U.S. patent application number 13/550211 was filed with the patent office on 2012-11-08 for orphan nuclear receptor.
Invention is credited to Stacey A. Jones, Steven A. Kliewer, Timothy M. Willson.
Application Number | 20120283412 13/550211 |
Document ID | / |
Family ID | 38196768 |
Filed Date | 2012-11-08 |
United States Patent
Application |
20120283412 |
Kind Code |
A1 |
Kliewer; Steven A. ; et
al. |
November 8, 2012 |
ORPHAN NUCLEAR RECEPTOR
Abstract
The present invention relates to a novel human orphan nuclear
receptor that binds to a cytochrome P-450 monooxygenase (CYP)
promoter and that is activated by compounds that induce CYP gene
expression. The invention further relates to nucleic acid sequences
encoding such a receptor, to methods of making the receptor and to
methods of using the receptor and nucleic acid sequences encoding
same. The invention also relates to non-human animals transformed
to express the human receptor and to methods of using such animals
to screen compounds for drug interactions and toxicities.
Inventors: |
Kliewer; Steven A.; (Cary,
NC) ; Jones; Stacey A.; (Wake Forest, NC) ;
Willson; Timothy M.; (Durham, NC) |
Family ID: |
38196768 |
Appl. No.: |
13/550211 |
Filed: |
July 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12877195 |
Sep 8, 2010 |
8221991 |
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13550211 |
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11766871 |
Jun 22, 2007 |
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12877195 |
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09276935 |
Mar 26, 1999 |
7238491 |
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11766871 |
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60079593 |
Mar 27, 1998 |
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Current U.S.
Class: |
530/350 ;
435/252.3; 435/254.2; 435/320.1; 435/325; 435/348; 435/370;
435/69.1; 536/23.5 |
Current CPC
Class: |
G01N 2500/00 20130101;
G01N 33/566 20130101 |
Class at
Publication: |
530/350 ;
536/23.5; 435/320.1; 435/69.1; 435/325; 435/348; 435/254.2;
435/252.3; 435/370 |
International
Class: |
C07K 14/705 20060101
C07K014/705; C12N 15/12 20060101 C12N015/12; C12N 1/21 20060101
C12N001/21; C12P 21/00 20060101 C12P021/00; C12N 5/10 20060101
C12N005/10; C12N 1/19 20060101 C12N001/19; C07K 19/00 20060101
C07K019/00; C12N 15/63 20060101 C12N015/63 |
Claims
1. An isolated human nuclear receptor that binds to a cytochrome
P-450 monooxygenase promoter, or a DNA binding or ligand binding
domain thereof.
2. The receptor according to claim 1 wherein the promoter is a
cytochrome P-450 monooxygenase 3A4 (CYP3A4) promoter.
3. The receptor according to claim 2 wherein said receptor is
hPXR.
4. An isolated human nuclear receptor having the amino acid
sequence given FIG. 1, or a fragment thereof, of at least 30
consecutive amino acids.
5. A fusion protein comprising a DNA binding or ligand binding
domain of hPXR and a non-hPXR-derived sequence.
6. An isolated nucleic acid comprising a sequence encoding the
receptor of claim 1.
7. A construct comprising the nucleic acid of claim 6 and a
vector.
8. A host cell comprising the construct of claim 7.
9. A method of making the receptor of claim 3, or fragment thereof,
comprising: culturing a host cell containing an expression
construct comprising a sequence encoding said receptor, or fragment
thereof, operably linked to a promoter, under conditions such that
said receptor, or fragment thereof, is produced, and isolating said
receptor, or fragment thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 12/877,195 filed Sep. 8, 2010, now issued as U.S. Pat. No.
8,221,991; which is a Divisional of U.S. application Ser. No.
11/766,871 filed Jun. 22, 2007, now abandoned; which is a
Divisional of U.S. application Ser. No. 09/276,935 filed Mar. 26,
1999, now issued as U.S. Pat. No. 7,238,491; which claims priority
from Provisional Application No. 60/079,593, filed Mar. 27, 1998,
the entire contents of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a novel human orphan
nuclear receptor that binds to a cytochrome P-450 monooxygenase
(CYP) promoter and that is activated by compounds that induce CYP
gene expression. The invention further relates to nucleic acid
sequences encoding such a receptor, to methods of making the
receptor and to methods of using the receptor and nucleic acid
sequences encoding same. The invention also relates to non-human
animals transformed to express the human receptor and to methods of
using such animals to screen compounds for drug interactions and
toxicities.
BACKGROUND OF THE INVENTION
[0003] Members of the cytochrome P-450 (CYP) family of hemoproteins
are critical in the oxidative metabolism of a wide variety of
endogenous substances and xenobiotics, including various
carcinogens and toxins (Nebert et al, Ann. Rev. Biochem. 56:945-993
(1987)). In man, the CYP3A4 monooxygenase plays a major role in the
biotransformation of drugs due to its abundance in liver and
intestine and its broad substrate specificity. CYP3A4 catalyzes the
metabolism of >60% of all drugs that are in use including
steroids, immunosuppressive agents, imidazole antimycotics, and
macrolide antibiotics (Maurel, P. in Cytochromes P450: metabolic
and toxicological aspects (ed. Ioannides, C.) 241-270 (CRC Press,
Inc., Boca Raton, Fla., 1996).
[0004] Expression of the CYP3A4 gene is markedly induced both in
vivo and in primary hepatocytes in response to treatment with a
variety of compounds. Many of the most efficacious inducers of
CYP3A4 expression are commonly used drugs such as the
glucocorticoid dexamethasone, the antibiotic rifampicin, the
antimycotic clotrimazole, and the hypocholesterolemic agent
lovastatin (Maurel, P. in Cytochromes P450: metabolic and
toxicological aspects (ed. Ioannides, C.) 241-270 (CRC Press, Inc.,
Boca Raton, Fla., 1996), Guzelian, P. S. in Microsomes and Drug
Oxidations (eds. Miners, J. O., Birkett, D. J., Drew, R. &
McManus, M.) 148-155 (Taylor and Francis, London, 1988). The
inducibility of CYP3A4 expression levels coupled with the broad
substrate specificity of the CYP3A4 protein represent the basis for
many drug interactions in patients undergoing combination drug
therapy. While attempts have been made to develop in vivo and in
vitro assays with which to profile the effects of compounds on
CYP3A expression levels, these efforts have been hampered by
species-specific effects that have limited the utility of using
animals and their tissues for testing purposes. Thus, analysis of
the effects of new compounds on CYP3A4 gene expression has been
largely restricted to laborious assays involving human liver
tissue.
[0005] Recently, efforts have been directed at understanding the
molecular basis for the induction of CYP3A4 gene expression. The
CYP3A4 promoter has been cloned and a 20 bp region residing
approximately 150 bp upstream of the transcription initiation site
shown to confer responsiveness to dexamethasone and rifampicin
(Hashimoto et al, Eur. J. Biochem. 218:585-595 (1993), Barwick et
al, Molec. Pharmacol. 50:10-16 (1996)). This region contains two
copies of the AG(G/T)TCA motif recognized by members of the nuclear
receptor superfamily, suggesting that a nuclear receptor might be
responsible for mediating at least some of the effects of the
chemical inducers of CYP3A4 expression. However, prior to the
present invention, proteins that bind to this response element had
not been characterized.
[0006] The present invention is based on the identification of a
novel orphan nuclear receptor that binds to a response element in
the CYP3A4 promoter and that is activated by a range of compounds
known to induce CYP3A4 expression. The identification of this
receptor makes possible assays that can be used to establish
whether drugs will interact in vivo.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a novel human orphan
nuclear receptor, designated the human pregnane X receptor (hPXR),
that binds to a CYP promoter, for example, the
rifampicin/dexamethasone response element in the cytochrome P-450
monooxygenase 3A4 (CYP3A4) promoter. The receptor is activated to
modulate transcription of a CYP (e.g., CYP3A4) gene. The present
invention further relates to nucleic acids encoding hPXR, including
expression vectors that can be used to effect expression of the
receptor in host cells. The invention also relates to host cells
transformed with such expression vectors and to methods of using
the receptor and receptor encoding sequences in assays designed to
screen compounds (e.g., drugs) for their ability to modulate CYP
(e.g., CYP3A4) gene expression. The invention also relates to
non-human animals transformed to express the human receptor and to
methods of using same in drug screens.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A-1D. Molecular cloning of hPXR. (FIG. 1 A)
Nucleotide (SEQ ID NO:13) and predicted amino acid (SEQ ID NO:14)
sequences of hPXR. (FIG. 1B) Amino acid sequence comparison between
hPXR, mPXR1, Xenopus orphan nuclear receptor 1 (xONR1) (Smith et
al, Nucl. Acids Res. 22:66-71 (1994)), and the human vitamin D
receptor (hVDR). Numbers indicate percent amino acid identity in
the DBDs and LBDs. (FIG. 1C) The hPXR clone encodes a functional
nuclear receptor. Transfection assays were performed with a
pSG5-hPXR expression vector containing the wild-type 5' region of
the hPXR cDNA and a reporter plasmid containing four copies of the
CYP3A1 DR3PXRE. Cells were treated with vehicle alone (0.1% DMSO)
or 10 .mu.M of dexamethasone-t-butylacetate. Cell extracts were
subsequently assayed for CAT activity. Data points represent the
mean of assays performed in duplicate. (FIG. 1D) Translation of the
full-length hPXR initiates at a non-AUG codon. In vitro
transcription and translation were performed with the pSG5-hPXR
expression vector containing the wild-type 5' region of the hPXR
cDNA or pSG5-hPXR AUG, in which the CUG codon at nucleotide
positions 304-306 was modified to AUG. The 50 kD product
synthesized when either template was used is indicated by the open
arrow and the asterisk. Two shorter products which are likely to
represent translation initiation at methionine-56 and methionine-69
within the DBD are indicated by closed arrows. A longer translation
product present at low levels is indicated by the bent arrow. Size
markers (in kD) are indicated at left.
[0009] FIG. 2. Northern blot analysis of hPXR expression pattern in
adult tissues (left to right, heart (1), brain (2), placenta (3),
lung (4), liver (5), skeletal muscle (6), kidney (7), pancreas (8),
spleen (9), thymus (10), prostate (11), testis (12), ovary (13),
small intestine (14), colon (15), PBL (16). RNA size markers (in
kb) are indicated at left.
[0010] FIGS. 3A-3C. hPXR activates transcription through an IR6
element in the CYP3A4 promoter. (FIG. 3A) CV-1 cells were
cotransfected with the (IR6).sub.3-tk-CAT reporter plasmid in
either the absence (-) or presence (+) of the pSG5-hPXR ATG
expression plasmid and treated with vehicle alone (open bars) or 10
.mu.M dexamethasone-t-butylacetate (closed bars). Cell extracts
were subsequently assayed for CAT activity. Data represent the mean
of assays performed in triplicate+/-S.E. (FIG. 3B) Oligonucleotides
used in band shift assays. The positions of nuclear receptor
half-site motifs and mutations are indicated. (FIG. 3C) Band shift
assays were performed with a radiolabeled oligonucleotide
containing the CYP3A4 IR6PXRE and hRXR and either hPXR (top panel)
or mPXR1 (bottom panel). Unlabeled competitor oligonucleotides were
added at a 10-fold or 50-fold molar excess as indicated.
[0011] FIGS. 4A-4C. hPXR is activated by structurally-distinct
inducers of CYP3A4 gene expression. (FIG. 4A) CV-1 cells were
transfected with the pSG5-hPXR ATG or pSG5-mPXR1 expression
plasmids and the (IR6).sub.3-tk-CAT reporter (left and middle
panels, respectively), or the RS-hGR expression plasmid (Giguere et
al, Cell 46:645-652 (1986)) and a reporter containing two copies of
a consensus glucocorticoid response element upstream of tk-CAT
(right panel). Cells were treated with 1 .mu.M mevastatin or
lovastatin, 100 .mu.M phenobarbital, or 10 .mu.M of the other
compounds. Cell extracts were subsequently assayed for CAT
activity. Data represent the mean of assays performed in
triplicate+/-S.E. (FIG. 4B) Structures of representative compounds
that activate hPXR. (FIG. 4C) CARLA was performed with
bacterially-expressed GST-hPXR or GST-mPXR1 and [.sup.35S]SRC1.14
synthesized in vitro. [.sup.35S]SRC1.14 was mixed with either
GST-hPXR or GST-mPXR1 in the presence of vehicle alone (1) (1%
DMSO) or 10 .mu.M of dexamethasone-t-butylacetate (2), rifampicin
(3), or clotrimazole (4). [.sup.35S]SRC1.14 complexed with GST-hPXR
(top panel) or GST-mPXR1 (bottom panel) was precipitated with
glutathione-sepharose beads.
[0012] FIG. 5. Reaction scheme for production of
[.sup.3H]GW-485801.
[0013] FIG. 6. Plot of specific binding vs. concentration of
[.sup.3H]GW-485801. Kd=370 nM.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention relates to a novel human nuclear
receptor, hPXR. The invention further relates to nucleic acid
sequences encoding hPXR, to constructs comprising such sequences,
to host cells containing the constructs and to a method of
producing hPXR using such host cells. The invention also relates to
non-human animals transformed to express hPXR. The invention
further relates to in vivo and in vitro assays that can be used to
identify compounds that induce CYP expression. While the disclosure
that follows makes specific reference to CYP3A4, it should be
appreciated that the details (e.g., methods) provided find
application in connection with other CYP genes as well.
[0015] hPXR is characterized as a protein comprising about 434
amino acids and having a molecular weight of about 49.7
kilodaltons. hPXR binds to a DNA response element in the CYP3A4
promoter as a heterodimer with the 9-cis retinoic acid receptor,
RXR. hPXR is activated by compounds known to modulate CYP3A4
expression. The receptor is most abundantly expressed in liver but
is also present in colon and small intestine.
[0016] One embodiment of the receptor of the invention has the
amino acid sequence set forth in FIG. 1, or an analog thereof
(wherein the term analog is intended to indicate a naturally
occurring human variant of the FIG. 1 sequence), or a fragment
thereof, including fragments having at least one functional
characteristic of hPXR (e.g., ligand binding or DNA binding).
Preferred fragments include portions of the FIG. 1 sequence at
least 30 consecutive amino acids in length, more preferably, at
least 50 consecutive amino acids in length, and most preferably, at
least 75 consecutive amino acids in length. Specific fragments
include the ligand binding domain (that is, amino acids 141 to 434
of the FIG. 1 sequence) and the DNA binding domain (that is, amino
acids 41 to 107 of the FIG. 1 sequence) as well as the domain that
is used for the ligand binding assay described in the Examples that
follow (that is, amino acids 130-434 of the FIG. 1 sequence). The
invention also includes a protein comprising a domain sharing at
least 80% amino acid sequence identity with the ligand binding
domain of the FIG. 1 sequence, more preferably, at least 85% amino
acid sequence identity and, most preferably, at least 90% or 95%,
96%, 97%, 98% or 99% amino acid sequence identity with the ligand
binding domain of the FIG. 1 sequence (% sequence identity being
determined, for example, by Basic Blast (version 2.0) available
through the NCBI website http://www.ncbi.nlm.nih.gov/), and,
advantageously, retaining the function of the FIG. 1 sequence.
[0017] The receptor of the invention, or fragment thereof, can bear
a detectable label (e.g., a radioactive or fluorescent label). The
receptor, or receptor fragment, can also be bound to a solid
support, e.g., a glass or plastic particle, a plate, or a
filter.
[0018] Nucleic acid sequences of the invention include DNA and RNA
sequences encoding hPXR, for example, hPXR having the amino acid
sequence given in FIG. 1, as well as nucleic acid sequences
encoding analogs and fragments of the FIG. 1 amino acid sequence as
defined above, and nucleic acid sequences encoding proteins
comprising a domain sharing at least 80% amino acid sequence
identify (more preferably, at least 85%, or at least 90%, or at
least 95%, or at least 96%, or at least 97%, or at least 98% or at
least 99%) with the ligand binding domain of the FIG. 1 sequence,
as described above. A specific nucleic acid sequence of the
invention is that shown in FIG. 1.
[0019] The hPXR encoding sequence can be present in a construct,
for example, in an expression construct, operably linked to a
promoter (e.g., the CMV, SV40, Taq, T7 or LacO promoter). Such
expression constructs are operative in a cell in culture (e.g.,
yeast, bacteria, insect or mammalian), to express the encoded hPXR,
or fragment thereof. Preferred expression vectors include pGEX,
pET, pFASTbacHT and pSG5.
[0020] The invention also relates to cells in culture (e.g., yeast,
bacteria or mammalian (for example, CV-1, HuH7, HepG2, or CaCo2
cells)) that are transformed with an above-described construct.
Transformation can be effected using any of a variety of standard
techniques. Such cells can be used in a method of making hPXR (or
fragment thereof) by culturing same under conditions suitable for
expression of the polypeptide product.
[0021] The invention further relates to chimeric receptors (or
fusion proteins having a receptor component) (and encoding
sequences) comprising at least a DNA-binding domain or a
ligand-binding domain of hPXR, and a non-hPXR derived sequence.
Non-hPXR derived sequences can be selected so as to be suitable for
the purpose to be served by the chimeric receptor. Examples of such
sequences include glutathione-S-transferase and the DNA binding
domain of yeast transcription factor GAL4 and other DNA binding
domains, e.g., DNA binding domains for the estrogen and
glucocorticoid receptors. The chimeric receptor can bear a
detectable label (e.g., a radioactive or fluorescent label). The
chimeric receptor can also be bound to a solid support, e.g., a
glass or plastic particle, a plate or a filter.
[0022] A further aspect of the invention relates to in vitro
(cell-free) and in vivo (cell-based) assays that can be used to
profile the effects of compounds (e.g. potential new drugs) on
CYP3A4 levels. The inducibility of CYP3A4 levels, coupled with the
broad substrate specificity of the CYP3A4 enzyme, represent the
basis for many drug-drug interactions in patients undergoing
multiple drug therapy. Ideally, new drugs would have little or no
effect on CYP3A4 expression levels.
[0023] The assays of the invention can take any of a variety of
forms. As compounds that activate hPXR function as inducers of
CYP3A4 gene expression, hPXR binding and activation assays provide
efficient means to identify compounds that can be expected to
activate CYP3A4.
[0024] Binding assays of the invention include cell free assays in
which hPXR, or the ligand binding domain thereof (alone or present
as a fusion protein), is incubated with a test compound which,
advantageously, bears a detectable label (e.g., a radioactive or
fluorescent label). The hPXR, or ligand binding domain thereof,
free or bound to test compound, is then separated from free test
compound using any of a variety of techniques (e.g., using gel
filtration chromatography (for example, on Sephadex G50 spin
columns) or through capture on a hydroxyapatite resin). The amount
of test compound bound to hPXR or ligand binding domain thereof, is
then determined (for example, by liquid scintillation counting in
the case of radiolabelled test compounds).
[0025] An alternative approach for detecting radiolabeled test
compound bound to hPXR, or ligand binding domain thereof, is a
scintillation proximity assay (SPA). In this assay, a bead (or
other particle) is impregnated with scintillant and coated with a
molecule that can capture the hPXR, or ligand binding domain
thereof (e.g., streptavidin-coated beads can be used to capture
biotinylated hPXR ligand binding domain). Radioactive counts are
detected only when the complex of radiolabeled test compound and
the hPXR, or ligand binding domain thereof, is captured on the
surface of the SPA bead, bringing the radioactive label into
sufficient proximity to the scintillant to emit a signal. This
approach has the advantage of not requiring the separation of free
test compound from bound (Nichols et al, Anal. Biochem. 257:112-119
(1998)).
[0026] Assays to determine whether a test compound interacts with
the hPXR ligand binding domain can also be performed via a
competition binding assay. In this assay, hPXR, or ligand binding
domain thereof, is incubated with a compound known to interact with
hPXR, which compound, advantageously, bears a detectable label
(e.g., a radioactive or fluorescent label (see Example 5--Crabtree
catalysts suitable for use in the synthetic approach described in
Example 5 include those reported by Chen et al, J. Labelled Compd.
Radiopharm. 39:291 (1997) and Crabtree et al, Inorg. Synth. 28:56
(1990))). A test compound is added to the reaction and assayed for
its ability to compete with the labeled compound for binding to
hPXR, or ligand binding domain thereof. A standard assay format
employing a step to separate free known (labeled) compound from
bound, or an SPA format, can be used to assess the ability of the
test compound to compete.
[0027] A further example of a binding assay in accordance with the
invention is based on the finding that hPXR ligands induce the
interaction of hPXR ligand binding domain with coactivators (e.g.,
SRC1, TIF-1, TIF-2 or ACTR, or fragment thereof). To determine if a
test compound activates hPXR, and thus induces CYP3A4 expression,
the ligand binding domain of hPXR is prepared (e.g., expressed) as
a fusion protein (e.g., with glutathione-S-transferase (GST), a
histidine tag or a maltose binding protein). The fusion protein and
coactivator (either or both advantageously labeled with a
detectable label, e.g., a radiolabel or fluorescent tag) are
incubated in the presence and absence of the test compound and the
extent of binding of the coactivator to the fusion protein
determined. The induction of interaction in the presence of the
test compound is indicative of an hPXR activator. hPXR activation
assays in accordance with the invention can be carried out using
full length hPXR and a reporter system comprising one or more
copies of the DNA binding site recognized by the hPXR binding
domain (see Example 3). Advantageously, however, the activation
assays are conducted using established chimeric receptor systems.
For example, the ligand binding domain of hPXR can be fused to the
DNA binding domain of, for example, yeast transcription factor
GAL4, or that of the estrogen or glucocorticoid receptor. An
expression vector for the chimera (e.g., the GAL4-hPXR chimera) can
be transfected into host cells (e.g., CV-1, HuH7, HepG2 or CaCo2
cells) together with a reporter construct. The reporter construct
can comprise one or more (e.g., 5) copies of the DNA binding site
recognized by the binding domain present in the chimera (e.g., the
GAL4 DNA binding site) driving expression of a reporter gene (e.g.,
CAT, SPAP or luciferase). Cells containing the constructs are then
treated with either vehicle alone or vehicle containing test
compound, and the level of expression of the reporter gene
determined. In accordance with this assay, enhancement of
expression of the reporter gene in the presence of the test
compound indicates that the test compound activates hPXR and thus
can function as an inducer of CYP3A4 gene expression. (See Example
4.)
[0028] Another format suitable for use in connection with the
present invention is the yeast two-hybrid assay. This is an
established approach to detect protein-protein interactions that is
performed in yeast. Protein #1, representing the bait, is expressed
in yeast as a chimera with a DNA binding domain (e.g., GAL4).
Protein #2, representing the predator, is expressed in the same
yeast cell as a chimera with a strong transcriptional activation
domain. The interaction of bait and predator results in the
activation of a reporter gene (e.g., luciferase or -galactosidase)
or the regulation of a selectable marker (e.g., LEU2 gene). This
approach can be used as a screen to detect, for example,
ligand-dependent interactions between hPXR1 and other proteins such
as coactivator proteins (e.g., SRC1, TIF1, TIF2, ACTR) or fragments
thereof. (Fields et al, Nature 340:245-246 (1989)).
[0029] Still another format is the ligand-induced complex formation
(LIC) assay. This is an approach to detect ligand-mediated effects
on nuclear receptor-DNA interactions. hPXR (or, minimally, the DNA
and ligand binding domains thereof) can be incubated with its
heterodimeric partner RXR in the presence of DNA representing an
established hPXR/RXR binding site. Test compounds can be assayed
for their ability to either enhance or interfere with binding of
the hPXR/RXR heterodimer to DNA (Forman et al, Proc. Natl. Acad.
Sci. USA 94:4312-4317 (1997)).
[0030] Compounds that bind PXR with a suitable pKi, for example
with a pKi>5, can be screened for selectivity for PXR versus
other nuclear receptors (e.g., RXR) using standard binding assays.
A compound that binds selectively to PXR (that is, has at least a
10 fold greater affinity for PXR, preferably, at least a 100 fold
greater affinity for PXR, than, for example, the glucocorticoid
receptor) and thereby affects the functional activity of PXR in a
cell (e.g., a cell in culture, a cell present in a tissue or a cell
present in a whole animal) can be used to associate PXR activity
with a mammalian disease state. For example, a compound that
activates PXR induces CYP3A. Thus, diseases in which CYP3A activity
is important are associated with PXR, and compounds that activate
or deactivate PXR may be useful in prevention or treatment of such
diseases. By using the associating methods of this invention, new
PXR-associated diseases can be discovered. Once these new
associations are discovered, new drugs for these diseases can be
identified by screening for compounds that activate or deactivate
PXR.
[0031] An example of a compound suitable for use in making disease
associations in accordance with the method described above is the
compound of formula I:
##STR00001## [0032] wherein each of R1, R2, R3 and R4 is,
independently, C.sub.1-C.sub.6alkyl (linear or branched),
preferably, C.sub.2 or C.sub.3alkyl (e.g., ethyl, n-propyl or
iso-propyl), more preferably, C.sub.2alkyl. The compounds can be
labelled with a detectable label, e.g., a radiolabel, e.g.,
tritium.
[0033] Another aspect of the invention relates to transgenic
animals that express hPXR. For example, transgenic mice can be
generated that express the hPXR gene as well as the endogenous
mouse PXR gene. Mice can also be generated in which the endogenous
PXR gene is knocked out and then replaced by the hPXR gene.
Transgenic aminals can be generated that express isoforms of hPXR
as well as mutant alleles of the gene. Transgenic animals developed
by these methods can be used to screen compounds for drug
interactions and toxicities, and to study the regulation of CYP3A
in vivo.
[0034] A further aspect of the present invention relates to
diagnostic assays that can be used to screen for mutations in hPXR
that alter the ability of the receptor to induce CYP3A4 gene
expression. These assays can be based on the sequencing of the hPXR
gene, on hybridization approaches designed to detect sequence
changes or polymorphisms, or the use of antibodies to distinguish
wild-type from mutant/polymorphic hPXR. Changes that result in
alteration of the DNA binding or ligand binding characteristics of
hPXR can be expected to have a significant impact on hPXR activity.
A mutation or polymorphism in hPXR can be indicative of a patient
at increased risk of suffering an adverse reaction to a drug as a
result of unusual rates of drug metabolism.
[0035] The invention also relates to antibodies, polyclonal or
monoclonal, that are specific for hPXR, and antigen binding
fragments thereof (e.g., Fab fragments). The antibodies can be
generated in accordance with standard techniques using intact hPXR
or a fragment thereof as defined above. The antibodies can be used,
for example, in assays to detect the presence of the receptor.
Further, the antibodies can be used in hPXR purification
protocols.
[0036] The invention also relates to kits suitable for use, for
example, in one or more method described above. The kits can
include hPXR (or fragment thereof) or nucleic acid encoding same or
antibodies as described above. The kit can also include compounds
that bind hPXR, such as GW-485801. The hPXR, nucleic acid and/or
antibody can be present in the kit disposed within a container
means. The kit can also include ancillary reagents and buffers,
etc., to facilitate practice of the specific method.
[0037] Certain aspects of the present invention are described in
greater detail in the non-limiting Examples that follow.
EXAMPLES
[0038] The following experimental details are relevant to the
specific Examples that follow.
Chemicals
[0039] Dexamethasone-t-butylacetate and RU486 were purchased from
Research Plus, Inc. (Bayonne, N.J.) and Biomol (Plymouth Meeting,
Pa.), respectively. All other compounds were purchased from either
Sigma Chemical Co. (St. Louis, Mo.) or Steraloids, Inc. (Wilton,
N.H.).
Molecular Cloning of hPXR cDNAs
[0040] An EST was identified in the Incyte database (clone
identification number 2211526) that contained nucleotides 444-2111
of the hPXR sequence. An oligonucleotide derived from this EST
sequence (5' CTGCTGCGCATCCAGGACAT 3') (SEQ ID NO:1) was used to
screen a pCMV-SPORT human liver cDNA library (Gibco/BRL) using Gene
Trapper solution hybridization cloning technology (Gibco/BRL). Two
clones were obtained that encoded hPXR, one containing nucleotides
1-2125, the other containing nucleotides 102-2118. The sequence of
the longer is shown in FIG. 1A. Sequences were aligned and analyzed
by the University of Wisconsin Genetics Computer Group
programs.
Plasmids
[0041] The expression vector pSG5-hPXR was generated by PCR
amplification and subcloning of nucleotides 1-1608 of the hPXR
clone into the pSG5 expression vector (Strategene). pSG5-hPXR ATG
was generated by PCR amplification of cDNA encoding amino acids
1-434 of hPXR using oligonucleotides
5'-GGGTGTGGGGAATCCACCACCATGGAGGTGAGACCCAAAGAAAGC-3' (SEQ ID NO:2)
(sense) and 5'-GGGTGTGGGGGATCCTCAGCTACCTGTGATGCCG-3' (SEQ ID NO:3)
(antisense) and insertion into EcoRI/BamHI-cut pSG5. The bacterial
expression vector pGEX-hPXR was generated by PCR amplification of
cDNA encoding amino acids 108-434 and insertion into pGEX-2T
(Pharmacia). The reporter plasmid (DR3).sub.4-tk-CAT was generated
by insertion of four copies of a double-stranded oligonucleotide
containing the CYP3A1 DR3PXRE
(5'-GATCAGACAGTTCATGAAGTTCATCTAGATC-3') (SEQ ID NO:4) into the
BamHI site of pBLCAT2 (Luckow et al, Nucl. Acids Res. 15:5490
(1987)). The reporter plasmid (IR6).sub.3-tk-CAT was generated by
insertion of three copies of the CYP3A4 IR6PXRE
(5'-GATCAATATGAACTCAAAGGAGGTCAGTG-3') (SEQ ID NO:5) into the BamHI
site of pBL2CAT. The pRSET-SRC1.14 expression plasmid has been
previously described (Kliewer, S. A., et al. Cell 92:73-82 (1998)).
All constructs were confirmed by sequence analysis.
Cotransfection Assays
[0042] CV-1 cells were plated in 24-well plates in DME medium
supplemented with 10% charcoal-stripped fetal calf serum at a
density of 1.2.times.10.sup.5 cells per well. In general,
transfection mixes contained 33 ng of receptor expression vector,
100 ng of reporter plasmid, 200 ng of -galactosidase expression
vector (pCH110, Pharmacia), and 166 ng of carrier plasmid. Cells
were transfected overnight by lipofection using Lipofectamine (Life
Technologies, Inc.), according to the manufacturer's instructions.
The medium was changed to DME medium supplemented with 10%
delipidated calf serum (Sigma) and cells were incubated for an
additional 24 hours. Cell extracts were prepared and assayed for
CAT and -galactosidase activities as previously described (Lehmann
et al, J. Biol. Chem. 270:12953-12956 (1995)).
Northern Analysis
[0043] An approximately 1.0 kb fragment encoding the LBD of hPXR
was [.sup.32P]-labeled by random priming and used to probe mouse
multiple tissue Northern blots (Clontech). Blots were hybridized in
ExpressHyb solution (Clontech) at 42.degree. C. overnight. Final
washes were performed with 0.1.times.SSC, 0.1% SDS at 58.degree.
C.
Band Shift Assays
[0044] hPXR, mPXR1, and hRXR were synthesized in vitro using the
TNT rabbit reticulocyte lysate coupled in vitro
transcription/translation system (Promega) according to the
manufacturer's instructions. Gel mobility shift assays (20 .mu.l)
contained 10 mM Tris (pH 8.0), 40 mM KCl, 0.05% NP-40, 6% glycerol,
1 mM DTT, 0.2 .mu.g of poly(dI-dC) and 2.5 .mu.l each of in vitro
synthesized PXR and RXR proteins. Competitor oligonucleotides were
included at a 10-fold or 50-fold excess. After a 10 min incubation
on ice, 10 ng of [.sup.32P]-labeled oligonucleotide was added and
the incubation continued for an additional 10 min. DNA-protein
complexes were resolved on a 4% polyacrylamide gel in 0.5.times.TBE
(1.times.TBE=90 mM Tris, 90 mM boric acid, 2 mM EDTA). Gels were
dried and subjected to autoradiography at -70.degree. C. The
following oligonucleotides were used as either radiolabeled probes
or competitors (sense strand is shown):
TABLE-US-00001 (SEQ ID NO: 6) CYP3A4 IR6: 5'
GATCAATATGAACTCAAAGGAGGTCAGTG 3' (SEQ ID NO: 7) CYP3A4 IR6m1 5'
GATCAATATGTTCTCAAAGGAGAACAGTG 3' (SEQ ID NO: 8) CYP3A4 IR6m2 5'
GATCAATAACAACTCAAAGGAGGTCAGTG 3' (SEQ ID NO: 9) CYP3A1 DR3: 5'
GATGCAGACAGTTCATGAAGTTCATCTAGATC 3'.
CARLA
[0045] GST-hPXR fusion protein was expressed in BL21(DE3)plysS
cells and bacterial extracts prepared by one cycle of freeze-thaw
of the cells in Protein Lysis Buffer containing 10 mM Tris, pH 8.0,
50 mM KCl, 10 mM DTT, and 1% NP-40 followed by centrifugation at
40,000.times.g for 30 minutes. Glycerol was added to the resulting
supernatant to a final concentration of 10%. Lysates were stored at
-80.degree. C. [.sup.35S]SRC1.14 was generated using the TNT rabbit
reticulocyte system (Promega) in the presence of Pro-Mix
(Amersham). Coprecipitation reactions included 25 .mu.l of lysate
containing GST-hPXR fusion protein, 25 .mu.l Incubation Buffer (50
mM KCl, 40 mM HEPES pH 7.5, 5 mM .beta.-mercaptoethanol, 1%
Tween-20, 1% non-fat dry milk), 5 .mu.l [.sup.35S]SRC1.14, and
vehicle (1% DMSO) or compounds as indicated. The mixtures were
incubated for 25 minutes at 4.degree. C. with gentle mixing prior
to the addition of 15 .mu.l of glutathione-sepharose 4B beads
(Pharmacia) that had been extensively washed with Protein Lysis
Buffer. Reactions were incubated with gentle mixing at 4.degree. C.
for an additional 25 min. The beads were pelleted at 3000 rpm in a
microfuge and washed 3 times with Protein Incubation Buffer
containing either vehicle alone, dexamethasone-t-butylacetate,
rifampicin, or clotrimazole. After the last wash, the beads were
resuspended in 25 .mu.l of 2.times.SDS-PAGE sample buffer
containing 50 mM DTT. Samples were heated at 100.degree. C. for 5
minutes and loaded onto a 10% Bis-Tris PAGE gel. Gels were dried
and subjected to autoradiography.
Example 1
Molecular Cloning and Tissue Expression Pattern of hPXR
[0046] A human EST was identified in the Incyte LifeSeq proprietary
database that was highly homologous to a region of mPXR1 (Kliewer
et al, Cell 92:73-82 (1998)). Two larger clones were isolated in a
screen of a human liver cDNA library using an oligonucleotide
within the EST as a probe. The longest of these clones was 2146 bp
in length (FIG. 1A) and encoded a new member of the nuclear
receptor superfamily that was 97% and 76% identical to mPXR1 in the
DNA binding domain (DBD) and ligand binding domain (LBD),
respectively (FIG. 1B). In terms of other members of the nuclear
receptor superfamily, hPXR was most closely related to the Xenopus
laevis orphan receptor ONR1 (Smith et al, Nucl. Acids Res. 22:66-71
(1994)) and the vitamin D receptor (FIG. 1B). Notably, the hPXR
sequence lacked an AUG initiator codon in between an in-frame stop
codon (nucleotides 205-207 in the hPXR sequence) and the start of
the region encoding the DBD. However, transfection experiments
performed in CV-1 cells with the hPXR clone and a reporter plasmid
containing four copies of an established mPXR binding site from the
rat CYP3A1 gene promoter inserted upstream of the minimal thymidine
kinase (tk) promoter and the chloramphenicol acetyltransferase
(CAT) gene (Kliewer et al, Cell 92:73-82 (1998)) demonstrated that
the hPXR clone encoded a functional nuclear receptor that was
activated efficiently by dexamethasone-t-butylacetate, a known
mPXR1 ligand (Kliewer et al, Cell 92:73-82 (1998)) (FIG. 1C).
[0047] Examination of the hPXR sequence revealed an in-frame CUG
codon (nucleotides 304-306) surrounded by a favorable Kozak
sequence (Kozak, J. Biol. Chem. 266:19867-19870 (1991)). There is
precedent for the use of CUG codons to initiate translation of
eukaryotic proteins, including the nuclear receptor RAR4 (Kozak, J.
Biol. Chem. 266:19867-19870 (1991), Nagpal et al, Proc. Natl. Acad.
Sci. USA 89:2718-2722 (1992)). Initiation of translation at this
CUG codon would yield a protein of 434 amino acids, three longer
than mPXR1, with a predicted MW of 49.7 kD. In order to determine
whether translation of the hPXR cDNA initiated at the CUG codon,
hPXR RNA containing the wild-type 5' region was translated in the
presence of [.sup.35S]methionine using rabbit reticulocyte lysates.
As a control, hPXR RNA, in which this CUG codon had been mutated to
the optimal AUG (hPXR AUG), was also translated in vitro.
Translation of the wild-type hPXR RNA resulted in an approximately
50 kD protein that co-migrated with the translation product of hPXR
AUG RNA (FIG. 1D, open arrow with asterisk). This 50 kD product was
not produced when hPXR antisense RNA was used in the translation
reaction. Much lower amounts of an approximately 53 kD translation
product were also produced in translation reactions performed with
hPXR RNA (FIG. 1D, bent arrow), indicating that a small amount of
translation initiated at other non-AUG codons upstream of the CUG
codon. However, the results indicate that the CUG codon represents
the principal translation initiation site for hPXR containing a
functional DBD.
[0048] The tissue expression pattern of hPXR was next examined via
Northern analysis using blots containing poly(A)+ RNA prepared from
multiple adult tissues. hPXR mRNA was expressed most abundantly in
liver and was also present in the colon and small intestine (FIG.
2). Three transcripts of different size were detected in each of
these tissues: a prominent 2.6 kb product and two less abundant
messages of approximately 4.3 kb and 5 kb. It was recently shown
that the mPXR gene is also abundantly expressed in liver and small
intestine (Kliewer et al, Cell 92:73-82 (1998)). Whereas mPXR
message was also detected at low levels in stomach and kidney, mRNA
for hPXR was not detected in these tissues (FIG. 2). Thus, both
hPXR and mPXR are most abundantly expressed in the liver and
tissues of the gastrointestinal tract; however, there are
differences in PXR expression patterns in mice and humans.
Example 2
hPXR Activates Transcription Through a Response Element in the
CYP3A4 Gene Promoter
[0049] Several lines of evidence have been provided that mPXR1
regulates CYP3A1 gene expression: mPXR1 was activated by compounds
known to activate CYP3A1 gene expression including glucocorticoids
and antiglucocorticoids, mPXR1 and CYP3A1 gene expression
colocalized in the liver and small intestine, and mPXR1 bound to a
response element in the CYP3A1 gene promoter that had previously
been determined to confer responsiveness to glucocorticoids and
antiglucocorticoids (Kliewer et al, Cell 92:73-82 (1998),
Quattrochi et al, J. Biol. Chem. 270:28917-28923 (1995), Huss et
al, J. Biol. Chem. 93:4666-4670 (1996)). The findings that the
CYP3A4 gene is also expressed in the liver and intestine and that
this expression is induced in response to glucocorticoids and
antiglucocorticoids (Molawa et al, Proc. Natl. Acad. Sci. USA
83:5311-5315 (1986), Kocarek et al, Drug Met. Dispos. 23:415-421
(1995)) led to the investigation of whether hPXR regulates CYP3A4
gene expression.
[0050] The induction of CYP3A4 expression in response to
dexamethasone and rifampicin has been localized to an approximately
20 bp region of the promoter that contains two copies of the
nuclear receptor half-site sequence AG(G/T)TCA organized as an
inverted repeat (IR) and separated by 6 base pairs, an IR6 motif
(Barwick et al, Molec. Pharmacol. 50:10-16 (1996)) (FIG. 3B). This
IR6 motif is highly conserved in the promoters of CYP3A gene family
members of several species (Barwick et al, Molec. Pharmacol.
50:10-16 (1996)). Interestingly, this half-site configuration is
very different from that found in the CYP3A1 PXR response element
(PXRE) which contains two half-sites organized as a direct repeat
(DR) with a 3 nucleotide spacer, a DR3 motif (Kliewer et al, Cell
92:73-82 (1998)). To determine whether hPXR could regulate
transcription through the IR6 motif, a reporter plasmid was
generated containing three copies of the CYP3A4 IR6 response
element upstream of the tk promoter and CAT gene. Cotransfection
assays were performed with the (IR6).sub.3-tk-CAT reporter and
pSG5-hPXR ATG expression plasmids in CV-1 cells that were either
treated with vehicle alone or 10 .mu.M
dexamethasone-t-butylacetate. hPXR induced reporter levels in the
presence of dexamethasone-t-butylacetate (FIG. 3A), demonstrating
that hPXR can activate transcription through the CYP3A4 IR6
motif.
[0051] In order to determine whether hPXR interacted directly with
the CYP3A4 IR6 response element, band shift assays were performed.
Since mPXR1 binds to DNA as a heterodimer with RXR (Kliewer et al,
Cell 92:73-82 (1998)), it was suspected that hPXR would require RXR
for high-affinity interactions with DNA. Neither hPXR nor RXR bound
to a radiolabeled oligonucleotide containing the CYP3A4 IR6 motif
on their own (FIG. 3C). However, hPXR and RXR bound efficiently as
a heterodimer to the IR6PXRE. The hPXR/RXR complex was competed
efficiently by unlabeled oligonucleotides encoding either the IR6
PXRE from the CYP3A4 promoter or the DR3PXRE from the CYP3A1
promoter that it was previously defined as a mPXR1/RXR binding site
(Kliewer et al, Cell 92:73-82 (1998)) (FIG. 3C). Thus, the hPXR/RXR
heterodimer interacted efficiently with two response elements with
remarkably different architecture. Little or no competition was
seen when competitor oligonucleotides were used that contained
mutations in either the 5' half-site or both half-site sequences of
the IR6PXRE (FIG. 3C). The same binding profile was observed when
the mPXR1 was substituted for hPXR (FIG. 3C). It was concluded from
these experiments that hPXR binds efficiently to the CYP3A4 IR6PXRE
as a heterodimer with RXR, and that hPXR and mPXR1 have very
similar DNA binding profiles.
Example 3
Differential Activation of Human and mPXR
[0052] CYP3A4 gene expression is induced in response to a
remarkable array of xenobiotics, including synthetic steroids
(Kocarek et al, Drug Met. Dispos. 23:415-421 (1995), Schuetz et al,
J. Biol. Chem. 259:2007-2012 (1984), Heuman et al, Mol. Pharmacol.
21:753-760 (1982), Schulte-Hermann et al, Cancer Res. 48:2462-2468
(1988)), macrolide antibiotics (Wrighton et al, Biochem.
24:2171-2178 (1985)), antimycotics (Hostetler et al, Mol.
Pharmacol. 35:279-285 (1989)), HMG-CoA reductase inhibitors
(statins) (Kocarek et al, Toxicol. Appl. Pharmacol. 120:298-307
(1993), Schuetz et al., Hepatology 18:1254-1262 (1993)), and
phenobarbital-like compounds (Heuman et al, Mol. Pharmacol.
21:753-760 (1982)). It was next determined whether hPXR might
mediate the effects of some or all of these compounds on CYP3A4
expression. CV-1 cells were cotransfected with the pSG5-hPXR ATG
expression plasmid and the (IR6).sub.3-tk-CAT reporter plasmid, and
the cells were treated with micromolar concentrations of a number
of compounds that are known to induce CYP3A gene expression in
humans and/or rodents. As shown in FIG. 4A, hPXR was activated by
the synthetic steroids dexamethasone, dexamethasone-t-butylacetate,
PCN, RU486, spironolactone, and cyproterone-acetate.
Dexamethasone-t-butylacetate and RU486 were the most efficacious
activators of hPXR among the synthetic steroids tested. Notably,
the antibiotic rifampicin and the antimycotic clotrimazole were
both efficacious activators of hPXR (FIG. 4A). The
antihypercholesterolemic drug lovastatin also activated hPXR as did
phenobarbital and the organochlorine pesticide transnonachlor (FIG.
4A). Thus, hPXR is activated by a remarkably diverse group of
synthetic compounds that are known to induce CYP3A4 gene expression
(FIG. 4B).
[0053] Several naturally-occurring C21 steroids were also tested on
hPXR that were previously shown to activate mPXR1 (Kliewer et al,
Cell 92:73-82 (1998)). Pregnenolone, progesterone, and 5
-pregnane-3,20-dione all activated hPXR roughly 4-fold. The
17-hydroxy derivatives of pregnenolone and progesterone were weak
activators of hPXR (FIG. 4A). These natural steroids all activated
hPXR in transient transfection assays with EC.sub.50 values >10
.mu.M, suggesting that they are unlikely to be natural hPXR
ligands. However, related pregnanes or pregnane metabolites may
serve as natural hPXR ligands.
[0054] Analyses of the effects of chemical inducers of CYP3A gene
expression in primary hepatocytes obtained from either rodents or
humans have revealed significant interspecies differences (Barwick
et al, Molec. Pharmacol. 50:10-16 (1996), Kocarek et al, Drug Met.
Dispos. 23:415-421 (1995)). For example, rifampicin is an
efficacious inducer of CYP3A4 gene expression in human hepatocytes
but has little or no effect on CYP3A1 levels in rat hepatocytes. In
contrast, PCN has marked effects on CYP3A levels in rat hepatocytes
but only modest effects in human hepatocytes. To examine whether
differences in PXR activation profiles might account for these
interspecies variations, the same panel of compounds was tested on
mPXR1. As shown in FIG. 4A, there were marked differences in the
response profiles of the mouse and human homologs of PXR. Whereas
rifampicin was an efficacious activator of hPXR, it was only a weak
activator of mPXR1 (FIG. 4A). Clotrimazole, lovastatin and
phenobarbital were also more efficacious activators of hPXR than
mPXR1. In contrast, PCN only activated hPXR approximately 3-fold
but activated mPXR1 roughly 9-fold (FIG. 4A). Taken together, these
data indicate that much of the interspecies variability in CYP3A
regulation may be due to differences in PXR activation
profiles.
[0055] The panel of chemicals that induce CYP3A expression was also
profiled on the human glucocorticoid receptor (GR). As shown in
FIG. 4A, only dexamethasone and dexamethasone-t-butylacetate were
efficacious activators of the GR. None of the other compounds
activated the GR>1.5-fold (FIG. 4A). In contrast to a recent
report (Calleja et al, Nature Med. 4:92-96 (1998)), activation of
the GR by rifampicin was not observed. Since this previous work was
performed in HepG2 cells, it may be that rifampicin is
differentially metabolized in various cell lines. As expected,
neither pregnenolone, progesterone, nor their 17-hydroxy
derivatives had an effect on GR activity (FIG. 4A). Thus, the broad
activation profile that was observed for the human and mouse
homologs of PXR with inducers of CYP3A gene expression is not a
general property of other steroid hormone receptors.
[0056] In the absence of high-affinity radioligands,
coactivator-based assays have been used as a biochemical means to
determine whether compounds that activate orphan nuclear receptors
do so through direct interactions with the protein (Kliewer et al,
Cell 92:73-82 (1998), Krey et al, Mol. Endocrinol. 11:779-791
(1997)). These assays are predicated on the finding that ligands
induce the interaction of nuclear receptors with accessory
proteins, termed coactivators (Krey et al, Mol. Endocrinol.
11:779-791 (1997)). It was recently demonstrated that several
steroidal activators of mPXR1, including
dexamethasone-t-butylacetate and PCN, promote the interaction of
the mPXR1 LBD with a 14 kD fragment of the steroid receptor
coactivator 1 (SRC1.14) (Kliewer et al, Cell 92:73-82 (1998)). In
order to examine whether the structurally-diverse compounds that
activate hPXR do so by acting as ligands, three of the more potent
activators representing different chemical classes were selected,
dexamethasone-t-butylacetate, rifampicin, and clotrimazole, for
testing in the coactivator-receptor ligand assay (CARLA). The LBDs
of hPXR and mPXR1 were expressed in E. coli as fusion proteins with
glutathione-S-transferase (GST), and SRC1.14 was synthesized in
vitro in the presence of [.sup.35S]methionine and
[.sup.35S]cysteine. As shown in FIG. 4C,
dexamethasone-t-butylacetate, rifampicin and clotrimazole each
promoted the interaction of [.sup.35S]SRC1.14 with GST-hPXR.
Consistent with the results of the transfection studies,
dexamethasone-t-butylacetate induced an efficient interaction
between GST-mPXR1 and [.sup.35S]SRC1.14 whereas rifampicin and
clotrimazole did not (FIG. 4C). Taken together, these data indicate
that structurally-divergent compounds can serve as hPXR ligands,
and that the human and mouse homologs of PXR differ significantly
in terms of their ligand binding properties.
Example 4
Transfection Assay
[0057] Plasmids: GAL4-hPXR chimera and UAS-tk-SPAP Reporters.
[0058] The GAL4-hPXR expression constructs contain the translation
initiation sequence and amino acids 1 to 147 of the yeast S.
crevisiae transcription factor GAL4 in the pSG5 expression vector
(Statagene). Amino acids 108 to 434 of hPXR are amplified by
polymerase chain reaction (PCR) using vent polymerase (New England
Biolads) and inserted C-terminal to the GAL4 sequences. The
UAS-tk-SPAP reporter contains 5 copies of the GAL4 binding site
upstream of the tk promoter and the CAT gene (Berger et al, Gene
66:1 (1988)).
[0059] Transfection Assay: SPAP Reporter.
[0060] CV-1 cells are plated in DME medium supplemented with 10%
delipidated fetal calf serum at a density of 2.4.times.10.sup.4
cells per well in a 96-well plate (Costar) 16-24 h before
transfection. In general, 8.0 ng of reporter plasmid, 25.0 ng of
-galactosidase expression vector (pCH110, Pharmacia), and 2.0 ng of
GAL4-hPXR expression vector are mixed with carrier DNA
(pBluescript, Stratagene) to a total of 80 ng of DNA per well in a
volume of 10 ml optiMEM I medium (Life Technologies). To this, a
second mix, containing 9.3 ml optiMEM I medium and 0.7 ml of
LIPOFECTAMINE.TM. (Life Technologies), is added. After 30 min., an
additional 80 ml of optiMEM I medium are added and the combined mix
is then applied to the cells. Sixteen hours later, the medium is
changed to DME medium supplemented with 10% delipidated and heat
inactivated fetal calf serum and the test compound at a
concentration of 10.sup.-5M. After incubation for 24 h, SPAP
activity and -galactosidase activity are measured by directly
adding to the medium 200 ml substrate mix (16 mM o-nitrophenyl
-D-galactopyranoside (Sigma), 120 mM fluorescein diphosphate
(Molecular Probes), 0.16% Triton X-100, 160 mM diethanolamine pH9,
44.8 mM NaCl, and 0.8 mM MgCl.sub.2). Alternatively, alkaline
phosphatase and -galactosidase activities are measured separately
using standard protocols. Briefly, cells are lysed by adding 25 ml
0.5% Triton X-100 to the supernatant. To 40 ml cell lysate, 200 ml
-galactosidase substrate reagent (36 mM o-nitrophenyl
-D-galactopyranoside, 1.25 mM MgCl.sub.2, 2.8 mM NaCl, 4.4M
-mercaptoethanol) or 200 ml alkaline phosphatase substrate reagent
(2.5 mM p-nitrophenyl phosphate, 0.5 mM MgCl.sub.2, 20 mM NaCl, 1 M
diethanolamine pH 9.85) are added and incubated for 1 h. Alkaline
phosphatase activity is expressed as fold activation relative to
that observed with vehicle alone (normalized to -galactosidase
activity which serves as internal control standard for transfection
efficiency).
Example 5
Synthesis of [.sup.3H]GW-485801
[0061] (i) The Preparation of [.sup.3H]3,5-Ditertbutyl-4-hydroxy
benzaldehyde.
[0062] 3,5-Diterbutyl-4-hydroxy benzaldehyde, 5 mg (20.6 .mu.mol)
and Crabtree catalyst, 7.5 mg (9.3 .mu.mol), were dissolved in 2 ml
dichloromethane and stirred under 10 Ci tritium gas for 5 hours.
The solution was then evaporated to dryness, and labile tritium was
removed by repeated evaporations from methanol. The residue was
redissolved in methanol, 10 ml, counted and analyzed.
[0063] Yield=800 mCi.
[0064] Radiochemical purity by TLC on silica in hexane:ethyl
acetate (80:20) was approximately 50%.
[0065] The crude material was evaporated to 1 ml and purified by
preparative plate chromatography on a single 500 .mu.m silica
plate, eluting in hexane:ethyl acetate (85:15). The plates were
viewed under UV, the band corresponding to required aldehyde was
collected and the product extracted into ethyl acetate. This was
evaporated to dryness and redissolved in dichloromethane, counted
and analyzed.
[0066] Yield=370 mCi.
[0067] TLC as above showed a singly labelled, specific activity 23
Ci/mmol.
(ii) The Preparation of [.sup.3H]GW-485801
[0068] The product from (i) above (370 mCi at 23 Ci/mmol, 16
.mu.mol) was evaporated to dryness, redissolved in THF, 1 ml, and
cooled in an ice bath with stirring. 1M Titanium (IV) chloride in
toluene, 55 .mu.l, 55 .mu.mol, was added, immediate yellow color
formed. Tetraethyl methylenediphosphonate, 75 .mu.l, of a THF
solution at 110 mg/ml, 28.6 .mu.mol, was added, followed by
N-methyl morpholine, 8.1 .mu.l, 7.5 mg, 74 .mu.mol. This caused a
deep blue color. The solution was then stirred at room temperature
for 4 hours.
[0069] TLC analysis on silica in ethyl acetate:methanol (90:10)
showed approximately 60% of the radioactivity to correspond to
inactive GW-485801.
(iii) The Purification of [.sup.3H]GW-485801
[0070] The crude product was purified by preparative plate
chromatography on 2.times.1 mm silica plates, eluting in ethyl
acetate:methanol (90:10). The plates were viewed under UV, the band
corresponding to required product was collected and the product
extracted into ethyl acetate:methanol (90:10). This was evaporated
to dryness and redissolved in nitrogen-flushed ethanol, 30 ml. This
was a yellow solution.
[0071] Yield=180 mCi.
(iv) The Analysis of [.sup.3H] GW-485801
[0072] The purified product resulting from (iii) was analyzed by
TLC, HPLC, mass spectroscopy and T-NMR.
[0073] TLC showed a radiochemical purity of 99%.
[0074] HPLC showed a radiochemical purity of 98.9%.
[0075] In both of the above systems, the radioactive peak co-eluted
with inactive GW-485801.
[0076] Mass spectroscopy showed a specific activity of 23 Ci/mmol,
the isotope distribution being 18.4% unlabelled, 81.6%
1.times..sup.3H. The spectrum of the radioactive material was
consistent with that of the inactive GW-485801.
[0077] T-NMR showed a single labelling position (peak split into
four signals by coupling to the phosphorus atoms) corresponding to
labelling in the vinylic position of GW-485801. This corresponds to
labelling in the aldehyde-H in the precursor.
[0078] A portion of the material was diluted to 1 mCi/ml with
nitrogen-flushed ethanol and dispensed as 1.times.2 mCi pack. The
remainder was stored at .about.20.degree. C. (approximately 170
mCi).
Example 6
Biotin-His6-PXR/RXRa Protein
[0079] The coding sequence representing amino acids 130-434 of
human PXR (Genbank AF061056) was subcloned into the pRSETa
expression vector (Invitrogen). Sequence encoding a polyhistidine
tag derived from an N-terminal PCR primer (MKKGHHHHHHG) (SEQ ID
NO:10) was fused in-frame. The resulting encoded His6-PXR sequence
was as follows:
TABLE-US-00002 (SEQ ID NO: 11)
MKKGHHHHHHGSERTGTQPLGVQGLTEEQRMMIRELMDAQMKTFDTTFSH
FKNFRLPGVLSSGCELPESLQAPSREEAAKWSQVRKDLCSLKVSLQLRGE
DGSVWNYKPPADSGGKEIFSLLPHMADMSTYMFKGIISFAKVISYFRDLP
IEDQISLLKGAAFELCQLRFNTVFNAETGTWECGRLSYCLEDTAGGFQQL
LLEPMLKFHYMLKKLQLHEEEYVLMQAISLFSPDRPGVLQHRVVDQLQEQ
FAITLKSYIECNRPQPAHRFLFLKIMAMLTELRSINAQHTQRLLRIQDIH
PFATPLMQELFGITGS.
[0080] Restriction enzymes Nde I and Hind III were used to release
the cDNA fragment encoding amino acids 225-462 of RXR from BB5508
(pRSETa). The fragment was ligated into the like-cut pET24a
expression plasmid (Novagen). The Bgl II, Hind III fragment
(contains T7 promoter, lac operator, RBS and RXRa) of this
construct was then cloned into the BamH I, Hind III sites (removes
tetracycline resistance) of pACYC184 (BB5114). This allows for
expression of RXR from the T7 promoter when grown in BL21(DE3)
cells and induced with IPTG. The resulting encoded RXR sequence was
as follows:
TABLE-US-00003 (SEQ ID NO: 12)
MKKGSANEDMPVERILEAELAVEPKTETYVEANMGLNPSSPNDPVTNICQ
AADKQLFTLVEWAKRIPHFSELPLDDQVILLRAGWNELLIASFSHRSIAV
KDGILLATGLHVHRNSAHSAGVGAIFDRVLTELVSKMRDMQMDKTELGCL
RAIVLENPDSKGLSNPAEVEALREKVYASLEAYCKHKYPEQPGRFAKLLL
RLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLEAPHQMT.
[0081] The His6-PXR/pRSETa and RXR/pACYC184 plasmids were
cotransformed into the BL21(DE3) E. coli strain. 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.degree. C. for 24 hours. The cells were
induced with 0.05 mM IPTG for 4-6 hours at 22.degree. C. then the
cells were harvested by centrifugation (20 minutes, 3500 g,
4.degree. C.). The cell pellet was stored at -80.degree. C. The
cell pellet was resuspended in 250 ml Buffer A (50 mM Tris-Cl
pH8.0, 250 mM NaCl, 50 mM imidazole pH7.5). Cells were sonicated
for 3-5 minutes on ice and the cell debris was removed by
centrifugation (45 minutes, 20,000 g, 4.degree. C.). The cleared
supernatant was filtered through a 0.45 mM filter and loaded on to
a 50 ml ProBond [Ni.sup.++ charged] chelation resin (Invitrogen).
After washing to baseline with Buffer A, the column was washed with
Buffer A containing 125 mM imidazle pH 7.5. The His6-PXR/RXR
complex was eluted from the column using Buffer A with 300 mM
imidazole pH 7.5. Column fractions were pooled and concentrated
using Centri-prep 30K (Amicon) units. The protein was subjected to
size exclusion, using a column (26 mm.times.90 cm) packed with
Sepharose 5-75 resin (Pharmacia) pre-equilibrated with 20 mM
Tris-Cl pH 8.0, 200 mM NaCl, 5 mM DTT, 2.5 mM EDTA, pH 8.0. Column
fractions were pooled and concentrated as before. The purified
His6-PXR/RXR was buffer exchanged by gel filtration into PBS,
resulting in an average total molar protein concentration of 45 mM.
A five-fold total molar excess of NHS-LC-Biotin (Pierce) was added
to this protein mixture in a minimal volume of PBS. This solution
was incubated with gentle mixing for 60 minutes at ambient
temperature, approximately 23.degree. C. The biotinylation
modification reaction was stopped by the addition of a 2000.times.
molar excess of Tris-HCl, pH 8. The biotin-His6-PXR/RXR was
dialyzed at 4.degree. C. against 3 buffer changes, each of at least
50 volumes, TBS pH 8 containing 5 mM DTT, 2 mM EDTA and 2% sucrose.
The biotin-H is 6-PXR/RXR was subjected to mass spectrometric
analysis to reveal the extent of modification by the reagent. The
biotinylated protein solution was frozen and stored at -80.degree.
C.
Example 7
PXR Scintillation Proximity Assay (SPA)
[0082] Streptavidin-PVT SPA beads (AmershamPharmacia cat #RPNQ0007)
were resuspended in assay buffer (50 mM Tris HCl pH 8.0, 50 mM KCl,
1 mM DTT, 0.1 mg/ml essentially fatty acid free bovine serum
albumin) at 0.5 mg/ml. Biotin-His6-PXR/RXR was added to the beads
to a final concentration of 50 nM. The receptors were allowed to
couple to the SPA beads for thirty minutes at room temperature. The
uncoupled receptor was removed by centrifuging the SPA beads at
3000 rpm for 5 minutes in a swinging bucket rotor of a Rupp &
Bowman Silencer centrifuge. The receptor coated SPA beads were then
resuspended in assay buffer to 3.3 mg/ml. 100 .mu.g (30 .mu.L) of
receptor coated SPA beads were added to each well of a 96-well
Optiplate (Packard cat #6005190). Each well also contained
[.sup.3H]GW-485801 at final concentrations ranging from 0.5 nM to
800 nM. Non-specific binding was determined by addition of 10 .mu.M
clotrimazole. The total volume in each well was 100 .mu.L. The
plates were sealed with TopSealA (Packard cat #6005185) and
agitated momentarily to ensure complete mixing. The plates were
then allowed to incubate at room temperature until equilibrium was
obtained. The plates were then counted on a TopCount liquid
scintillation counter (Packard) using a protocol optimized for
.sup.3H PVT SPA. Triplicate samples in the absence (T samples) or
presence (NS samples) of clotrimazole were averaged and specific
binding was calculated using the equation:
specific binding=T-NS
Plots of specific binding vs concentration of [.sup.3H]GW-485801
were generated (FIG. 6). Kd values were determined using non-linear
regression when the data were fit to the equation of a rectangular
hyperbola.
[0083] Test compounds were dissolved in DMSO at 10 mM and diluted
1:10 in DMSO before serially diluting in assay buffer. Compounds
were typically tested at concentrations ranging from 100 .mu.M to
0.3 nM. Streptavidin-PVT SPA beads (AmershamPharmacia cat
#RPNQ0007) were resuspended in assay buffer (50 mM Tris HCl pH 8.0,
50 mM KCl, 1 mM DTT, 0.1 mg/ml essentially fatty acid free bovine
serum albumin) at 0.5 mg/ml. Biotin-His6-PXR/RXR was added to the
beads to a final concentration of 50 nM. The receptors were allowed
to couple to the SPA beads for thirty minutes at room temperature.
The uncoupled receptor was removed by centrifuging the SPA beads at
3000 rpm for 5 minutes in a swinging bucket rotor of a Rupp &
Bowman Silencer centrifuge. The receptor coated SPA beads were then
resuspended in assay buffer to 3.3 mg/ml. 100 .mu.g (30 .mu.L) of
receptor coated SPA beads was added to each well of a 96-well
Optiplate (Packard cat #6005190). Each well also contained
[.sup.3H]GW-485801 at a final concentration of 25 nM and test
compound or an equal volume of assay buffer. Non-specific binding
was determined by addition of 10 .mu.M clotrimazole. The total
volume in each well was 100 .mu.L. The plates were sealed with
TopSealA (Packard cat #6005185) and agitated momentarily to ensure
complete mixing. The plates were then allowed to incubate at room
temperature until equilibrium was obtained, approximately 1.5
hours. The plates were then counted on a TopCount liquid
scintillation counter (Packard) using a protocol optimized for
.sup.3H PVT SPA and programmed to correct for color quenching.
Values for "% [.sup.3H]GW-485801 Bound" were calculated using the
following equation:
% [.sup.3H]GW-485801
Bound=100*[(C.sub.DPM-NS.sub.DPM)/(T.sub.DPM-NS.sub.DPM)]
where C.sub.DPM is the DPM value from a well containing a test
compound, NS.sub.DPM is the average of the DPM values from the
"non-specific" wells which contained 10 .mu.M clotrimazole,
T.sub.DPM is the average of the DPM values from the "total" wells
which contained no added compounds. Graphs of % [.sup.3H]GW-485801
Bound vs concentration were generated for each test compound and
IC50 values were determined using non-linear regression (see Table
1).
TABLE-US-00004 TABLE 1 Compound IC50 (.mu.M) GW-485801 0.58
Clotrimazole 1.3 Rifampicin 2.4 5b-pregnane-3,20-dione 1.0
[0084] All documents cited above are hereby incorporated in their
entirety by reference.
[0085] One skilled in the art will appreciate from a reading of
this disclosure that various changes in form and detail can be made
without departing from the true scope of the invention.
Sequence CWU 1
1
14120DNAArtificial SequenceProbe 1ctgctgcgca tccaggacat
20245DNAArtificial SequenceProbe 2gggtgtgggg aatccaccac catggaggtg
agacccaaag aaagc 45334DNAArtificial SequenceProbe 3gggtgtgggg
gatcctcagc tacctgtgat gccg 34431DNAArtificial SequenceProbe
4gatcagacag ttcatgaagt tcatctagat c 31529DNAArtificial
SequenceProbe 5gatcaatatg aactcaaagg aggtcagtg 29629DNAArtificial
SequenceProbe 6gatcaatatg aactcaaagg aggtcagtg 29729DNAArtificial
SequenceProbe 7gatcaatatg ttctcaaagg agaacagtg 29829DNAArtificial
SequenceProbe 8gatcaataac aactcaaagg aggtcagtg 29932DNAArtificial
SequenceProbe 9gatgcagaca gttcatgaag ttcatctaga tc
321011PRTArtificial Sequencepoly histadine tag 10Met Lys Lys Gly
His His His His His His Gly1 5 1011316PRTArtificial
SequenceHis6-PXR Fusion Protein 11Met Lys Lys Gly His His His His
His His Gly Ser Glu Arg Thr Gly1 5 10 15Thr Gln Pro Leu Gly Val Gln
Gly Leu Thr Glu Glu Gln Arg Met Met 20 25 30Ile Arg Glu Leu Met Asp
Ala Gln Met Lys Thr Phe Asp Thr Thr Phe 35 40 45Ser His Phe Lys Asn
Phe Arg Leu Pro Gly Val Leu Ser Ser Gly Cys 50 55 60Glu Leu Pro Glu
Ser Leu Gln Ala Pro Ser Arg Glu Glu Ala Ala Lys65 70 75 80Trp Ser
Gln Val Arg Lys Asp Leu Cys Ser Leu Lys Val Ser Leu Gln 85 90 95Leu
Arg Gly Glu Asp Gly Ser Val Trp Asn Tyr Lys Pro Pro Ala Asp 100 105
110Ser Gly Gly Lys Glu Ile Phe Ser Leu Leu Pro His Met Ala Asp Met
115 120 125Ser Thr Tyr Met Phe Lys Gly Ile Ile Ser Phe Ala Lys Val
Ile Ser 130 135 140Tyr Phe Arg Asp Leu Pro Ile Glu Asp Gln Ile Ser
Leu Leu Lys Gly145 150 155 160Ala Ala Phe Glu Leu Cys Gln Leu Arg
Phe Asn Thr Val Phe Asn Ala 165 170 175Glu Thr Gly Thr Trp Glu Cys
Gly Arg Leu Ser Tyr Cys Leu Glu Asp 180 185 190Thr Ala Gly Gly Phe
Gln Gln Leu Leu Leu Glu Pro Met Leu Lys Phe 195 200 205His Tyr Met
Leu Lys Lys Leu Gln Leu His Glu Glu Glu Tyr Val Leu 210 215 220Met
Gln Ala Ile Ser Leu Phe Ser Pro Asp Arg Pro Gly Val Leu Gln225 230
235 240His Arg Val Val Asp Gln Leu Gln Glu Gln Phe Ala Ile Thr Leu
Lys 245 250 255Ser Tyr Ile Glu Cys Asn Arg Pro Gln Pro Ala His Arg
Phe Leu Phe 260 265 270Leu Lys Ile Met Ala Met Leu Thr Glu Leu Arg
Ser Ile Asn Ala Gln 275 280 285His Thr Gln Arg Leu Leu Arg Ile Gln
Asp Ile His Pro Phe Ala Thr 290 295 300Pro Leu Met Gln Glu Leu Phe
Gly Ile Thr Gly Ser305 310 31512242PRTArtificial SequenceRXR Alpha
Protein 12Met Lys Lys Gly Ser Ala Asn Glu Asp Met Pro Val Glu Arg
Ile Leu1 5 10 15Glu Ala Glu Leu Ala Val Glu Pro Lys Thr Glu Thr Tyr
Val Glu Ala 20 25 30Asn Met Gly Leu Asn Pro Ser Ser Pro Asn Asp Pro
Val Thr Asn Ile 35 40 45Cys Gln Ala Ala Asp Lys Gln Leu Phe Thr Leu
Val Glu Trp Ala Lys 50 55 60Arg Ile Pro His Phe Ser Glu Leu Pro Leu
Asp Asp Gln Val Ile Leu65 70 75 80Leu Arg Ala Gly Trp Asn Glu Leu
Leu Ile Ala Ser Phe Ser His Arg 85 90 95Ser Ile Ala Val Lys Asp Gly
Ile Leu Leu Ala Thr Gly Leu His Val 100 105 110His Arg Asn Ser Ala
His Ser Ala Gly Val Gly Ala Ile Phe Asp Arg 115 120 125Val Leu Thr
Glu Leu Val Ser Lys Met Arg Asp Met Gln Met Asp Lys 130 135 140Thr
Glu Leu Gly Cys Leu Arg Ala Ile Val Leu Phe Asn Pro Asp Ser145 150
155 160Lys Gly Leu Ser Asn Pro Ala Glu Val Glu Ala Leu Arg Glu Lys
Val 165 170 175Tyr Ala Ser Leu Glu Ala Tyr Cys Lys His Lys Tyr Pro
Glu Gln Pro 180 185 190Gly Arg Phe Ala Lys Leu Leu Leu Arg Leu Pro
Ala Leu Arg Ser Ile 195 200 205Gly Leu Lys Cys Leu Glu His Leu Phe
Phe Phe Lys Leu Ile Gly Asp 210 215 220Thr Pro Ile Asp Thr Phe Leu
Met Glu Met Leu Glu Ala Pro His Gln225 230 235 240Met
Thr132146DNAArtificial SequenceProbe 13tgaaatatag gtgagagaca
agattgtctc atatccgggg aaatcataac ctatgactag 60gacgggaaga ggaagcactg
cctttacttc agtgggaatc tcggcctcag cctgcaagcc 120aagtgttcac
agtgagaaaa gcaagagaat aagctaatac tcctgtcctg aacaaggcag
180cggctccttg gtaaagctac tccttgatcg atcctttgca ccggattgtt
caaagtggac 240cccaggggag aagtcggagc aaagaactta ccaccaagca
gtccaagagg cccagaagca 300aacctggagg tgagacccaa agaaagctgg
aaccatgctg actttgtaca ctgtgaggac 360acagagtctg ttcctggaaa
gcccagtgtc aacgcagatg aggaagtcgg aggtccccaa 420atctgccgtg
tatgtgggga caaggccact ggctatcact tcaatgtcat gacatgtgaa
480ggatgcaagg gctttttcag gagggccatg aaacgcaacg cccggctgag
gtgccccttc 540cggaagggcg cctgcgagat cacccggaag acccggcgac
agtgccaggc ctgccgcctg 600cgcaagtgcc tggagagcgg catgaagaag
gagatgatca tgtccgacga ggccgtggag 660gagaggcggg ccttgatcaa
gcggaagaaa agtgaacgga cagggactca gccactggga 720gtgcaggggc
tgacagagga gcagcggatg atgatcaggg agctgatgga cgctcagatg
780aaaacctttg acactacctt ctcccatttc aagaatttcc ggctgccagg
ggtgcttagc 840agtggctgcg agttgccaga gtctctgcag gccccatcga
gggaagaagc tgccaagtgg 900agccaggtcc ggaaagatct gtgctctttg
aaggtctctc tgcagctgcg gggggaggat 960ggcagtgtct ggaactacaa
acccccagcc gacagtggcg ggaaagagat cttctccctg 1020ctgccccaca
tggctgacat gtcaacctac atgttcaaag gcatcatcag ctttgccaaa
1080gtcatctcct acttcaggga cttgcccatc gaggaccaga tctccctgct
gaagggggcc 1140gctttcgagc tgtgtcaact gagattcaac acagtgttca
acgcggagac tggaacctgg 1200gagtgtggcc ggctgtccta ctgcttggaa
gacactgcag gtggcttcca gcaacttcta 1260ctggagccca tgctgaaatt
ccactacatg ctgaagaagc tgcagctgca tgaggaggag 1320tatgtgctga
tgcaggccat ctccctcttc tccccagacc gcccaggtgt gctgcagcac
1380cgcgtggtgg accagctgca ggagcaattc gccattactc tgaagtccta
cattgaatgc 1440aatcggcccc agcctgctca taggttcttg ttcctgaaga
tcatggctat gctcaccgag 1500ctccgcagca tcaatgctca gcacacccag
cggctgctgc gcatccagga catacacccc 1560tttgctacgc ccctcatgca
ggagttgttc ggcatcacag gtagctgagc ggctgccctt 1620gggtgacacc
tccgagaggc agccagaccc agagccctct gagccgccac tcccgggcca
1680agacagatgg acactgccaa gagccgacaa tgccctgctg gcctgtctcc
ctagggaatt 1740cctgctatga cagctggcta gcattcctca ggaaggacat
gggtgccccc cacccccagt 1800tcagtctgta gggagtgaag ccacagactc
ttacgtggag agtgcactga cctgtaggtc 1860aggaccatca gagaggcaag
gttgcccttt ccttttaaaa ggccctgtgg tctggggaga 1920aatccctcag
atcccactaa agtgtcaagg tgtggaaggg accaagcgac caaggatagg
1980ccatctgggg tctatgccca catacccacg tttgttcgct tcctgagtct
tttcattgct 2040acctctaata gtcctgtctc ccacttccca ctcgttcccc
tcctcttccg agctgctttg 2100tgggctccag gcctgtactc atcggcaggt
gcatgagtat ctgtgg 214614434PRTHomo Sapien 14Leu Glu Val Arg Pro Lys
Glu Ser Trp Asn His Ala Asp Phe Val His1 5 10 15Cys Glu Asp Thr Glu
Ser Val Pro Gly Lys Pro Ser Val Asn Ala Asp 20 25 30Glu Glu Val Gly
Gly Pro Gln Ile Cys Arg Val Cys Gly Asp Lys Ala 35 40 45Thr Gly Tyr
His Phe Asn Val Met Thr Cys Glu Gly Cys Lys Gly Phe 50 55 60Phe Arg
Arg Ala Met Lys Arg Asn Ala Arg Leu Arg Cys Pro Phe Arg65 70 75
80Lys Gly Ala Cys Glu Ile Thr Arg Lys Thr Arg Arg Gln Cys Gln Ala
85 90 95Cys Arg Leu Arg Lys Cys Leu Glu Ser Gly Met Lys Lys Glu Met
Ile 100 105 110Met Ser Asp Glu Ala Val Glu Glu Arg Arg Ala Leu Ile
Lys Arg Lys 115 120 125Lys Ser Glu Arg Thr Gly Thr Gln Pro Leu Gly
Val Gln Gly Leu Thr 130 135 140Glu Glu Gln Arg Met Met Ile Arg Glu
Leu Met Asp Ala Gln Met Lys145 150 155 160Thr Phe Asp Thr Thr Phe
Ser His Phe Lys Asn Phe Arg Leu Pro Gly 165 170 175Val Leu Ser Ser
Gly Cys Glu Leu Pro Glu Ser Leu Gln Ala Pro Ser 180 185 190Arg Glu
Glu Ala Ala Lys Trp Ser Gln Val Arg Lys Asp Leu Cys Ser 195 200
205Leu Lys Val Ser Leu Gln Leu Arg Gly Glu Asp Gly Ser Val Trp Asn
210 215 220Tyr Lys Pro Pro Ala Asp Ser Gly Gly Lys Glu Ile Phe Ser
Leu Leu225 230 235 240Pro His Met Ala Asp Met Ser Thr Tyr Met Phe
Lys Gly Ile Ile Ser 245 250 255Phe Ala Lys Val Ile Ser Tyr Phe Arg
Asp Leu Pro Ile Glu Asp Gln 260 265 270Ile Ser Leu Leu Lys Gly Ala
Ala Phe Glu Leu Cys Gln Leu Arg Phe 275 280 285Asn Thr Val Phe Asn
Ala Glu Thr Gly Thr Trp Glu Cys Gly Arg Leu 290 295 300Ser Tyr Cys
Leu Glu Asp Thr Ala Gly Gly Phe Gln Gln Leu Leu Leu305 310 315
320Glu Pro Met Leu Lys Phe His Tyr Met Leu Lys Lys Leu Gln Leu His
325 330 335Glu Glu Glu Tyr Val Leu Met Gln Ala Ile Ser Leu Phe Ser
Pro Asp 340 345 350Arg Pro Gly Val Leu Gln His Arg Val Val Asp Gln
Leu Gln Glu Gln 355 360 365Phe Ala Ile Thr Leu Lys Ser Tyr Ile Glu
Cys Asn Arg Pro Gln Pro 370 375 380Ala His Arg Phe Leu Phe Leu Lys
Ile Met Ala Met Leu Thr Glu Leu385 390 395 400Arg Ser Ile Asn Ala
Gln His Thr Gln Arg Leu Leu Arg Ile Gln Asp 405 410 415Ile His Pro
Phe Ala Thr Pro Leu Met Gln Glu Leu Phe Gly Ile Thr 420 425 430Gly
Ser
* * * * *
References