U.S. patent application number 11/648961 was filed with the patent office on 2007-05-31 for protein having pge2 synthase activity and use thereof.
This patent application is currently assigned to Chugai Seiyaku Kabushiki Kaisha, a Japanese corporation. Invention is credited to Ichiro Kudo, Makoto Murakami, Sachiko Oh-Ishi.
Application Number | 20070122866 11/648961 |
Document ID | / |
Family ID | 18557268 |
Filed Date | 2007-05-31 |
United States Patent
Application |
20070122866 |
Kind Code |
A1 |
Kudo; Ichiro ; et
al. |
May 31, 2007 |
Protein having PGE2 synthase activity and use thereof
Abstract
A single protein which is the substance of the PGE2 synthesis
activity in brain soluble fractions of LPS administered rats has
been purified and identified. The protein has an activity of
synthesizing PGE2 from PGH2, and further, has an activity of
synthesizing PGE2 from arachidonic acid in combination with
COX.
Inventors: |
Kudo; Ichiro; (Tokyo,
JP) ; Murakami; Makoto; (Tokyo, JP) ; Oh-Ishi;
Sachiko; (Tokyo, JP) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Chugai Seiyaku Kabushiki Kaisha, a
Japanese corporation
Tokyo
JP
Ichiro Kudo
Tokyo
JP
|
Family ID: |
18557268 |
Appl. No.: |
11/648961 |
Filed: |
January 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10182233 |
Dec 27, 2002 |
7169580 |
|
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PCT/JP00/05758 |
Aug 25, 2000 |
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11648961 |
Jan 3, 2007 |
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Current U.S.
Class: |
435/25 ; 435/189;
435/320.1; 435/325; 435/69.1; 514/573; 536/23.2 |
Current CPC
Class: |
C12N 9/90 20130101; A61K
38/00 20130101; A61P 29/00 20180101 |
Class at
Publication: |
435/025 ;
435/189; 435/069.1; 435/320.1; 435/325; 536/023.2; 514/573 |
International
Class: |
C12Q 1/26 20060101
C12Q001/26; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 9/02 20060101 C12N009/02; A61K 31/557 20060101
A61K031/557 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 3, 2000 |
JP |
2000-32704 |
Claims
1-19. (canceled)
20. A host cell harboring a vector comprising a nucleotide sequence
encoding a COX enzyme and a vector comprising a nucleotide sequence
encoding a polypeptide selected from the group consisting of: (a) a
polypeptide comprising SEQ ID NO: 1; (b) a polypeptide comprising
SEQ ID NO: 1 with between one and thirty amino acid substitutions,
deletions, additions, or insertions, wherein the polypeptide has
PGE2 synthase activity; (c) a polypeptide encoded by a DNA that
hybridizes under stringent conditions to a DNA consisting of the
complement of SEQ ID NO: 2; and (d) PGES-2.
21. The host cell of claim 20, wherein the polypeptide comprises
SEQ ID NO: 1 and the COX enzyme is COX-1.
22. The host cell of claim 20, wherein the polypeptide comprises
SEQ ID NO: 1 with between one and thirty amino acid substitutions,
deletions, additions, or insertions, wherein the polypeptide has
PGE2 synthase activity, and the COX enzyme is COX-1.
23. The host cell of claim 20, wherein the polypeptide is encoded
by a DNA that hybridizes under stringent conditions to a DNA
consisting of the complement of SEQ ID NO: 2, and the COX enzyme is
COX-1.
24. The host cell of claim 20, wherein the polypeptide is PGES-2
and the COX enzyme is COX-2.
25. A method of screening for a PGH2 synthase inhibitor, the method
comprising the steps of: (1) contacting a test compound and PGH2
with a host cell harboring a vector comprising a nucleotide
sequence encoding a polypeptide selected from the group consisting
of: (a) a polypeptide comprising SEQ ID NO: 1; (b) a polypeptide
comprising SEQ ID NO: 1 with between one and thirty amino acid
substitutions, deletions, additions, or insertions, wherein the
polypeptide has PGE2 synthase activity; and (c) a polypeptide
encoded by a DNA that hybridizes under stringent conditions to a
DNA consisting of the complement of SEQ ID NO: 2; (2) detecting
PGE2 produced by the host cell; and (3) selecting a test compound
that reduces the level of PGE2 produced as compared with that
produced in the absence of the test compound.
26. The method of claim 25, wherein the polypeptide comprises SEQ
ID NO: 1.
27. The method of claim 25, wherein the polypeptide comprises SEQ
ID NO: 1 with between one and thirty amino acid substitutions,
deletions, additions, or insertions, wherein the polypeptide has
PGE2 synthase activity.
28. The method of claim 25, wherein the polypeptide is encoded by a
DNA that hybridizes under stringent conditions to a DNA consisting
of the complement of SEQ ID NO: 2.
29. A method of screening for a PGE2 synthase inhibitor, comprising
the steps of: (1) contacting the host cell of claim 21 with a test
compound and arachidonic acid; (2) detecting the level of PGE2
produced by the host cell; and (3) selecting a test compound that
reduces the level of PGE2 produced as compared with that produced
in the absence of the test compound.
30. A method of screening for a PGE2 synthase inhibitor, comprising
the steps of: (1) contacting the host cell of claim 22 with a test
compound and arachidonic acid; (2) detecting the level of PGE2
produced by the host cell; and (3) selecting a test compound that
reduces the level of PGE2 produced as compared with that produced
in the absence of the test compound.
31. A method of screening for a PGE2 synthase inhibitor, comprising
the steps of: (1) contacting the host cell of claim 23 with a test
compound and arachidonic acid; (2) detecting the level of PGE2
produced by the host cell; and (3) selecting a test compound that
reduces the level of PGE2 produced as compared with that produced
in the absence of the test compound.
32. A method of screening for a PGE2 synthase inhibitor, comprising
the steps of: (1) contacting the host cell of claim 24 with a test
compound and arachidonic acid: (2) detecting the level of PGE2
produced by said host cell; and (3) selecting a test compound that
reduces the level of PGE2 produced as compared with that produced
in the absence of the test compound.
33. A kit for screening for a PGE2 synthase inhibitor, the kit
comprising: PGH2 and a host cell harboring a vector comprising a
nucleotide sequence encoding a polypeptide selected from the group
consisting of: (a) a polypeptide comprising SEQ ID NO: 1; (b) a
polypeptide comprising SEQ ID NO: 1 with between one and thirty
amino acid substitutions, deletions, additions, or insertions,
wherein the polypeptide has PGE2 synthase activity; and (c) a
polypeptide encoded by a DNA that hybridizes under stringent
conditions to a DNA consisting of the complement of SEQ ID NO:
2.
34. The kit of claim 33, wherein the polypeptide comprises SEQ ID
NO: 1.
35. The kit of claim 33, wherein the polypeptide comprises SEQ ID
NO: 1 with between one and thirty amino acid substitutions,
deletions, additions, or insertions, wherein the polypeptide has
PGE2 synthase activity.
36. The kit of claim 33, wherein the polypeptide is encoded by a
DNA that hybridizes under stringent conditions to a DNA consisting
of the complement of SEQ ID NO: 2.
37. A kit for screening for a PGE2 synthase inhibitor, the kit
comprising the host cell of claim 21 and arachidonic acid.
38. A kit for screening for a PGE2 synthase inhibitor, the kit
comprising the host cell of claim 22 and arachidonic acid.
39. A kit for screening for a PGE2 synthase inhibitor, the kit
comprising the host cell of claim 23 and arachidonic acid.
40. A kit for screening for a PGE2 synthase inhibitor, the kit
comprising the host cell of claim 24 and arachidonic acid.
Description
TECHNICAL FIELD
[0001] This invention relates to proteins having PGE2 synthase
activity and use thereof.
BACKGROUND ART
[0002] Nonsteroidal anti-inflammatory drugs (NSAIDs), represented
by aspirin and piroxicam, are used widely as antipyretic analgesic
antiphlogistic drugs and are considered to manifest
anti-inflammatory effects, such as antipyretic, analgesic, and
antiphlogistic effects, by suppressing the production of
prostaglandins through inhibitory effects on COX (cyclooxygenase).
COXs are enzymes that produce PGH2 (prostaglandin H2) from
arachidonic acid, and 2 types of enzymes, referred to as
constitutive COX-1 and inducible COX-2, are known to exist.
Depending on the cell or tissue of production, PGH2 produced by COX
is enzymatically converted to PGE2, PGD2, PGF2.alpha., PGI2
(prostacyclin), or TXA2 (thromboxane A2) (Vane J R and Botting R M,
Inflammation Research. 44:1, 1995).
[0003] In particular, suppression of PGE2 production among the
above-mentioned prostanoids is suggested to be important for NSAIDs
to show the effect of the drug, due to the fact that PEG2 among
these prostanoids is believed to be deeply involved with
inflammatory processes, such as pain generation, fever, and edema,
and that it exists as the highest concentration at the site of
inflammation (Vane J R and Botting R M, Inflammation Research.
44:1, 1995); moreover, the drug efficacies of anti-PGE2 antibody
and NSAIDs are reported to be nearly equal in a rat inflammatory
model (Portanova J P, Zhang Y, Anderson G D, Hauser S D, Masferrer
J L, Seibert K, Gregory S A, Isakson P C, Journal of Experimental
Medicine. 184:883-91, 1996).
[0004] On the other hand, NSAIDs, apart from PGE2, also suppress
the production of PGD2, PGF2.alpha., PGI2, and TXA2 by inhibiting
COX, and thus, may exhibit not only anti-inflammatory effects but
also effects based on the suppression of the production of these
other prostanoids. For example, childbirth is known to be delayed
by the inhibition of uterine contraction at the time of delivery
due to suppression of PGF2.alpha. production, and blood coagulation
is known to be delayed by the suppression of TXA2 production (AHFS
Drug Information 98, p 1571 McEnvoy G K Ed., American Society of
Health-System Pharmacists, 1998).
[0005] Therefore, substances that specifically inhibit the action
of PGE2, such as PGE2 synthase (PGES) inhibitors, are expected to
serve as excellent anti-inflammatory drugs with lower side effects,
by specifically suppressing PGE2 production without suppressing the
production of other prostanoids.
[0006] To date, although synthases specific for PGD2, PGF2.alpha.,
PGI2, and TXA2, respectively, have been identified, those for PGE2
synthase have been suggested to exist but have not yet been
identified.
[0007] Recently, Per-Johan Jakobsson et al. identified a
membrane-bound human PGE2 synthase for the first time (Proc. Natl.
Acad. Sci. U.S.A., 96:7220-7225, 1999) (herein, the enzyme is
referred to as "PGES-2").
[0008] On the other hand, the existence of other enzymes with
characteristics different from the PGES-2 has been also suggested.
Specifically, Ogorochi et al. have estimated that PGES-2 and a
protein of pI5.4 with PEGS activity existing in the cytoplasm of
human brain are identical, because the proteins are purified
together with glutathione S-transferase (GST) (Ogorochi T, Ujihara
M., and Narumiya S., J. Neurochem., 48:900-909, 1987).
DISCLOSURE OF THE INVENTION
[0009] The object of the present invention is to identify proteins
having PGE2 synthase activity, and to provide a use for the
proteins. According to one embodiment, the present invention
provides methods for producing PGE2 using the protein. In another
embodiment, methods for screening PGE2 synthase inhibitors using
cells expressing the protein are provided. According to a preferred
embodiment, the invention provides methods for screening PGE2
synthase inhibitors using cells co-expressing PGE2 synthase and
COX.
[0010] The present inventors discovered that the ability to
synthesize PGE2 is remarkably induced in the soluble fraction of
LPS-administered rat brain. Based on this finding, the present
inventors vigorously conducted research to identify the substance
responsible for the PGE2 synthesis activity in these fractions. And
as a result, the inventors succeeded in identifying and purifying a
single protein, which is the substance responsible for the
activity.
[0011] A database search using the amino acid sequence of the
obtained protein demonstrated a surprising result, which
demonstrated the protein to be identical to a protein reported as
human progesterone receptor complex component protein (Johnson J L
et al., Mol. Cell. Biol., 14:1956-63, 1994).
[0012] Further, a cDNA encoding the obtained protein was inserted
into an expression vector to transfect HEK293 cells. A glutathione
(GSH)-dependent PGE2 synthesis activity (PGES activity) was
observed in the cell lysate of the transfected cells, and the
activity was suppressed by 1-chloro-2,4-dinitrobenzene (CDNB), a
GST inhibitor. Thus, the cDNA isolated by the present inventors was
confirmed to encode the object PGES (the protein was dubbed
"PGES-1"). The protein, which had been reported as progesterone
receptor complex component protein, was demonstrated to be a
protein belonging to PGES.
[0013] In addition, the present inventors discovered that screening
for PGES inhibitors is enabled by utilizing a system that produces
PGE2 from PGH2 using cells made to express PGES-1. However, PGH2, a
direct substrate of PGES, is extremely chemically unstable and
decomposes non-enzymatically. Thus, construction of a system for
screening PGES inhibitors, which is more stable than that wherein
PGH2 is added to PGES-expressing cells, is desired in the art. The
present inventors presumed that a stable screening system may be
constructed by producing human cells that simultaneously express
human PGES and human COX, and adding arachidonic acid, a relatively
stable substrate of COX, to these cells.
[0014] Accordingly, first, the relationship of PGES-1 to COX-1 and
COX-2 were examined. The inventors observed that PGE2 production
levels increased drastically in the presence of arachidonic acid in
cells prepared by transfecting human PGES-1 cDNA to HEK293 cells
that express human COX-1 as compared cells without human PGES-1
cDNA transfection. In contrast, human COX-2-expressing HEK293 cells
that were made to express PGES-1 did not show an increase in PGE2
production. Cooperative function (coupled function) of COX-1 and
PGES-1 was demonstrated. Furthermore, a phenomenon where both
enzymes function cooperatively (coupling) was also confirmed in
cells expressing both COX-2 and PGES-2. Thus, the present inventors
succeeded in establishing a system that produces PGE2 from
arachidonic acid by utilizing cells made to express both COX and
PGES. This system enables the efficient screening of PGES
inhibitors. Compounds isolated by such screening are expected to be
applicable as anti-inflammatory drugs and such.
[0015] The present invention relates to proteins having PGE2
synthase activity, as well as to methods for producing PGE2 and
screening PGE2 synthase inhibitors utilizing the PGE2 synthase
activity. Specifically, the present invention provides:
[0016] (1) a protein having PGE2 synthase activity, comprising the
amino acid sequence of SEQ ID NO: 1;
[0017] (2) a protein having PGE2 synthase activity selected from
the group of:
[0018] (a) a protein comprising the amino acid sequence of SEQ ID
NO: 1 in which one or more amino acids are substituted, deleted,
added, and/or inserted; and
[0019] (b) a protein encoded by a DNA that hybridizes under
stringent conditions to a DNA consisting of the nucleotide sequence
of SEQ ID NO: 1;
[0020] (3) the protein of (1) or (2), which is used to synthesize
PGE2;
[0021] (4) a DNA that encodes the protein of (1) or (2);
[0022] (5) a vector containing the DNA of (4);
[0023] (6) a transformant carrying the vector of (5);
[0024] (7) a method for producing the proteins of (1) or (2),
comprising the steps of cultivating the transformant of (6), and
collecting the expressed protein from said transformant or from the
culture supernatant thereof;
[0025] (8) a method for producing PGE2, wherein the protein of (1)
or (2) is acted on PGH2;
[0026] (9) a method for producing PGE2, wherein COX and PGE2
synthase are acted on arachidonic acid;
[0027] (10) the method of (9), wherein COX is COX-1 and PGE2
synthase is the protein of (1) or (2);
[0028] (11) the method of (9), wherein COX is COX-2 and PGE2
synthase is PGES-2;
[0029] (12) a PGE2 synthesizing agent containing the protein of (1)
or (2) as the active ingredient;
[0030] (13) a transformant carrying a vector containing a DNA
encoding COX and a vector containing a DNA encoding PGE2
synthase;
[0031] (14) the transformant of (13), wherein COX is COX-1 and PGE2
synthase is a protein of (1) or (2);
[0032] (15) the transformant of (13), wherein COX is COX-2 and PGE2
synthase is PGES-2;
[0033] (16) a method of screening for PGE2 synthase inhibitors,
comprising the steps of:
[0034] (a) contacting the transformant of (6) with a test sample
and PGH2;
[0035] (b) detecting the level of PGE2 produced by said
transformant; and
[0036] (c) selecting the compound that reduces the level of PGE2
produced as compared with that produced in the absence of the test
sample;
[0037] (17) a method for screening PGE2 synthase inhibitors,
comprising the steps of:
[0038] (a) contacting the transformant of any one of (13) to (15)
with a test sample and arachidonic acid;
[0039] (b) detecting the level of PGE2 produced by said
transformant; and
[0040] (c) selecting the compound that reduces the level of PGE2
produced as compared with that produced in the absence of the test
sample;
[0041] (18) a PGE2 synthase inhibitor that can be isolated by the
screening method of (16) or (17); and
[0042] (19) an anti-inflammatory drug containing the PGE2 synthase
inhibitor of (18) as the active ingredient.
[0043] Herein, the term "PGE2 synthase" refers to enzymes that have
the activity to produce PGE2 using PGH2 as substrate. Accordingly,
herein, "PGE2 synthase activity" refers to an activity to produce
PGE2 using PGH2 as substrate. Moreover, the term "COX" herein
refers to enzymes that have the activity to produce PGH2 using
arachidonic acid as substrate.
[0044] The present invention provides a PGES-1 protein having PGE2
synthase activity. The amino acid sequence of the PGES-1 protein
isolated by the present inventors is indicated in SEQ ID NO: 1, and
the nucleotide sequence of a cDNA encoding the protein is indicated
in SEQ ID NO: 2. The PGES-1 protein was isolated by purifying
proteins from soluble fractions of LPS-administered rat brain using
the PGE2 synthesizing activity as an index. The primary structure
of the PGES-1 protein is identical to the protein reported as human
progesterone receptor complex component protein (Johnson J L et
al., Mol. Cell. Biol., 14:1956-63, 1994); however, the present
inventors were the first to find that this protein has PGE2
synthase activity. As described later, the PGES-1 may be utilized
to produce PGE2 and to screen PGE2 synthase inhibitors due to the
PGE2 synthase activity thereof.
[0045] The present invention includes those proteins that are
structurally similar to the PGES-1 protein (SEQ ID NO: 1), so long
as they retain the PGE2 synthase activity. Such proteins may
include, for example, mutants of the PGES-1 protein, allele
variants, homologs, and such.
[0046] One method well known to those skilled in the art for
preparing functionally equivalent proteins is to introduce
mutations into proteins. For example, one skilled in the art can
prepare mutants that retain PGE2 synthase activity of human PGES-1
proteins by introducing appropriate mutations into the amino acid
sequence of the protein (SEQ ID NO: 1), by using site-specific
mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275;
Zoller, M J, and Smith, M. (1983) Methods Enzymol. 100, 468-500;
Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer
W, and Fritz H J (1987) Methods Enzymol. 154, 350-367; Kunkel, TA
(1985) Proc Natl Acad Sci USA. 82, 488-492; Kunkel (1988) Methods
Enzymol. 85, 2763-2766), and such. Mutation of amino acids may
occur in nature as well. Thus, a protein comprising the amino acid
sequence of human PGES-1 protein (SEQ ID NO:1) in which one or more
amino acids are mutated is also included in the present invention,
so long as it has PGE2 synthase activity. In such a mutant protein,
the number of amino acids mutated is typically 30 residues or less,
preferably 10 residues or less, more preferably 5 residues or less
(for example 3 residues or less).
[0047] It is preferable to mutate an amino acid residue into one
that allows the properties of the amino acid side-chain to be
conserved. Examples of properties of amino acid side chains
include: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),
hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and
amino acids comprising the following side chains: aliphatic
side-chains (G, A, V, L, I, P); hydroxyl group-containing
side-chains (S, T, Y); sulfur atom-containing side-chains (C, M);
carboxylic acid- and amide-containing side-chains (D, N, E, Q);
base-containing side-chain (R, K, H); and aromatic-containing
side-chains (H, F, Y, W) (The letters within parenthesis indicate
the one-letter codes of amino acids).
[0048] It is well known that a protein having deletion, addition,
and/or substitution of one or more amino acid residues in the
sequence of the protein can retain the original biological activity
(Mark, D. F. et al. Proc. Natl. Acad. Sci. U.S.A. 81:5662-5666
(1984); Zoller, M. J. and Smith, M. Nucleic Acids Res. 10:6487-6500
(1982); Wang, A. et al. Science 224:1431-1433; Dalbadie-McFarland,
G. et al. Proc. Natl. Acad. Sci. U.S.A. 79:6409-6413 (1982)).
[0049] In the Example of the present invention, it was demonstrated
that the substitution of tyrosine, the 9.sup.th residue from
N-terminus of the PGES-1 protein, with an aspartic acid reduces the
PGE2 synthase activity. Therefore, this tyrosine residue is
suggested to be important for maintaining the PGE2 synthase
activity.
[0050] An alternative method well known to those skilled in the art
for preparing functionally equivalent proteins is, for example, the
method utilizing the hybridization technique (Sambrook, J. et al.,
Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab.
Press, 1989). Generally, one skilled in the art can isolate DNA
highly homologous to the whole or part of a DNA sequence encoding
human PGES-1 protein (SEQ ID NO: 2), and then isolate a protein
functionally equivalent to the human PGES-1 protein from those
isolated DNAs. The present invention includes proteins encoded by
DNAs that hybridize under stringent conditions to a DNA encoding
the human PGES-1 protein, so long as the protein has PGE2 synthase
activity. These proteins include, for example, non-human mammalian
homologues (e.g. proteins encoded by genes of mice, rats, rabbits,
cattle, and such).
[0051] Stringent hybridization conditions for isolating a DNA
encoding a protein functionally equivalent to human PGES-1 protein
may be appropriately selected by a person skilled in the art. For
example, prehybridization is performed at 68.degree. C. for 30 min
or more using "Rapid-hyb buffer" (Amersham LIFE SCIENCE). A labeled
probe is added thereto, and hybridization is conducted by warming
at 68.degree. C. for one hour or more. Then, washing in
2.times.SSC, 0.01% SDS at room temperature for 20 min three times;
in 1.times.SSC, 0.1% SDS at 37.degree. C. for 20 min three times;
and then in 1.times.SSC, 0.1% SDS at 50.degree. C. for 20 min
twice. However, several factors, such as temperature and salt
concentration, can influence the stringency of hybridization and
one skilled in the art can suitably select the factors to
accomplish a similar stringency.
[0052] In place of hybridization, gene amplification methods using
primers synthesized based on the sequence information of the DNA
(SEQ ID NO: 2) encoding the human PGES-1 proteins, for example, the
polymerase chain reaction (PCR) method, can be utilized for the
isolation.
[0053] A protein encoded by a DNA isolated through the above
hybridization techniques or gene amplification techniques normally
has a high homology to the amino acid sequence of the human PGES-1
protein (SEQ ID NO: 1). The proteins of the present invention also
include proteins that have a high homology to the amino acid
sequence of the human PGES-1 protein, so long as the protein has a
PGE2 synthase activity. "Highly homologous" refers to, normally an
identity of at least 25% or higher, preferably 40% or higher, more
preferably 60% or higher, even more preferably 80% or higher (for
example, 90% or higher, 95% or higher) at the amino acid level. The
homology of a protein can be determined by following the algorithm
in "Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad. Sci.
USA 80:726-730".
[0054] Whether a prepared protein has the PGES synthase activity or
not can be detected, for example, by a method described in Example
1.
[0055] The proteins of the present invention may have variations in
the amino acid sequence, molecular weight, isoelectric point, the
presence or absence of sugar chains, form, and so on, depending on
the cell or host used to produce it or the purification method
utilized. Nevertheless, so long as the obtained protein has PGE2
synthase activity, it is within the scope of the present invention.
For example, if a protein of the present invention is expressed in
a prokaryotic cell, such as E. coli, the protein includes a
methionine residue at the N-terminus in addition to the natural
amino acid sequence of the protein. Such proteins are also included
in the proteins of the present invention.
[0056] The proteins of the present invention can be prepared as
recombinant proteins or naturally occurring proteins, using methods
commonly known in the art. Alternatively, a protein of the present
invention can be synthesized artificially. When the protein is a
recombinant protein, it may be produced by inserting a DNA (for
example, a DNA having the nucleotide sequence of SEQ ID NO: 2)
encoding a protein of the present invention into an appropriate
expression vector, collecting the transformant obtained by
introducing the vector into an appropriate host cell, obtaining an
extract, and then purifying and preparing the protein using
chromatography, such as ion exchange, reverse phase, or gel
filtration; or affinity chromatography using a column immobilized
with antibodies against the protein of the invention; or by
combining these columns. Alternatively, when a protein of the
invention is expressed in host cells (e.g., animal cells or E.
coli) as a fusion protein with glutathione S transferase protein,
or a recombinant protein with multiple histidine residues, the
expressed recombinant protein can be purified using a glutathione
column or nickel column. After the fusion protein is purified, if
necessary, regions of the fusion protein (apart from the desired
protein) can be digested and removed with thrombin, factor Xa,
etc.
[0057] The native protein of the invention can be isolated by
methods well known in the art, for example, by purifying an extract
of tissues or cells that express a protein of the invention with an
affinity column bound using antibodies that bind to a protein of
the present invention. The antibodies may be polyclonal or
monoclonal antibodies.
[0058] The proteins of the invention can be used as PGE2
synthesizing agents. The term "PGE2 synthesizing agents" herein
refers to reagents for industrial synthesis of PGE2 or for
synthesis of PGE2 for research, and also includes pharmaceutical
agents for administration to living bodies.
[0059] In addition to being utilized in the above-described in vivo
or in vitro production of a protein of the present invention, a DNA
encoding a protein of the present invention may also be applied,
for example, in the therapy of diseases caused by an aberration in
the PGES synthase activity of a protein of the present invention.
Any type of DNA, such as cDNA synthesized from mRNA, genomic DNA,
or chemical synthetic DNA, can be used, so long as the DNA encodes
a protein of the present invention. Further, so long as they can
encode a protein of the present invention, DNAs comprising
arbitrary sequences based on the degeneracy of the genetic code are
also included.
[0060] The DNA of the present invention can be prepared using
methods known in the art. For example, a cDNA library can be
constructed from cells expressing a protein of the present
invention and hybridization can be conducted using a part of the
DNA sequence of the present invention (for example, SEQ ID NO: 2)
as a probe. The cDNA library may be prepared, for example,
according to the method described by Sambrook J. et al. (Molecular
Cloning, Cold Spring Harbor Laboratory Press (1989)), or instead,
commercially available DNA libraries may be used. Alternatively, a
DNA of the present invention can be obtained by preparing RNA from
cells expressing a protein of the present invention, synthesizing
cDNA therefrom using a reverse transcriptase, synthesizing
oligo-DNA based on a DNA sequence of the present invention (for
example, SEQ ID NO: 2), and amplifying the cDNA encoding a protein
of the present invention by PCR using the oligo-DNA as primers.
[0061] From the nucleotide sequence of the obtained cDNA, one can
determine an open reading frame, and thereby, obtain the amino acid
sequence of a protein of the invention. The cDNA obtained may also
be used as a probe for screening a genomic DNA library to isolate
genomic DNA.
[0062] More specifically, mRNA may first be isolated from a cell,
tissue, or organ in which a protein of the invention is expressed.
Known methods can be used to isolate mRNA; for instance, total RNA
may be prepared by the guanidine ultracentrifugation (Chirgwin, J.
M. et al., Biochemistry 18:5294-5299 (1979)) or the AGPC method
(Chomczynski, P. and Sacchi, N., Anal. Biochem. 162:156-159
(1987)), and mRNA may be purified from total RNA using mRNA
Purification Kit (Pharmacia) and such. Alternatively, mRNA may be
directly prepared by QuickPrep mRNA Purification Kit
(Pharmacia).
[0063] The obtained mRNA is used to synthesize cDNA using reverse
transcriptase. cDNA may be synthesized using a kit, such as AMV
Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku
Kogyo). Alternatively, cDNA may be synthesized and amplified
following the 5'-RACE method (Frohman, M. A. et al., Proc. Natl.
Acad. Sci. U.S.A. 85:8998-9002 (1988); Belyavsky, A. et al.,
Nucleic Acids Res. 17:2919-2932 (1989)) that uses primers and such
described herein; using 5'-Ampli FINDER RACE Kit (Clontech); and by
polymerase chain reaction (PCR).
[0064] A desired DNA fragment is prepared from the obtained PCR
products and linked to a vector DNA. The recombinant vector is used
to transform E. coli and such, and the desired recombinant vector
is prepared from a selected colony. The nucleotide sequence of the
desired DNA can be verified by conventional methods, such as
dideoxynucleotide chain termination.
[0065] A DNA of the invention may be designed to have a sequence
that is expressed more efficiently by taking into account the
frequency of codon usage in the host used for expression (Grantham,
R. et al., Nucleic Acids Res. 9:r43-74 (1981)). The DNA of the
present invention may be altered by a commercially available kit or
a conventional method. For instance, the DNA may be altered by
digestion with restriction enzymes, insertion of a synthetic
oligonucleotide or an appropriate DNA fragment, addition of a
linker, or insertion of the initiation codon (ATG) and/or the stop
codon (TAA, TGA, or TAG), etc.
[0066] The vectors of the present invention are useful in
maintaining the DNA of the present invention within the host cell,
or expressing a protein of the present invention. When E. coli is
used as the host cell, there is no limitation other than that the
vector should have an "ori" to amplify and mass-produce the vector
in E. coli (e.g., JM109, DH5.alpha., HB101, or XL1Blue), and such;
and a marker gene for selecting the transformed E. coli (e.g., a
drug-resistance gene selected by a drug (e.g., ampicillin,
tetracycline, kanamycin, or chloramphenicol)). For example,
M13-series vectors, pUC-series vectors, pBR322, pBluescript,
pCR-Script, and such can be used. Besides the vectors, pGEM-T,
pDIRECT, pT7, and so on can also be used for subcloning and
excision of the cDNA as well. When a vector is used to produce a
protein of the present invention, an expression vector is
especially useful. When the expression vector is expressed, for
example, in E. coli, it should have the above characteristics in
order to be amplified in E. coli. Additionally, when E. coli, such
as JM109, DH5.alpha., HB101, or XL1-Blue, are used as the host
cell, the vector should have a promoter, e.g., the lacZ promoter
(Ward et. al. (1989) Nature 341:544-546; (1992) FASEB J.
6:2422-2427), the araB promoter (Better et al., (1988) Science
240:1041-1043), or the T7 promoter, that can efficiently promote
the expression of the desired gene in E. coli. Other examples of
the vectors are pGEX-5x-1 (Pharmacia), "QIAexpress system"
(QIAGEN), pEGFP, and pET (for this vector, BL21, a strain
expressing T7 RNA polymerase, is preferably used as the host).
[0067] Further, the vector may comprise a signal sequence that
induces secretion of the polypeptide. For producing the protein in
the periplasm of E. coli, the pelB signal sequence (Lei, S. P. et
al., J. Bacteriol. 169:4379 (1987)) may be used as the signal
sequence for protein secretion. For example, the calcium chloride
method or electroporation may be used to introduce the vector into
host cells.
[0068] Examples of vectors used to produce the proteins of the
present invention include, for example, expression vectors other
than E. coli, such as expression vectors derived from mammals
(e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids Res. (1990)
18(17):5322), pEF, pCDM8), insect cells (e.g., "Bac-to-BAC
baculovirus expression system" (GIBCO-BRL), pBacPAK8), plants (e.g.
pMH1, pMH2), animal viruses (e.g., pHSV, pMV, pAdexLcw),
retroviruses (e.g., pZIPneo) yeasts (e.g., "Pichia Expression Kit"
(Invitrogen), pNV11, SP-Q01) and Bacillus subtilis (e.g., pPL608,
pKTH50).
[0069] In order to express proteins in animal cells, such as CHO,
COS, and NIH3T3 cells, the vector should include a promoter
necessary for expression in such cells (e.g., the SV40 promoter
(Mulligan et al., (1979) Nature 277:108), the MMLV-LTR promoter,
the EF1.alpha. promoter (Mizushima et al., (1990) Nucleic Acids
Res. 18:5322), the CMV promoter, etc.). It is more preferable for
the vector to additionally have a marker gene that enables
selection of the transformants (for example, a drug resistance gene
selected by a drug (e.g., neomycin, G418, etc.)). Examples of
vectors with such characteristics include pMAM, pDR2, pBK-RSV,
pBK-CMV, pOPRSV, pOP13, and so on.
[0070] Furthermore, in order to stably express the gene and to
amplify the copy number in cells, the method using CHO cells
deficient in nucleic acid synthetic pathways as the host,
incorporating-into the CHO cells a vector (such as pCHOI) having a
DHFR gene that compensates for the deficiency, and amplifying the
vector with methotrexate (MTX) can be used. Furthermore, for
transiently expressing a gene, the method that transforms COS cells
that have the gene for SV40 T antigen on the chromosome with a
vector (such as pcD) having the SV40 replication origin can be
mentioned. The replication origin may be that of a polyomavirus,
adenovirus, bovine papilloma virus (BPV), and the like. Further, to
amplify the gene copy number in the host cells, selection markers,
such as aminoglycoside transferase (APH) gene, thymidine kinase
(TK) gene, E. coli xanthine-guanine phosphoribosyl transferase
(Ecogpt) gene, and dihydrofolate reductase (dhfr) gene may be
comprised in the expression vector.
[0071] A DNA of the present invention can be expressed in animals
by, for example, inserting a DNA of the invention into an
appropriate vector and introducing the vector into a living body by
the retrovirus method, the liposome method, the cationic liposome
method, the adenovirus method, and so on. Thus, it is possible to
perform gene therapy of diseases caused by a mutation in a gene of
the present invention. The vectors used in these methods include,
but are not limited to, adenovirus vectors (e.g. pAdexlcw),
retrovirus vectors (e.g. pZIPneo), and so on. General techniques
for gene manipulation, such as insertion of the DNA of the
invention into a vector, can be performed according to conventional
methods (Molecular Cloning, 5.61-5.63). Administration to the
living body may be performed according to ex vivo methods or in
vivo methods.
[0072] The host cell into which the vector of the invention is
introduced is not particularly limited. For example, E. coli,
various animal cells, and such, can be used. The host cell of the
present invention can be used, for example, as a production system
to produce and express a protein of the present invention. Protein
production systems include in vitro and in vivo systems. Such
production systems using eukaryotic cells or prokaryotic cells can
be given as in vitro production systems.
[0073] As eukaryotic host cells, for example, animal cells, plant
cells, and fungi cells can be used. Mammalian cells, for example,
CHO (J. Exp. Med. (1995) 108:945), COS, 3T3, myeloma, BHK (baby
hamster kidney), HeLa, Vero, amphibian cells (e.g. platanna oocytes
(Valle et al., (1981) Nature 291:358-340), and insect cells (e.g.
Sf9, Sf21, Tn5) are known as animal cells. Among CHO cells, those
deficient in the DHFR gene, dhfr-CHO (Proc. Natl. Acad. Sci. USA
(1980) 77:4216-4220) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968)
60:1275), are particularly preferable. Among animal cells, CHO
cells are particularly preferable for mass expression. A vector can
be introduced into a host cell by, for example, the calcium
phosphate method, the DEAE-dextran method, methods using cationic
liposome DOTAP (Boehringer-Mannheim), electroporation, lipofection,
etc.
[0074] As plant cells, for example, plant cells originating from
Nicotiana tabacum are known as protein producing systems and may be
used as callus cultures. As fungal cells, yeast cells such as
Saccharomyces, including Saccharomyces cerevisiae, or filamentous
fungi such as Aspergillus, including Aspergillus niger, are
known.
[0075] Useful prokaryotic cells include bacterial cells. Bacterial
cells, such as E. coli, for example, JM109, DH5.alpha., HB101, and
such, as well as Bacillus subtilis are known.
[0076] These cells are transformed by a desired DNA, and the
resulting transformants are cultured in vitro to obtain the
protein. Transformants can be cultured using known methods. For
example, culture medium, such as DMEM, MEM, RPMI1640, or IMDM, may
be used with or without serum supplements, such as fetal calf serum
(FCS) as culture medium for animal cells. The pH of the culture
medium is preferably between about 6 and 8. Such cells are
typically cultured at about 30 to 40.degree. C. for about 15 to 200
hr, and the culture medium may be replaced, aerated, or stirred if
necessary.
[0077] Animal and plant hosts may be used for in vivo production.
For example, a desired DNA can be introduced into an animal or
plant host. Encoded proteins are produced in vivo, and then
recovered. These animal and plant hosts are included in the "host"
of the present invention.
[0078] Animals to be used for the production system described above
include mammals and insects. Mammals, such as goats, pigs, sheep,
mice, and cattle, may be used (Vicki Glaser, SPECTRUM Biotechnology
Applications (1993)). Alternatively, the mammals may be transgenic
animals.
[0079] For instance, a desired DNA may be prepared as a fusion gene
with a gene such as goat .beta. casein gene that encodes a protein
specifically produced into milk. DNA fragments comprising the
fusion gene are injected into goat embryos, which are then
introduced back to female goats. Desired proteins are then
recovered from milk produced by the transgenic goats (i.e., those
born from the goats that had received the modified embryos) or by
their offspring. To increase the amount of milk containing the
proteins produced by transgenic goats, appropriate hormones may be
administered to the transgenic goats (Ebert, K. M. et al., (1994)
Bio/Technology 12:699-702).
[0080] Alternatively, insects, such as silkworm, may be used.
Baculoviruses into which a DNA encoding a desired protein has been
inserted can be used to infect silkworms, and the desired protein
can be recovered from the body fluid (Susumu, M. et al., (1985)
Nature 315:592-594).
[0081] As plants, for example, tobacco can be used. When using
tobacco, a DNA encoding a desired protein may be inserted into a
plant expression vector, such as pMON 530, which is introduced into
bacteria, such as Agrobacterium tumefaciens. Then, the bacteria can
be used to infect tobacco, such as Nicotiana tabacum, and the
desired polypeptide can be recovered from the leaves (Julian, K.-C.
Ma et al., (1994) Eur. J. Immunol. 24:131-138).
[0082] A protein of the present invention obtained as above may be
isolated from inside or outside of host (medium, etc.), and
purified as a substantially pure homogeneous protein. The method
for protein isolation and purification is not limited to any
specific method; in fact, any standard method may be used. For
instance, column chromatography, filters, ultrafiltration, salting
out, solvent precipitation, solvent extraction, distillation,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis,
isoelectric point electrophoresis, dialysis, and recrystallization
may be appropriately selected and combined to isolate and purify
the protein.
[0083] For chromatography, for example, affinity chromatography,
ion-exchange chromatography, hydrophobic chromatography, gel
filtration chromatography, reverse phase chromatography, adsorption
chromatography, and such may be used (Strategies for Protein
Purification and Characterization: A Laboratory Course Manual. Ed.
Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press
(1996)). These chromatographies may be performed by liquid
chromatographies such as HPLC and FPLC. Thus, the present invention
encompasses highly purified proteins produced by the above
methods.
[0084] A protein may be optionally modified or partially deleted by
treating it with an appropriate protein-modifying enzyme before or
after purification. For example, trypsin, chymotrypsin,
lysylendopeptidase, protein kinase, glucosidase, and such are used
as protein-modifying enzymes.
[0085] Further, the present invention provides methods for
producing PGE2 utilizing a protein of this invention having the
PGE2 synthase activity. According to an embodiment, the method is
characterized by the treatment of PGH2 with a protein having the
PGE2 synthase activity of the invention.
[0086] A protein having the PGE2 synthase activity used to produce
PGE2 may be a naturally derived protein, a recombinant protein, or
an artificially synthesized protein. Moreover, it may be a purified
protein or a protein in an intracellularly expressed form.
Furthermore, the protein may be immobilized in a reaction system.
Since a PGE2 synthase of this invention demonstrates a glutathione
(GSH) dependent activity, addition of glutathione to the reaction
system is preferred. The PGH2 used for this reaction may be, for
example, a commercial product (manufactured by Cayman).
[0087] Further, a preferred embodiment of the method for producing
PGE2 utilizing the protein of this invention having a PGE2 synthase
activity is a method characterized by reacting COX and PGE2
synthase with arachidonic acid. The method takes into consideration
the fact that PGH2, which is the direct substrate of PGE2 synthase,
is chemically extremely unstable and decomposes non-enzymatically,
and thus, produces PGE2 from arachidonic acid, which is a
relatively stable precursor of PGH2. Specifically, production of
PGE2 from arachidonic acid is achieved by a cooperative action
using COX, that produces PGH2 from arachidonic acid, and PGE2
synthase, that produces PGE2 from PGH2.
[0088] A PGE2 synthase of this invention and COX used to produce
PGE2 may be naturally derived proteins, recombinant proteins, or
artificially synthesized proteins. Furthermore, they may be
purified proteins or proteins in intracellularly expressed form.
Furthermore, the proteins may be immobilized in a reaction system.
Since a PGE2 synthase of this invention demonstrates a glutathione
(GSH) dependent activity, addition of glutathione to the reaction
system is preferred.
[0089] PGE2 was demonstrated in the Example to be produced from
arachidonic acid by the cooperation of PGES-1 with COX-1, or PGES-2
with COX-2. Therefore, preferred enzymes for use in PGE2 production
include the combination of PGES-1 and COX-1, and the combination of
PGES-2 and COX-2.
[0090] The produced PGE2 may be purified by normal means of
purification, for example, distillation under normal pressure or
reduced pressure; high-speed liquid chromatography using silica gel
or magnesium silicate; thin layer chromatography; column
chromatography, washing; recrystallization; and such.
[0091] Furthermore, the present invention provides methods of
screening for PGE2 synthase inhibitors. According to an embodiment,
the screening method of this invention comprises the steps of: (a)
contacting a transformant which carries a vector containing a
PGES-encoding DNA with a test sample and PGH2; (b) detecting the
production level of PGE2 of the cell; and (c) selecting the
compound that reduces the production level of PGE2 with that
observed in the absence of the contact with the test sample.
[0092] The transformant used for the screening can be prepared, for
example, by inserting a DNA encoding a PGE2 synthase into a vector,
such as pcDNA3.1, and then by introducing this into cells, such as
HEK293 cells.
[0093] Test samples to be contacted with the transformant include,
for example, cell extracts, cell culture supernatants,
microorganism fermentation products, marine organism extracts,
plant extracts, purified or crude proteins, peptides, non-peptide
compounds, synthetic low-molecular-weight compounds, and natural
compounds; but are not limited to these examples. The test sample
may include antibodies binding to a PGE2 synthase, or antisense
oligonucleotides suppressing the expression of the enzyme.
[0094] According to a preferred embodiment of the screening method
of this invention, the method comprises the steps of: (a)
contacting the transformant which carries a vector containing a
COX-encoding DNA and a vector containing a PGES-encoding DNA, with
a test sample and arachidonic acid; (b) detecting the production
level of PGE2 of the transformant; and (c) selecting the compound
that reduces the production level of PGE2 compared with that
observed in the absence of the contact with the test sample.
[0095] The transformant used for the screening can be prepared, for
example, by inserting a DNA encoding a PGE2 synthase and a DNA
encoding COX, respectively, into vectors, such as pcDNA3.1, and
then by co-transfecting them into cells, such as HEK293 cells. The
genes to be cotransfected preferably are exemplified by the
combination of PGES-1 and COX-1, and the combination of PGES-2 and
COX-2. Arachidonic acid to be contacted with the transformant may
be, for example, a commercial product (manufactured by Sigma).
[0096] Similar to the screening method mentioned above, the test
samples include, for example, cell extracts, cell culture
supernatants, microorganism fermentation products, marine organism
extracts, plant extracts, purified or crude proteins, peptides,
non-peptide compounds, synthetic low-molecular-weight compounds,
and natural compounds; but are not limited to these examples.
Further, the test sample may be antibodies binding to a PGE2
synthase, or antisense oligonucleotides suppressing the expression
of the enzyme.
[0097] The PGE2 production level during the screening of the
present invention can be measured, for example, by Enzyme
Immunoassay (a kit by Cayman) As a result, if the PGE2 production
level is reduced by the contact of a test sample with the
transformant, compared with that observed in the absence of the
contact, the test sample is suggested to be a compound that
inhibits the PGE2 synthase activity.
[0098] A PGE2 synthase inhibitor, which may be isolated by a
screening method of this invention, may be utilized as an
anti-inflammatory drug. NSAIDs (nonsteroidal anti-inflammatory
drugs), represented by aspirin and piroxicam, which have been used
widely as antipyretic analgesic antiphlogistic drugs, suppress the
production of PGD2, PGF2.alpha., PGI2, and TXA2, in addition to
PGE2 by inhibiting COX. Therefore, NSAIDs not only possess
anti-inflammatory effects but also are likely to express effects
based on the suppression of the production of these prostanoids
(other than the PGE2). On the other hand, since the compounds that
can be isolated by the screening of this invention are expected to
specifically inhibit a PGE2 synthase, development of medicaments
with fewer side effects is enabled.
[0099] Furthermore, cells that co-express COX and PGE2 synthase
have significantly faster proliferation rates as compared to the
control cells, taking on a form similar to a transformant lacking
contact inhibition ability, and thus, can be considered as a model
system for human clinical cancer suggested to be related to COX2.
Therefore, compounds that can be isolated by the screening of this
invention may be utilized as anti-cancer drugs.
[0100] A compound isolated by the screening of this invention can
be as a pharmaceutical agent for humans and other mammals, such as
mice, rats, guinea-pigs, rabbits, chicken, cats, dogs, sheep, pigs,
cattle, monkeys, baboons, and chimpanzees. Specifically, the
isolated compound can either itself be directly administered to
subjects or it can be formulated into a pharmaceutical composition
using known pharmaceutical preparation methods for administration.
For example, according to the need, the drugs can be taken orally,
as sugar coated tablets, capsules, elixirs, and microcapsules; or
non-orally, in the form of injections of sterile solutions or
suspensions with water or any other pharmaceutically acceptable
liquid. For example, the compounds can be formulated by mixing
appropriately with pharmacologically acceptable carriers or medium,
such as, sterilized water, physiological saline, plant-oil,
emulsifiers, suspending agents, surfactants, stabilizers, flavoring
agents, excipients, vehicles, preservatives, and binders, in a unit
dose form required for generally accepted drug implementation. The
amount of active ingredient in these preparations makes a suitable
dosage within the indicated range acquirable.
[0101] Examples of additives that can be mixed for tablets and
capsules are, binders such as gelatin, corn starch, tragacanth gum,
and arabic gum; excipients such as crystalline cellulose; swelling
agents such as corn starch, gelatin, and alginic acid; lubricants
such as magnesium stearate; sweeteners such as sucrose, lactose, or
saccharin; flavoring agents such as peppermint, Gaultheria
adenothrix oil, and cherry. When the unit dosage form is a capsule,
a liquid carrier, such as oil, can also be included in the above
ingredients. Sterile composites for injections can be formulated
following normal drug implementations using vehicles such as
distilled water used for injections.
[0102] Physiological saline, glucose, and other isotonic liquids
including adjuvants, such as D-sorbitol, D-mannnose, D-mannitol,
and sodium chloride, can be used as aqueous solutions for
injections. These can be used in conjunction with suitable
solubilizers, such as alcohol, specifically ethanol, polyalcohols
such as propylene glycol and polyethylene glycol, non-ionic
surfactants, such as Polysorbate 80 (TM) and HCO-50.
[0103] Sesame oil or Soy-bean oil can be used as a oleaginous
liquid and may be used in conjunction with benzyl benzoate or
benzyl alcohol as a solubilizer or may be formulated with a buffer
such as phosphate buffer and sodium acetate buffer, a pain-killer
such as procaine hydrochloride, a stabilizer such as benzyl
alcohol, phenol, or an anti-oxidant. The prepared injection is
generally filled into a suitable ampule.
[0104] Methods well known to one skilled in the art may be used to
administer a pharmaceutical compound to patients, for example as
intraarterial, intravenous, subcutaneous injections and also as
intranasal, transbronchial, intramuscular, percutaneous, or oral
administrations. The dosage varies according to the body-weight and
age of a patient, and the administration method; however, one
skilled in the art can suitably select the dosage. If said compound
can be encoded by a DNA, the DNA can be inserted into a vector for
gene therapy to perform the therapy. The dosage and method of
administration vary according to the body-weight, age, and symptoms
of a patient, but one skilled in the art can select them
suitably.
[0105] Although varying according to the symptoms, the dose of a
PGE2 synthase inhibitor that can be isolated by the screening
methods of this invention is generally in the range of about 0.1 mg
to 1 g, preferably about 1.0 to 100 mg, and more preferably about
1.0 to 20 mg per day for adults (body weight: 60 kg) in the case of
an oral administration. Although varying according to the subject,
target organ, symptoms, and method of administration, a single dose
of a compound for parenteral administration is advantageous, for
example, when administered intravenously to normal adults (60 kg
body weight) in the form of injection, in the range of about 0.01
to 300 mg, preferably about 0.1 to 100 mg, and more preferably
about 0.1 to 10 mg per day. Doses converted to 60 kg body weight or
per body surface area can be administered to other animals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0106] FIG. 1 depicts a graph demonstrating the PGES activity in
ammonium sulfate precipitated fraction of the rat brain soluble
fraction.
[0107] FIG. 2 depicts graphs demonstrating the elution profiles of
the PGES activity by DEAE-Sephacel ion exchange column
chromatography.
[0108] FIG. 3 depicts graphs demonstrating the elution profiles of
the PGES activity by Superdex 200 gel filtration column
chromatography (top), and photographs demonstrating the results of
SDS-PAGE analyses (bottom).
[0109] FIG. 4 depicts a graph demonstrating the effect of CDNB on
the PGES activity in each fraction obtained by Superdex 200 gel
filtration column chromatography.
[0110] FIG. 5 depicts the nucleotide sequence of p23 (PGES-1) cDNA
and the amino acid sequence thereof.
[0111] FIG. 6 depicts graphs demonstrating the in vitro enzyme
profile of the recombinant PGES-1 and PGES-2 proteins expressed in
HEK293 cells. PGES-1 (left panel) was demonstrated to be GSH
dependent and CDNB sensitive; whereas PGES-2 (right panel) was
demonstrated to be GSH dependent and CDNB insensitive.
[0112] FIG. 7 depicts a graph demonstrating the PGES activity of a
mutant protein (PGES-1 Y9N), wherein the ninth tyrosine from the
N-terminus (Tyr.sup.9) is mutated to asparagine. Tyr.sup.9 was
demonstrated to be essential for the PGES-1 activity.
[0113] FIG. 8 depicts graphs (left and middle panels) and
photographs (right two panels) demonstrating the effect on
PGE.sub.2 production of the introduced PGES-1 antisense expression
vector into 3Y1 cells.
[0114] FIG. 9 depicts graphs demonstrating the conversion of added
arachidonic acid into PGE.sub.2 by PGES-1 or PGES-2. PGES-1 is
demonstrated to function by selective coupling with COX-1, and
PGES-2 is demonstrated to exhibit an enhanced function by the
coupling with COX-2 than with COX-1.
[0115] FIG. 10 depicts a graph demonstrating the conversion of
endogenous arachidonic acid into PGE.sub.2 by PGES-2 in HEK293
transfect cells stimulated with IL-1.beta..
[0116] FIG. 11 depicts photographs demonstrating the intracellular
distribution of COX-2 and PGES-2. The top panel shows COX-2
distribution, the center panel shows PGES-2 distribution, and the
lower panel is an overlay of both of these distributions.
[0117] FIG. 12 depicts graphs demonstrating the effect of the
coexpression of COX-2 and PGES-2 to enhance PGE.sub.2 production
and cell proliferation.
BEST MODE FOR CARRYING OUT THE INVENTION
[0118] The present invention will be described in more detail below
by way of Examples, but should not construed as being limited to
these Examples.
Example 1
Identification of the PGES-1 Protein
[0119] PGES activity was measured as follows: Cells suspended in 20
mmol/L Tris-hydrochloride buffer (pH 7.4) were homogenized using
ultrasonic homogenizer (Branson Sonifier Model 200) and centrifuged
at 800.times.g for 5 minutes at 4.degree. C., and the supernatant
was used as the cell lysate in the following. 0.5 .mu.g of PGH2 was
added to the lysate, which was suspended with 0.1 mol/L
Tris-hydrochloride buffer (pH 8.0) containing 1 mmol/L GSH, with a
protein content of 10 .mu.g, was incubated for 30 seconds at
24.degree. C., and then 8 mmol/L FeCl.sub.2 was added to terminate
the reaction. The amount of produced PGE2 was measured by Enzyme
Immunoassay Kit (Cayman), and PGES activity was expressed as the
amount of PGE2 produced per 30 seconds, per 10 .mu.g of protein
(ng/30 sec/10 .mu.g protein).
[0120] 150 .mu.g/kg body LPS (Salmonella minnesota: Re595) was
administered intravenously from rat tail. Rat brain was removed 2
days after the injection and was homogenized using Potter's
homogenizer at 4.degree. C. The homogenate was centrifuged at
10,000.times.g at 4.degree. C. for 20 minutes, and the supernatant
was collected. The supernatant was centrifuged again at
100,000.times.g at 4.degree. C. for 60 minutes to recover the
supernatant. 60 to 80% saturated ammonium sulfate precipitation
fractions having PGES activity (FIG. 1) were collected from the
supernatant, and fractions having the PGES activity eluted at an
NaCl concentration of around 0.5 mol/L to around 0.8 mol/L by
DEAE-Sephacel ion exchange column chromatography were collected
(FIG. 2).
[0121] Furthermore, these fractions were subjected to Superdex-200
column gel filtration FPLC (elution buffer: 20 mmol/L
Tris-hydrochloride buffer (pH 7.4), 150 mmol/L NaCl), to yield 3
peaks demonstrating PGES activity: A (molecular weight of
approximately 300 kDa); B (molecular weight of approximately 100
kDa); and C (molecular weight of approximately 50 kDa). No
significant differences in the peaks A and B were observed in the
PGES activity between LPS-administered rats and normal rats;
whereas, about 6 times higher PGES activity of peak C was observed
in LPS-administered rats compared to normal rats (FIG. 3, top
panel).
[0122] A protein of a molecular weight of about 27 kDa,
corresponding with the PGES activity (PGES-1), was obtained by SDS
electrophoretic analysis of peak C (FIG. 3, bottom). The production
of this protein was predicted to elevate in response to
inflammatory stimulus due to the fact that large amount of the
protein was detected in LPS-administered rats than in normal rats.
Furthermore, in contrast to peak A and B, peak C was strongly
inhibited by CDNB (FIG. 4). Therefore, the protein was suggested to
be a GST-like isozyme.
[0123] Peptide mapping of PGES-1 protein was carried out using V8
protease, and 3 fragments among the peptide fragments were analyzed
by N-terminal amino acid sequence analyzer to determine the partial
amino acid sequence thereof. According to GenBank database search
with one of these amino acid sequences, the sequence completely
matched to a portion of a known sequence reported as the human
progesterone receptor complex component protein p23 (Johnson, J L
et al., Mol. Cell. Biol., 14:1956-63, 1994). Therefore, referring
to the reported cDNA sequence, the cDNA of full-length human
progesterone receptor complex component protein p23 was isolated by
RT-PCR method using HeLa cell mRNA as the material (FIG. 5). PGES
activity dependent to GSH was observed in the cell lysate by
inserting the cDNA into expression vector pcDNA3.1, and
transfecting HEK293 cells using lipofectamine, which activity was
suppressed in the coexistence of CDNB (FIG. 6, left panel). Thus,
the cDNA was confirmed to encode the desired PGES. In addition, the
mutant protein, wherein the 9th tyrosine residue from the
N-terminus was substituted by point mutation to asparagine, did not
demonstrate the PGES activity, which elucidated the tyrosine
residue to be essential for the expression of the activity (FIG.
7). On the other hand, when pcDNA3.1-hygro, which has the entire
PGES-1-encoding region cloned in reverse, was transfected to human
fibroblast 3Y1, PGES-1 synthesis was inhibited and PGE2 production
and PGES activity was decreased demonstrating the inhibition of
PGE2 production by antisense RNA (FIG. 8).
[0124] Although a GSH-dependent PGES activity was confirmed in
HEK293 cells transfected with PGE2 synthase (PGES-2), an enzyme
reported by Per-Johan Jakobsson et al., the activity was not
suppressed by the coexistence of CDNB (FIG. 6, right panel) which
demonstrates the difference between PGES-1 and PGES-2.
[0125] The PGES-1 protein is an enzyme that converts PGH2 produced
by COX, particularly by COX-1, into PGE2, and is thought to have an
important role in inflammatory reactions where PGE2 plays a major
role.
Example 2
Preparation of Cells Coexpressing COX and PGES
[0126] Human kidney-derived HEK293 cells (human, embryo, kidney,
transformed with adenovirus 5 DNA) were purchased from ATCC (ATCC
Number: CRL-1573).
[0127] Expression vectors wherein human COX-1 and COX-2 cDNAs were
inserted into pcDNA3.1, respectively, were transfected into HEK293
cells using lipofectamine to produce HEK293 cells that express
either COX-1 or COX-2. HEK293 cells expressing PGES-1 or PGES-2
alone, or coexpressing COX-1 and PGES-1, COX-1 and PGES-2, COX-2
and PGES-1, or COX-2 and PGES-2 were constructed by similarly
transfecting expression vectors wherein either human PGES-1 cDNA or
human PGES-2 cDNA was inserted into pcDNA3.1 into constructed COX-1
or COX-2 expressing cells and into HEK293 cells of the parent
strain, and these cells were used in the following experiment.
Evaluation of cell proliferation was performed by seeding the cell
on RPMI1640 media in a 6-well plate (Iwaki) at 1.5.times.10.sup.5/3
mL/well, incubating in carbon dioxide gas incubator at 5% CO.sub.2
for 4 days, and then, calculating the number of living cells by the
trypan blue staining method using a cytometric plate.
[0128] The PGE2 production level of these cells under a condition
adding 2 .mu.mol/L of arachidonic acid to the culture medium was
examined. The PGE2 production level of cells coexpressing COX-1 and
PGES-1 largely increased compared to cells expressing COX-1 alone,
whereas no increase in PGE2 production was observed in cells
coexpressing human COX-2 and PGES-1 (FIG. 9, left panel) This
suggests the coupling (cooperative functioning) of PGES-1 and
COX-1. On the other hand, large amounts of PGE2 was produced by the
expression of PGES-2 together with COX-2 (FIG. 9, right panel),
suggesting the coupling (cooperative functioning) of PGES-2 and
COX-2. A more remarkable increase in PGE2 production by coupling of
PGES-2 and COX-2 was demonstrated by stimulating cells with 1 ng/mL
human Interleukin-1.beta. (IL-1.beta.; Genzyme) under conditions
without the addition of arachidonic acid to the culture medium
(FIG. 10).
[0129] Furthermore, a close relation between COX-2 and PGES-2 was
also demonstrated by a similar distribution of the enzymes detected
by an indirect immunostaining using a combination of mouse
monoclonal antibody against FLAG with FITC-labeled anti-mouse IgG
antibody, or goat antisera against COX-2 (Santa Cruz, N-20 goat
polyclonal Ab) with Cy3-labeled anti-goat IgG antibody on cells,
which cells were prepared to coexpress COX-2 and a fusion protein,
consisting of PGES-2 and FLAG, by similarly transfecting an
expression vector containing PGES-2 cDNA and FLAG cDNA to cells
that express COX-2 alone (FIG. 11).
[0130] Not only PGES but also COX is greatly involved in the
pathway to produce PGE2 via the arachidonic acid metabolic pathway.
Therefore, when screening for PGES inhibitors using cells, it is
preferable to use cells expressing not only PGES but those
simultaneously expressing COX. In addition, a phenomenon where both
enzymes function cooperatively (coupling) was demonstrated in cells
expressing both of COX-1 and PGES-1, and in cells expressing both
of COX-2 and PGES-2. Accordingly, actually in vivo, during PGE2
production induced by the stimulus accompanying inflammation, both
of COX-2 and PGES-2, or both of COX-1 and PGES-1, are indicated to
have an important role functioning cooperatively. Thus, cells
expressing these combinations seem to reproduce the situation
caused by an inflammatory stimulus in a stabilized form.
Furthermore, cell proliferation in addition to PGE2 production is
increased by the coexpression of PGES-2 and COX-2 (FIG. 12), which
may reflect an aspect of a process where normal cells transform
into cancer cells.
INDUSTRIAL APPLICABILITY
[0131] The present invention provides a PGES-1 protein having PGE2
synthase activity and genes encoding the protein, which enables the
efficient production of PGE2 as well as the efficient screening of
PGE2 synthase inhibitors that are useful as anti-inflammatory
drugs, and such. Specifically, efficient and accurate screening of
PGE2 synthase inhibitors is enabled by cells simultaneously
expressing the PGE2 synthase and COX.
Sequence CWU 1
1
2 1 160 PRT Homo sapiens 1 Met Gln Pro Ala Ser Ala Lys Trp Tyr Asp
Arg Arg Asp Tyr Val Phe 1 5 10 15 Ile Glu Phe Cys Val Glu Asp Ser
Lys Asp Val Asn Val Asn Phe Glu 20 25 30 Lys Ser Lys Leu Thr Phe
Ser Cys Leu Gly Gly Ser Asp Asn Phe Lys 35 40 45 His Leu Asn Glu
Ile Asp Leu Phe His Cys Ile Asp Pro Asn Asp Ser 50 55 60 Lys His
Lys Arg Thr Asp Arg Ser Ile Leu Cys Cys Leu Arg Lys Gly 65 70 75 80
Glu Ser Gly Gln Ser Trp Pro Arg Leu Thr Lys Glu Arg Ala Lys Leu 85
90 95 Asn Trp Leu Ser Val Asp Phe Asn Asn Trp Lys Asp Trp Glu Asp
Asp 100 105 110 Ser Asp Glu Asp Met Ser Asn Phe Asp Arg Phe Ser Glu
Met Met Asn 115 120 125 Asn Met Gly Gly Asp Glu Asp Val Asp Leu Pro
Glu Val Asp Gly Ala 130 135 140 Asp Asp Asp Ser Gln Asp Ser Asp Asp
Glu Lys Met Pro Asp Leu Glu 145 150 155 160 2 483 DNA Homo sapiens
CDS (1)...(480) 2 atg cag cct gct tct gca aag tgg tac gat cga agg
gac tat gtc ttc 48 Met Gln Pro Ala Ser Ala Lys Trp Tyr Asp Arg Arg
Asp Tyr Val Phe 1 5 10 15 att gaa ttt tgt gtt gaa gac agt aag gat
gtt aat gta aat ttt gaa 96 Ile Glu Phe Cys Val Glu Asp Ser Lys Asp
Val Asn Val Asn Phe Glu 20 25 30 aaa tcc aaa ctt aca ttc agt tgt
ctc gga gga agt gat aat ttt aag 144 Lys Ser Lys Leu Thr Phe Ser Cys
Leu Gly Gly Ser Asp Asn Phe Lys 35 40 45 cat tta aat gaa att gat
ctt ttt cac tgt att gat cca aat gat tcc 192 His Leu Asn Glu Ile Asp
Leu Phe His Cys Ile Asp Pro Asn Asp Ser 50 55 60 aag cat aaa aga
acg gac aga tca att tta tgt tgt tta cga aaa gga 240 Lys His Lys Arg
Thr Asp Arg Ser Ile Leu Cys Cys Leu Arg Lys Gly 65 70 75 80 gaa tct
ggc cag tca tgg cca agg tta aca aaa gaa agg gca aag ctt 288 Glu Ser
Gly Gln Ser Trp Pro Arg Leu Thr Lys Glu Arg Ala Lys Leu 85 90 95
aat tgg ctt agt gtc gac ttc aat aat tgg aaa gac tgg gaa gat gat 336
Asn Trp Leu Ser Val Asp Phe Asn Asn Trp Lys Asp Trp Glu Asp Asp 100
105 110 tca gat gaa gac atg tct aat ttt gat cgt ttc tct gag atg atg
aac 384 Ser Asp Glu Asp Met Ser Asn Phe Asp Arg Phe Ser Glu Met Met
Asn 115 120 125 aac atg ggt ggt gat gag gat gta gat tta cca gaa gta
gat gga gca 432 Asn Met Gly Gly Asp Glu Asp Val Asp Leu Pro Glu Val
Asp Gly Ala 130 135 140 gat gat gat tca caa gac agt gat gat gaa aaa
atg cca gat ctg gag 480 Asp Asp Asp Ser Gln Asp Ser Asp Asp Glu Lys
Met Pro Asp Leu Glu 145 150 155 160 taa 483
* * * * *