman et aL, (1999) Annu. Rev. Biochem. 68: 559-81. These additional strands form the fourth ( ⁇ 1 , residues 210 to 217) and fifth ( ⁇ 1' f residues 221 to 226) strands of a five-stranded anti-parallel ⁇ - sheet. An "insertion domain" containing roughly the same number of residues was engineered out of the VDR prior to crystallization and structure determination (Rochel et aL, (2000) Mol. Cell 5:173-79).
  • the ligand binding cavity of the hPXR-LBD is largely hydrophobic and might be flexible in nature, in order to accommodate both small and large ligands.
  • the 28 amino acid residues desolvated by the binding of SR12813, which are presented in Table 3, are considered to be lining the cavity.
  • the structures of the apo and the ligand bound cavities are similar, exhibiting a 1.12 A RMSD over all atoms in these 28 residues.
  • Table 2 shows that the binding cavity volume of 1 ,150 A 3 , making it larger than most known nuclear receptor ligand binding cavities.
  • GIu-321 , His-327, His-407 and Arg- 410 Twenty of the cavity-lining residues are hydrophobic, four are polar (Ser-208, Ser-247, Cys-284 and Gln-285), and four are charged or potentially charged (GIu-321 , His-327, His-407 and Arg- 410). This observation is depicted via graphics in Figures 2 and 3 and is additionally presented in Table 3. As described further herein below, GIu-321 and Arg-410 are involved in a salt bridge, effectively neutralizing their charged character adjacent to the ligand binding cavity. Thus, an electrostatic view of the inner surface of this ligand binding cavity reveals a relatively uncharged and hydrophobic environment, as seen in Figures 2 and 3.
  • This highly mobile region spans the space between the C-terminus of ⁇ 4 to the N-terminus of ⁇ 7, and exhibits a mean thermal displacement parameter of 82.3 A 2 over main-chain atoms despite persistent electron density.
  • Nine of the thirteen residues in this loop are completely conserved in the mammalian PXR molecules of known sequence (highlighted in bold in SEQ ID NO: 5 above, and depicted in Figures 2 and 3), including the solvent-exposed hydrophobic residues of SEQ ID NO: 5, Phe- 315, Leu-318, Leu-319 and Leu-320.
  • the 309-321 loop is linked to the ligand binding cavity of hPXR by a non-solvent accessible pore. This observation is depicted in Figures 2 and 3.
  • binding of a particularly large and hydrophobic ligand in the binding cavity might force the pore to this loop region to open, enlarging the ligand binding region of hPXR and lining it with additional hydrophobic amino acid side chains.
  • positions 1 , 2 and 3 were observed in the ligand binding cavity of hPXR, shown in Figures 4-9. These orientations were identified during structural refinement and were rigorously confirmed using difference maps involving data obtained from crystals containing an iodinated form of SR12813 (I- SR12813 in Figures 4-9). As shown in Figures 4-9, each orientation forms distinct interactions with residues that line the ligand binding cavity of PXR. While ligand position 3 forms the most hydrophilic interactions with the protein, positions 1 and 2 were clearly indicated in the detailed examination of difference maps and in refinement. Indeed, when any one of the orientations is not considered in an unbiased refinement, clear positive electron density appears in difference maps to indicate its presence in the binding cavity of the protein.
  • the nuclear receptor PXR serves as a key component of the body's defense mechanism against xenobiotics by detecting these compounds and regulating the transcription of genes involved in their metabolism (Waxman,
  • PXR Unlike other nuclear receptors, which evolved to interact selectively with their cognate hormone, PXR evolved the ability to interact promiscuously with a structurally diverse collection of hydrophobic compounds.
  • the volume of the PXR ligand binding cavity is >1 ,100 A 3 , which is substantially larger than that of many other nuclear receptors including the progesterone, estrogen, retinoid, and thyroid hormone receptors, an observation represented in Table 2.
  • the PXR structure disclosed in the present invention also suggests that the ligand binding cavity may be capable of expanding to an even larger size.
  • a stretch of conserved residues 309-321 (identified in Figures 1-3) loop out and away from the ligand binding cavity of PXR in the structures reported here, exposing conserved hydrophobic residues to solvent. This loop is connected to the existing ligand binding cavity by a solvent-inaccessible pore.
  • PXR Proliferative X receptor
  • the entry site appears to be located on the back of the molecule between ⁇ 7 and ⁇ 10.
  • the putative entrance-exit path to the ligand binding cavity in hPXR might be gated by two salt bridges (i.e. between Asp-205 and Arg-413, and GIu-321 and Arg-410), as shown in Figure 10.
  • Mutagenesis studies disclosed in the present invention show that Arg-410 and Asp-205, which are in van der Waals contact with each other, are critical to defining the appropriate basal activity level for PXR.
  • the drop in basal activity level in the Asp-205-Ala mutant might also indicate that this form of the protein is less stable than the wildtype. Asp-205 could be critical for ordering the 198-210 region of the molecule; the loss of this residue could introduce flexibility to PXR that affects its stability and basal activation level.
  • the ligand binding cavity of PXR is relatively smooth and hydrophobic, but contains a small number of key polar residues. Twenty of the 28 residues lining the binding cavity are hydrophobic; the remaining polar residues are spaced roughly evenly throughout the cavity, offering the potential of a small number of hydrogen bonds.
  • the character of the PXR ligand cavity mirrors the character of the majority of the ligands known to activate PXR, such as phenobarbital, clotrimazole, RU486, hyperforin, PCN and lovastatin, which are largely hydrophobic and uncharged, but which contain oneto four functional groups capable of forming hydrogen bonds.
  • a combination of hydrophobic and shuffled polar interactions allows the potent PXR agonist SR12813 to bind to the hPXR ligand binding cavity in three distinct orientations.
  • SR12813 occupies essentially the same portion of the cavity in each of the three binding modes, each mode is facilitated by a different set of hydrogen bonding and hydrophobic interactions ( Figures 4-6).
  • Ser-247 forms a hydrogen bond with the phenolic hydroxyl group on SR12813 in one binding mode, and a hydrogen bond with a phosphate group in another.
  • PXR does not define a specific ligand binding surface that will drive the binding of ligands in one mode.
  • PXR offers an essentially smooth and uncharged ligand binding surface, with evenly spaced hydrogen bond donors and acceptors ( Figures 2 and 3). Such an arrangement not only permits PXR to bind to structurally distinct ligands, but also allows single ligands to bind in multiple modes. This binding mode stands in sharp contrast to other nuclear receptor-ligand interactions, which have evolved to be highly specific.
  • Transfected cells were treated with increasing concentrations of either SR12813 or rifampicin, as illustrated in the plots of Figure 11.
  • Mutation of Asp-205 resulted in a marked decrease in the basal (ligand-independent) transcriptional activity of PXR, while mutation of Arg-413 resulted in a more modest reduction in PXR basal activity.
  • mutation of GIu-321 resulted in a marked increase in the basal activity of PXR; conversely, the Arg-410-Ala mutant had reduced basal activity.
  • Trp-223 side chains from each monomer are locked across the dimer interface, and thus are completely protected from solvent, permitting them to form an "offset-edge" stacking interaction frequently observed between tryptophans (Samanta et al., (1999) Acta Crystalog. D 55: 1421-27).
  • Tyr-225 is also involved in this interface and becomes buried in the dimer, packing against Pro-175 from the region leading up to residue 177.
  • the position of Trp-223 is further stabilized by a 3.0 A hydrogen bond between the main-chain carbonyl oxygen of Pro-175 and the indol ring nitrogen of the tryptophan side chain.
  • a model for a putative heterotetrameric complex formed by the ligand binding domains of hPXR and RXR ⁇ was generated by superimposing PXR on PPAR ⁇ in the heterodimeric structure of PPAR ⁇ and RXR ⁇ (Gampe et al., (2000) Mol Cell 5: 545-55).
  • the interface between PXR and RXR ⁇ generated in this fashion is nearly ideal, requiring no manual optimization and producing no van der Waals overlaps between atoms.
  • the homodimerization of PXR does not interfere with the PXR/RXR ⁇ interface in the heterotetramer, suggesting that a heterotetramer of PXR and RXR ⁇ can or could occur in vivo.
  • mouse PXR is activated more efficiently by the synthetic steroid PCN than either the human or rabbit orthologs (Jones et al., (2000) Mol. Endocrinol. 14: 27-39; Savas et aL, (2000) Drug Metab Dispos. 28: 529-37).
  • mouse PXR was "humanized" - that is, its selectivity was altered such that it would respond to SR12813 but not to PCN.
  • PXR is promiscuous in that it binds a wide variety of endogenous compounds and xenobiotics. However, this receptor also exhibits specificity as demonstrated by the fact that PXRs from different species show distinct activation profiles. To test the hypothesis that key polar residues determine specificity, the crystal structures of hPXR disclosed in the present invention were employed to design an altered form of PXR.
  • the present invention discloses a substantially pure PXR LBD polypeptide in crystalline form.
  • PXR is crystallized with bound ligand.
  • Crystals are formed from PXR LBD polypeptides that are usually expressed by a cell culture, such as E. coli.
  • BYomo-, iodo- and substitutions can be included during the preparation of crystal forms and can act as heavy atom substitutions in PXR ligands and in crystals of PXR and the PXR LBD. This method can be advantageous for the phasing of the crystal, which is a crucial, and sometimes limiting, step in solving the three-dimensional structure of a crystallized entity.
  • the need for generating the heavy metal derivatives traditionally employed in crystallography might be eliminated.
  • the resultant three-dimensional structure can be used in computational methods to design synthetic ligands for PXR and other PXR polypeptide fragments. Further activity structure relationships can be determined through routine testing, using assays disclosed herein and known in the art.
  • the three-dimensional structure of ligand binding hPXR is unprecedented and will greatly aid in the development of new synthetic ligands for a PXR polypeptide, such as PXR agonists and antagonists, including those that bind exclusively to any one of the PXR orthologs.
  • PXR is well suited to modern methods, including three-dimensional structure elucidation and combinatorial chemistry, such as those disclosed in U.S. Patent No. 5,463,564, incorporated herein by reference. Structure determination using X-ray crystallography is possible because of the solubility properties of the PXR orthologs. Computer programs that use crystallography data when practicing the present invention will enable the rational design of ligands to these receptors.
  • RASMOL Biomolecular Structures Group, Glaxo Wellcome Research & Development Stevenage, Hertfordshire, UK Version 2.6, August 1995, Version 2.6.4, December 1998, Copyright ⁇ Roger Sayle 1992-1999
  • Computer programs such as those sold under the registered trademark INSIGHT II ® and such as GRASP (Nicholls et aL, (1991) Proteins 11 : 281-96) allow for further manipulations and the ability to introduce new structures.
  • high throughput binding and bioactivity assays can be devised using purified recombinant protein and modern reporter gene transcription assays known to those of skill in the art in order to refine the activity of a designed ligand.
  • a method of identifying modulators of the activity of a PXR polypeptide using rational drug design comprises designing a potential modulator for a PXR polypeptide of the present invention that will form non-covalent bonds with amino acids in the ligand binding cavity based upon the crystalline structure of the hPXR LBD polypeptide; synthesizing the modulator; and determining whether the potential modulator modulates the activity of the PXR polypeptide.
  • the modulator is designed for a hPXR polypeptide.
  • the hPXR polypeptide comprises the amino acid sequence of SEQ ID NO: 2
  • the hPXR LBD comprises the amino acid sequence SEQ ID NO: 4.
  • the determination of whether the modulator modulates the biological activity of a PXR polypeptide is made in accordance with the screening methods disclosed herein, or by other screening methods known to those of skill in the art. Modulators can be synthesized using techniques known to those of ordinary skill in the art.
  • a method of designing a modulator of a PXR polypeptide in accordance with the present invention comprising: (a) selecting a candidate PXR ligand; (b) determining which amino acid or amino acids of a PXR polypeptide interact with the ligand using a three-dimensional model of a crystallized hPXR LBD; (c) identifying in a biological assay for PXR activity a degree to which the ligand modulates the activity of the PXR polypeptide,; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the PXR polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) performing the chemical modification on the ligand to form a modified ligand; (f) contacting the modified ligand with the PXR polypeptide; (g) identifying in a biological assay for PXR activity a degree to which the modified ligand modulates the biological activity of the PX
  • the present invention permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including modulatory compounds, capable of binding to the ligand binding cavity or an accessory binding site of hPXR and the hPXR LBD, in whole or in part.
  • the present invention also provides for the application of similar techniques in the design of modulators of any PXR polypeptide.
  • the structure coordinates of a crystalline hPXR LBD can be used to design compounds that bind to a PXR LBD (more preferably a hPXR LBD) and alter the properties of a PXR LBD (for example, the dimerization or ligand binding ability) in different ways.
  • a PXR LBD more preferably a hPXR LBD
  • alter the properties of a PXR LBD for example, the dimerization or ligand binding ability
  • One aspect of the present invention provides for the design of compounds that act as competitive inhibitors of a PXR polypeptide by binding to all, or a portion of, the binding sites on a PXR LBD.
  • the present invention also provides for the design of compounds that can act as uncompetitive inhibitors of a PXR LBD.
  • These compounds can bind to all, or a portion of, an accessory binding site of a PXR that is already binding its ligand and can, therefore, be more potent and less non-specific than known competitive inhibitors that compete only for the PXR ligand binding cavity.
  • non-competitive inhibitors that bind to and inhibit PXR LBD activity, whether or not it is bound to another chemical entity can be designed using the PXR LBD structure coordinates of this invention.
  • a second design approach is to probe a PXR or PXR LBD (preferably a hPXR or hPXR LBD) crystal with molecules comprising a variety of different chemical entities to determine optimal sites for interaction between candidate PXR or PXR LBD modulators and the polypeptide.
  • a PXR or PXR LBD preferably a hPXR or hPXR LBD
  • molecules comprising a variety of different chemical entities to determine optimal sites for interaction between candidate PXR or PXR LBD modulators and the polypeptide.
  • high resolution X-ray diffraction data collected from crystals saturated with solvent allows the determination of the site where each type of solvent molecule adheres. Small molecules that bind tightly to those sites can then be designed, synthesized and tested for their hPXR modulator activity.
  • assays can be used to establish its efficacy of the ligand as a modulator of PXR (preferably hPXR) activity.
  • the ligands can be further refined by generating intact PXR, or PXR LBD, crystals with a ligand bound to the PXR.
  • the structure of the ligand can then be further refined using the chemical modification methods described herein and known to those of skill in the art, in order to improve the modulation activity or the binding affinity of the ligand. This process can lead to second generation ligands with improved properties.
  • Ligands also can be selected that modulate PXR responsive gene transcription by the method of altering the interaction of co-activators and co- repressors with their cognate PXR.
  • agonistic ligands can be selected that block or dissociate a co-repressor from interacting with the PXR, and/or that promote binding or association of a co-activator.
  • Antagonistic ligands can be selected that block co-activator interaction and/or promote co- repressor interaction with a target receptor. Selection can be done via binding assays that screen for designed ligands having the desired modulatory properties. Preferably, interactions of a hPXR polypeptide are targeted. Suitable assays for screening that can be employed, mutatis mutandis in the present invention, are described in published PCT international applications WO 00/037077 and WO 00/025134, incorporated herein by reference in their entirety.
  • the compound must be able to assume a conformation that allows it to associate with a PXR LBD. Although certain portions of the compound will not directly participate in this association with a PXR LBD, those portions can still influence the overall conformation of the molecule. This, in turn, can have a significant impact on potency.
  • Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., the ligand binding cavity or an accessory binding site of a PXR LBD, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with a PXR LBD.
  • the potential modulatory or binding effect of a chemical compound on a PXR LBD can be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques that employ the coordinates of a crystalline hPXR LBD polypeptide of the present invention. If the theoretical structure of the given compound suggests insufficient interaction and association between it and a PXR LBD, synthesis and testing of the compound is obviated. However, if computer modeling indicates a strong interaction, the molecule can then be synthesized and tested for its ability to bind and modulate the activity of a PXR LBD. In this manner, synthesis of unproductive or inoperative compounds can be avoided.
  • hPXR LBD hPXR LBD
  • One of several methods can be used to screen chemical entities or fragments for their ability to associate with a PXR LBD and, more particularly, with the individual binding sites of a PXR LBD, such as ligand binding cavity or an accessory binding site.
  • This process can begin by visual inspection of, for example, the ligand binding cavity on a computer screen based on the hPXR LBD atomic coordinates in Table 4. Selected fragments or chemical entities can then be positioned in a variety of orientations, or docked, within an individual binding site of a hPXR LBD as defined herein above.
  • Docking can be accomplished using software programs such as those available under the tradenames QUANTATM (Molecular Simulations Inc., San Diego, California) and SYBYLTM (Tripos, Inc., St. Louis, Missouri), followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARM (Brooks et al friction (1983) J. Comp. Chem., 8: 132) and AMBER 5 (Case et aL, (1997), AMBER 5, University of California, San Francisco; Pearlman et aL, (1995) Comput. Phys. Commun. 91 : 1-41).
  • QUANTATM Molecular Simulations Inc., San Diego, California
  • SYBYLTM Tripos, Inc., St. Louis, Missouri
  • Specialized computer programs can also assist in the process of selecting fragments or chemical entities. These include:
  • GRIDTM program version 17 (Goodford, (1985) J. Med. Chem. 28: 849-57), which is available from Molecular Discovery Ltd., Oxford, UK; 2. MCSSTM program (Miranker & Karplus, (1991) Proteins 11 : 29-34), which is available from Molecular Simulations, Inc., San Diego, California;
  • LUDITM program (Bohm, (1992) J. Comput. Aid. Mol. Des., 6: 61- 78), which is available from Molecular Simulations, Inc., San Diego, California.
  • 3D Database systems such as MACCS-3DTM system program, which is available from MDL Information Systems, San Leandro, California.
  • modulatory or other binding compounds can be designed as a whole or de novo using the structural coordinates of a crystalline hPXR LBD polypeptide of the present invention and either an empty binding site or optionally including some portion(s) of a known modulator(s).
  • Applicable methods can employ the following software programs: 1. LUDiTM program (Bohm, (1992) J. Comput. Aid. Mol. Des., 6: 61- 78), which is available from Molecular Simulations, Inc., San Diego, California;
  • LEGENDTM program (Nishibata & Itai, (1991 ) Tetrahedron 47: 8985); and 3. LEAPFROGTM, which is available from Tripos Associates, St. Louis,
  • a compound that has been designed or selected to function as a hPXR LBD modulator should also preferably traverse a volume not overlapping that occupied by the binding site when it is bound to its native ligand.
  • an effective PXR LBD modulator should preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding).
  • the most efficient PXR LBD modulators should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, and preferably, not greater than 7 kcal/mole. It is possible for PXR LBD modulators to interact with the polypeptide in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free compound and the average energy of the conformations observed when the modulator binds to the polypeptide.
  • a compound designed or selected as binding to a PXR polypeptide can be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target polypeptide.
  • Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.
  • the sum of all electrostatic interactions between the modulator and the polypeptide when the modulator is bound to a PXR LBD preferably make a neutral or favorable contribution to the enthalpy of binding.
  • Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include:
  • Gaussian 98TM which is available from Gaussian, Inc., Pittsburgh, Pennsylvania
  • AMBERTM program version 6.0, which is available from the
  • substitutions can then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided.
  • substituted chemical compounds can then be analyzed for efficiency of fit to a PXR LBD binding site using the same computer-based approaches described in detail above. VII.B.
  • the present invention discloses the ability to generate new synthetic ligands to distinguish between PXR isoforms and orthologs.
  • computer-designed ligands can be generated that distinguish between binding isoforms and orthologs, thereby allowing the generation of species specific, tissue specific or function specific ligands.
  • the atomic structural coordinates disclosed in the present invention reveal structural details unique to hPXR. These structural details can be exploited when a novel ligand is designed using the methods of the present invention or other ligand design methods known in the art.
  • the structural features that differentiate a hPXR from a mouse PXR and one isoform from another can be targeted in ligand design.
  • a ligand can be designed that will recognize a particular PXR isoform or ortholog, while not interacting with other PXR isoforms or orthologs, or even with moieties having similar structural features.
  • a detailed understanding of the differences between PXR orthoforms and the ability to target a particular PXR isoform or ortholog was unattainable.
  • a candidate substance identified according to a screening assay of the present invention has an ability to modulate the biological activity of a PXR polypeptide or a PXR LBD polypeptide.
  • a candidate compound can have utility in the treatment of disorders and conditions associated with the biological activity of a hPXR or a hPXR LBD polypeptide, including, but not limited to, hPXR and hPXR LBD-based drug- drug interactions, hPXR and hPXR LBD-based drug resistance, individualized treatment of disease due to polymorphisms, liver cholestasis and other degenerative liver disorders.
  • the method comprises the steps of establishing a control system comprising a hPXR polypeptide and a ligand which is capable of binding to the polypeptide; establishing a test system comprising a hPXR polypeptide, the ligand, and a candidate compound; and determining whether the candidate compound modulates the activity of the polypeptide by comparison of the test and control systems.
  • a representative ligand comprises a fatty acid or other small molecule, and in this embodiment, the biological activity or property screened includes binding affinity.
  • a form of a hPXR polypeptide or a catalytic or immunogenic fragment or oligopeptide thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such a screening can be affixed to a solid support.
  • the formation of binding complexes, between a hPXR polypeptide and the agent being tested, will be detected.
  • the hPXR polypeptide has an amino acid sequence of SEQ ID NO: 2.
  • a preferred embodiment will include a hPXR polypeptide having the amino acid sequence of SEQ ID NO: 4.
  • Another technique for drug screening which can be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO 84/03564, herein incorporated by reference.
  • this method as applied to a polypeptide of the present invention, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with the polypeptide, or fragments thereof. Bound polypeptide is then detected by methods well known to those of skill in the art. The polypeptide can also be placed directly onto plates for use in the aforementioned drug screening techniques.
  • a method of screening for a modulator of a hPXR polypeptide or a hPXR LBD polypeptide comprises: providing a library of test samples; contacting a hPXR polypeptide or a hPXR LBD polypeptide with each test sample; detecting an interaction between a test sample and a hPXR polypeptide or a hPXR LBD polypeptide; identifying a test sample that interacts with a hPXR polypeptide or a hPXR LBD polypeptide; and isolating a test sample that interacts with a hPXR polypeptide or a hPXR LBD polypeptide.
  • an interaction can be detected spectrophotometrically, radiologically or immunologically.
  • An interaction between a hPXR polypeptide or a hPXR LBD polypeptide and a test sample can also be quantified using methodology known to those of skill in the art.
  • the hPXR polypeptide and the hPXR LBD is in crystalline form.
  • This screening method comprises separately contacting each of a plurality of substantially identical samples with a hPXR polypeptide or a hPXR LBD and detecting a resulting binding complex.
  • the plurality of samples preferably comprises more than about 10 4 samples, or more preferably comprises more than about 5 x 10 4 samples.
  • an assay method for identifying a compound that inhibits binding of a ligand to a PXR polypeptide is disclosed.
  • a known ligand of hPXR can be used in the assay method as the ligand against which the inhibition by a test compound is gauged.
  • SR12813 is a preferred ligand in the assay method.
  • the method comprises (a) incubating a PXR polypeptide with a ligand in the presence of a test inhibitor compound; (b) determining an amount of ligand that is bound to the PXR polypeptide, wherein decreased binding of ligand to the PXR polypeptide in the presence of the test inhibitor compound relative to binding in the absence of the test inhibitor compound is indicative of inhibition; and (c) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed.
  • the ligand is SR12813.
  • the disclosed assay method can be used in the structural refinement of candidate PXR inhibitors. For example, multiple rounds of optimization can be followed by gradual structural changes in a strategy of inhibitor design. A strategy such as this is made possible by the disclosure of the coordinates of the hPXR LBD and the disclosure of the orientation of a ligand of PXR, SR12813.
  • the hPXR crystal structure of the present invention can be used to generate modulators of other PXR isoforms or orthologs, such as mouse PXR (Swiss-Prot Accession No. 054915). Analysis of the disclosed crystal structure can provide a guide for designing modulators of PXR isoforms or orthologs. Purely for purposes of explanation, the development of a mouse PXR modulator will be considered herein below. It will be apparent to those of skill in the art, and explicitly noted here, that the following discussion will be applicable mutatis mutandis to PXR isoforms and other PXR orthologs, including rat PXR (Swiss-Prot Accession No. Q9R1A7).
  • mice PXR modulators Absent the crystal structure of the present invention, researchers would be required to design mouse PXR modulators de novo.
  • the present invention addresses this problem by providing insights into the binding cavity of hPXR, which can be extended, due to significant structural similarity with other PXR isoforms and orthologs, to the binding cavity of, for example, mouse PXR.
  • An evaluation of the binding cavity of hPXR indicates that a potential mouse PXR modulator would meet a broad set of general criteria.
  • a potent mouse PXR ligand would require several general features including: (a) a hydrophobic binding cavity; and (b) the ability to adopt a conformation that is complementary to the shape of the binding cavity.
  • a mouse PXR modulator can be designed. For example, based on an evaluation of a homology model of mouse PXR, which is derived from the hPXR crystal structure, it is expected that a potent ligand would need similar characteristics as listed above for a compound recognized by hPXR. Additional modifications can be included, based on the disclosed structure, which are predicted to further define a modulator specific for mouse PXR over other orthologs. Thus, the disclosed crystal structure of hPXR can be useful when designing modulators of mouse PXR and other orthologs and isoforms.
  • An additional aspect of the present invention is to provide a technique for predicting the differences in the metabolism of a drug between mice (or other species of interest) and humans.
  • the "humanization" of the mouse PXR via four point mutations demonstrate that the structures disclosed herein can be employed as a predictor of mouse PXR function.
  • a mouse PXR model based on the structure of human PXR, as disclosed herein and co-crystallized and refined with known pharmacophores, can provide needed insight into PXR activity and mechanisms.
  • a "humanized" mouse PXR can provide valuable insight into the metabolism of drugs in humans.
  • the ability to generate such a system offers the potential to generate PXR drug metabolism data in a mouse system that is more predictive for human systems, than PXR metabolic data generated based only on a mouse model.
  • a "humanized" mouse PXR also facilitates insights into existent drug metabolism, and the development of human drugs and therapeutics with greater efficacies, using a convenient mouse model system.
  • the present invention provides for the generation of PXR and PXR mutants (preferably hPXR and hPXR LBD mutants), and the ability to solve the crystal structures of those that crystallize. More particularly, through the provision of the three-dimensional structure of a hPXR LBD, desirable sites for mutation can be identified, based on analysis of the three-dimensional hPXR LBD structure coordinates provided herein.
  • the structure coordinates of a hPXR LBD provided in accordance with the present invention also facilitate the identification of related proteins or enzymes analogous to hPXR in function, structure or both, (for example, a mouse PXR), which can lead to novel therapeutic modes for treating or preventing a range of disease states.
  • a further aspect of the present invention is that sterically similar compounds can be formulated to mimic the key portions of a PXR LBD structure. Such compounds are functional equivalents.
  • the generation of a structural functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art and described herein. Modeling and chemical design of PXR and PXR LBD structural equivalents can be based on the structure coordinates of a crystalline hPXR LBD polypeptide of the present invention. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • chimeric PXR polypeptides can comprise a PXR LBD polypeptide or a portion of a PXR LBD, (e.g. a hPXR LBD) which is fused to a candidate polypeptide or a suitable region of the candidate polypeptide, for example a PXR expressed in mouse or other species.
  • a chimeric polypeptide can comprise a PXR LBD polypeptide or a portion of a PXR LBD, (e.g. a hPXR LBD) which is fused to a candidate polypeptide or a suitable region of the candidate polypeptide, for example a PXR expressed in mouse or other species.
  • mutant encompass not only mutants of a PXR LBD polypeptide but chimeric proteins generated using a PXR LBD as well. It is thus intended that the following discussion of mutant PXR LBDs apply mutatis mutandis to chimeric PXR and PXR LBD polypeptides and to structural
  • a mutation can be directed to a particular site or combination of sites of a wild-type PXR LBD.
  • an accessory binding site or the binding cavity can be chosen for mutagenesis.
  • a residue having a location on, at or near the surface of the polypeptide can be replaced, resulting in an altered surface charge of one or more charge units, as compared to the wild-type PXR and PXR LBD.
  • an amino acid residue in a PXR or a PXR LBD can be chosen for replacement based on its hydrophilic or hydrophobic characteristics.
  • Such mutants can be characterized by any one of several different properties as compared with the wild-type PXR LBD.
  • mutants can have an altered surface charge of one or more charge units, or can have an increase in overall stability.
  • Other mutants can have altered substrate specificity in comparison with, or a higher specific activity than, a wild-type PXR or PXR LBD.
  • PXR and PXR LBD mutants of the present invention can be generated in a number of ways.
  • the wild-type sequence of a PXR or a PXR LBD can be mutated at those sites identified using this invention as desirable for mutation, by means of oligonucleotide-directed mutagenesis or other conventional methods, such as deletion.
  • mutants of a PXR or a PXR LBD can be generated by the site-specific replacement of a particular amino acid with an unnaturally occurring amino acid.
  • PXR or PXR LBD mutants can be generated through replacement of an amino acid residue, for example, a particular cysteine or methionine residue, with selenocysteine or selenomethionine.
  • Mutations can be introduced into a DNA sequence coding for a PXR or a PXR LBD using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites. Mutations can be generated in the full-length DNA sequence of a PXR or a PXR LBD or in any sequence coding for polypeptide fragments of a PXR or a PXR LBD.
  • a mutated PXR or PXR LBD DNA sequence produced by the methods described above, or any alternative methods known in the art can be expressed using an expression vector.
  • An expression vector typically includes elements that permit autonomous replication in a host cell independent of the host genome, and one or more phenotypic markers for selection purposes. Either prior to or after insertion of the DNA sequences surrounding the desired PXR or PXR LBD mutant coding sequence, an expression vector also will include control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes and a signal for termination.
  • nucleotides encoding a "signal sequence" can be inserted prior to a PXR or a PXR LBD mutant coding sequence.
  • a desired DNA sequence For expression under the direction of the control sequences, a desired DNA sequence must be operatively linked to the control sequences; that is, the sequence must have an appropriate start signal in front of the DNA sequence encoding the PXR or PXR LBD mutant, and the correct reading frame to permit expression of that sequence under the control of the control sequences and production of the desired product encoded by that PXR or PXR LBD sequence must be maintained.
  • any of a wide variety of well-known available expression vectors can be useful to express a mutated PXR or PXR LBD coding sequences of this invention and generated as described in Laboratory Example 3.
  • These expression vectors can be used in the techniques disclosed in Laboratory Examples 1 and 3 and can include, for example, vectors comprising segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40, known bacterial plasmids, e.g., plasmids from E.
  • coli including col E1 , pCR1 , pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs, e.g., the numerous derivatives of phage ⁇ , e.g., NM 989, and other DNA phages, e.g., M13 and filamentous single stranded DNA phages, yeast plasmids and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences.
  • the E phage DNAs
  • other DNA phages e.g., M13 and filamentous single stranded DNA phages
  • yeast plasmids and vectors derived from combinations of plasmids and phage DNAs such as plasmids
  • coli vector pRSETA including a T7-based expression system
  • any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence when operatively linked to it — can be used in these vectors to express the mutated DNA sequences according to this invention.
  • useful expression control sequences include, for example, the early and late promoters of SV40 for animal cells, the lac system, the trp system the TAC or TRC system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, all for E.
  • the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes the promoters of acid phosphatase, e.g., Pho5
  • the promoters of the yeast ⁇ -mating factors for yeast and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • hosts are also useful for producing mutated hPXR and hPXR LBD polypeptides according to this invention.
  • These hosts include, for example, bacteria, such as E. coli, Bacillus and Streptomyces, fungi, such as yeasts, and animal cells, such as CHO and COS-1 cells, plant cells, insect cells, such as Sf9 cells, and transgenic host cells.
  • an expression control sequence a variety of factors should also be considered. These include, for example, the relative strength of the system, its controllability and its compatibility with the DNA sequence encoding a modified PXR or PXR LBD polypeptide of this invention, with particular regard to the formation of potential secondary and tertiary structures.
  • Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of a modified PXR or PXR LBD to them, their ability to express mature products, their ability to fold proteins correctly, their fermentation requirements, the ease of purification of a modified PXR or PXR LBD and safety.
  • one of skill in the art can select various vector/expression control system/host combinations that will produce useful amounts of a mutant PXR or PXR LBD.
  • a mutant PXR or PXR LBD produced in these systems can be purified by a variety of conventional steps and strategies, including those used to purify the wild-type PXR or PXR LBD.
  • mutants can be tested for any one of several properties of interest. For example, mutants can be screened for an altered charge at physiological pH. This is determined by measuring the mutant PXR or PXR LBD isoelectric point (pi) and comparing the observed value with that of the wild-type parent. Isoelectric point can be measured by gel-electrophoresis according to the method of Wellner (Wellner, (1971) Anal. Chem. 43: 597).
  • a mutant PXR or PXR LBD polypeptide containing a replacement amino acid located at the surface of the enzyme, as provided by the structural information of this invention, can lead to an altered surface charge and an altered pi.
  • a unique PXR or PXR LBD polypeptide can be generated. Such a mutant can facilitate purification and can facilitate the study of the ligand binding abilities of a PXR polypeptide.
  • engineered PXR LDB refers to polypeptides having amino acid sequences which contain at least one mutation in the wild-type sequence. The terms also refer to PXR and PXR
  • LBD polypeptides which are capable of exerting a biological effect in that they comprise all or a part of the amino acid sequence of an engineered PXR or PXR LBD mutant polypeptide of the present invention, or cross-react with antibodies raised against an engineered PXR or PXR LBD mutant polypeptide, or retain all or some or an enhanced degree of the biological activity of the engineered PXR or PXR LBD mutant amino acid sequence or protein.
  • Such biological activity can include lipid binding in general, and fatty acid binding in particular.
  • engineered PXR LBD and "PXR LBD mutant” also includes analogs of an engineered PXR LBD or PXR LBD mutant polypeptide.
  • analog is intended that a DNA or polypeptide sequence can contain alterations relative to the sequences disclosed herein, yet retain all or some or an enhanced degree of the biological activity of those sequences. Analogs can be derived from genomic nucleotide sequences or from other organisms, or can be created synthetically. Those of skill in the art will appreciate that other analogs, as yet undisclosed or undiscovered, can be used to design and/or construct PXR LBD or PXR LBD mutant analogs.
  • engineered PXR LBD or PXR LBD mutant polypeptide there is no need for an engineered PXR LBD or PXR LBD mutant polypeptide to comprise all or substantially all of the amino acid sequence of SEQ ID NOs: 2 or 4. Shorter or longer sequences are anticipated to be of use in the invention; shorter sequences are herein referred to as “segments”.
  • engineered PXR LBD and PXR LBD mutant also includes fusion, chimeric or recombinant engineered PXR LBD or PXR LBD mutant polypeptides and proteins comprising sequences of the present invention. Methods of preparing such proteins are disclosed herein above and are known in the art.
  • the term "substantially similar” means that a particular sequence varies from nucleic acid sequence of SEQ ID NOs: 1 or 3, or the amino acid sequence of SEQ ID NOs: 2 or 4 by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the natural gene, gene product, or sequence.
  • sequences include "mutant” or “polymorphic” sequences, or sequences in which the biological activity and/or the physical properties are altered to some degree but retains at least some or an enhanced degree of the original biological activity and/or physical properties.
  • nucleic acid sequences In determining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequences or substitution of equivalent amino acids to create biologically functional equivalents.
  • Nucleic acids that are substantially identical to a nucleic acid sequence of an engineered PXR or PXR LBD mutant of the present invention e.g. allelic variants, genetically altered versions of the gene, etc., bind to an engineered PXR or PXR LBD mutant sequence under stringent hybridization conditions.
  • probes, particularly labeled probes of DNA sequences one can isolate homologous or related genes.
  • the source of homologous genes can be any species, e.g. primate species; rodents, such as rats and mice, canines, felines, bovines, equines, yeast, nematodes, etc.
  • homologs have substantial sequence similarity, i.e. at least 75% sequence identity between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which can be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nucleotides(nt) long, more usually at least about 30 nt long, and can extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., (1990) J. Mol. Biol. 215: 403-10.
  • Percent identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group.
  • the GAP program utilizes the alignment method of Needleman et aL, (1970) J. Mol. Biol. 48: 443, as revised by Smith et al., (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps.
  • similarity is contrasted with the term “identity”. Similarity is defined as above; "identity”, however, means a nucleic acid or amino acid sequence having the same amino acid at the same relative position in a given family member of a gene family. Homology and similarity are generally viewed as broader terms than the term identity. Biochemically similar amino acids, for example leucine/isoleucine or glutamate/aspartate, can be present at the same position — these are not identical per se, but are biochemically "similar.” As disclosed herein, these are referred to as conservative differences or conservative substitutions. This differs from a conservative mutation at the DNA level, which changes the nucleotide sequence without making a change in the encoded amino acid, e.g. TCC to TCA, both of which encode serine.
  • DNA analog sequences are "substantially identical" to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived from coding regions of the nucleic acid sequence shown in SEQ ID NOs: 1 or 3; or (b) the DNA analog sequence is capable of hybridization with DNA sequences of (a) under stringent conditions and which encode a biologically active hPXR or hPXR LBD gene product; or (c) the DNA sequences are degenerate as a result of alternative genetic code to the DNA analog sequences defined in (a) and/or (b).
  • Substantially identical analog proteins and nucleic acids will have between about 70% and 80%, preferably between about 81% to about 90% or even more preferably between about 91% and 99% sequence identity with the corresponding sequence of the native protein or nucleic acid. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
  • stringent conditions means conditions of high stringency, for example 6X SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 ⁇ g/ml salmon sperm DNA and 15% formamide at 68°C.
  • preferred conditions are salt concentration of about 200 mM and temperature of about 45°C.
  • One example of such stringent conditions is hybridization at 4X SSC, at 65°C, followed by a washing in 0.1X SSC at 65°C for one hour.
  • Another exemplary stringent hybridization scheme uses 50% formamide, 4X SSC at 42°C.
  • sequence identity can be determined by hybridization under lower stringency conditions, for example, at 50°C or higher and 0.1X SSC (9 mM NaCI/0.9 mM sodium citrate) and the sequences will remain bound when subjected to washing at 55°C in 1X SSC.
  • complementary sequences means nucleic acid sequences which are base-paired according to the standard Watson- Crick complementarity rules.
  • the present invention also encompasses the use of nucleotide segments that are complementary to the sequences of the present invention.
  • Hybridization can also be used for assessing complementary sequences and/or isolating complementary nucleotide sequences.
  • nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of about 30°C, typically in excess of about 37°C, and preferably in excess of about 45°C.
  • Stringent salt conditions will ordinarily be less than about 1 ,000 mM, typically less than about 500 mM, and preferably less than about 200 mM.
  • the term "functionally equivalent codon” is used to refer to codons that encode the same amino acid, such as the ACG and AGU codons for serine.
  • Human PXR or hPXR LBD-encoding nucleic acid sequences comprising SEQ ID NOs: 1 and 3, which have functionally equivalent codons, are covered by the present invention.
  • applicants are in possession of amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience.
  • amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' nucleic acid sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence retains biological protein activity where polypeptide expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences which can, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or can include various internal sequences, i.e., introns, which are known to occur within genes.
  • the present invention envisions and includes biological equivalents of an engineered PXR or PXR LBD mutant polypeptide of the present invention.
  • biological equivalent refers to proteins having amino acid sequences which are substantially identical to the amino acid sequence of an engineered PXR LBD mutant of the present invention and which are capable of exerting a biological effect in that they are capable of binding DNA moieties or cross-reacting with anti-PXR or PXR LBD mutant antibodies raised against an engineered mutant PXR or PXR LBD polypeptide of the present invention.
  • certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive capacity with, for example, structures in the nucleus of a cell.
  • Bioly equivalent polypeptides are polypeptides in which certain, but not most or all, of the amino acids can be substituted.
  • SEQ ID NOs: 1 and 3 applicants envision substitution of codons that encode biologically equivalent amino acids, as described herein, into the sequence example of SEQ ID NOs: 1 and 3, respectively.
  • amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience.
  • functionally equivalent proteins or peptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged, e.g.
  • Amino acid substitutions such as-those which might be employed in modifying an engineered PXR or PXR LBD mutant polypeptide of the present invention are generally, but not necessarily, based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.
  • arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
  • Other biologically functionally equivalent changes will be appreciated by those of skill in the art. It is implicit in the above discussion, however, that one of skill in the art can appreciate that a radical, rather than a conservative substitution is warranted in a given situation.
  • Non-conservative substitutions in engineered mutant PXR or PXR LBD polypeptides of the present invention are also an aspect of the present invention.
  • hydropathic index of amino acids can be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, (1982), J. Mol. Biol. 157: 105-132, incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ⁇ 2 of the original value is preferred, those which are within ⁇ 1 of the original value are particularly preferred, and those within ⁇ 0.5 of the original value are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartate (+ 3.0 ⁇ 1); glutamate (+ 3.0 ⁇ 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1 ); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (- 1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • Recombinant vectors and isolated DNA segments can therefore variously include an engineered hPXR or hPXR LBD mutant polypeptide-encoding region itself, include coding regions bearing selected alterations or modifications in the basic coding region, or include larger polypeptides which nevertheless comprise a hPXR or hPXR LBD mutant polypeptide-encoding regions or can encode biologically functional equivalent proteins or polypeptides which have variant amino acid sequences.
  • Biological activity of an engineered hPXR or hPXR LBD mutant polypeptide can be determined, for example, by Iipid-binding assays known to those of skill in the art.
  • the nucleic acid segments of the present invention regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters, enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length can be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid fragments can be prepared which include a short stretch complementary to a nucleic acid sequence set forth in SEQ ID NOs: 1 and 3, such as about 10 nucleotides, and which are up to 10,000 or 5,000 base pairs in length.
  • DNA segments with total lengths of about 4,000, 3,000, 2,000, 1 ,000, 500, 200, 100, and about 50 base pairs in length are also useful.
  • the DNA segments of the present invention encompass biologically functional equivalents of engineered PXR or PXR LBD mutant polypeptides. Such sequences can rise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or polypeptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. Changes can be introduced through the application of site- directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test variants of an engineered PXR or PXR LBD mutant of the present invention in order to examine the degree of lipid- binding activity, or other activity at the molecular level.
  • site-directed mutagenesis techniques are known to those of skill in the art and can be employed in the present invention.
  • the invention further encompasses fusion proteins and peptides wherein an engineered PXR or PXR LBD mutant coding region of the present invention is aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes.
  • Recombinant vectors form important further aspects of the present invention.
  • Particularly useful vectors are those in which the coding portion of the DNA segment is positioned under the control of a promoter.
  • the promoter can be that naturally associated with a PXR gene, as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology and/or other methods known in the art, in conjunction with the compositions disclosed herein.
  • a recombinant or heterologous promoter is a promoter that is not normally associated with a PXR gene in its natural environment.
  • Such promoters can include promoters isolated from bacterial, viral, eukaryotic, or mammalian cells. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression.
  • promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology (See, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, specifically incorporated herein by reference).
  • the promoters employed can be constitutive or inducible and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
  • One preferred promoter system contemplated for use in high-level expression is a T7 promoter-based system.
  • the PXR mutants disclosed herein have a variety of applications, including in the screening of components for PXR/SXR activation using the cell-free reporter gene assay methods disclosed herein above, and using whole animal models.
  • the PXR mutants can also be used in cell-free, cell- based and whole animal assay methods for bioavailability of compounds and for toxicology analysis. Additionally, PXR mutants can be employed in crystallizations, screening for changes in ligand activation, screening for species-specific changes in ligand activation and screening for changes in oligomerization state both with and without ligand.
  • a mutant PXR comprising a "humanized” mouse can find particular utility as a model for the study of drug metabolism in humans. Such a model can be more informative regarding drug metabolism in humans than studies using unmodified mouse PXR as a predictive model for human drug metabolism. It also offers the convenience and advantages of employing a mouse model system to study a human protein.
  • a method of evaluating a candidate therapeutic agent in humans using a mouse model system comprises the following steps.
  • Atomic coordinates of a human PXR ligand binding domain are provided to a computerized modeling system. Preferred coordinates are supplied in Table 4 of the present disclosure.
  • a candidate therapeutic agent that fits spatially into a binding cavity or on the surface of a human PXR ligand binding domain is modeled.
  • Modeling of a candidate therapeutic agent can conveniently employ a computer-based approach.
  • Computer programs, such as the INSIGHT II ® program (Molecular Simulations, Inc., San Diego, California) can be employed to create three-dimensional structures of candidate agents.
  • a computer program (which can comprise the appropriate design and structure evaluation modules), can be employed to preliminarily evaluate the candidate agent for use as a therapeutic.
  • the candidate agent can be docked with a site on the PXR, for example, and the efficiency of the interaction can be computationally evaluated for various physical properties, such as steric considerations and efficiency of binding.
  • a mouse PXR polypeptide is provided, and one or more mutations to be introduced into the mouse PXR amino acid sequence of the mouse PXR polypeptide are selected.
  • the mutations are preferably selected so as to alter the mouse PXR polypeptide to be similar to a human PXR polypeptide.
  • preferred mutations are selected from the group consisting of an arginine to leucine substitution at residue 203 of the mouse PXR polypeptide, a protein to serine substitution at residue 205 of the mouse PXR polypeptide, a glutamine to histidine substitution at residue 404 of the mouse PXR polypeptide and a glutamine to arginine substitution at residue 407 of the mouse PXR polypeptide.
  • the selected one or more mutations is/are introduced into a mouse PXR polypeptide and a mutant mouse PXR polypeptide comprising the selected one or more mutations selected is provided.
  • the mutant mouse PXR polypeptide is expressed and purified using methods that are disclosed herein and will be apparent to those of skill in the art, upon consideration of the present disclosure. The effect of the candidate therapeutic agent on the mutant mouse
  • PXR polypeptide is determined.
  • the effect can be determined by employing any appropriate reporter system.
  • the biological activity of a PXR polypeptide can be employed to determine an interaction.
  • the potential of the candidate therapeutic agent for use in humans is evaluated based on the effect of the candidate therapeutic agent on the mutant mouse PXR polypeptide.
  • the suitability of a candidate therapeutic for human disorders and conditions is conveniently assessed in a mouse model system. This system offers the ability to assess the biological response of a candidate therapeutic in vivo, which can add an additional degree of confidence to a conclusion made regarding the candidate therapeutic.
  • the structural coordinates of a hPXR LBD, or portions thereof, as provided by the present invention are particularly useful in solving the structure of other crystal forms of hPXR and the crystalline forms of other PXRs.
  • the coordinates provided in the present invention can also be used to solve the structure of PXR or PXR LBD mutants (such as those described in Section VIII above), PXR LDB co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of PXR.
  • a PXR or a PXR LBD mutant or a PXR or a PXR LBD polypeptide complexed with another compound (a "co-complex"), or the crystal of some other protein with significant amino acid sequence homology to any functional region of the a hPXR LBD, can be determined using the hPXR LBD structure coordinates provided in Table 4. This method provides an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
  • PXR or PXR mutants can be crystallized in complex with known modulators.
  • the crystal structures of a series of such complexes can then be solved by molecular replacement and compared with that of wild-type hPXR or the wild-type hPXR LBD. Potential sites for modification within the various binding sites of the enzyme can thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between the hPXR LBD and a chemical entity or compound.
  • All of the complexes referred to in the present disclosure can be studied using X-ray diffraction techniques (See, e.g., Blundell & Johnson (1985) Method. Enzymol., 114A & 115B, (Wyckoff et al., eds.), Academic Press) and can be refined using computer software, such as the X-PLORTM program (Br ⁇ nger, (1992) X-PLOR, Version 3.1. A System for X-ray Crystallography and NMR, Yale University Press, New Haven, Connecticut; X-PLOR is available from Molecular Simulations, Inc., San Diego, California). This information can thus be used to optimize known classes of PXR and PXR LBD modulators, and more importantly, to design and synthesize novel classes of PXR and PXR LBD modulators. Laboratory Examples
  • the PXR LBD expression construct was engineered as an N-terminal polyhistidine tagged fusion protein with residues 130-434 from the human PXR.
  • the fusion insert (SEQ ID NO: 10) was subcloned into the pRSETA expression vector (Invitrogen, Carlsbad, California).
  • DNA encoding residues 623-710 of the human SRC-1 gene (Onate et aL, (1995) Science 270: 1354- 57) (SEQ ID NO: 12) were subcloned into the bacterial vector pACYC184 (American Type Culture Collection #37033) along with a T7 promoter (Nolte et aL, (1998) Nature 395: 137-43).
  • the hPXRLBD/pRSETA and the SRC- 1/pACYCI84 plasmids were co-transformed into the BL21(DE3) strain of E. coli.
  • One-liter shake flask liquid cultures containing standard Luria-Bertani (LB) broth with 0.05 mg/ml ampicillin and 0.05 mg/ml chloramphenicol were inoculated and grown at 22°C for 20 hours.
  • the cells were harvested by centrifugation (20 minutes, 3500g, 4°C) and the cell pellet was stored at - 80°C.
  • the cell pellet was resuspended in 250 ml of Buffer A (50 mM Tris-CI pH 7.8, 250 mM NaCL, 50 mM imidazole pH 7.5, 5% glycerol). Cells were sonicated for 3-5 minutes on ice and the cell debris was removed by centrifugation (45 minutes, 20,000g, 4°C). The cleared supernatant was filtered through a 0.45 ⁇ M filter and loaded on to a 50 ml PROBONDTM nickel- chelating resin (Invitrogen, Carlsbad, California). After washing to baseline with Buffer A, the column was washed with Buffer A containing 125 mM, imidazole pH 7.5.
  • Buffer A 50 mM Tris-CI pH 7.8, 250 mM NaCL, 50 mM imidazole pH 7.5, 5% glycerol.
  • the PXR-LBD/SRC-1 complex was eluted from the column using Buffer A with 500 mM imidazole pH 7.5. Column fractions were pooled and concentrated using CENTRI-PREPTM 30K (Amicon/Millipore, Bedford, Massachusetts) units. The protein was subjected to size exclusion, using a column (26 mm X 90 cm) packed with SEPHAROSETM S-75 resin (Amersham Pharmacia Biotech, Piscataway, New Jersey) pre-equilibrated with 20mM Tris-CI pH 7.8, 250 mM NaCl, 5 mM DTT, 2.5 mM EDTA pH 8.0,5% glycerol.
  • the human PXR ligand binding domain/SRC-1 complex (hPXR- LBD/SRC-1 ) was concentrated in the presence of 10-fold molar excesses of the SR12813 or I-SR12813 compounds to final concentrations of 4 and 5 mg/mL, respectively.
  • the apo complex was concentrated to 5 mg/mL.
  • Non-identical side chains were trimmed prior to rotation and translation function searches in AmoRe (Navaza & Saludjian, (1997) Method Enzymol.
  • Each map was calculated to 3.0 A resolution and both showed a consistent set of significant positive difference density peaks in the ligand binding cavity, which were interpreted to be the positions of the iodine atoms on ISR12813. These peaks were the highest difference density peaks in both maps. It was found that three SR12813 ligands optimally satisfied the electron density in the ligand binding cavity.
  • the first SR12813 (position 1 ) was placed in part based on two 7 ⁇ difference density peaks at appropriate positions in both maps.
  • the second SR12813 (position 3) was positioned using two 5 ⁇ difference density peaks at appropriate positions.
  • the third SR12813 (position 2) was placed based on 4 ⁇ iodine difference density peaks and residual difference density in the maps after the refinement of positions 1 and 3 together.
  • Standard simulated density maps [(

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Abstract

A solved three-dimensional crystal structure of a human PXR ligand binding domain polypeptide is disclosed, along with a crystal form of the PXR ligand binding domain. Orientations of the ligand SR12813 in the binding cavity are also disclosed. Additionally, methods of designing modulators of the biological activity of PXR, and other PXR ligand binding domain polypeptides, are also disclosed.

Description

Description
CRYSTALLIZED HUMAN XENOBIOTIC NUCLEAR RECEPTOR PXR/SXR
LIGAND BINDING DOMAIN POLYPEPTIDE AND
SCREENING METHODS EMPLOYING SAME
Technical Field The present invention relates generally to the structure of the ligand binding domain of PXR, and more particularly to the crystalline structure of the ligand binding domain of PXR. The invention further relates to methods by which modulators and ligands of PXR can be identified.
Abbreviations
ATP adenosine triphosphate
ADP adenosine diphosphate
APS Advanced Photon Source
BSA bovine serum albumin
CBP CREB-binding protein cDNA complementary DNA
CI chemical ionization
CPS counts per second
CYP cytochrome P450
DBD DNA binding domain
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
El electron impact ionization
ER estrogen receptor
FRET fluorescent resonance energy transfer
GC gas chromatography
GC/MS gas chromatography/mass spectrometry
HEPES N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid hPXR human pregnane X receptor
HRE hormone response element kDa kilodalton(s)
LBD ligand binding domain
MAD multiwavelength anomalous diffraction
MODY mature onset diabetes of the young
MS mass spectrometry
NDP nucleotide diphosphate nt nucleotide
NTP nucleotide triphosphate
PAGE polyacrylamide gel electrophoresis
PAR pregnane-activated receptor
PCN pregnenolone 16a-carbonitrile
PCR polymerase chain reaction
Pi isoelectric point
PPAR peroxisome proliferator-activated receptor
PR progesterone receptor
PXR pregnane X receptor
RAR retinoic acid receptor
RIF rifampicin
RMSD root-mean-square deviation
RXR retinoid X receptor
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SIRAS single isomorphous replacement anomalous scattering
SR SR12813 SXR steroid and xenobiotic receptor TIC total ion chromatogram TR thyroid hormone receptor TTR plasma transthyretin
VDR vitamin D receptor vHNF variant hepatocyte nuclear factor
WT wildtype
Amino Acid Abbreviations
Single-Letter Code Three-Letter Code Name
A Ala Alanine
V Val Valine
L Leu Leucine
I lie Isoleucine
P Pro Praline
F Phe Phenylalanine
W Trp Tryptophan
M Met Methionine
G Gly Glycine
S Ser Serine
T Thr Threonine
C Cys Cysteine
Y Tyr Tyrosine
N Asn Asparagine
Q Gin Glutamine
D Asp Aspartic Acid
E Glu Glutamic Acid
K Lys Lysine
R Arg Arginine
H His Histidine
Functionally Equivalent Codons
Amino Acid Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU Aspartic Acid Asp D GAC GAU
Glumatic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine He I AUA AUC AUU
Lysine Lys K AAA AAG
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gin Q CAA CAG
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Trp w UGG
Tyrosine Tyr Y UAC UAU
Leucine Leu L UUA UUG CUA CUC CUG CUU
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S ACG AGU UCA UCC UCG UCU
Background Art
Nuclear receptors represent a superfamily of proteins that specifically bind a physiologically relevant small molecule, such as a hormone or vitamin. As a result of a molecule binding to a nuclear receptor, the nuclear receptor changes the ability of a cell to transcribe DNA, i.e. nuclear receptors modulate the transcription of DNA. However they can also have transcription independent actions. Unlike integral membrane receptors and membrane-associated receptors, nuclear receptors reside in either the cytoplasm or nucleus of eukaryotic cells. Thus nuclear receptors comprise a class of intracellular, soluble ligand-regulated transcription factors. Nuclear receptors include but are not limited to receptors for giucocorticoids, androgens, mineralcorticoids, progestins, estrogens, thyroid hormones, vitamin D retinoids, and icosanoids. Many nuclear receptors, identified by either sequence homology to known receptors (See, Drewes et al., (1996) Mol. Cell. Biol. 16:925-31) or based on their affinity for specific DNA binding sites in gene promoters (See, Sladek et a , Genes Dev. 4:2353-65), have unascertained ligands and are therefore termed "orphan receptors".
Structurally, nuclear receptors are generally characterized by two distinct structural elements. First, nuclear receptors comprise a central DNA binding domain that targets the receptor to specific DNA sequences, which are known as hormone response elements (HREs). The DNA binding domains of these receptors are related in structure and sequence, and are located within the middle of the receptor. Second, the C-terminal region of nuclear receptors encompasses the ligand binding domain (LBD). Upon binding a ligand, the receptor shifts to a transcriptionally active state.
The cytochrome P450 (CYP) family of heme-containing proteins play critical roles in the oxidative metabolism of drugs and other xenobiotics in the liver and small intestine. In particular, the CYP3A gene products bind and hydroxylate a wide variety of chemical structures, including >50% of all drugs. Maurel, (1996) in Cytochrome P450: Metabolic and Toxicological Aspects (lonnides, ed.) CRC Press, Inc., Boca Raton, Florida, 241-70. Expression of CYP3A is induced at the level of transcription by a variety of xenobiotics, including many that are metabolized by CYP3A. The pregnane X receptor (PXR; NR112), a member of the nuclear receptor family of ligand-activated transcription factors, is a key regulator of CYP3A gene expression in mammalian liver and small intestine. Kliewer et al., (1998) Cell 92: 73-82; Lehmann et al., (1998) J. Clin. Invest. 102: 1016- 23; Bertilsson et al., (1998) Proc. Nat. Acad. Sci. U.S.A. 95: 12208-13; Blumberg et al., (1998) Gene Dev. 12:3195-205; Xie et al., (2000) Nature 406: 435-39. The human ortholog of PXR is alternatively known as pregnane activated receptor (PAR) and steroid and xenobiotic receptor (SXR). Bertilsson et al., (1998) Proc. Nat. Acad. Sci. U.S.A. 95: 12208-13; Blumberg et al., (1998) Gene Dev. 12: 3195-205. PXR is activated by most of the xenobiotics that are known to induce CYP3A gene expression, including commonly used drugs such as the antibiotic rifampicin, the glucocorticoid dexamethasone, and the herbal therapy St. John's wort. Kliewer et al., (1998) Ce/792: 73-82; Lehmann et al., (1998) J. Clin. Invest. 102: 1016-23; Bertilsson et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95: 12208-13; Blumberg et al., (1998) Gene Dev. 12: 3195-205; Jones et al., (2000) Mol. Endocrinol. 14: 27- 39; Moore et al., (2000) Proc. Nat. Acad.Sci. U.S.A. 97: 7500-502. Like other nuclear receptors, PXR contains both a DNA binding domain and a ligand binding domain (LBD). PXR binds to the xenobiotic response elements in the regulatory regions of CYP3A genes as a heterodimer with the 9-cis retinoic acid receptor (RXR). Kliewer et al., (1998) Cell 92: 730-82; Lehmann et al., (1998) J. Clin. Invest. 102: 1016-23; Blumberg et al., (1998) Gene Dev. 12: 3195-205.
PXR mediates potentially dangerous drug-drug interactions by upregulating CYP3A expression in response to one compound, which in turn can then lead to the metabolism of other drugs vital to survival. For example, patients taking the herbal anti-depressant St. John's wort have exhibited a dramatic drop in serum levels of other critical drugs, including the antiretroviral drug Indinavir (also known as CRIXIVAN®) and the immunosuppressant compound cyclosporin. Piscitelli et al., (2000) Lancet 355: 547-48; Ruschitzka et al., (2000) Lancet 355: 548-49. The molecular basis of this effect was traced to PXR. Moore et al., (2000) Proc. Nat. Acad.Sci. U.S.A. 97: 7500-502; Wentworth et al., (2000) J. Endocrinol. 166: R11-R16. Hyperforin, a constituent of St. John's wort, activates PXR and upregulates CYP3A expression, which leads to the metabolism of these vital drugs. Unraveling the molecular basis of PXR activation by ligands could prove critical in preventing such potentially deadly cross-reactivities between drugs. Unlike the steroid, retinoid, and thyroid hormone receptors, which are highly selective for their cognate hormone, PXR has evolved to detect structurally-diverse compounds. These include drugs and xenobiotics, as well as endogenous compounds, including the toxic bile acid lithocholic acid and certain C21 steroids (pregnanes). Kliewer et al., (1998) Cell 92: 730-82; Bertilsson et al., (1998) Proc. Nat. Acad. Sci. U.S.A. 95: 12208-13; Jones et aL, (2000) Mol. Endocrinol. 14: 27-39. Although these diverse interactions imply a degree of promiscuity, PXR also exhibits specificity. This is illustrated by the fact there are marked differences in the pharmacologic activation profile of PXR across species. For instance, human PXR is activated efficiently by rifampicin and the cholesterol-lowering drug SR12813 (Berkhout et al„ (1996) J. Biol. Chem. 271 : 14376-82; Berkhout et al., (1997) Atherosclerosis 133: 203-21 ), whereas mouse PXR is not (Jones et al., (2000) Mol. Endocrinol. 14: 27-39). The mouse version of this receptor is activated by the synthetic steroid pregnenolone 16α-carbonitrile (PCN), whereas the human receptor is not. These data suggest that PXR has evolved in order to detect the different xenobiotic challenges faced by different species. By binding diverse but precise arrays of compounds, PXR exhibits directed promiscuity.
Unraveling the structural basis of how PXR recognizes an array of different endogenous and exogenous compounds, including both small and large ligands, is critical to understanding how harmful compounds are cleared from the body. Such knowledge can also improve our ability to predict and avoid dangerous drug-drug interactions.
Polypeptides, including the ligand binding domain of PXR, have a three-dimensional structure determined by the primary amino acid sequence and the environment surrounding the polypeptide. This three-dimensional structure establishes the polypeptide's activity, stability, binding affinity, binding specificity, and other biochemical attributes. Thus, knowledge of a protein's three-dimensional structure can provide much guidance in designing agents that mimic, inhibit, or improve its biological activity in soluble or membrane bound forms. The three-dimensional structure of a polypeptide can be determined in a number of ways. Many of the most precise methods employ X-ray crystallography (See, e.g., Van Holde, (1971) Physical Biochemistry, Prentice- Hall, N. J., 221-39). This technique relies on the ability of crystalline lattices to diffract X-rays or other forms of radiation. Diffraction experiments suitable for determining the three-dimensional structure of macromolecules typically require high-quality crystals. Unfortunately, such crystals have been unavailable for the ligand binding domain of PXR, as well as many other proteins of interest. Thus, high-quality diffracting crystals of the ligand binding domain of PXR would greatly assist in the elucidation of PXR's three- dimensional structure, and would provide insight into the ligand binding properties of PXR. Clearly, the solved crystal structure of the PXR ligand binding domain would be useful in the design of modulators of activity mediated by all PXR isoforms. Evaluation of the available sequence data has made it clear that PXR shows structural homology with the three-dimensional fold of other proteins. A solved PXR-ligand crystal structure would provide structural details and insights necessary to design a modulator of PXR that maximizes preferred requirements for any modulator, i.e. potency and specificity. By exploiting the structural details obtained from a PXR-ligand crystal structure, it would be possible to design a PXR modulator that, despite PXR's similarity with other proteins, exploits the unique structural features of PXR. A PXR modulator developed using structure-assisted design would take advantage of heretofore unknown PXR structural considerations and thus be more effective than a modulator developed using homology-based design. Potential or existent homology models cannot provide the necessary degree of specificity. A PXR modulator designed using the structural coordinates of a crystalline form of PXR would also provide a starting point for the development of modulators of other structurally similar proteins.
What is needed, therefore, is a crystallized form of a PXR LBD polypeptide, preferably in complex with a ligand. Acquisition of crystals of the PXR LBD polypeptide will permit the three dimensional structure of the PXR LBD to be determined. Knowledge of this three dimensional structure will facilitate the design of modulators of PXR activity. Such modulators can lead to therapeutic compounds to treat a wide range of conditions, including conditions associated with toxic xenobiotics.
Summary of the Invention A substantially pure PXR ligand binding domain polypeptide in crystalline form is disclosed. Preferably, the crystalline form has lattice constants of a = 91.6 A, b = 91.6 A, c = 85.0 A, α = 90°, β = 90°, γ = 90°. More preferably, the crystalline form is a tetragonal crystalline form. Even more preferably, the crystalline form has a space group of P432ι2. Still more preferably, the PXR ligand binding domain polypeptide has the amino acid sequence shown in SEQ ID NO: 4.
In a preferred embodiment, the PXR ligand binding domain polypeptide is in complex with a ligand. More preferably, the ligand is a hypocholesterolemic drug. Even more preferably, the hypocholesterolemic drug is SR12813.
A method for determining the three-dimensional structure of a crystallized PXR ligand binding domain polypeptide to a resolution of about 3.0 A or better is also disclosed. The method comprises (a) crystallizing a PXR ligand binding domain polypeptide; and (b) analyzing the PXR ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized PXR ligand binding domain polypeptide, whereby the three- dimensional structure of a crystallized PXR ligand binding domain polypeptide is determined to a resolution of about 3.0 A or better.
A method of designing a modulator of a PXR polypeptide is also disclosed. The method comprises (a) designing a potential modulator of a PXR polypeptide that will form bonds with amino acids in a ligand binding site based upon a crystalline structure of a PXR ligand binding domain polypeptide; (b) synthesizing the modulator; and (c) determining whether the potential modulator modulates the activity of the PXR polypeptide, whereby a modulator of a PXR polypeptide is designed.
In an alternative embodiment, a method of designing a modulator that selectively modulates the activity of a PXR polypeptide in accordance with the present invention comprises: (a) obtaining a crystalline form of a PXR ligand binding domain polypeptide; (b) evaluating the three-dimensional structure of the crystallized PXR ligand binding domain polypeptide; and (c) synthesizing a potential modulator based on the three-dimensional crystal structure of the crystallized PXR ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of a PXR polypeptide is designed. Preferably, the method further comprises contacting a PXR ligand binding domain polypeptide with the potential modulator; and assaying the PXR ligand binding domain polypeptide for binding of the potential modulator, for a change in activity of the PXR ligand binding domain polypeptide, or both. More preferably, the crystalline form is such that the three-dimensional structure of the crystallized PXR ligand binding domain polypeptide can be determined to a resolution of about 3.0 A or better.
In yet another embodiment, a method of designing a modulator of a PXR polypeptide in accordance with the present invention comprises: (a) selecting a candidate PXR ligand; (b) determining which amino acid or amino acids of a PXR polypeptide interact with the ligand using a three-dimensional model of a crystallized protein comprising a PXR LBD; (c) identifying in a biological assay for PXR activity a degree to which the ligand modulates the activity of the PXR polypeptide; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the PXR polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) performing the chemical modification on the ligand to form a modified ligand; (f) contacting the modified ligand with the PXR polypeptide; (g) identifying in a biological assay for PXR activity a degree to which the modified ligand modulates the biological activity of the PXR polypeptide; and (h) comparing the biological activity of the PXR polypeptide in the presence of modified ligand with the biological activity of the PXR polypeptide in the presence of the unmodified ligand, whereby a modulator of a PXR polypeptide is designed. Preferably, the PXR polypeptide is a PXR polypeptide. More preferably, the three-dimensional model of a crystallized protein is a PXR LBD polypeptide with a bound ligand. Even more preferably, the method further comprises repeating steps (a) through (f), if the biological activity of the PXR polypeptide in the presence of the modified ligand varies from the biological activity of the PXR polypeptide in the presence of the unmodified ligand.
A method for identifying a PXR modulator is also disclosed. The method comprises (a) providing atomic coordinates of a PXR ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits spatially into a binding cavity or on the surface of the PXR ligand binding domain to thereby identify a PXR modulator. Preferably, the method further comprises identifying in an assay for PXR-mediated activity a modeled ligand that increases or decreases the activity of the PXR.
A method of identifying a PXR modulator that selectively modulates the activity of a PXR polypeptide compared to other polypeptides is disclosed. The method comprises (a) providing atomic coordinates of a PXR ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits spatially into a binding cavity or on the surface of a PXR ligand binding domain and that interacts with conformationally constrained residues of a PXR that are conserved among PXR isoforms to thereby identify a PXR modulator. Preferably, the method further comprises identifying in a biological assay for PXR-mediated activity a modeled ligand that selectively binds to the PXR ligand binding domain and increases or decreases the activity of the PXR.
An assay method for identifying a compound that inhibits binding of a ligand to a PXR polypeptide is disclosed. The assay method comprises: (a) designing a test inhibitor compound capable of modulating PXR activity, based on the atomic coordinates of a PXR ligand binding domain; (b) synthesizing the test inhibitor compound; (c) incubating a PXR polypeptide with a ligand in the presence of a test inhibitor compound; (d) determining an amount of ligand that is bound to the PXR polypeptide, wherein decreased binding of ligand to the PXR protein in the presence of the test inhibitor compound relative to binding of ligand in the absence of the test inhibitor compound is indicative of inhibition; and (e) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed, whereby a compound that inhibits binding of a ligand to a PXR polypeptide is identified. Preferably, the ligand is a hypocholesterolemic drug. More preferably, the hypocholesterolemic drug is SR12813.
A method of evaluating a candidate therapeutic agent in humans using mouse model system is disclosed. In a preferred embodiment, the method comprises: (a) providing atomic coordinates of a human PXR ligand binding domain to a computerized modeling system; (b) modeling a candidate therapeutic agent that fits spatially into a binding cavity or on the surface of a human PXR ligand binding domain; (c) providing a mouse PXR polypeptide; (d) selecting one or more mutations to be introduced into an amino acid sequence of the mouse PXR polypeptide, the mutations being selected so as to alter the mouse PXR polypeptide to be similar to a human PXR polypeptide; (e) providing a mutant mouse PXR polypeptide comprising the one or more mutations selected in step (d); (f) contacting the candidate therapeutic agent modeled in step (b) with the mutant mouse PXR polypeptide; (g) determining an effect of the candidate therapeutic agent on the mutant mouse PXR polypeptide; and (h) evaluating the potential of the candidate therapeutic agent for use in humans based on the effect of the candidate therapeutic agent on the mutant mouse PXR polypeptide, whereby the candidate therapeutic agent in humans using a mouse model system is evaluated.
Accordingly, it is an object of the present invention to provide a three dimensional structure of the ligand binding domain of PXR. The object is achieved in whole or in part by the present invention. An object of the invention having been stated hereinabove, other objects will be evident as the description proceeds, when taken in connection with the accompanying Drawings and Laboratory Examples as best described hereinbelow.
Brief Description of the Drawings
Figure 1 is a ribbon diagram depicting the structure of the ligand binding domain of the human xenobiotic receptor PXR. Figure 2 is a cut-away view of the ligand binding cavity of the human xenobiotic PXR from a first perspective.
Figure 3 is a cut-away view of the ligand binding cavity of the human xenobiotic PXR from a second perspective. Figure 4 is an experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown directly. Amino acid side chains are shown in blue. SR12813 is depicted as a wireframe model and interacting atoms are presented as a spacefilling model comprising light gray spheres. Figure 5 is an experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown schematically, van der Waals contacts are indicated by solid arrows and hydrogen bonds with dashed arrows.
Figure 6 is a second experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown directly. Amino acid side chains are shown in blue.
SR12813 is depicted as a wireframe model and interacting atoms are presented as a spacefilling model comprising light gray spheres.
Figure 7 is a second experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown schematically, van der Waals contacts are indicated by solid arrows and hydrogen bonds with dashed arrows.
Figure 8 is a third experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown directly. Amino acid side chains are shown in blue. SR12813 is depicted as a wireframe model and interacting atoms are presented as a spacefilling model comprising light gray spheres.
Figure 9 is a third experimentally-observed position of SR12813 in the ligand binding cavity of human PXR. In this figure, intermolecular interactions are shown schematically, van der Waals contacts are indicated by solid arrows and hydrogen bonds with dashed arrows. Figure 10 is a ribbon diagram depicting two salt bridges (Glu-321 to Arg-410 and Asp-205 to Arg-413) adjacent to the ligand binding cavity of human PXR.
Figures 11A-11 D are a series of plots representing the luciferase (normalized luciferase activity counts per second (CPS) x 1000, y axis) and alkaline phosphatase activity of several PXR mutants in the presence of rifampicin or SR12813 (concentration in M, x axis). To generate this plot, CV- 1 cells were transfected with expression plasmids for hPXR, D205A, R413A, E321A, or R410A. 0=wildtype(WT); Δ=D205A mutant; H=R413A mutant, in addition to the XREM-CYP3A4-luciferase reporter.
Figure 12 is a computer-generated model depicting the homodimerization interaction observed in human PXR. The various distances between interacting residues are presented in the figure. The individual monomers are denoted by blue and green coloring. Figure 13 is a ribbon diagram depicting an overview of the crystallographically-observed homodimer of human PXR. Individual monomers are denoted by red and gold coloring, with green and blue coloring representing interacting structure.
Figure 14 is a plot representing the luciferase and alkaline phosphatase activity (normalized luciferase activity counts per second (CPS) x 1000, y axis) of PXR mutants in the presence of rifampicin(RIF) or SR12813(SR) (concentration in M, x axis). To generate this plot, CV-1 cells were transfected with expression plasmids encoding wildtype hPXR or mutant hPXR comprising the mutations W223A, Y225A and the XREM-CYP3A4- luciferase reporter. 0=WT-SR; D=W223A, Y225A-RIF; •=WT-SR; ■= W223A, Y225A-RIF.
Figures 15A-15C are a series of bar graphs demonstrating that four point mutants "humanize" mouse PXR's sensitivity to ligands. CV-1 cells were transfected with expression plasmids for mouse PXR (Fig. 15A), human PXR (Fig. 15B), or R203L, P205S, Q404H, Q407R (mouse→human) mouse PXR (Fig. 15C) and the XREM-CYP3A4-luciferase reporter. On the horizontal axis, bars from left to right represent vehicle, PCN and SR12813. Brief Description of the Sequences in the Sequence Listing SEQ ID NO: 1 is a DNA sequence encoding a full-length human PXR polypeptide (Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
SEQ ID NO: 2 is an amino acid sequence of a full-length human PXR polypeptide and is derived from the DNA sequence of SEQ ID NO: 1 (Swiss- Prot Accession No. 075469; GenBank Accession No. AF061056).
SEQ ID NO: 3 is a DNA sequence encoding the ligand binding domain of human PXR. The sequence codes for residues 130-434, which corresponds to the ligand binding domain of PXR (Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
SEQ ID NO: 4 is an amino acid sequence of the PXR ligand binding domain and is derived from the DNA sequence of SEQ ID NO: 3 (Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
SEQ ID NO: 5 is an amino acid sequence of the loop involving residues 309-321 in hPXR (Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
SEQ ID NO: 6 is a DNA sequence encoding a full-length human VDR polypeptide (Swiss-Prot Accession No. P11473; GenBank Accession No. J03258).
SEQ ID NO: 7 is an amino acid sequence of a full-lenth human VDR polypeptide and is derived from the DNA sequence of SEQ ID NO: 6 (Swiss- Prot Accession No. P11473; GenBank Accession No. J03258). SEQ ID NO: 8 is a DNA sequence encoding the ligand binding domain of human VDR. The sequence codes for residues 192-427, which corresponds to the ligand binding domain of PXR (Swiss-Prot Accession No. P11473; GenBank Accession No. J03258).
SEQ ID NO: 9 is an amino acid sequence of the VDR ligand binding domain and is derived from the DNA seqence of SEQ ID NO: 8 (Swiss-Prot Accession No. P11473; GenBank Accession No. J03258). SEQ ID NO: 10 is a DNA sequence encoding an N-terminal polyhistidine tagged PXR ligand binding domain fusion protein comprising residues 130-434 from human PXR (fragment from Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056). SEQ ID NO: 11 is an amino acid sequence of the N-terminal polyhistidine tagged fusion protein comprising residues 130-434 from human PXR and is derived from the DNA seqence of SEQ ID NO: 10 (fragment from Swiss-Prot Accession No. 075469; GenBank Accession No. AF061056).
SEQ ID NO: 12 is a DNA sequence encoding residues 623-710 of the human SRC-1 gene (fragment from GenBank Accession No. U59302).
SEQ ID NO: 13 is an amino acid sequence of residues 623-710 of the human SRC-1 gene and is derived from the DNA sequence of SEQ ID NO: 12 (fragment from GenBank Accession No. U59302).
Detailed Description of the Invention
Until disclosure of the present invention presented herein, the ability to obtain crystalline forms of a PXR LBD has not been realized. And until disclosure of the present invention presented herein, a detailed three- dimensional crystal structure of a PXR polypeptide has not been solved. In addition to providing structural information, crystalline polypeptides provide other advantages. For example, the crystallization process itself further purifies the polypeptide, and satisfies one of the classical criteria for homogeneity. In fact, crystallization frequently provides unparalleled purification quality, removing impurities that are not removed by other purification methods such as HPLC, dialysis, conventional column chromatography, etc. Moreover, crystalline polypeptides are often stable at ambient temperatures and free of protease contamination and other degradation associated with solution storage. Crystalline polypeptides can also be useful as pharmaceutical preparations. Finally, crystallization techniques in general are largely free of problems such as denaturation associated with other stabilization methods (e.g., lyophilization). Once crystallization has been accomplished, crystallographic data provides useful structural information that can assist the design of compounds that can serve as agonists or antagonists, as described herein below. In addition, the crystal structure provides information useful to map a receptor-binding domain, which could then be mimicked by a small non-peptide molecule that would serve as an antagonist or agonist.
I Definitions
Following long-standing patent law convention, the terms "a" and "an" mean "one or more" when used in this application, including the claims. As used herein, the term "mutation" carries its traditional connotation and means a change, inherited, naturally occurring or introduced, in a nucleic acid or polypeptide sequence, and is used in its sense as generally known to those of skill in the art.
As used herein, the term "labeled" means the attachment of a moiety, capable of detection by spectroscopic, radiologic or other methods, to a probe molecule.
As used herein, the term "target cell" refers to a cell, into which it is desired to insert a nucleic acid sequence or polypeptide, or to otherwise effect a modification from conditions known to be standard in the unmodified cell. A nucleic acid sequence introduced into a target cell can be of variable length.
Additionally, a nucleic acid sequence can enter a target cell as a component of a plasmid or other vector or as a naked sequence.
As used herein, the term "transcription" means a cellular process involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene. The process includes, but is not limited to the following steps: (a) the transcription initiation, (b) transcript elongation, (c) transcript splicing, (d) transcript capping, (e) transcript termination, (f) transcript polyadenylation, (g) nuclear export of the transcript, (h) transcript editing, and (i) stabilizing the transcript.
As used herein, the term "expression" generally refers to the cellular processes by which a polypeptide is produced from RNA. As used herein, the term "transcription factor" means a cytoplasmic or nuclear protein which binds to a gene, or binds to an RNA transcript of a gene, or binds to another protein which binds to a gene or an RNA transcript or another protein which in turn binds to a gene or an RNA transcript, so as to thereby modulate expression of the gene. Such modulation can additionally be achieved by other mechanisms; the essence of a "transcription factor for a gene" pertains to a factor that alters the level of transcription of the gene in some way.
As used herein, the term "hybridization" means the binding of a probe molecule, a molecule to which a detectable moiety has been bound, to a target sample.
As used herein, the term "detecting" means confirming the presence of a target entity by observing the occurrence of a detectable signal, such as a radiologic or spectroscopic signal that will appear exclusively in the presence of the target entity.
As used herein, the term "sequencing" means determining the ordered linear sequence of nucleic acids or amino acids of a DNA or protein target sample, using conventional manual or automated laboratory techniques.
As used herein, the term "isolated" means oligonucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which they can be associated, such association being either in cellular material or in a synthesis medium. The term can also be applied to polypeptides, in which case the polypeptide will be substantially free of nucleic acids, carbohydrates, lipids and other undesired polypeptides. As used herein, the term "substantially pure" means that the polynucleotide or polypeptide is substantially free of the sequences and molecules with which it is associated in its natural state, and those molecules used in the isolation procedure. The term "substantially free" means that the sample is at least 50%, preferably at least 70%, more preferably 80% and most preferably 90% free of the materials and compounds with which is it associated in nature. As used herein, the term "primer" means a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and more preferably more than eight and most preferably at least about 20 nucleotides of an exonic or intronic region. Such oligonucleotides are preferably between ten and thirty bases in length.
As used herein, the term "DNA segment" means a DNA molecule that has been isolated free of total genomic DNA of a particular species. In a preferred embodiment, a DNA segment encoding a PXR polypeptide refers to a DNA segment that contains SEQ ID NO: 1 , but can optionally comprise fewer or additional nucleic acids, yet is isolated away from, or purified free from, total genomic DNA of a source species, such as Homo sapiens. Included within the term "DNA segment" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. As used herein, the phrase "enhancer-promoter" means a composite unit that contains both enhancer and promoter elements. An enhancer- promoter is operatively linked to a coding sequence that encodes at least one gene product.
As used herein, the phrase "operatively linked" means that an enhancer-promoter is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter. Techniques for operatively linking an enhancer-promoter to a coding sequence are well known in the art; the precise orientation and location relative to a coding sequence of interest is dependent, inter alia, upon the specific nature of the enhancer-promoter.
As used herein, the terms "candidate substance" and "candidate compound" are used interchangeably and refer to a substance that is believed to interact with another moiety, for example a given ligand that is believed to interact with a complete, or a fragment of, a PXR polypeptide, and which can be subsequently evaluated for such an interaction. Representative candidate substances or compounds include "xenobiotics", such as drugs and other therapeutic agents, carcinogens and environmental pollutants, natural products and extracts, as well as "endobiotics", such as steroids, fatty acids and prostaglandins. Other examples of candidate compounds that can be investigated using the methods of the present invention include, but are not restricted to, agonists and antagonists of a PXR polypeptide, toxins and venoms, viral epitopes, hormones (e.g., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, co-factors, lectins, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins, small molecules and monoclonal antibodies.
As used herein, the term "biological activity" means any observable effect flowing from interaction between a PXR polypeptide and a ligand. Representative, but non-limiting, examples of biological activity in the context of the present invention include dimerization of a PXR and association of a PXR with DNA.
As used herein, the term "modified" means an alteration from an entity's normally occurring state. An entity can be modified by removing discrete chemical units or by adding discrete chemical units. The term "modified" encompasses detectable labels as well as those entities added as aids in purification.
As used herein, the terms "structure coordinates" and "structural coordinates" mean mathematical coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a molecule in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal.
Those of skill in the art understand that a set of structure coordinates determined by X-ray crystallography is not without standard error. For the purpose of this invention, any set of structure coordinates for PXR or a PXR mutant that have a root mean square deviation (RMSD) from ideal of preferably no more than 1.5 A, more preferably no more than 1.0 A, and most preferably no more than 0.5 A when superimposed, using the polypeptide backbone atoms, on the structure coordinates listed in Table 4 shall be considered identical.
As used herein, the term "space group" means the arrangement of symmetry elements of a crystal.
As used herein, the term "molecular replacement" means a method that involves generating a preliminary model of the wild-type PXR ligand binding domain, or a PXR mutant crystal whose structure coordinates are unknown, by orienting and positioning a molecule whose structure coordinates are known within the unit cell of the unknown crystal so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal. See, e.g., Lattman, (1985) Method Enzymol., 115: 55-77; Rossmann, ed, (1972) The Molecular Replacement Method, Gordon & Breach, New York.) Using the structure coordinates of the ligand binding domain of hPXR provided by this invention, molecular replacement can be used to determine the structure coordinates of a crystalline mutant or homologue of the hPXR ligand binding domain, or of a different crystal form of the hPXR ligand binding domain.
As used herein, the term "isomorphous replacement" means a method of using heavy atom derivative crystals to obtain the phase information necessary to elucidate the three-dimensional structure of a native crystal (Blundell et al., (1976) Protein Crystallography, Academic Press; Otwinowski, (1991), in Isomorphous Replacement and Anomalous Scattering, (Evans & Leslie, eds.), 80-86, Daresbury Laboratory, Daresbury, United Kingdom). The phrase "heavy-atom derivatization" is synonymous with the term "isomorphous replacement".
As used herein, the terms "β-sheet" and "beta-sheet" mean the conformation of a polypeptide chain stretched into an extended zig-zig conformation. Portions of polypeptide chains that run "parallel" all run in the same direction. Polypeptide chains that are "antiparallel" run in the opposite direction from the parallel chains.
As used herein, the terms " -helix" and "alpha-helix" mean the conformation of a polypeptide chain wherein the polypeptide backbone is wound around the long axis of the molecule in a left-handed or right-handed direction, and the R groups of the amino acids protrude outward from the helical backbone, wherein the repeating unit of the structure is a single turnoff the helix, which extends about 0.56 nm along the long axis.
As used herein, the term "unit cell" means a basic parallelepiped shaped block. The entire volume of a crystal can be constructed by regular assembly of such blocks. Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal. Thus, the term "unit cell" means the fundamental portion of a crystal structure that is repeated infinitely by translation in three dimensions. A unit cell is characterized by three vectors a, b, and c, not located in one plane, which form the edges of a parallelepiped. Angles α, β and γ define the angles between the vectors: angle α is the angle between vectors b and c; angle β is the angle between vectors a and c; and angle γ is the angle between vectors a and b. The entire volume of a crystal can be constructed by regular assembly of unit cells; each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal.
As used herein, the term "tetragonal unit cell" means a unit cell wherein a = b ≠ c; and = β = γ = 90°. The vectors a, b and c describe the unit cell edges and the angles α, β, and γ describe the unit cell angles. As used herein, the term "crystal lattice" means the array of points defined by the vertices of packed unit cells.
As used herein, the term "ligand binding site" and "ligand binding domain" are used interchangeably and mean that site in a polypeptide where substrate binding occurs. For hPXR, the ligand binding domain comprises the residues 130-434 of the full-length human PXR protein.
As used herein, the term "PXR" means nucleic acids encoding a pregnane X receptor (PXR) nuclear receptor polypeptide that can bind DNA and/or one or more ligands, and/or has the ability to form multimers. The term "PXR" includes invertebrate homologs; however, preferably, PXR nucleic acids and polypeptides are isolated from vertebrate sources. "PXR" further includes vertebrate homologs of PXR family members, including, but not limited to, mammalian and avian homologs. Representative mammalian homologs of PXR family members include, but are not limited to, murine and human homologs.
As used herein, the terms "PXR gene product", "PXR protein", "PXR polypeptide", and "PXR peptide" are used interchangeably and mean peptides having amino acid sequences which are substantially identical to native amino acid sequences from an organism of interest and which are biologically active in that they comprise all or a part of the amino acid sequence of a PXR polypeptide, or cross-react with antibodies raised against a PXR polypeptide, or retain all or some of the biological activity (e.g., DNA or ligand binding ability and/or dimerization ability) of the native amino acid sequence or protein. Such biological activity can include immunogenicity.
As used herein, the terms "PXR gene product", "PXR protein", "PXR polypeptide", and "PXR peptide" also include analogs of a PXR polypeptide. By "analog" is intended that a DNA or peptide sequence can contain alterations relative to the sequences disclosed herein, yet retain all or some of the biological activity of those sequences. Analogs can be derived from genomic nucleotide sequences as are disclosed herein or from other organisms, or can be created synthetically. Those skilled in the art will appreciate that other analogs, as yet undisclosed or undiscovered, can be used to design and/or construct PXR analogs. There is no need for a "PXR gene product", "PXR protein", "PXR polypeptide", or "PXR peptide" to comprise all or substantially all of the amino acid sequence of a PXR polypeptide gene product. Shorter or longer sequences are anticipated to be of use in the invention; shorter sequences are herein referred to as "segments". Thus, the terms "PXR gene product", "PXR protein", "PXR polypeptide", and "PXR peptide" also include fusion, chimeric or recombinant PXR polypeptides and proteins comprising sequences of the present invention. Methods of preparing such proteins are disclosed herein and are known in the art.
As used herein, the term "polypeptide" means any polymer comprising any of the 20 protein amino acids, regardless of its size. Although "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides and proteins, unless otherwise noted. As used herein, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product.
As used herein, the term "modulate" means an increase, decrease, or other alteration of any, or all, chemical and biological activities or properties of a wild-type or mutant PXR polypeptide. The term "modulation" as used herein refers to both upregulation (i.e., activation or stimulation) and downregulation (i.e. inhibition or suppression) of a response.
As used herein, the terms "PXR gene" and "recombinant PXR gene" mean a nucleic acid molecule comprising an open reading frame encoding a PXR polypeptide of the present invention, including both exon and (optionally) intron sequences. As used herein, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences. Preferred embodiments of genomic and cDNA sequences are disclosed herein. As used herein, the term "DNA sequence encoding a PXR polypeptide" can refer to one or more coding sequences within a particular individual. Moreover, certain differences in nucleotide sequences can exist between individual organisms, which are called alleles. It is possible that such allelic differences might or might not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity. As is well known, genes for a particular polypeptide can exist in single or multiple copies within the genome of an individual. Such duplicate genes can be identical or can have certain modifications, including nucleotide substitutions, additions or deletions, all of which still code for polypeptides having substantially the same activity.
As used herein, the term "intron" means a DNA sequence present in a given gene that is not-translated into protein.
As used herein, the term "interact" means detectable interactions between molecules, such as can be detected using, for example, a yeast two hybrid assay. The term "interact" is also meant to include "binding" interactions between molecules. Interactions can, for example, be protein- protein or protein-nucleic acid in nature.
As used herein, the terms "cells," "host cells" or "recombinant host cells" are used interchangeably and mean not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny might not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
As used herein, the term "agonist" means an agent that supplements or potentiates the bioactivity of a functional PXR gene or protein, of a polypeptide encoded by a gene that is up- or down-regulated by a PXR polypeptide, and/or a polypeptide encoded by a gene that contains a PXR binding site in its promoter region.
As used herein, the term "antagonist" means an agent that decreases or inhibits the bioactivity of a functional PXR gene or protein, or that supplements or potentiates the bioactivity of a naturally occurring or engineered non-functional PXR gene or protein. Alternatively, an antagonist can decrease or inhibit the bioactivity of a functional gene or polypeptide encoded by a gene that is up- or down-regulated by a PXR polypeptide and/or contains a PXR binding site in its promoter region. An antagonist can also supplement or potentiate the bioactivity of a naturally occurring or engineered non-functional gene or polypeptide encoded by a gene that is up- or down- deregulated by a PXR polypeptide, and/or contains a PXR binding site in its promoter region.
As used herein, the terms "chimeric protein" or "fusion protein" are used interchangeably and mean a fusion of a first amino acid sequence encoding a PXR polypeptide with a second amino acid sequence defining a polypeptide domain foreign to, and not homologous with, any domain of one of a PXR polypeptide. A chimeric protein can present a foreign domain that is found in an organism that also expresses the first protein, or it can be an "interspecies" or "intergenic" fusion of protein structures expressed by different kinds of organisms. In general, a fusion protein can be represented by the general formula X — PXR — Y, wherein PXR represents a portion of the protein which is derived from a PXR polypeptide, and X and Y are independently absent or represent amino acid sequences which are not related to a PXR sequence in an organism, which includes naturally occurring mutants. The term "chimeric gene" refers to a nucleic acid construct that encodes a "chimeric protein" or "fusion protein" as defined herein.
As used herein, the term "therapeutic agent" is a chemical entity intended to effectuate a change in an organism. Preferably, but not necessarily, the organism is a human being. It is not necessary that a therapeutic agent be known to effectuate a change in an organism; chemical entities that are suspected, predicted or designed to effectuate a change in an organism are therefore encompassed by the term "therapeutic agent." The effectuated change can be of any kind, observable or unobservable, and can include, for example, a change in the biological activity of a protein. Representative therapeutic compounds include small molecules, proteins and peptides, oligonucleotides of any length, "xenobiotics", such as drugs and other therapeutic agents, carcinogens and environmental pollutants, natural products and extracts, as well as "endobiotics", such as steroids, fatty acids and prostaglandins. Other examples of therapeutic agents can include, but are not restricted to, agonists and antagonists of a PXR polypeptide, toxins and venoms, viral epitopes, hormones (e.g., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, co-factors, lectins, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins, small molecules and monoclonal antibodies.
IL Description of Tables Table 1 is a table summarizing the crystal and data statistics obtained from the crystallized ligand binding domain of human PXR. Data on the unit cell are presented, including data on the crystal space group, unit cell dimensions, molecules per asymmetric cell and crystal resolution.
Table 2 is a table comparing human PXR-LBD with nuclear receptor LBDs of known structure.
Table 3 is a table summarizing the calculated buried solvent-accessible surface area of amino acid residues that line the ligand binding cavity of human PXR.
Table 4 is a table of the atomic structure coordinate data obtained from X-ray diffraction from the ligand binding domain of human PXR in complex with a ligand.
Table 5 is a table of the atomic structure coordinate data obtained from X-ray diffraction from human vitamin D receptor that was used in the molecular replacement solution of the human PXR ligand binding domain.
General Considerations
Expression of the CYP3A genes is induced at the level of transcription by a variety of xenobiotics, including many that are metabolized by CYP3A. This transcriptional regulation of CYP3A expression provides a mechanism for amplifying the physiologic response during periods of prolonged xenobiotic challenge. The induction of CYP3A expression also represents the basis for an important class of drug-drug interactions. Recently, a member of the nuclear receptor family of ligand-activated transcription facts, the pregnane X receptor (PXR) was shown to serve as a key regulator of CYP3A gene expression. The human ortholog of PXR is alternately referred to as the pregnane-activated receptor (PAR) or the steroid and xenobiotic receptor (SXR). Like other nuclear receptors PXR contains a DNA binding domain (DBD) and a ligand binding domain (LBD). PXR binds to xenobiotic response elements in the regulatory regions of CYP3A genes as a heterodimer with the 9-cis retinoic receptor (RXR). Notably, PXR is activated by most of the xenobiotics that are known to induce CYP3A gene expression, including commonly used drugs such as the antibiotic rifampicin and the glucocorticoid dexamethasone. Certain endogenous compounds also activate PXR, including the toxic bile acid lithocholic acid and certain C21 steroids (pregnanes). Thus, unlike the steroid, retinoid and thyroid hormone receptors, which are highly selective for their cognate hormone, PXR has evolved in order to detect structurally diverse substrates. Interestingly, there are marked differences in the pharmacologic activation profile of PXR across species. For instance, human PXR is activated efficiently by the rifampicin and the hypocholesterolemic drug SR12813, whereas mouse PXR is not. By contrast, mouse PXR is activated by the synthetic steroid pregnenolone 16a- carbonitrile (PCN), whereas the human receptor is not. These data suggest that PXR has evolved to detect the different xenobiotic challenges faced by different species.
The present invention will usually be applicable mutatis mutandis to all PXR polypeptides, as discussed herein based, in part, on the patterns of PXR structure and modulation that have emerged as a consequence of determining the three dimensional structure of human PXR in complex with a ligand. Generally speaking, PXR homologs and orthologs display substantial regions of amino acid homology. Additionally, the PXRs display an overall structural motif comprising three modular domains: 1 ) a variable amino-terminal domain;
2) a highly conserved DNA-binding domain (DBD); and
3) a less conserved carboxy-terminal ligand binding domain (LBD). The modularity of PXR permits different domains of each protein to separately accomplish different functions, although the domains can influence each other. The separate function of a domain is usually preserved when a particular domain is isolated from the remainder of the protein. Using conventional protein chemistry techniques, a modular domain can sometimes be separated from the parent protein. Using conventional molecular biology techniques, each domain can usually be separately expressed with its original function intact or, as discussed herein below, chimeric proteins comprising two different proteins can be constructed, wherein the chimeric proteins retain the properties of the individual functional domains of the respective polypeptides from which the chimeric proteins were generated.
The amino terminal domain of PXR is the least conserved of the three domains. This domain is involved in transcriptional activation and, in some cases, its uniqueness can dictate selective receptor-DNA binding and activation of target genes by PXR.
The DBD is the most conserved structure in PXR. It typically contains about 70 amino acids that fold into two zinc finger motifs, wherein a zinc ion coordinates four cysteines. The DBD generally contains two perpendicularly oriented α-helices that extend from the base of the first and second zinc fingers. The two zinc fingers function in concert along with non-zinc finger residues to direct the PXRs to specific target sites on DNA. Various amino acids in the DBD influence spacing between two half-sites (which usually comprises six nucleotides) for receptor homodimerization. The optimal spacings facilitate cooperative interactions between DBDs, and D box residues are part of the dimerization interface. Other regions of the DBD facilitate DNA-protein and protein-protein interactions required for PXR-RXR heterodimerization.
The LBD is the second most highly conserved domain in these receptors. Whereas the integrity of several different LBD sub-domains is important for ligand binding, truncated molecules containing only the LBD can retain normal ligand binding activity. This domain also participates in other functions, including dimerization, nuclear translocation and transcriptional regulation activities. Importantly, this domain can bind a ligand and can undergo ligand-induced conformational changes. Ligand binding allows the activation domain to serve as an interaction site for essential co-activator proteins that function to stimulate or inhibit transcription. The carboxy-terminal activation subdomain is in close three- dimensional proximity in the LBD to the ligand, so as to allow for ligands bound to the LBD to coordinate (or interact) with amino acid(s) in the activation subdomain. As disclosed herein, the LBD of PXR is expressed, crystallized and its three dimensional structure determined. Computational and other methods for the design of ligands to the LBD are also disclosed.
IV. Production of PXR Polypeptides
The native and mutated PXR polypeptides, and fragments thereof, of the present invention can be chemically synthesized in whole or part using techniques that are well-known in the art (See, e.g., Creighton, (1983) Proteins: Structures and Molecular Principles, W.H. Freeman & Co., New York, incorporated herein in its entirety). Alternatively, methods that are well known to those skilled in the art can be used to construct expression vectors containing a partial or the entire native or mutated PXR polypeptide coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, and Ausubel et al., (1989) Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York, both incorporated herein in their entirety.
A variety of host-expression vector systems can be utilized to express a PXR coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing a PXR coding sequence; yeast transformed with recombinant yeast expression vectors containing a PXR coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a PXR coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a PXR coding sequence; or animal cell systems. The expression elements of these systems vary in their strength and specificities.
Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. When cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter can be used. When cloning in plant cell systems, promoters derived from the genome of plant cells, such as heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) can be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. When generating cell lines that contain multiple copies of the tyrosine kinase domain DNA, SV40-, BPV- and EBV-based vectors can be used with an appropriate selectable marker.
V. Formation of PXR Ligand Binding Domain Crystals
In one embodiment, the present invention provides crystals of PXR. The crystals were obtained using the methodology disclosed in the Laboratory Examples. The PXR crystals, which can be native crystals, derivative crystals or co-crystals, have tetragonal unit cells (a tetragonal unit cell is a unit cell wherein a = b ≠ c, and wherein α = β = γ = 90°) and space group symmetry P43212. There is one PXR molecule in the asymmetric unit. In the PXR crystalline form, the unit cell has dimensions of a= b= 91.6 A, c= 85.0 A, and = β = γ = 90°.
The structures of the apo and SR12813-bound forms of PXR were solved by molecular replacement using the structure of the human vitamin D receptor as a search model (Rochel et al., (2000) Mol. Cell 5: 173-79, PDB ID: 1DB1 ; GenBank Accession No. XM007046; available online at http://www.rcsb.org/pdb/). The apo form was refined to a resolution of about 2.5 A. The ligand-bound form was refined to a resolution of about 2.75 A. The heavy atom derivatized form of the PXR LBD-ligand structure can be solved using single isomorphous replacement anomalous scattering (SIRAS) techniques and/or multiwavelength anomalous diffraction (MAD) techniques. In the SIRAS method of solving protein crystals, a derivative crystal is prepared that contains an atom that is heavier than the other atoms of the sample. Heavy atom derivative crystals are commonly prepared by soaking a crystal in a solution containing a selected heavy atom salt. For example, some heavy atom derivative crystals have been prepared by soaking a crystalline form of the protein of interest in a solution of methyl mercury chloride (MeHgCI). Another representative heavy atom that can be incorporated into a derivative crystal is iodine. Heavy atoms can associate with the protein of interest, or can be localized in a ligand that associates with a protein of interest. In the present invention, the latter approach was taken. Specifically, an iodine-containing form of the ligand SR12813 was co-crystallized with the PXR LBD and was used to assist in the positioning of the ligand relative to the PXR LBD.
Analysis of derivative crystals takes advantage of differences in the reflections from the derivative crystal as compared to the underivatized crystal. Symmetry-related reflections in the X-ray diffraction pattern, which are usually identical, are altered by the anomalous scattering contribution of the heavy atoms. The measured differences in symmetry-related reflections are used to determine the position of the heavy atoms, leading to an initial estimation of the diffraction phases, and subsequently, an electron density map is prepared. The prepared electron density map is then used to identify the position of the other atoms in the sample. VA Preparation of PXR Crystals The native and derivative co-crystals, and fragments thereof, disclosed in the present invention can be obtained by a variety of techniques, including batch, liquid bridge, dialysis, vapor diffusion and hanging drop methods (See, e.g., McPherson, (1982) Preparation and Analysis of Protein Crystals, John Wiley, New York.; McPherson, (1990) Eur. J. Biochem. 189:1-23.; Weber, (1991 ) Adv. Protein Chem. 41 :1-36). In a preferred embodiment, the vapor diffusion and hanging drop methods are used for the crystallization of PXR polypeptides and fragments thereof.
In general, native crystals of the present invention are grown by dissolving substantially pure PXR LBD polypeptide or a fragment thereof in an aqueous buffer containing a precipitant at a concentration just below that necessary to precipitate the protein. Water is removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases. In a preferred embodiment of the invention, native crystals are grown by vapor diffusion (See, e.g., McPherson, (1982) Preparation and Analysis of Protein Crystals, John Wiley, New York; McPherson, (1990) Eur. J. Biochem. 189:1-23). In this method, the polypeptide/precipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir having a precipitant concentration optimal for producing crystals. Generally, less than about 25 μL of PXR LBD polypeptide solution is mixed with an equal volume of reservoir solution, giving a precipitant concentration about half that required for crystallization. This solution is suspended as a droplet underneath a coverslip, which is sealed onto the top of the reservoir. The sealed container is allowed to stand, until crystals grow. Crystals generally form within two to six weeks, and are suitable for data collection within approximately seven to ten weeks. Of course, those of skill in the art will recognize that the above- described crystallization procedures and conditions can be varied. V.B. Preparation of Derivative Crystals
Derivative crystals of the present invention, e.g. heavy atom derivative crystals, can be obtained by soaking native crystals in mother liquor containing salts of heavy metal atoms. Alternatively, a ligand comprising a heavy atom can be associated with a protein, and subsequently co- crystallized. Such derivative crystals are useful for phase analysis in the solution of crystals of the present invention. This mechanism provides derivative crystals suitable for use as isomorphous replacements in determining the X-ray crystal structure of a PXR polypeptide. Additional reagents useful for the preparation of the derivative crystals of the present invention will be apparent to those of skill in the art after review of the disclosure of the present invention presented herein.
V.C. Preparation of Co-crystals Co-crystals of the present invention can be obtained by soaking a native crystal in mother liquor containing compounds known or predicted to bind the LBD of a PXR, or a fragment thereof. Alternatively, co-crystals can be obtained by co-crystallizing a PXR LBD polypeptide or a fragment thereof in the presence of one or more compounds known or predicted to bind the polypeptide. In a preferred embodiment of the present invention, for example, the ligand SR12831 , or an iodinated form of the ligand SR12831 , is co- crystallized with a PXR-SRC-1 complex.
V.D. Solving a Crystal Structure of the Present Invention Crystal structures of the present invention can be solved using a variety of techniques including, but not limited to, isomorphous replacement anomalous scattering or molecular replacement methods. Computer software packages will also be helpful in solving a crystal structure of the present invention. Applicable software packages include but are not limited to X- PLOR™ program (Brϋnger, (1992) X-PLOR, Version 3.1. A System for X-ray Crystallography and NMR, Yale University Press, New Haven, Connecticut; X-PLOR is available from Molecular Simulations, Inc., San Diego, California), Xtal View (McRee, (1992) J. Mol. Graphics 10: 44-47; X-tal View is available from the San Diego Supercomputer Center), SHELXS 97 (Sheldrick (1990) Acta Cryst. A46: 467; SHELX 97 is available from the Institute of Inorganic Chemistry, Georg-August-Universitat, Gottingen, Germany), HEAVY (Terwilliger, Los Alamos National Laboratory) can be used and SHAKE-AND- BAKE (Hauptman, (1997) Curr. Opin. Struct. Biol. 7: 672-80; Weeks et al., (1993) >Acfa Cryst. D49: 179; available from the Hauptman-Woodward Medical Research Institute, Buffalo, New York). See also, Ducruix & Geige, (1992) Crystallization of Nucleic Acids and Proteins: A Practical Approach, IRL Press, Oxford, England, and references cited therein.
VI. Summary of Results for the PXR Ligand binding Domain
Two structures of the LBD of human PXR are disclosed in the present invention. First, a 2.5 A apo structure and second, a 2.75 A structure in complex with the high affinity ligand SR12813. More specifically, a 1 :1 complex, comprising residues 130-434 of human PXR and residues 623-710 of the human transcriptional co-activator protein SRC-1 , was incubated in the crystallization mixture. The mixture was crystallized. The 88-amino acid co- activator peptide SRC-1 was not observed in the crystal structure, and the examination of extensively washed and dissolved crystals by SDS-PAGE revealed that this co-activator fragment was not present in the crystals of the PXR LBD. Residues 142-177 and 198-431 of human PXR LBD were traced in the structures reported in an aspect of the present invention, while ordered electron density of the remaining amino acids, including the 20-residue stretch from 178-197, was not observed at any time during refinement.
The hydrophobic ligand binding cavity of human PXR contains a small number of polar residues, permitting the ligand SR12813 to bind in three distinct orientations. Despite the promiscuity of this receptor, human and mouse PXR are activated by different collections of xenobiotics. Critical residues responsible for this directed promiscuity are disclosed in the present invention. Interestingly, the mutation of only four residues "humanizes" murine PXR. That is, the mutation of four residues is observed to change the binding profile of murine PXR from a murinic profile to that of the human PXR. The crystal structures of the present invention disclose important insights into how human PXR detects xenobiotics and thus, can be useful in predicting and avoiding drug-drug interactions.
VI Overall Structure of the Human PXR Ligand Binding Domain Two structures of the ligand binding domain (LBD) of hPXR were determined by molecular replacement (Navaza & Saludjian, (1997) Method Enzymol. 276A: 581-94) and were refined with CNS (Brϋnger et al., (1998) Acta Crystallogr. D 54: 905-21). As noted herein above, the solved structures disclosed in the present invention include the human PXR LBD apo structure, which was solved to a resolution of 2.5 A, and the structure of human PXR in complex with the high-affinity (Kd = 41 nM) ligand SR12813, which was solved to a resolution of 2.75 A. The apo structure was refined to final R and Rfree values of 0.209 and 0.284 respectively. The ligand-bound structure (PXR- SR12813) was refined to R and Rfree values of 0.213 and 0.274, respectively. These and other calculated and observed crystallographic data are summarized in Table 1. Residues 142-177 and 198-431 of the human PXR LBD were traced in the structures reported in the present invention. The human PXR LBD is an "α-helical sandwich" comprising three layers: α1/α3, α7/α10, and a middle layer comprising α-helices 4, 5 and 8. See Figures 1 and 13. This region of the molecule is similar to nuclear receptor ligand binding domains having known structure (Moras & Gronenmeyer, (1998) Curr. Opin. Cell Biol. 10: 384-91 ; Weatherman et al„ (1999) Annu. Rev. Biochem. 68: 559-81 ). The standard three-stranded β- sheet is expanded to a five-stranded antiparallel β-sheet in the human PXR LBD, comprising β-strands 1 , 1', 2, 3 and 4, and the ligand binding cavity is localized at the bottom of the molecule. This structural feature is shown in Figure 1. The structures of the apo and ligand-bound forms of the hPXR LBD are essentially identical, exhibiting a root-mean-square deviation (RMSD) of 0.68 A over the Cα positions, and 0.89 A over all atoms. In both the apo and SR12813-bound structures, the activation function 2 helix (α-AF2), which plays a critical role in transcriptional activation by nuclear receptors, is packed against the body of the receptor in a position that appears permissive for coactivator interactions.
The human PXR (SEQ ID NOs: 1 and 2) LBD (SEQ ID NOs: 3 and 4) is most closely related in structure to the vitamin D receptor (VDR) (Rochel et aL, (2000) Mol. Cell 5: 173-79), with which it shares 45% sequence identity (SEQ ID NOs: 8 and 9) and exhibits a 1.8 A RMSD over 225 equivalent Cα positions. Table 2 illustrates this similarity. More limited structural similarity, between 2.4 and 2.9 A RMSD, is shared with the ligand binding domains of the retinoid x receptor (RXR), peroxisome proliferator-activated receptor γ (PPARγ), and the estrogen and progesterone receptors (Bourget et aL, (1995) Nature 375: 377-82; Renaud et al., (1995) Nature 378: 681-89; Nolte et al., (1998) Nature 395: 137-43; Williams & Sigler, (1998) Nature 393: 392-96; Shiau et al., (1998) Ce/795: 927-37; Brzozowski et al., (1997) Nature 389: 753-58). This observation is also presented in Table 2.
VLB. Novel Structural Features of the Human PXR LBD While human PXR is similar to known nuclear receptor ligand binding domains, it contains several distinct features that appear critical to its function as a promiscuous xenobiotic receptor. First, the variable region typically found between 1 and cc3 in other nuclear receptors is replaced by a four- residue turn in human PXR, as depicted in Figure 1. In the peroxisome proliferator-activated receptors (PPARs), this region contains α2 and is the proposed ligand access site for the binding cavity (Nolte et al., (1998) Nature 395: 137-43; Gampe et al., (2000) Mol. Cell 5: 545-55. This region cannot be the access site in hPXR because it is occluded by the turn formed by residues 171-174. These structural features suggest that a distinct ligand entry site exists in hPXR.
Second, helix α6, which is common to many nuclear receptors, is replaced in hPXR by a conserved, flexible loop composed of residues 309- 321 (See Figures 1 and 13). This region lies adjacent in space to the ligand binding cavity of hPXR. This feature might be related to the ability of the hPXR receptor to accommodate both small and large ligands in the binding cavity.
Third, hPXR has two additional β-strands that have not previously been observed in a nuclear receptor ligand binding domain. This feature is depicted in Figure 1. Weatherman et aL, (1999) Annu. Rev. Biochem. 68: 559-81. These additional strands form the fourth (β1 , residues 210 to 217) and fifth (β1'f residues 221 to 226) strands of a five-stranded anti-parallel β- sheet. An "insertion domain" containing roughly the same number of residues was engineered out of the VDR prior to crystallization and structure determination (Rochel et aL, (2000) Mol. Cell 5:173-79). However, small angle x-ray scattering (Rochel et al., Eur. J. Biochem. 268: 971-79) suggests that the 51 -amino acid insertion domain in VDR (residues 164-216) is unstructured, and shares only 12% sequence identity with the analogous 45- amino acid (residues 184 to 229) in PXR. β1', at the base of this five- stranded beta-sheet, forms a novel homodimerization interaction in hPXR. As discussed herein below, these structural features appear to play a role in ligand binding, accessibility of the binding cavity, and the formation of higher- order oligomers of hPXR.
VI.C. The Ligand Binding Cavity of Human PXR
The ligand binding cavity of the hPXR-LBD is largely hydrophobic and might be flexible in nature, in order to accommodate both small and large ligands. The 28 amino acid residues desolvated by the binding of SR12813, which are presented in Table 3, are considered to be lining the cavity. The structures of the apo and the ligand bound cavities are similar, exhibiting a 1.12 A RMSD over all atoms in these 28 residues. Table 2 shows that the binding cavity volume of 1 ,150 A3, making it larger than most known nuclear receptor ligand binding cavities. Twenty of the cavity-lining residues are hydrophobic, four are polar (Ser-208, Ser-247, Cys-284 and Gln-285), and four are charged or potentially charged (GIu-321 , His-327, His-407 and Arg- 410). This observation is depicted via graphics in Figures 2 and 3 and is additionally presented in Table 3. As described further herein below, GIu-321 and Arg-410 are involved in a salt bridge, effectively neutralizing their charged character adjacent to the ligand binding cavity. Thus, an electrostatic view of the inner surface of this ligand binding cavity reveals a relatively uncharged and hydrophobic environment, as seen in Figures 2 and 3. Spaced throughout this cavity are five critical polar residues which form key binding interactions with SR12813 (Ser-208, Ser-247, Gln-285, His-407 and Arg-410, also indicated in Figures 2 and 3). These residues are capable of hydrogen bonding and are spaced evenly throughout the upper portion of the ligand binding cavity. The precise placement of polar residues is found to be critical for defining the activation profile of mouse PXR with respect to specific ligands. hPXR is able to bind both small and large ligands. The loop involving residues 309-321 in hPXR (comprising the sequence EDTAGGFQQLLLE, SEQ ID NO: 5) replaces 6 observed previously in reported nuclear receptor ligand binding domain structures. This highly mobile region spans the space between the C-terminus of β4 to the N-terminus of α7, and exhibits a mean thermal displacement parameter of 82.3 A2 over main-chain atoms despite persistent electron density. Nine of the thirteen residues in this loop are completely conserved in the mammalian PXR molecules of known sequence (highlighted in bold in SEQ ID NO: 5 above, and depicted in Figures 2 and 3), including the solvent-exposed hydrophobic residues of SEQ ID NO: 5, Phe- 315, Leu-318, Leu-319 and Leu-320. The 309-321 loop is linked to the ligand binding cavity of hPXR by a non-solvent accessible pore. This observation is depicted in Figures 2 and 3. The binding of a particularly large and hydrophobic ligand in the binding cavity (e.g. rifampicin) might force the pore to this loop region to open, enlarging the ligand binding region of hPXR and lining it with additional hydrophobic amino acid side chains.
VI.D. Three Orientations of SR12813 in the Ligand binding Cavity Three distinct binding modes of the high-affinity ligand SR12813
(positions 1 , 2 and 3) were observed in the ligand binding cavity of hPXR, shown in Figures 4-9. These orientations were identified during structural refinement and were rigorously confirmed using difference maps involving data obtained from crystals containing an iodinated form of SR12813 (I- SR12813 in Figures 4-9). As shown in Figures 4-9, each orientation forms distinct interactions with residues that line the ligand binding cavity of PXR. While ligand position 3 forms the most hydrophilic interactions with the protein, positions 1 and 2 were clearly indicated in the detailed examination of difference maps and in refinement. Indeed, when any one of the orientations is not considered in an unbiased refinement, clear positive electron density appears in difference maps to indicate its presence in the binding cavity of the protein.
Of the nineteen residues involved in contacting these orientations of SR12813, only Phe-288 interacts with all three ligand conformations. A phenylalanine residue is conserved at position 288 in the known mammalian PXR sequences. The remainder of the hydrophobic residues contact either two orientations (Leu-209, Phe-251 , Phe-281 , Trp-299) or one orientation (Leu-206, Val-211 , Leu-240, Met-243, Met-246, Met-323, Leu-324, Phe-420) of the ligand. Among the polar side chains, two (Ser-247, His-407) interact with two orientations of the ligand, while the remaining four (Ser-208, Cys- 284, Gln-285, Arg-410) interact with only one orientation. All but five of these residues are conserved by either identity or similarity in the mammalian PXR sequences reported to date, an observation presented in Table 3.
VI.D.1. The Ligand Binding Promiscuity of PXR
The nuclear receptor PXR serves as a key component of the body's defense mechanism against xenobiotics by detecting these compounds and regulating the transcription of genes involved in their metabolism (Waxman,
(1999) Arch. Biochem Biophys. 369: 11-23; Savas et al„ (1999) Mol.
Pharmacol. 56: 851-57). Unlike other nuclear receptors, which evolved to interact selectively with their cognate hormone, PXR evolved the ability to interact promiscuously with a structurally diverse collection of hydrophobic compounds. The x-ray crystal structures of the apo and SR12813-bound PXR-LBD reveal several important features underlying PXR's promiscuous ligand binding properties.
First, the volume of the PXR ligand binding cavity is >1 ,100 A3, which is substantially larger than that of many other nuclear receptors including the progesterone, estrogen, retinoid, and thyroid hormone receptors, an observation represented in Table 2. The PXR structure disclosed in the present invention also suggests that the ligand binding cavity may be capable of expanding to an even larger size. A stretch of conserved residues 309-321 (identified in Figures 1-3) loop out and away from the ligand binding cavity of PXR in the structures reported here, exposing conserved hydrophobic residues to solvent. This loop is connected to the existing ligand binding cavity by a solvent-inaccessible pore. These observations suggest that PXR might rearrange its ligand binding cavity when confronted by larger ligands, such as rifampicin. If the pore were to open between the ligand binding cavity and the 309-321 loop, the conserved hydrophobic residues currently exposed to solvent might be used to line a larger hydrophobic binding cavity. Thus, structural flexibility in the ligand binding cavity can contribute to PXR's ability to bind both to small hydrophobic ligands as well as to the much larger compound rifampicin. Second, the position of the ligand entry site appears to be unique in
PXR. Instead of being near the AF2-helix as in the steroid, retinoid, and thyroid hormone receptors or in the front of the molecule as in the PPARs, the entry site appears to be located on the back of the molecule between α7 and α10. The putative entrance-exit path to the ligand binding cavity in hPXR might be gated by two salt bridges (i.e. between Asp-205 and Arg-413, and GIu-321 and Arg-410), as shown in Figure 10. Mutagenesis studies disclosed in the present invention show that Arg-410 and Asp-205, which are in van der Waals contact with each other, are critical to defining the appropriate basal activity level for PXR. When Asp-205 is mutated to alanine, the basal activity level of PXR drops dramatically in cell-based reporter gene assays. In contrast, changing Arg-410 to alanine increases the basal activity level of PXR activity, as indicated in Figure 11. The interactions of GIu-321 and Arg- 413 with Arg-410 and Asp-205, respectively, might be necessary simply to define the locations of Asp-205 and Arg-410; the correct positioning of these two residues appears to tune the transcriptional activity of PXR. Perhaps during the opening and closing of this gate to allow for the movement of ligands, a vital intermediate is formed by the interaction of Arg-410 with Asp- 205. The drop in basal activity level in the Asp-205-Ala mutant might also indicate that this form of the protein is less stable than the wildtype. Asp-205 could be critical for ordering the 198-210 region of the molecule; the loss of this residue could introduce flexibility to PXR that affects its stability and basal activation level.
Third, the ligand binding cavity of PXR is relatively smooth and hydrophobic, but contains a small number of key polar residues. Twenty of the 28 residues lining the binding cavity are hydrophobic; the remaining polar residues are spaced roughly evenly throughout the cavity, offering the potential of a small number of hydrogen bonds. The character of the PXR ligand cavity mirrors the character of the majority of the ligands known to activate PXR, such as phenobarbital, clotrimazole, RU486, hyperforin, PCN and lovastatin, which are largely hydrophobic and uncharged, but which contain oneto four functional groups capable of forming hydrogen bonds. A combination of hydrophobic and shuffled polar interactions allows the potent PXR agonist SR12813 to bind to the hPXR ligand binding cavity in three distinct orientations. Although SR12813 occupies essentially the same portion of the cavity in each of the three binding modes, each mode is facilitated by a different set of hydrogen bonding and hydrophobic interactions (Figures 4-6). For example, Ser-247 forms a hydrogen bond with the phenolic hydroxyl group on SR12813 in one binding mode, and a hydrogen bond with a phosphate group in another. Thus, PXR does not define a specific ligand binding surface that will drive the binding of ligands in one mode. Instead, PXR offers an essentially smooth and uncharged ligand binding surface, with evenly spaced hydrogen bond donors and acceptors (Figures 2 and 3). Such an arrangement not only permits PXR to bind to structurally distinct ligands, but also allows single ligands to bind in multiple modes. This binding mode stands in sharp contrast to other nuclear receptor-ligand interactions, which have evolved to be highly specific.
VI.E. Electrostatic Interactions Adjacent to the Ligand binding Cavity Two salt-bridges occur across the region of the ligand binding cavity that is closest to the surface of hPXR and are depicted in Figure 10: Arg-410 with GIu-321 , and Arg-413 with Asp-205. Asp-205, GIu-321 and Arg-413 are almost completely conserved in the PXR molecules of known sequence; rabbit PXR contains a glutamic acid at position 205. Arg-410, however, is less conserved; in human and rabbit PXR, this residue is an arginine or lysine, while in rat and mouse it is a glutamine.
To examine the functional relevance of the Asp-205/Arg-413 and Glu- 321/Arg-410 salt bridges, each of these residues was individually mutated to alanine. Expression plasmids for each of the four mutant PXRs were co- transfected into CV-1 cells together with the XREM-CYP3A4-LUC reporter plasmid containing the distal enhancer and proximal promoter of CYP3A4, which include two high-affinity PXR binding sites, driving expression of the luciferase gene (Goodwin et aL, (1999) Mol. Pharmacol. 56: 1329-39). Transfected cells were treated with increasing concentrations of either SR12813 or rifampicin, as illustrated in the plots of Figure 11. Mutation of Asp-205 resulted in a marked decrease in the basal (ligand-independent) transcriptional activity of PXR, while mutation of Arg-413 resulted in a more modest reduction in PXR basal activity. In the other salt bridge, mutation of GIu-321 resulted in a marked increase in the basal activity of PXR; conversely, the Arg-410-Ala mutant had reduced basal activity. Taken together, these data demonstrate that the residues that form these salt bridges are important in determining the basal activity of the PXR. Moreover, the data highlight the asymmetry in the functions of the residues comprising the salt bridges: mutation of either Arg-410 or Asp-205 has a more dramatic effect than alteration of their salt bridge partners, suggesting that these two residues are particularly critical for determining the basal transcriptional activity of PXR. The mutant PXR LBD polypeptides Asp-205-Ala, Arg-413-Ala and Glu- 321-Ala were all activated by both SR12813 and rifampicin, although the dynamic range of the response was compressed relative to the wildtype receptor for the GIu-321 -Ala mutant. Interestingly, rifampicin was a more potent activator of the Asp-205-Ala mutant than wildtype PXR. This effect was not seen with SR12813, suggesting that SR12813 and rifampicin bind to PXR in different ways. These results also indicate that mutation of residues involved in salt bridges can affect the potency and selectivity with which compounds activate PXR.
VI. F. hPXR Forms a Novel Homodimer
A remarkable observation arising from these structural studies was that the ligand binding domain of hPXR forms a homodimer through the β-sheet structure that is unique to PXR. β1' from the β 1 -β 1 ' region of this structure forms an ideal anti-parallel β-sheet interaction with β1' from a crystallographically-related monomer, as shown in Figure 12. The dimer interface appears to be stabilized in two ways. First, six 2.9-3.0 A main-chain to main-chain hydrogen bonds in an anti-parallel β-sheet pattern occur across the interface. This generates a ten-stranded anti-parallel β-sheet between the two monomers, highlighted in Figure 13. Second, two aromatic side chains are observed to be buried which lends a large degree of specificity to this interaction. Notably, the Trp-223 side chains from each monomer are locked across the dimer interface, and thus are completely protected from solvent, permitting them to form an "offset-edge" stacking interaction frequently observed between tryptophans (Samanta et al., (1999) Acta Crystalog. D 55: 1421-27). Tyr-225 is also involved in this interface and becomes buried in the dimer, packing against Pro-175 from the region leading up to residue 177. The position of Trp-223 is further stabilized by a 3.0 A hydrogen bond between the main-chain carbonyl oxygen of Pro-175 and the indol ring nitrogen of the tryptophan side chain. In all, this highly specific dimer interface buries about 1 ,600 A2 of solvent accessible surface area on each monomer. Trp-223, Tyr-225 and Pro-175 are conserved in all the reported mammalian PXR sequences. The evolutionary conservation of the residues involved in this interaction supports its role in the biological function of PXR.
To assess whether this homodimerization interface is functionally relevant in vivo, reporter gene experiments were performed with a PXR double mutant in which Trp-223 and Tyr-225 were mutated to alanine. The Trp-223-Ala/Tyr-225-Ala mutant hPXR polypeptide had dramatically reduced basal activity and was only very weakly responsive to either SR12813 or rifampicin, as shown in Figure 14. Gel mobility shift assays revealed that this double mutant retains the ability to bind to DNA as a heterodimer with RXRα. Taken together, these data suggest an unprecedented role for a novel homodimerization interface in PXR-mediated transcriptional activation.
A model for a putative heterotetrameric complex formed by the ligand binding domains of hPXR and RXRα was generated by superimposing PXR on PPARγ in the heterodimeric structure of PPARγ and RXRα (Gampe et al., (2000) Mol Cell 5: 545-55). The interface between PXR and RXRα generated in this fashion is nearly ideal, requiring no manual optimization and producing no van der Waals overlaps between atoms. Importantly, the homodimerization of PXR does not interfere with the PXR/RXRα interface in the heterotetramer, suggesting that a heterotetramer of PXR and RXRα can or could occur in vivo.
VI. G Structure-Based Receptor Design: Altering the Ligand
Specificity of Mouse PXR By Four Targeted Site-Directed
Mutants Marked differences in the pharmacological activation profiles of PXR occur across species. For example, human and rabbit PXR are activated efficiently by SR12813 whereas mouse PXR is not. Conversely, mouse PXR is activated more efficiently by the synthetic steroid PCN than either the human or rabbit orthologs (Jones et al., (2000) Mol. Endocrinol. 14: 27-39; Savas et aL, (2000) Drug Metab Dispos. 28: 529-37). To verify the importance of polar residues in the selective PXR-SR12813 interaction, mouse PXR was "humanized" - that is, its selectivity was altered such that it would respond to SR12813 but not to PCN.
Four residues that interact with SR12813 in the hPXR crystal structure and differ between human and mouse PXR were chosen for mutagenesis: Leu-206/Arg-203, Ser-208/Pro-205, His-407/Gln-404, Arg-410/Gln-407 (human residues in each pair are listed first). Each of these residues was mutated in the context of a mouse PXR expression plasmid to the corresponding amino acid in the hPXR (R203L, P205S, Q404H, Q407R; using the mouse numbering scheme). As expected, wildtype mouse PXR responded to PCN and was only weakly activated by SR12813 in reporter assays (Figure 5). However, the R203L, P205S, Q404H, Q407R mutant (mouse→human PXR) was no longer activated by PCN but was activated efficiently by SR12813, mirroring the hPXR activation profile, as shown by the bar graphs presented in Figure 15. Thus, mouse PXR's response to particular ligands was "humanized" by employing the crystal structure to design targeted mutants. These results show that the selectivity of PXR for certain ligands resides in the number and position of only a few polar residues within the molecule's large, hydrophobic ligand binding cavity.
VI.G.1. Species-Specific Activation by Ligand in the PXRs
PXR is promiscuous in that it binds a wide variety of endogenous compounds and xenobiotics. However, this receptor also exhibits specificity as demonstrated by the fact that PXRs from different species show distinct activation profiles. To test the hypothesis that key polar residues determine specificity, the crystal structures of hPXR disclosed in the present invention were employed to design an altered form of PXR. Mutation of only four residues in the mouse PXR ligand binding cavity (Arg-203, Pro-205, Gln-404, Gln-407) to the corresponding residues in the human receptor (Leu-206, Ser- 208, His-407, Arg-410, respectively) results in a mouse- human PXR that is activated efficiently by SR12813 but not by PCN, which a rodent-selective PXR ligand (Figure 15). Thus, a "humanized" mouse PXR was created by altering four polar amino acids. This indicates that the relative positions of critical polar residues are sufficient for achieving selectivity in this receptor. Promiscuity appears to arise from the general hydrophobic nature and relatively small number of polar group within the cavity.
VI.H. Generation of Easily-Solved PXR Crystals
The present invention discloses a substantially pure PXR LBD polypeptide in crystalline form. In a preferred embodiment, exemplified in the Figures and Laboratory Examples, PXR is crystallized with bound ligand. Crystals are formed from PXR LBD polypeptides that are usually expressed by a cell culture, such as E. coli. BYomo-, iodo- and substitutions can be included during the preparation of crystal forms and can act as heavy atom substitutions in PXR ligands and in crystals of PXR and the PXR LBD. This method can be advantageous for the phasing of the crystal, which is a crucial, and sometimes limiting, step in solving the three-dimensional structure of a crystallized entity. Thus, the need for generating the heavy metal derivatives traditionally employed in crystallography might be eliminated. After the three- dimensional structure of a PXR or PXR LBD with or without a ligand bound is determined, the resultant three-dimensional structure can be used in computational methods to design synthetic ligands for PXR and other PXR polypeptide fragments. Further activity structure relationships can be determined through routine testing, using assays disclosed herein and known in the art.
VII. Uses of PXR Crystals and the Three-Dimensional Structure of the Ligand Binding Domain of PXR
VILA. Design and Development of PXR Modulators The knowledge of the structure of the hPXR ligand binding domain, an aspect of the present invention, provides a tool for investigating the mechanism of action of hPXR and other PXR polypeptides in a subject. For example, various computer models, as described herein, can predict the binding of various substrate molecules to the LBD of hPXR. Upon discovering that such binding in fact takes place, knowledge of the protein structure then allows design and synthesis of small molecules that mimic the functional binding of the substrate to the LBD of hPXR. This is the method of "rational" drug design, further described herein.
Use of the isolated and purified hPXR crystalline structure of the present invention in rational drug design is thus provided in accordance with the present invention. Additional rational drug design techniques are described in U.S. Patent Nos. 5,834,228 and 5,872,011 , incorporated herein in their entirety.
Thus, in addition to the compounds described herein, other sterically similar compounds can be formulated to mimic the key structural regions of PXRs in general, or of hPXR in particular. The generation of a structural functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art and described herein. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
VII.A.1. Rational Drug Design
The three-dimensional structure of ligand binding hPXR is unprecedented and will greatly aid in the development of new synthetic ligands for a PXR polypeptide, such as PXR agonists and antagonists, including those that bind exclusively to any one of the PXR orthologs. In addition, PXR is well suited to modern methods, including three-dimensional structure elucidation and combinatorial chemistry, such as those disclosed in U.S. Patent No. 5,463,564, incorporated herein by reference. Structure determination using X-ray crystallography is possible because of the solubility properties of the PXR orthologs. Computer programs that use crystallography data when practicing the present invention will enable the rational design of ligands to these receptors. Programs such as RASMOL (Biomolecular Structures Group, Glaxo Wellcome Research & Development Stevenage, Hertfordshire, UK Version 2.6, August 1995, Version 2.6.4, December 1998, Copyright © Roger Sayle 1992-1999) can be used with the atomic structural coordinates from crystals of the present invention, crystals generated by practicing the invention or crystals used to practice the invention by generating three-dimensional models and/or determining the structures involved in ligand binding. Computer programs such as those sold under the registered trademark INSIGHT II® and such as GRASP (Nicholls et aL, (1991) Proteins 11 : 281-96) allow for further manipulations and the ability to introduce new structures. In addition, high throughput binding and bioactivity assays can be devised using purified recombinant protein and modern reporter gene transcription assays known to those of skill in the art in order to refine the activity of a designed ligand.
A method of identifying modulators of the activity of a PXR polypeptide using rational drug design is thus provided in accordance with the present invention. The method comprises designing a potential modulator for a PXR polypeptide of the present invention that will form non-covalent bonds with amino acids in the ligand binding cavity based upon the crystalline structure of the hPXR LBD polypeptide; synthesizing the modulator; and determining whether the potential modulator modulates the activity of the PXR polypeptide. In a preferred embodiment, the modulator is designed for a hPXR polypeptide. Preferably, the hPXR polypeptide comprises the amino acid sequence of SEQ ID NO: 2, and the hPXR LBD comprises the amino acid sequence SEQ ID NO: 4. The determination of whether the modulator modulates the biological activity of a PXR polypeptide is made in accordance with the screening methods disclosed herein, or by other screening methods known to those of skill in the art. Modulators can be synthesized using techniques known to those of ordinary skill in the art.
In an alternative embodiment, a method of designing a modulator of a PXR polypeptide in accordance with the present invention is disclosed comprising: (a) selecting a candidate PXR ligand; (b) determining which amino acid or amino acids of a PXR polypeptide interact with the ligand using a three-dimensional model of a crystallized hPXR LBD; (c) identifying in a biological assay for PXR activity a degree to which the ligand modulates the activity of the PXR polypeptide,; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the PXR polypeptide and the ligand is predicted to be modulated by the chemical modification; (e) performing the chemical modification on the ligand to form a modified ligand; (f) contacting the modified ligand with the PXR polypeptide; (g) identifying in a biological assay for PXR activity a degree to which the modified ligand modulates the biological activity of the PXR polypeptide ; and (h) comparing the biological activity of the PXR polypeptide in the presence of modified ligand with the biological activity of the PXR polypeptide in the presence of the unmodified ligand, whereby a modulator of a PXR polypeptide is designed.
Vll.A.2. Methods for Using the hPXR LBD Structural Coordinates
For Molecular Design For the first time, the present invention permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including modulatory compounds, capable of binding to the ligand binding cavity or an accessory binding site of hPXR and the hPXR LBD, in whole or in part. Correspondingly, the present invention also provides for the application of similar techniques in the design of modulators of any PXR polypeptide.
In accordance with a preferred embodiment of the present invention, the structure coordinates of a crystalline hPXR LBD can be used to design compounds that bind to a PXR LBD (more preferably a hPXR LBD) and alter the properties of a PXR LBD (for example, the dimerization or ligand binding ability) in different ways. One aspect of the present invention provides for the design of compounds that act as competitive inhibitors of a PXR polypeptide by binding to all, or a portion of, the binding sites on a PXR LBD. The present invention also provides for the design of compounds that can act as uncompetitive inhibitors of a PXR LBD. These compounds can bind to all, or a portion of, an accessory binding site of a PXR that is already binding its ligand and can, therefore, be more potent and less non-specific than known competitive inhibitors that compete only for the PXR ligand binding cavity. Similarly, non-competitive inhibitors that bind to and inhibit PXR LBD activity, whether or not it is bound to another chemical entity, can be designed using the PXR LBD structure coordinates of this invention.
A second design approach is to probe a PXR or PXR LBD (preferably a hPXR or hPXR LBD) crystal with molecules comprising a variety of different chemical entities to determine optimal sites for interaction between candidate PXR or PXR LBD modulators and the polypeptide. For example, high resolution X-ray diffraction data collected from crystals saturated with solvent allows the determination of the site where each type of solvent molecule adheres. Small molecules that bind tightly to those sites can then be designed, synthesized and tested for their hPXR modulator activity.
Once a computationally-designed ligand is synthesized using the methods of the present invention or other methods known to those of skill in the art, assays can be used to establish its efficacy of the ligand as a modulator of PXR (preferably hPXR) activity. After such assays, the ligands can be further refined by generating intact PXR, or PXR LBD, crystals with a ligand bound to the PXR. The structure of the ligand can then be further refined using the chemical modification methods described herein and known to those of skill in the art, in order to improve the modulation activity or the binding affinity of the ligand. This process can lead to second generation ligands with improved properties.
Ligands also can be selected that modulate PXR responsive gene transcription by the method of altering the interaction of co-activators and co- repressors with their cognate PXR. For example, agonistic ligands can be selected that block or dissociate a co-repressor from interacting with the PXR, and/or that promote binding or association of a co-activator. Antagonistic ligands can be selected that block co-activator interaction and/or promote co- repressor interaction with a target receptor. Selection can be done via binding assays that screen for designed ligands having the desired modulatory properties. Preferably, interactions of a hPXR polypeptide are targeted. Suitable assays for screening that can be employed, mutatis mutandis in the present invention, are described in published PCT international applications WO 00/037077 and WO 00/025134, incorporated herein by reference in their entirety.
VII.A.3. Methods of Designing PXR LBD Modulator Compounds The design of candidate substances, also referred to as "compounds" or "candidate compounds", that bind to or inhibit PXR LBD-mediated activity according to the present invention generally involves consideration of two factors. First, the compound must be capable of physically and structurally associating with a PXR LBD. Non-covalent molecular interactions important in the association of a PXR LBD with its substrate include hydrogen bonding, van der Waals interactions and hydrophobic interactions.
Second, the compound must be able to assume a conformation that allows it to associate with a PXR LBD. Although certain portions of the compound will not directly participate in this association with a PXR LBD, those portions can still influence the overall conformation of the molecule. This, in turn, can have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., the ligand binding cavity or an accessory binding site of a PXR LBD, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with a PXR LBD.
The potential modulatory or binding effect of a chemical compound on a PXR LBD can be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques that employ the coordinates of a crystalline hPXR LBD polypeptide of the present invention. If the theoretical structure of the given compound suggests insufficient interaction and association between it and a PXR LBD, synthesis and testing of the compound is obviated. However, if computer modeling indicates a strong interaction, the molecule can then be synthesized and tested for its ability to bind and modulate the activity of a PXR LBD. In this manner, synthesis of unproductive or inoperative compounds can be avoided. A modulatory or other binding compound of a PXR LBD polypeptide
(preferably a hPXR LBD) can be computationally evaluated and designed via a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the individual binding sites or other areas of a crystalline hPXR LBD polypeptide of the present invention.
One of several methods can be used to screen chemical entities or fragments for their ability to associate with a PXR LBD and, more particularly, with the individual binding sites of a PXR LBD, such as ligand binding cavity or an accessory binding site. This process can begin by visual inspection of, for example, the ligand binding cavity on a computer screen based on the hPXR LBD atomic coordinates in Table 4. Selected fragments or chemical entities can then be positioned in a variety of orientations, or docked, within an individual binding site of a hPXR LBD as defined herein above. Docking can be accomplished using software programs such as those available under the tradenames QUANTA™ (Molecular Simulations Inc., San Diego, California) and SYBYL™ (Tripos, Inc., St. Louis, Missouri), followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARM (Brooks et al„ (1983) J. Comp. Chem., 8: 132) and AMBER 5 (Case et aL, (1997), AMBER 5, University of California, San Francisco; Pearlman et aL, (1995) Comput. Phys. Commun. 91 : 1-41).
Specialized computer programs can also assist in the process of selecting fragments or chemical entities. These include:
1. GRID™ program, version 17 (Goodford, (1985) J. Med. Chem. 28: 849-57), which is available from Molecular Discovery Ltd., Oxford, UK; 2. MCSS™ program (Miranker & Karplus, (1991) Proteins 11 : 29-34), which is available from Molecular Simulations, Inc., San Diego, California;
3. AUTODOCK™ 3.0 program (Goodsell & Olsen, (1990) Proteins 8: 195-202), which is available from the Scripps Research Institute, La Jolla, California; 4. DOCK™ 4.0 program (Kuntz et aL, (1992) J. Mol. Biol. 161 : 269-
88), which is available from the University of California, San Francisco, California; 5. FLEX-X™ program (See, Rarey et aL, (1996) J. Comput. Aid. Mol.
Des. 10:41-54), which is available from Tripos, Inc., St. Louis, Missouri;
6. MVP program (Lambert, (1997) in Practical Application of Computer-Aided Drug Design, (Charifson, ed.) Marcel-Dekker, New York, pp. 243-303); and
7. LUDI™ program (Bohm, (1992) J. Comput. Aid. Mol. Des., 6: 61- 78), which is available from Molecular Simulations, Inc., San Diego, California.
Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or modulator. Assembly can proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of a hPXR LBD. Manual model building using software such as QUANTA™ or SYBYL™ typically follows.
Useful programs to aid one of ordinary skill in the art in connecting the individual chemical entities or fragments include:
1. CAVEAT™ program (Bartlett et aL, (1989) Special Pub., Royal Chem. Soc. 78: 182-96), which is available from the University of California, Berkeley, California;
2. 3D Database systems, such as MACCS-3D™ system program, which is available from MDL Information Systems, San Leandro, California.
This area is reviewed in Martin, (1992) J. Med. Chem. 35: 2145-54; and
3. HOOK™ program (Eisen et aL, (1994). Proteins 19: 199-221), which is available from Molecular Simulations, Inc., San Diego, California.
Instead of proceeding to build a PXR LBD modulator (preferably a hPXR LBD modulator) in a step-wise fashion one fragment or chemical entity at a time as described above, modulatory or other binding compounds can be designed as a whole or de novo using the structural coordinates of a crystalline hPXR LBD polypeptide of the present invention and either an empty binding site or optionally including some portion(s) of a known modulator(s). Applicable methods can employ the following software programs: 1. LUDi™ program (Bohm, (1992) J. Comput. Aid. Mol. Des., 6: 61- 78), which is available from Molecular Simulations, Inc., San Diego, California;
2. LEGEND™ program (Nishibata & Itai, (1991 ) Tetrahedron 47: 8985); and 3. LEAPFROG™, which is available from Tripos Associates, St. Louis,
Missouri.
Other molecular modeling techniques can also be employed in accordance with this invention. See, e.g., Cohen et a , (1990) J. Med. Chem. 33: 883-94. See also, Navia & Murcko, (1992) Curr. Opin. Struc. Biol. 2: 202- 10; U.S. Patent No. 6,008,033, herein incorporated by reference.
Once a compound has been designed or selected by the above methods, the efficiency with which that compound can bind to a PXR LBD can be tested and optimized by computational evaluation. By way of particular example, a compound that has been designed or selected to function as a hPXR LBD modulator should also preferably traverse a volume not overlapping that occupied by the binding site when it is bound to its native ligand. Additionally, an effective PXR LBD modulator should preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient PXR LBD modulators should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, and preferably, not greater than 7 kcal/mole. It is possible for PXR LBD modulators to interact with the polypeptide in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free compound and the average energy of the conformations observed when the modulator binds to the polypeptide.
A compound designed or selected as binding to a PXR polypeptide (preferably a hPXR LBD polypeptide) can be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target polypeptide. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the modulator and the polypeptide when the modulator is bound to a PXR LBD preferably make a neutral or favorable contribution to the enthalpy of binding. Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction. Examples of programs designed for such uses include:
1. Gaussian 98™, which is available from Gaussian, Inc., Pittsburgh, Pennsylvania; 2. AMBER™ program, version 6.0, which is available from the
University of California at San Francisco;
3. QUANTA™ program, which is available from Molecular Simulations, Inc., San Diego, California;
4. CHARMm® program, which is available from Molecular Simulations, Inc., San Diego, California; and
4. INSIGHT II® program, which is available from Molecular Simulations, Inc., San Diego, California.
These programs can be implemented using a suitable computer system. Other hardware systems and software packages will be apparent to those skilled in the art after review of the disclosure of the present invention presented herein.
Once a PXR LBD modulating compound has been optimally selected or designed, as described above, substitutions can then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds can then be analyzed for efficiency of fit to a PXR LBD binding site using the same computer-based approaches described in detail above. VII.B. Distinguishing Between PXR Isoforms and Orthologs The present invention discloses the ability to generate new synthetic ligands to distinguish between PXR isoforms and orthologs. As described herein, computer-designed ligands can be generated that distinguish between binding isoforms and orthologs, thereby allowing the generation of species specific, tissue specific or function specific ligands. The atomic structural coordinates disclosed in the present invention reveal structural details unique to hPXR. These structural details can be exploited when a novel ligand is designed using the methods of the present invention or other ligand design methods known in the art. The structural features that differentiate a hPXR from a mouse PXR and one isoform from another can be targeted in ligand design. Thus, for example, a ligand can be designed that will recognize a particular PXR isoform or ortholog, while not interacting with other PXR isoforms or orthologs, or even with moieties having similar structural features. Prior to the disclosure of the present invention, a detailed understanding of the differences between PXR orthoforms and the ability to target a particular PXR isoform or ortholog was unattainable.
VII.C. Method of Screening for Chemical and Biological Modulators of the Biological Activity of hPXR
A candidate substance identified according to a screening assay of the present invention has an ability to modulate the biological activity of a PXR polypeptide or a PXR LBD polypeptide. In a preferred embodiment, such a candidate compound can have utility in the treatment of disorders and conditions associated with the biological activity of a hPXR or a hPXR LBD polypeptide, including, but not limited to, hPXR and hPXR LBD-based drug- drug interactions, hPXR and hPXR LBD-based drug resistance, individualized treatment of disease due to polymorphisms, liver cholestasis and other degenerative liver disorders. In a cell-free system, the method comprises the steps of establishing a control system comprising a hPXR polypeptide and a ligand which is capable of binding to the polypeptide; establishing a test system comprising a hPXR polypeptide, the ligand, and a candidate compound; and determining whether the candidate compound modulates the activity of the polypeptide by comparison of the test and control systems. A representative ligand comprises a fatty acid or other small molecule, and in this embodiment, the biological activity or property screened includes binding affinity.
In another embodiment of the invention, a form of a hPXR polypeptide or a catalytic or immunogenic fragment or oligopeptide thereof, can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such a screening can be affixed to a solid support. The formation of binding complexes, between a hPXR polypeptide and the agent being tested, will be detected. In a preferred embodiment, the hPXR polypeptide has an amino acid sequence of SEQ ID NO: 2. When a hPXR LBD polypeptide is employed, a preferred embodiment will include a hPXR polypeptide having the amino acid sequence of SEQ ID NO: 4.
Another technique for drug screening which can be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO 84/03564, herein incorporated by reference. In this method, as applied to a polypeptide of the present invention, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with the polypeptide, or fragments thereof. Bound polypeptide is then detected by methods well known to those of skill in the art. The polypeptide can also be placed directly onto plates for use in the aforementioned drug screening techniques.
In yet another embodiment, a method of screening for a modulator of a hPXR polypeptide or a hPXR LBD polypeptide comprises: providing a library of test samples; contacting a hPXR polypeptide or a hPXR LBD polypeptide with each test sample; detecting an interaction between a test sample and a hPXR polypeptide or a hPXR LBD polypeptide; identifying a test sample that interacts with a hPXR polypeptide or a hPXR LBD polypeptide; and isolating a test sample that interacts with a hPXR polypeptide or a hPXR LBD polypeptide.
In each of the foregoing embodiments, an interaction can be detected spectrophotometrically, radiologically or immunologically. An interaction between a hPXR polypeptide or a hPXR LBD polypeptide and a test sample can also be quantified using methodology known to those of skill in the art. In another embodiment, the hPXR polypeptide and the hPXR LBD is in crystalline form.
In accordance with the present invention there is also provided a rapid and high throughput screening method that relies on the methods described above. This screening method comprises separately contacting each of a plurality of substantially identical samples with a hPXR polypeptide or a hPXR LBD and detecting a resulting binding complex. In such a screening method the plurality of samples preferably comprises more than about 104 samples, or more preferably comprises more than about 5 x 104 samples.
VII.D. Method of Identifying Compounds Which Inhibit Ligand Binding Using the crystal structures and ligand orientations, disclosed for the first time in the present invention, it is possible to design test compounds that inhibit binding of ligands normally bound by a PXR polypeptide.
In one aspect of the present invention, an assay method for identifying a compound that inhibits binding of a ligand to a PXR polypeptide is disclosed. A known ligand of hPXR can be used in the assay method as the ligand against which the inhibition by a test compound is gauged. SR12813 is a preferred ligand in the assay method. The method comprises (a) incubating a PXR polypeptide with a ligand in the presence of a test inhibitor compound; (b) determining an amount of ligand that is bound to the PXR polypeptide, wherein decreased binding of ligand to the PXR polypeptide in the presence of the test inhibitor compound relative to binding in the absence of the test inhibitor compound is indicative of inhibition; and (c) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed. Preferably, the ligand is SR12813. ln another aspect of the present invention, the disclosed assay method can be used in the structural refinement of candidate PXR inhibitors. For example, multiple rounds of optimization can be followed by gradual structural changes in a strategy of inhibitor design. A strategy such as this is made possible by the disclosure of the coordinates of the hPXR LBD and the disclosure of the orientation of a ligand of PXR, SR12813.
VILE. Design of PXR Isoform and Ortholog Modulators
The hPXR crystal structure of the present invention can be used to generate modulators of other PXR isoforms or orthologs, such as mouse PXR (Swiss-Prot Accession No. 054915). Analysis of the disclosed crystal structure can provide a guide for designing modulators of PXR isoforms or orthologs. Purely for purposes of explanation, the development of a mouse PXR modulator will be considered herein below. It will be apparent to those of skill in the art, and explicitly noted here, that the following discussion will be applicable mutatis mutandis to PXR isoforms and other PXR orthologs, including rat PXR (Swiss-Prot Accession No. Q9R1A7).
Absent the crystal structure of the present invention, researchers would be required to design mouse PXR modulators de novo. The present invention, however, addresses this problem by providing insights into the binding cavity of hPXR, which can be extended, due to significant structural similarity with other PXR isoforms and orthologs, to the binding cavity of, for example, mouse PXR. An evaluation of the binding cavity of hPXR indicates that a potential mouse PXR modulator would meet a broad set of general criteria. Broadly, it can be stated that, based on the crystal structure of hPXR, a potent mouse PXR ligand would require several general features including: (a) a hydrophobic binding cavity; and (b) the ability to adopt a conformation that is complementary to the shape of the binding cavity.
Using the discerned structural similarities and differences between PXR isoforms and orthologs, as represented and predicted based on the crystal structure of the present invention and homology models, a mouse PXR modulator can be designed. For example, based on an evaluation of a homology model of mouse PXR, which is derived from the hPXR crystal structure, it is expected that a potent ligand would need similar characteristics as listed above for a compound recognized by hPXR. Additional modifications can be included, based on the disclosed structure, which are predicted to further define a modulator specific for mouse PXR over other orthologs. Thus, the disclosed crystal structure of hPXR can be useful when designing modulators of mouse PXR and other orthologs and isoforms.
An additional aspect of the present invention, then, is to provide a technique for predicting the differences in the metabolism of a drug between mice (or other species of interest) and humans. As described further herein, the "humanization" of the mouse PXR via four point mutations demonstrate that the structures disclosed herein can be employed as a predictor of mouse PXR function. A mouse PXR model, based on the structure of human PXR, as disclosed herein and co-crystallized and refined with known pharmacophores, can provide needed insight into PXR activity and mechanisms. Notably, a "humanized" mouse PXR can provide valuable insight into the metabolism of drugs in humans. Particularly, the ability to generate such a system offers the potential to generate PXR drug metabolism data in a mouse system that is more predictive for human systems, than PXR metabolic data generated based only on a mouse model. A "humanized" mouse PXR also facilitates insights into existent drug metabolism, and the development of human drugs and therapeutics with greater efficacies, using a convenient mouse model system.
VilL Design, Preparation and Structural Analysis of hPXR and hPXR LBD Mutants and Structural Equivalents
The present invention provides for the generation of PXR and PXR mutants (preferably hPXR and hPXR LBD mutants), and the ability to solve the crystal structures of those that crystallize. More particularly, through the provision of the three-dimensional structure of a hPXR LBD, desirable sites for mutation can be identified, based on analysis of the three-dimensional hPXR LBD structure coordinates provided herein. The structure coordinates of a hPXR LBD provided in accordance with the present invention also facilitate the identification of related proteins or enzymes analogous to hPXR in function, structure or both, (for example, a mouse PXR), which can lead to novel therapeutic modes for treating or preventing a range of disease states.
VINA Sterically Similar Compounds
A further aspect of the present invention is that sterically similar compounds can be formulated to mimic the key portions of a PXR LBD structure. Such compounds are functional equivalents. The generation of a structural functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art and described herein. Modeling and chemical design of PXR and PXR LBD structural equivalents can be based on the structure coordinates of a crystalline hPXR LBD polypeptide of the present invention. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
Vlll.B. PXR Polypeptides
The generation of chimeric PXR polypeptides is also an aspect of the present invention. Such a chimeric polypeptide can comprise a PXR LBD polypeptide or a portion of a PXR LBD, (e.g. a hPXR LBD) which is fused to a candidate polypeptide or a suitable region of the candidate polypeptide, for example a PXR expressed in mouse or other species. Throughout the present disclosure it is intended that the term "mutant" encompass not only mutants of a PXR LBD polypeptide but chimeric proteins generated using a PXR LBD as well. It is thus intended that the following discussion of mutant PXR LBDs apply mutatis mutandis to chimeric PXR and PXR LBD polypeptides and to structural equivalents thereof.
In accordance with the present invention, a mutation can be directed to a particular site or combination of sites of a wild-type PXR LBD. For example, an accessory binding site or the binding cavity can be chosen for mutagenesis. Similarly, a residue having a location on, at or near the surface of the polypeptide can be replaced, resulting in an altered surface charge of one or more charge units, as compared to the wild-type PXR and PXR LBD. Alternatively, an amino acid residue in a PXR or a PXR LBD can be chosen for replacement based on its hydrophilic or hydrophobic characteristics. Such mutants can be characterized by any one of several different properties as compared with the wild-type PXR LBD. For example, such mutants can have an altered surface charge of one or more charge units, or can have an increase in overall stability. Other mutants can have altered substrate specificity in comparison with, or a higher specific activity than, a wild-type PXR or PXR LBD.
PXR and PXR LBD mutants of the present invention can be generated in a number of ways. For example, the wild-type sequence of a PXR or a PXR LBD can be mutated at those sites identified using this invention as desirable for mutation, by means of oligonucleotide-directed mutagenesis or other conventional methods, such as deletion. Alternatively, mutants of a PXR or a PXR LBD can be generated by the site-specific replacement of a particular amino acid with an unnaturally occurring amino acid. In addition, PXR or PXR LBD mutants can be generated through replacement of an amino acid residue, for example, a particular cysteine or methionine residue, with selenocysteine or selenomethionine. This can be achieved by growing a host organism capable of expressing either the wild-type or mutant polypeptide on a growth medium depleted of either natural cysteine or methionine (or both) but enriched in selenocysteine or selenomethionine (or both). Mutations can be introduced into a DNA sequence coding for a PXR or a PXR LBD using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites. Mutations can be generated in the full-length DNA sequence of a PXR or a PXR LBD or in any sequence coding for polypeptide fragments of a PXR or a PXR LBD. According to the present invention, a mutated PXR or PXR LBD DNA sequence produced by the methods described above, or any alternative methods known in the art, can be expressed using an expression vector. An expression vector, as is well known to those of skill in the art, typically includes elements that permit autonomous replication in a host cell independent of the host genome, and one or more phenotypic markers for selection purposes. Either prior to or after insertion of the DNA sequences surrounding the desired PXR or PXR LBD mutant coding sequence, an expression vector also will include control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes and a signal for termination. In some embodiments, where secretion of the produced mutant is desired, nucleotides encoding a "signal sequence" can be inserted prior to a PXR or a PXR LBD mutant coding sequence. For expression under the direction of the control sequences, a desired DNA sequence must be operatively linked to the control sequences; that is, the sequence must have an appropriate start signal in front of the DNA sequence encoding the PXR or PXR LBD mutant, and the correct reading frame to permit expression of that sequence under the control of the control sequences and production of the desired product encoded by that PXR or PXR LBD sequence must be maintained.
Any of a wide variety of well-known available expression vectors can be useful to express a mutated PXR or PXR LBD coding sequences of this invention and generated as described in Laboratory Example 3. These expression vectors can be used in the techniques disclosed in Laboratory Examples 1 and 3 and can include, for example, vectors comprising segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40, known bacterial plasmids, e.g., plasmids from E. coli including col E1 , pCR1 , pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs, e.g., the numerous derivatives of phage λ, e.g., NM 989, and other DNA phages, e.g., M13 and filamentous single stranded DNA phages, yeast plasmids and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences. In a preferred embodiment of this invention, the E. coli vector pRSETA, including a T7-based expression system, is employed. ln addition, any of a wide variety of expression control sequences — sequences that control the expression of a DNA sequence when operatively linked to it — can be used in these vectors to express the mutated DNA sequences according to this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40 for animal cells, the lac system, the trp system the TAC or TRC system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, all for E. coli, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors for yeast, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
A wide variety of hosts are also useful for producing mutated hPXR and hPXR LBD polypeptides according to this invention. These hosts include, for example, bacteria, such as E. coli, Bacillus and Streptomyces, fungi, such as yeasts, and animal cells, such as CHO and COS-1 cells, plant cells, insect cells, such as Sf9 cells, and transgenic host cells.
It should be understood that not all expression vectors and expression systems function in the same way to express mutated DNA sequences of this invention, and to produce modified PXR and PXR LBD polypeptides or PXR or PXR LBD mutants. Neither do all hosts function equally well with the same expression system. One of skill in the art can, however, make a selection among these vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, an important consideration in selecting a vector will be the ability of the vector to replicate in a given host. The copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the system, its controllability and its compatibility with the DNA sequence encoding a modified PXR or PXR LBD polypeptide of this invention, with particular regard to the formation of potential secondary and tertiary structures.
Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of a modified PXR or PXR LBD to them, their ability to express mature products, their ability to fold proteins correctly, their fermentation requirements, the ease of purification of a modified PXR or PXR LBD and safety. Within these parameters, one of skill in the art can select various vector/expression control system/host combinations that will produce useful amounts of a mutant PXR or PXR LBD. A mutant PXR or PXR LBD produced in these systems can be purified by a variety of conventional steps and strategies, including those used to purify the wild-type PXR or PXR LBD.
Once a PXR LBD mutation(s) has been generated in the desired location, such as an ligand binding or dimerization site, the mutants can be tested for any one of several properties of interest. For example, mutants can be screened for an altered charge at physiological pH. This is determined by measuring the mutant PXR or PXR LBD isoelectric point (pi) and comparing the observed value with that of the wild-type parent. Isoelectric point can be measured by gel-electrophoresis according to the method of Wellner (Wellner, (1971) Anal. Chem. 43: 597). A mutant PXR or PXR LBD polypeptide containing a replacement amino acid located at the surface of the enzyme, as provided by the structural information of this invention, can lead to an altered surface charge and an altered pi.
Vlll.C. Generation of an Engineered PXR or PXR LBD Mutant In another aspect of the present invention, a unique PXR or PXR LBD polypeptide can be generated. Such a mutant can facilitate purification and can facilitate the study of the ligand binding abilities of a PXR polypeptide.
As used in the following discussion, the terms "engineered PXR",
"engineered PXR LDB", "PXR mutant", and "PXR LBD mutant" refers to polypeptides having amino acid sequences which contain at least one mutation in the wild-type sequence. The terms also refer to PXR and PXR
LBD polypeptides which are capable of exerting a biological effect in that they comprise all or a part of the amino acid sequence of an engineered PXR or PXR LBD mutant polypeptide of the present invention, or cross-react with antibodies raised against an engineered PXR or PXR LBD mutant polypeptide, or retain all or some or an enhanced degree of the biological activity of the engineered PXR or PXR LBD mutant amino acid sequence or protein. Such biological activity can include lipid binding in general, and fatty acid binding in particular.
The terms "engineered PXR LBD" and "PXR LBD mutant" also includes analogs of an engineered PXR LBD or PXR LBD mutant polypeptide. By "analog" is intended that a DNA or polypeptide sequence can contain alterations relative to the sequences disclosed herein, yet retain all or some or an enhanced degree of the biological activity of those sequences. Analogs can be derived from genomic nucleotide sequences or from other organisms, or can be created synthetically. Those of skill in the art will appreciate that other analogs, as yet undisclosed or undiscovered, can be used to design and/or construct PXR LBD or PXR LBD mutant analogs. There is no need for an engineered PXR LBD or PXR LBD mutant polypeptide to comprise all or substantially all of the amino acid sequence of SEQ ID NOs: 2 or 4. Shorter or longer sequences are anticipated to be of use in the invention; shorter sequences are herein referred to as "segments". Thus, the terms "engineered PXR LBD" and "PXR LBD mutant" also includes fusion, chimeric or recombinant engineered PXR LBD or PXR LBD mutant polypeptides and proteins comprising sequences of the present invention. Methods of preparing such proteins are disclosed herein above and are known in the art.
VIII.D. Sequence Similarity and Identity
As used herein, the term "substantially similar" means that a particular sequence varies from nucleic acid sequence of SEQ ID NOs: 1 or 3, or the amino acid sequence of SEQ ID NOs: 2 or 4 by one or more deletions, substitutions, or additions, the net effect of which is to retain at least some of biological activity of the natural gene, gene product, or sequence. Such sequences include "mutant" or "polymorphic" sequences, or sequences in which the biological activity and/or the physical properties are altered to some degree but retains at least some or an enhanced degree of the original biological activity and/or physical properties. In determining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequences or substitution of equivalent amino acids to create biologically functional equivalents.
VIII. D.1. Sequences That are Substantially Identical to an
Engineered PXR or PXR LBD Mutant Sequence of the Present Invention Nucleic acids that are substantially identical to a nucleic acid sequence of an engineered PXR or PXR LBD mutant of the present invention, e.g. allelic variants, genetically altered versions of the gene, etc., bind to an engineered PXR or PXR LBD mutant sequence under stringent hybridization conditions. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes can be any species, e.g. primate species; rodents, such as rats and mice, canines, felines, bovines, equines, yeast, nematodes, etc.
Between mammalian species, e.g. human and mouse, homologs have substantial sequence similarity, i.e. at least 75% sequence identity between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which can be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 nucleotides(nt) long, more usually at least about 30 nt long, and can extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., (1990) J. Mol. Biol. 215: 403-10. Percent identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group. The GAP program utilizes the alignment method of Needleman et aL, (1970) J. Mol. Biol. 48: 443, as revised by Smith et al., (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps. See, e.g., Schwartz et aL, eds., (1979), Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 357-358, and Gribskov et al., (1986) Nucl. Acids. Res. 14: 6745.
The term "similarity" is contrasted with the term "identity". Similarity is defined as above; "identity", however, means a nucleic acid or amino acid sequence having the same amino acid at the same relative position in a given family member of a gene family. Homology and similarity are generally viewed as broader terms than the term identity. Biochemically similar amino acids, for example leucine/isoleucine or glutamate/aspartate, can be present at the same position — these are not identical per se, but are biochemically "similar." As disclosed herein, these are referred to as conservative differences or conservative substitutions. This differs from a conservative mutation at the DNA level, which changes the nucleotide sequence without making a change in the encoded amino acid, e.g. TCC to TCA, both of which encode serine.
As used herein, DNA analog sequences are "substantially identical" to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived from coding regions of the nucleic acid sequence shown in SEQ ID NOs: 1 or 3; or (b) the DNA analog sequence is capable of hybridization with DNA sequences of (a) under stringent conditions and which encode a biologically active hPXR or hPXR LBD gene product; or (c) the DNA sequences are degenerate as a result of alternative genetic code to the DNA analog sequences defined in (a) and/or (b). Substantially identical analog proteins and nucleic acids will have between about 70% and 80%, preferably between about 81% to about 90% or even more preferably between about 91% and 99% sequence identity with the corresponding sequence of the native protein or nucleic acid. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.
As used herein, "stringent conditions" means conditions of high stringency, for example 6X SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 μg/ml salmon sperm DNA and 15% formamide at 68°C. For the purposes of specifying additional conditions of high stringency, preferred conditions are salt concentration of about 200 mM and temperature of about 45°C. One example of such stringent conditions is hybridization at 4X SSC, at 65°C, followed by a washing in 0.1X SSC at 65°C for one hour. Another exemplary stringent hybridization scheme uses 50% formamide, 4X SSC at 42°C.
In contrast, nucleic acids having sequence similarity are detected by hybridization under lower stringency conditions. Thus, sequence identity can be determined by hybridization under lower stringency conditions, for example, at 50°C or higher and 0.1X SSC (9 mM NaCI/0.9 mM sodium citrate) and the sequences will remain bound when subjected to washing at 55°C in 1X SSC.
VIII.D.2. Complementarity and Hybridization to an Engineered
PXR or PXR LBD Mutant Sequence As used herein, the term "complementary sequences" means nucleic acid sequences which are base-paired according to the standard Watson- Crick complementarity rules. The present invention also encompasses the use of nucleotide segments that are complementary to the sequences of the present invention.
Hybridization can also be used for assessing complementary sequences and/or isolating complementary nucleotide sequences. As discussed above, nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of about 30°C, typically in excess of about 37°C, and preferably in excess of about 45°C. Stringent salt conditions will ordinarily be less than about 1 ,000 mM, typically less than about 500 mM, and preferably less than about 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur & Davidson, (1968) J. Mol. Biol. 31 : 349-70. Determining appropriate hybridization conditions to identify and/or isolate sequences containing high levels of homology is well known in the art. See, e.g., Sambrook et aL, (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York.
VIII.D.3. Functional Equivalents of an Engineered PXR or PXR LBD Mutant Nucleic Acid Sequence of the Present Invention
As used herein, the term "functionally equivalent codon" is used to refer to codons that encode the same amino acid, such as the ACG and AGU codons for serine. Human PXR or hPXR LBD-encoding nucleic acid sequences comprising SEQ ID NOs: 1 and 3, which have functionally equivalent codons, are covered by the present invention. Thus, when referring to the sequence example presented in SEQ ID NOs: 1 and 3, applicants contemplate substitution of functionally equivalent codons into the sequence example of SEQ ID NOs: 1 and 3. Thus, applicants are in possession of amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience.
It will also be understood by those of skill in the art that amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' nucleic acid sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence retains biological protein activity where polypeptide expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which can, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or can include various internal sequences, i.e., introns, which are known to occur within genes.
VIII.D.4. Biological Equivalents
The present invention envisions and includes biological equivalents of an engineered PXR or PXR LBD mutant polypeptide of the present invention. The term "biological equivalent" refers to proteins having amino acid sequences which are substantially identical to the amino acid sequence of an engineered PXR LBD mutant of the present invention and which are capable of exerting a biological effect in that they are capable of binding DNA moieties or cross-reacting with anti-PXR or PXR LBD mutant antibodies raised against an engineered mutant PXR or PXR LBD polypeptide of the present invention. For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive capacity with, for example, structures in the nucleus of a cell. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or the nucleic acid sequence encoding it) to obtain a protein with the same, enhanced, or antagonistic properties. Such properties can be achieved by interaction with the normal targets of the protein, but this need not be the case, and the biological activity of the invention is not limited to a particular mechanism of action. It is thus in accordance with the present invention that various changes can be made in the amino acid sequence of an engineered PXR or PXR LBD mutant polypeptide of the present invention or its underlying nucleic acid sequence without appreciable loss of biological utility or activity.
Biologically equivalent polypeptides, as used herein, are polypeptides in which certain, but not most or all, of the amino acids can be substituted. Thus, when referring to the sequence examples presented in SEQ ID NOs: 1 and 3, applicants envision substitution of codons that encode biologically equivalent amino acids, as described herein, into the sequence example of SEQ ID NOs: 1 and 3, respectively. Thus, applicants are in possession of amino acid and nucleic acids sequences which include such substitutions but which are not set forth herein in their entirety for convenience. Alternatively, functionally equivalent proteins or peptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged, e.g. substitution of lie for Leu. Changes designed by man can be introduced through the application of site- directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test an engineered PXR or PXR LBD mutant polypeptide of the present invention in order to modulate DNA-binding, lipid- binding or other activity, at the molecular level.
Amino acid substitutions, such as-those which might be employed in modifying an engineered PXR or PXR LBD mutant polypeptide of the present invention are generally, but not necessarily, based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents. Other biologically functionally equivalent changes will be appreciated by those of skill in the art. It is implicit in the above discussion, however, that one of skill in the art can appreciate that a radical, rather than a conservative substitution is warranted in a given situation. Non-conservative substitutions in engineered mutant PXR or PXR LBD polypeptides of the present invention are also an aspect of the present invention.
In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, (1982), J. Mol. Biol. 157: 105-132, incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 of the original value is preferred, those which are within ±1 of the original value are particularly preferred, and those within ±0.5 of the original value are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101 , incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. As detailed in U.S. Patent No. 4,554,101 , the following hydrophilicity values have been assigned to amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartate (+ 3.0±1); glutamate (+ 3.0±1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5±1 ); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (- 1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 of the original value is preferred, those which are within ±1 of the original value are particularly preferred, and those within ±0.5 of the original value are even more particularly preferred.
While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes can be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons can code for the same amino acid.
Thus, it will also be understood that this invention is not limited to the particular amino acid and nucleic acid sequences of SEQ ID NOs: 1-4. Recombinant vectors and isolated DNA segments can therefore variously include an engineered hPXR or hPXR LBD mutant polypeptide-encoding region itself, include coding regions bearing selected alterations or modifications in the basic coding region, or include larger polypeptides which nevertheless comprise a hPXR or hPXR LBD mutant polypeptide-encoding regions or can encode biologically functional equivalent proteins or polypeptides which have variant amino acid sequences. Biological activity of an engineered hPXR or hPXR LBD mutant polypeptide can be determined, for example, by Iipid-binding assays known to those of skill in the art. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters, enhancers, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length can be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments can be prepared which include a short stretch complementary to a nucleic acid sequence set forth in SEQ ID NOs: 1 and 3, such as about 10 nucleotides, and which are up to 10,000 or 5,000 base pairs in length. DNA segments with total lengths of about 4,000, 3,000, 2,000, 1 ,000, 500, 200, 100, and about 50 base pairs in length are also useful. The DNA segments of the present invention encompass biologically functional equivalents of engineered PXR or PXR LBD mutant polypeptides. Such sequences can rise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or polypeptides can be created via the application of recombinant DNA technology, in which changes in the protein structure can be engineered, based on considerations of the properties of the amino acids being exchanged. Changes can be introduced through the application of site- directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test variants of an engineered PXR or PXR LBD mutant of the present invention in order to examine the degree of lipid- binding activity, or other activity at the molecular level. Various site-directed mutagenesis techniques are known to those of skill in the art and can be employed in the present invention.
The invention further encompasses fusion proteins and peptides wherein an engineered PXR or PXR LBD mutant coding region of the present invention is aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes.
Recombinant vectors form important further aspects of the present invention. Particularly useful vectors are those in which the coding portion of the DNA segment is positioned under the control of a promoter. The promoter can be that naturally associated with a PXR gene, as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology and/or other methods known in the art, in conjunction with the compositions disclosed herein.
In other embodiments, certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is a promoter that is not normally associated with a PXR gene in its natural environment. Such promoters can include promoters isolated from bacterial, viral, eukaryotic, or mammalian cells. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology (See, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, specifically incorporated herein by reference). The promoters employed can be constitutive or inducible and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. One preferred promoter system contemplated for use in high-level expression is a T7 promoter-based system.
IX. Uses of PXR Mutants
The PXR mutants disclosed herein have a variety of applications, including in the screening of components for PXR/SXR activation using the cell-free reporter gene assay methods disclosed herein above, and using whole animal models. The PXR mutants can also be used in cell-free, cell- based and whole animal assay methods for bioavailability of compounds and for toxicology analysis. Additionally, PXR mutants can be employed in crystallizations, screening for changes in ligand activation, screening for species-specific changes in ligand activation and screening for changes in oligomerization state both with and without ligand.
IX.A. Evaluating a Candidate Therapeutic Agent in Humans Using a
Mouse Model System
A mutant PXR comprising a "humanized" mouse can find particular utility as a model for the study of drug metabolism in humans. Such a model can be more informative regarding drug metabolism in humans than studies using unmodified mouse PXR as a predictive model for human drug metabolism. It also offers the convenience and advantages of employing a mouse model system to study a human protein.
In a preferred embodiment, a method of evaluating a candidate therapeutic agent in humans using a mouse model system comprises the following steps. Atomic coordinates of a human PXR ligand binding domain are provided to a computerized modeling system. Preferred coordinates are supplied in Table 4 of the present disclosure. Based on the human PXR atomic coordinates, a candidate therapeutic agent that fits spatially into a binding cavity or on the surface of a human PXR ligand binding domain is modeled. Modeling of a candidate therapeutic agent can conveniently employ a computer-based approach. Computer programs, such as the INSIGHT II® program (Molecular Simulations, Inc., San Diego, California) can be employed to create three-dimensional structures of candidate agents. After a candidate therapeutic agent structure is generated, a computer program (which can comprise the appropriate design and structure evaluation modules), can be employed to preliminarily evaluate the candidate agent for use as a therapeutic. The candidate agent can be docked with a site on the PXR, for example, and the efficiency of the interaction can be computationally evaluated for various physical properties, such as steric considerations and efficiency of binding.
A mouse PXR polypeptide is provided, and one or more mutations to be introduced into the mouse PXR amino acid sequence of the mouse PXR polypeptide are selected. The mutations are preferably selected so as to alter the mouse PXR polypeptide to be similar to a human PXR polypeptide. Although any mutations can be selected, preferred mutations are selected from the group consisting of an arginine to leucine substitution at residue 203 of the mouse PXR polypeptide, a protein to serine substitution at residue 205 of the mouse PXR polypeptide, a glutamine to histidine substitution at residue 404 of the mouse PXR polypeptide and a glutamine to arginine substitution at residue 407 of the mouse PXR polypeptide.
The selected one or more mutations is/are introduced into a mouse PXR polypeptide and a mutant mouse PXR polypeptide comprising the selected one or more mutations selected is provided. The mutant mouse PXR polypeptide is expressed and purified using methods that are disclosed herein and will be apparent to those of skill in the art, upon consideration of the present disclosure. The effect of the candidate therapeutic agent on the mutant mouse
PXR polypeptide is determined. The effect can be determined by employing any appropriate reporter system. For example, the biological activity of a PXR polypeptide can be employed to determine an interaction.
The potential of the candidate therapeutic agent for use in humans is evaluated based on the effect of the candidate therapeutic agent on the mutant mouse PXR polypeptide. Thus, in one aspect of the present invention, the suitability of a candidate therapeutic for human disorders and conditions is conveniently assessed in a mouse model system. This system offers the ability to assess the biological response of a candidate therapeutic in vivo, which can add an additional degree of confidence to a conclusion made regarding the candidate therapeutic.
X. The Role of the Three-Dimensional Structure of the hPXR LDB in Solving Additional PXR Crystals Because polypeptides can crystallize in more than one crystal form, the structural coordinates of a hPXR LBD, or portions thereof, as provided by the present invention, are particularly useful in solving the structure of other crystal forms of hPXR and the crystalline forms of other PXRs. The coordinates provided in the present invention can also be used to solve the structure of PXR or PXR LBD mutants (such as those described in Section VIII above), PXR LDB co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of PXR.
IX.A. Determining the Three-Dimensional Structure of a Polypeptide
Using the Three-Dimensional Structure of the hPXR LBD as a Template in Molecular Replacement One method that can be employed for the purpose of solving additional PXR crystal structures is molecular replacement. See generally, Rossmann, ed, (1972) The Molecular Replacement Method, Gordon & Breach, New York. In the molecular replacement method, the unknown crystal structure, whether it is another crystal form of a PXR or a PXR LBD, (i.e. a PXR or a PXR LBD mutant), or a PXR or a PXR LBD polypeptide complexed with another compound (a "co-complex"), or the crystal of some other protein with significant amino acid sequence homology to any functional region of the a hPXR LBD, can be determined using the hPXR LBD structure coordinates provided in Table 4. This method provides an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
In addition, in accordance with this invention, PXR or PXR mutants (preferably hPXR or hPXR LBD mutants) can be crystallized in complex with known modulators. The crystal structures of a series of such complexes can then be solved by molecular replacement and compared with that of wild-type hPXR or the wild-type hPXR LBD. Potential sites for modification within the various binding sites of the enzyme can thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between the hPXR LBD and a chemical entity or compound.
All of the complexes referred to in the present disclosure can be studied using X-ray diffraction techniques (See, e.g., Blundell & Johnson (1985) Method. Enzymol., 114A & 115B, (Wyckoff et al., eds.), Academic Press) and can be refined using computer software, such as the X-PLOR™ program (Brϋnger, (1992) X-PLOR, Version 3.1. A System for X-ray Crystallography and NMR, Yale University Press, New Haven, Connecticut; X-PLOR is available from Molecular Simulations, Inc., San Diego, California). This information can thus be used to optimize known classes of PXR and PXR LBD modulators, and more importantly, to design and synthesize novel classes of PXR and PXR LBD modulators. Laboratory Examples
The following Laboratory Examples have been included to illustrate preferred modes of the invention. Certain aspects of the following Laboratory
Examples are described in terms of techniques and procedures found or contemplated by the present inventors to work well in the practice of the invention. These Laboratory Examples are exemplified through the use of standard laboratory practices of the inventors. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Laboratory Examples are intended to be exemplary only and that numerous changes, modifications and alterations can be employed without departing from the spirit and scope of the invention.
Laboratory Example 1 PXR Expression and Purification The PXR LBD expression construct was engineered as an N-terminal polyhistidine tagged fusion protein with residues 130-434 from the human PXR. The fusion insert (SEQ ID NO: 10) was subcloned into the pRSETA expression vector (Invitrogen, Carlsbad, California). DNA encoding residues 623-710 of the human SRC-1 gene (Onate et aL, (1995) Science 270: 1354- 57) (SEQ ID NO: 12) were subcloned into the bacterial vector pACYC184 (American Type Culture Collection #37033) along with a T7 promoter (Nolte et aL, (1998) Nature 395: 137-43). The hPXRLBD/pRSETA and the SRC- 1/pACYCI84 plasmids were co-transformed into the BL21(DE3) strain of E. coli. One-liter shake flask liquid cultures containing standard Luria-Bertani (LB) broth with 0.05 mg/ml ampicillin and 0.05 mg/ml chloramphenicol were inoculated and grown at 22°C for 20 hours. The cells were harvested by centrifugation (20 minutes, 3500g, 4°C) and the cell pellet was stored at - 80°C. The cell pellet was resuspended in 250 ml of Buffer A (50 mM Tris-CI pH 7.8, 250 mM NaCL, 50 mM imidazole pH 7.5, 5% glycerol). Cells were sonicated for 3-5 minutes on ice and the cell debris was removed by centrifugation (45 minutes, 20,000g, 4°C). The cleared supernatant was filtered through a 0.45 μM filter and loaded on to a 50 ml PROBOND™ nickel- chelating resin (Invitrogen, Carlsbad, California). After washing to baseline with Buffer A, the column was washed with Buffer A containing 125 mM, imidazole pH 7.5. The PXR-LBD/SRC-1 complex was eluted from the column using Buffer A with 500 mM imidazole pH 7.5. Column fractions were pooled and concentrated using CENTRI-PREP™ 30K (Amicon/Millipore, Bedford, Massachusetts) units. The protein was subjected to size exclusion, using a column (26 mm X 90 cm) packed with SEPHAROSE™ S-75 resin (Amersham Pharmacia Biotech, Piscataway, New Jersey) pre-equilibrated with 20mM Tris-CI pH 7.8, 250 mM NaCl, 5 mM DTT, 2.5 mM EDTA pH 8.0,5% glycerol. Column fractions containing the PXR/SRC-1 complex were pooled and diluted five fold with the dilution buffer (20 mM Tris-CI pH 7.8, 5mM DTT, 2.5mM EDT A pH 8.0, 5% glycerol) and loaded on to a 50 ml Q SEPHAROSE™ FAST FLOW™ column (Amersham Pharmacia Biotech, Piscataway, New Jersey). The column was washed to baseline with 20mM Tris-CI pH 7.8,50 mM NaCL, 5 rnM DTT, 2.5 mM EDTA pH 8.0, 5% glycerol, and the protein complex was eluted from the column utilizing an increased salt gradient. The appropriate column fractions were pooled and stored on ice.
Laboratory Example 2 Crystallization of a PXR Ligand binding Domain
The human PXR ligand binding domain/SRC-1 complex (hPXR- LBD/SRC-1 ) was concentrated in the presence of 10-fold molar excesses of the SR12813 or I-SR12813 compounds to final concentrations of 4 and 5 mg/mL, respectively. The apo complex was concentrated to 5 mg/mL. Crystallization of hPXR-LBD was achieved by hanging-drop vapor diffusion against the following conditions at 22°C: SRI2813: 50 mM imidazole at pH 7.2,10% 2-propanol (v/v); I-SRI2813: 50 mM imidazole at pH 7.2, 11 % 2- propanol (v/v); apo: 50 mM imidazole at pH 7.4,10% 2-propanol (v/v). Laboratory Example 3
Structure Determination
The structures of an apo (unliganded) and SRI28l3-bound form of the ligand binding domain of human PXR were determined by molecular replacement using the crystal structure of the vitamin D receptor (VDR) as search models (SEQ ID NO: 7, structural coordinates presented in Table 5).
Non-identical side chains were trimmed prior to rotation and translation function searches in AmoRe (Navaza & Saludjian, (1997) Method Enzymol.
276A: 581-94); clear solutions were obtained for each in the proper enantiomorphic space group, P432ι2. The structures were refined using the torsion angle protocol in CNS with the maximum likelihood function as a target, and included an overall anisotropic B-factor and a bulk solvent correction (Brϋnger et al., (1998) Λcfa Crystallog. D 54: 905-21 ).
For both structures, 10% of the observed data were set aside for cross- validation using the free-R statistic prior to any structural refinement (Brϋnger, (1993) Acta Crystallog. D 49: 24-36). Manual adjustments and rebuilding of the model were performed using O (Jones et aL, (1991 ) Acta Crystallog. A 47:110-19) and σA-weighted electron density maps (Read, (1986) Acta Crystallog. A 42: 140-49). At the later stages of refinement 281 and 166 period solvent sites were added to the apo and SR12813 complexes, respectively. Structures exhibit good geometry with no Ramachandran outliers, as shown in Table 1. Molecular graphics figures were created with MOLSCRIPT (Kraulis, (1991) J. Applied Crystallogr. 24: 946-50), RASTER-3D (Merritt & Bacon, (1997) Method Enzymol. 277: 505-24), GRASP (Nicholls et aL, (1991 ) Proteins 11 : 281-96) and CS CHEMDRAW PRO™, available from CambridgeSoft of Cambridge, Massachusetts.
Laboratory Example 4
Positioning of SR12813 Ligands Clear electron density was evident in the ligand binding cavity of PXR at the earliest stages of refinement. The positioning of single SR12813 molecules into this electron density, however, resulted in significant positive electron density peaks elsewhere in the cavity, suggesting that more than one conformation of the ligand was present. This was confirmed using difference density generated from crystals of hPXR-LBD containing an iodinated form of SR12813 (I-SR12813). The two t-butyl groups of SR12813 were replaced with iodine in I-SR12813; this ligand was found to activate PXR as well as the parent SR12813 compound. Two difference density maps were used to guide' the positioning of SR12813 ligands: [(| FobsISR12813 I - I Fobs SR12813 I), φca,cPROTE,N] and [(| Fobs ISR12813 1 - 1 Fobs PROTEIN|), φCaioPROTE,N].
Each map was calculated to 3.0 A resolution and both showed a consistent set of significant positive difference density peaks in the ligand binding cavity, which were interpreted to be the positions of the iodine atoms on ISR12813. These peaks were the highest difference density peaks in both maps. It was found that three SR12813 ligands optimally satisfied the electron density in the ligand binding cavity. The first SR12813 (position 1 ) was placed in part based on two 7σ difference density peaks at appropriate positions in both maps. The second SR12813 (position 3) was positioned using two 5σ difference density peaks at appropriate positions. The third SR12813 (position 2) was placed based on 4σ iodine difference density peaks and residual difference density in the maps after the refinement of positions 1 and 3 together.
Standard simulated density maps, [(| FobsPROTEIN I - I Fcaιc PROTEIN|), φcaicPR0TEIN], at 2.75 A resolution were used to guide the positioning of the remainder of each SR12813 ligand and to assess the quality of the fits to the density (Brϋnger et aL, (1990) Acta Crystallog. A 46: 585-93). No positive or negative difference density peaks above 30' were present in the binding cavity after the refinement of the three ligand positions together. Occupancies of each ligand position were estimated based on iodine difference density and standard electron density peaks, and were fixed at 0.5, 0.2 and 0.3 for positions 1 , 2 and 3, respectively. In addition, the R and R-free after the positioning of the three ligand positions were improved over each conformation alone, or over pairs of each conformation. Laboratory Example 5 Mutagenesis The human and mouse PXR mutants were generated using the QUIKCHANGE™ mutagenesis kit (Stratagene, La Jolla, California) according to the manufacturer's instructions. All mutants were confirmed by sequence analysis.
Laboratory Example 6
Cotransfection Assays Transient cotransfection experiments were performed using CV -1 cells and the XREM-CYP3A4-LUC reporter, containing the enhancer and promoter of CYP3A4 driving luciferase expression, as described in Moore et aL, (2000) J. Biol. Chem. 275: 15122-27.
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TABLE 1
CRYSTALLOGRAPHIC DATA AND REFINEMENT
APO SR12813 l-SR12813∞
Resolution (A; highest shell) 20-2.5 (2.54-2.5) 20-2.75 (2.8-2.75) 20-3.0 (3.05-3.0)
Space Group P43212 P432.,2 P432!2
Cell Constants (A) A=91.6; c=85.0 a=91.3; c=85.3 a=91.1 ; c=84.5
Total Reflections 179,671 133,315 91 ,752
Unique Reflections 12,611 9,512 7,269
Mean Redundancy 14.2 14 12.6
Rsym* (%; highest shell) 7.3 (39.0) 9.8 (40.0) 9.7 (51.0)
Completeness (%; highest 99.9 (100) 99.6 (100) 99.9 (100) shell)
Mean l/σ (highest shell) 40.8 (7.9) 25.9 (3.7) 31.4 (5.4) αvst1^ (%; highest shell) 20.9 (25.9) 21.3 (26.1) - Rfree* (%; highest shell) 28.4 (32.1) 27.4 (40.5) —
RMSD§ Bond Lengths (A) 0.0059 0.0093 -
RMSD§ Bond Angles (°) 1.11 1.29 -
RMSD§ Dihedrals (°) 20.54 21.33 -
RMSD§ Impropers (°) 0.71 0.91 —
Number Protein Atoms 2141 2141 —
Number Solvent Sites 281 166 —
Number Ligand Atoms - 99 -
Λ Data from crystals containing I-SR12813 were used only to guide and confirm
Figure imgf000091_0001
TABLE 2
COMPARISON OF HUMAN PXR-LBD WITH NUCLEAR RECEPTOR LBDs
OF KNOWN STRUCTURE
Number Residues Sequence RMSD (A) Ligand binding Superimposed Identity Cavity Volume* (A3)
(%)
PXR 1 ,150
Vitamin D Receptor 225 45 1.8 871
RXRα 178 22 2.8 687
PPARγ 203 25 2.6 1 ,619
Progesterone Receptor 201 19 2.9 557
Estrogen Receptor 180 18 2.8 476
* The cavity volumes were calculated with GRASP (Nicholls et al., (1991) Proteins 11 : 281- 96) using a grid spacing of 0.2 A, probe radius of 1.4 A and the atomic radii of Bondi (H=1.2 A, C=1.7 A, N=1.55 A, 0=1.52 A, S=1.8 A) (Bondi, (1964) J. Phys. Chem. 68: 441-51), where hydrogens were treated explicitly. Openings in the PXR, PPARγ and RXRα pockets were closed by covering these proteins with an external layer of water molecules prior to calculation of the molecular surface and cavities. TABLE 3
CALCULATED BURIED SOLVENT-ACCESSIBLE SURFACE AREA OF AMINO ACID RESIDUES THAT LINE THE LIGAND BINDING CAVITY OF
HPXR
Residue Buried Contacts Human Rabbit Rat Mouse
Surface SR12813
(A*) Ligand?
206 9.8 Yes L L R S
208 15.8 Yes S T P P
209 30.5 Yes L M M M
211 16.3 Yes V L 1 1
240 14.8 Yes L L L L
243 43.6 Yes M L L L
244 1.9 No A A A A
246 6.1 Yes M M V V
247 30.4 Yes S S s s
251 11.2 Yes F F F F
281 29.3 Yes F L F F
284 9.6 Yes C C C C
285 23.0 Yes Q L 1 1
288 29.6 Yes F F F F
299 28.6 Yes W W W W
306 12.9 No Y Y Y Y
308 3.7 No L V F F
321 4.2 No E D D D
323 20.3 Yes M L L L
324 12.0 Yes L L M M
327 2.5 No H H H H
407 41.6 Yes H H Q Q
410 14.7 Yes R R Q Q
411 33.1 No L L L L
414 11.5 No 1 1 1 1
420 9.3 Yes F F F F
425 6.2 No M M M M
429 3.6 No F F F F
Boldface indicates identity; ; italics indicate similarity. The buried surface area upon ligand binding was calculated using CNS (Brϋnger et al., (1998) Acta Crystallogr. D 54: 905-21).
The four residues underlined were mutated to examine the ligand specificity of mouse and human PXR. TABLE 4
ATOMIC COORDINATE DATA FOR CRYSTALLIZED HUMAN PXR
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
TABLE 5
ATOMIC COORDINATE DATA FOR HUMAN VITAMIN D RECEPTOR
EMPLOYED IN THE MOLECULAR REPLACEMENT SOLUTION OF HUMAN
PXR LIGAND BINDING DOMAIN CRYSTALS
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
It will be understood that various details of the invention can be changed without departing from the scope of the invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation — the invention being defined by the claims.

Claims

CLAIMS What is claimed is:
1. A substantially pure PXR ligand binding domain polypeptide in crystalline form.
2. The polypeptide of claim 1 , wherein the crystalline form has lattice constants of a = 91.6 A, b = 91.6 A, c = 85.0 A, α = 90°, β = 90°, γ = 90°.
3. The polypeptide of claim 1 or 2, wherein the crystalline form is a tetragonal crystalline form.
4. The polypeptide of claim 1 or 2, wherein the crystalline form has a space group of P432-i2.
5. The polypeptide of claim 1 or 2, wherein the PXR ligand binding domain polypeptide has the amino acid sequence shown in SEQ ID NO: 4.
6. The polypeptide of claim 1 or 2, wherein the PXR ligand binding domain polypeptide is in complex with a ligand.
7. The polypeptide of claim 6, wherein the ligand is a drug.
8. The polypeptide of claim 7, wherein the drug is SR12813.
9. The polypeptide of claim 1 or 2, wherein the PXR ligand binding domain has a crystalline structure further characterized by the coordinates corresponding to Table 4.
10. The polypeptide of claim 1 or 2, wherein the crystalline form contains one PXR ligand binding domain polypeptide in the asymmetric unit.
11. The polypeptide of claim 1 or 2, wherein the crystalline form is such that the three-dimensional structure of the crystallized hPXR ligand binding domain polypeptide can be determined to a resolution of about 2.75 A or better.
12. The polypeptide of claim 1 or 2, wherein the crystalline form contains one or more atoms having an atomic weight of 40 grams/mol or greater.
13. A method for determining the three-dimensional structure of a crystallized hPXR ligand binding domain polypeptide to a resolution of about 3.0 A or better, the method comprising:
(a) crystallizing a hPXR ligand binding domain polypeptide; and
(b) analyzing the hPXR ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized hPXR ligand binding domain polypeptide, whereby the three- dimensional structure of a crystallized hPXR ligand binding domain polypeptide is determined to a resolution of about 2.75 A or better.
14. The method of claim 13, wherein the analyzing is by X-ray diffraction.
15. The method of claim 13, wherein the crystallization is accomplished by the hanging drop vapor diffusion method, and wherein the
PXR ligand binding domain is mixed with an equal volume of reservoir.
16. The method of claim 15, wherein the reservoir comprises 250 mM NaCl, 20 mM Tris-HCl pH 7.8, 5% glycerol (v/v), 5 mM DTT, 2.5 mM EDTA.
17. The method of claim 15, wherein the reservoir 50 mM imidazole, pH 7.4, 10% 2-propanol (v/v).
18. A method of generating a crystallized PXR ligand binding domain polypeptide, the method comprising:
(a) incubating a solution comprising a PXR ligand binding domain with an equal volume of reservoir; and
(b) crystallizing the PXR ligand binding domain polypeptide using the hanging drop method, whereby a crystallized PXR ligand binding domain polypeptide is generated.
19. A crystallized PXR ligand binding domain polypeptide produced by the method of claim 18.
20. A method of designing a modulator of a PXR polypeptide, the method comprising:
(a) designing a potential modulator of a PXR polypeptide that will form bonds with amino acids in a ligand binding site based upon a crystalline structure of a PXR ligand binding domain polypeptide;
(b) synthesizing the modulator; and
(c) determining whether the potential modulator modulates the activity of the PXR polypeptide, whereby a modulator of a PXR polypeptide is designed.
21. A method of designing a modulator that selectively modulates the activity of a human PXR polypeptide, the method comprising:
(a) obtaining a crystalline form of a human PXR ligand binding domain polypeptide; (b) evaluating the three-dimensional structure of the crystallized human PXR ligand binding domain polypeptide; and (c) synthesizing a potential modulator based on the three- dimensional crystal structure of the crystallized human PXR ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of a human PXR polypeptide is designed.
22. The method of claim 21 , wherein the method further comprises contacting a human PXR ligand binding domain polypeptide with the potential modulator; and assaying the human PXR ligand binding domain polypeptide for binding of the potential modulator, for a change in activity of the human PXR ligand binding domain polypeptide, or both.
23. The method of claim 21 , wherein the crystalline form is in tetragonal form.
24. The method of claim 23, wherein the crystalline form is such that the three-dimensional structure of the crystallized human PXR ligand binding domain polypeptide can be determined to a resolution of about 2.75 A or better.
25. A method for identifying a PXR modulator, the method comprising:
(a) providing atomic coordinates of a PXR ligand binding domain to a computerized modeling system; and (b) modeling a ligand that fits spatially into a binding cavity or on the surface of the PXR ligand binding domain, whereby a PXR modulator is identified.
26. The method of claim 25, wherein the method further comprises identifying in an assay for PXR-mediated activity a modeled ligand that increases or decreases the activity of the PXR.
27. A method of identifying a PXR modulator that selectively modulates the activity of a PXR polypeptide compared to other polypeptides, the method comprising:
(a) providing atomic coordinates of a PXR ligand binding domain to a computerized modeling system; and
(b) modeling a ligand that fits spatially into a binding cavity or on the surface of a PXR ligand binding domain and that interacts with conformationally constrained residues of a PXR that are conserved among PXR orthologs and isoforms, whereby a PXR modulator is identified.
28. The method of claim 27, wherein the method further comprises identifying in a biological assay for PXR-mediated activity a modeled ligand that selectively binds to the PXR ligand binding domain and increases or decreases the activity of the PXR.
29. A method of designing a modulator of a PXR polypeptide, the method comprising:
(a) selecting a candidate PXR ligand; (b) determining which amino acid or amino acids of a PXR polypeptide interact with the ligand using a three-dimensional model of a crystallized protein comprising a PXR LBD;
(c) identifying in a biological assay for PXR activity a degree to which the ligand modulates the activity of the PXR polypeptide; (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the PXR polypeptide and the ligand is predicted to be modulated by the chemical modification;
(e) performing the chemical modification on the ligand to form a modified ligand;
(f) contacting the modified ligand with the PXR polypeptide;
(g) identifying in a biological assay for PXR activity a degree to which the modified ligand modulates the biological activity of the PXR polypeptide; and (h) comparing the biological activity of the PXR polypeptide in the presence of modified ligand with the biological activity of the PXR polypeptide in the presence of the unmodified ligand, whereby a modulator of a PXR polypeptide is designed.
30. The method of claim 29, wherein the PXR polypeptide is a human PXR polypeptide.
31. The method of claim 29, wherein the three-dimensional model of a crystallized protein is a human PXR LBD polypeptide with a bound ligand.
32. The method of claim 31 , wherein the ligand is a drug.
33. The method of claim 32, wherein the drug is SR12813.
34. The method of claim 29, wherein the method further comprises repeating steps (a) through (f), if the biological activity of the PXR polypeptide in the presence of the modified ligand varies from the biological activity of the PXR polypeptide in the presence of the unmodified ligand.
35. An assay method for identifying a compound that inhibits binding of a ligand to a PXR polypeptide, the assay method comprising: (a) designing a test inhibitor compound capable of modulating PXR activity, based on the atomic coordinates of a PXR ligand binding domain;
(b) synthesizing the test inhibitor compound;
(c) incubating a PXR polypeptide with a ligand in the presence of a test inhibitor compound;
(d) determining an amount of ligand that is bound to the PXR polypeptide, wherein decreased binding of ligand to the PXR protein in the presence of the test inhibitor compound relative to binding of ligand in the absence of the test inhibitor compound is indicative of inhibition; and (e) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed, whereby a compound that inhibits binding of a ligand to a PXR polypeptide is identified.
36. The method of claim 35, wherein the ligand is a drug.
37. The method of claim 36, wherein the drug is SR12813.
38. A method of evaluating a candidate therapeutic agent in humans using a mouse model system, the method comprising:
(a) providing atomic coordinates of a human PXR ligand binding domain to a computerized modeling system;
(b) modeling a candidate therapeutic agent that fits spatially into a binding cavity or on the surface of a human PXR ligand binding domain;
(c) providing a mouse PXR polypeptide; (d) selecting one or more mutations to be introduced into an amino acid sequence of the mouse PXR polypeptide, the mutations being selected so as to alter the mouse PXR polypeptide to be similar to a human PXR polypeptide;
(e) providing a mutant mouse PXR polypeptide comprising the one or more mutations selected in step (d);
(f) contacting the candidate therapeutic agent modeled in step (b) with the mutant mouse PXR polypeptide;
(f) determining an effect of the candidate therapeutic agent on the mutant mouse PXR polypeptide; and (g) evaluating the potential of the candidate therapeutic agent for use in humans based on the effect of the candidate therapeutic agent on the mutant mouse PXR polypeptide, whereby the candidate therapeutic agent in humans using a mouse model system is evaluated.
39. The method of claim 38, further comprising repeating steps (a) through (h) a desired number of times.
40. The method of claim 38, wherein the one or more mutations is selected from the group consisting of an arginine to leucine substitution at residue 203 of the mouse PXR polypeptide, a protein to serine substitution at residue 205 of the mouse PXR polypeptide, a glutamine to histidine substitution at residue 404 of the mouse PXR polypeptide and a glutamine to arginine substitution at residue 407 of the mouse PXR polypeptide.
41. The method of claim 38, wherein the mouse PXR polypeptide comprises a mouse PXR ligand binding domain (LBD).
42. The method of claim 38, wherein the mutant mouse PXR polypeptide is expressed by:
(a) introducing the selected mutation into the mouse PXR DNA sequence, thereby forming a mutant mouse PXR DNA sequence;
(b) transforming cells with the mutant mouse PXR DNA sequence; and
(c) expressing the mutant mouse PXR DNA sequence, thereby forming a mutant mouse PXR polypeptide.
43. The method of claim 42, wherein the mutant mouse PXR polypeptide is expressed in cells.
44. The method of claim 43, wherein the cells are CV-1 cells.
45. The method of claim 42, wherein the mutant mouse PXR DNA sequence is cotransformed with a reporter gene.
46. The method of claim 45, wherein the reporter gene is an XREM- CYP3A4-LUC reporter.
47. The method of claim 45, wherein the mouse PXR polypeptide comprises a mouse PXR ligand binding domain (LBD).
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Cited By (2)

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US7973135B2 (en) 2006-07-12 2011-07-05 Oncotx, Inc. Compositions and methods for targeting cancer-specific transcription complexes
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Title
DATABASE PROTEIN [online] 8 May 2001 (2001-05-08), WATKINS ET AL.: "Crystal structure of human pregame X receptor ligand binding domain bound to Sr12813", XP002958151, Database accession no. 1ILH_A *
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Cited By (2)

* Cited by examiner, † Cited by third party
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US7973135B2 (en) 2006-07-12 2011-07-05 Oncotx, Inc. Compositions and methods for targeting cancer-specific transcription complexes
CN113811542A (en) * 2018-12-21 2021-12-17 非营利性组织佛兰芒综合大学生物技术研究所 Fusion proteins comprising a cytokine and a scaffold protein

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