WO2004033692A1

WO2004033692A1 - Human prostaglandin g/h synthase 2 splice variant

Info

Publication number: WO2004033692A1
Application number: PCT/EP2003/011057
Authority: WO
Inventors: Zhimin Zhu
Original assignee: Bayer Healthcare AG
Current assignee: Bayer AG
Priority date: 2002-10-08
Filing date: 2003-10-07
Publication date: 2004-04-22
Anticipated expiration: 2005-04-08
Also published as: AU2003271692A1

Abstract

Reagents that regulate human prostaglandin G/H synthase 2 precursor and reagents which bind to human prostaglandin G/H synthase 2 precursor gene products can play a role in preventing or ameliorating pain.

Description

HUMAN PROSTAGLANDIN G/H SYNTHASE 2 SPLICE VARIANT

FIELD OF THE INVENTION

The invention relates to the regulation of human prostaglandin G/H synthase 2 precursor.

BACKGROUND OF THE INVENTION

It is generally accepted by those skilled in the art that the pharmacology of nonsteroidal antiinflammatory drugs, also called NSAIDs, results from the inhibition of prostaglandin G H synthase in arachidonic acid metabolism. U.S. Patent 5,571,825. Prostaglandin G/H synthase has also been called cyclooxygenase. Prostaglandin synthase activity results in the formation of prostaglandins and other arachidonic acid metabolites that are important inflammatory agents. These metabolites also have cytoprotective properties and are important for the maintenance of tissue physiology and homeostasis.

It has been recently discovered that there are two distinct isoforms of prostaglandin G/H synthase. The two forms have been called prostaglandin G/H synthase- 1 and prostaglandin G/H synthase-2.

Prostaglandin G/H synthase- 1 is a constitutive enzyme expressed in a variety of tissues and ap- pears to be the isoform important for physiological and homeostatic processes. In contrast, prostaglandin G/H synthase-2 is upregulated by a variety of agents, including proinflammatorycytokines, endotoxins, mitogens, growth factors and hormones and is downregulated by glucocorticoids.

It is believed that prostaglandin G/H synthase-2 is responsible for the formation of prostaglandins and other arachidonic acid metabolites which contribute to the pathology of inflammation and other diseases.

In general, the nonsteroidal antiinflammatory drugs that are currently used to treat patients inhibit both prostaglandin G/H synthase- 1 and prostaglandin G/H synthase-2, and therefore, have mechanism-based side effects due to the inhibition of prostaglandin G/H synthase- 1 and the resulting interference with tissue homeostasis.

Compounds which inhibit prostaglandin G/H synthase-2, but have little or no effect on prostaglandin G/H synthase- 1 can provide effective antiinflammatory, analgesic and antipyretic activity without causing side effects such as gastrointestinal and renal toxicities which are seen when using traditional nonsteroidal antiinflammatory drugs. There is a need in the art to identify related enzymes, which can be regulated to provide therapeutic effects.

It is an object of the invention to provide reagents and methods of regulating a human prostaglandin G/H synthase 2 precursor. This and other objects of the mvention are provided by one or more of the embodiments described below.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the DNA-sequence encoding a prostaglandin g/H synthase 2 precursor Polypeptide (SEQ ID NO:l).

Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig.l (SEQ ID NO:2).

Fig. 3 shows the DNA-sequence encoding a prostaglandin g H synthase 2 precursor Polypeptide (SEQ ID NO: 3).

Fig. 4 shows the DNA-sequence encoding a prostaglandin g/H synthase 2 precursor Polypeptide (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide from the group consisting of:

a) a polynucleotide encoding a Prostaglandin g/H synthase 2 precursor polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 95% identical to

the amino acid sequence shown in SEQ ID NO: 2; and

the amino acid sequence shown in SEQ ID NO: 2.

b) a polynucleotide comprising the sequence of SEQ ID NO: 1 ;

c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a Prostaglandin g/H synthase 2 precursor polypeptide; d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a Prostaglandin g/H synthase 2 precursor polypeptide; and

e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a Prostaglandin g/H synthase 2 precursor polypeptide.

The protein of the present invention is a splice variant human prostaglandin G/H synthase 2 precursor. Human prostaglandin G/H synthase 2 precursor comprises the amino acid sequence shown in SEQ ID NO:2. A coding sequence for human prostaglandin G H synthase 2 precursor is shown in SEQ ID NO: l . This sequence is contained within the longer sequence shown in SEQ ID NO: 3. This sequence is located on chromosome lq31.1.

Human prostaglandin G/H synthase 2 precursor of the invention is expected to be useful for the same purposes as previously identified prostaglandin G/H synthase 2 precursor enzymes. Human prostaglandin G/H synthase 2 precursor is believed to be useful in therapeutic methods to treat pain. Human prostaglandin G/H synthase 2 precursor also can be used to screen for human prostaglandin G/H synthase 2 precursor activators and inhibitors.

One embodiment of the present invention is an expression vector containing any polynucleotide of the present invention.

Yet another embodiment of the present invention is a host cell containing any expression vector of the present invention.

Still another embodiment of the present invention is a substantially purified Prostaglandin g/H synthase 2 precursor polypeptide encoded by any polynucleotide of the present invention.

Even another embodiment of the present invention is a method of producing a Prostaglandin g/H synthase 2 precursor polypeptide of the present invention, wherein the method comprises the following steps:

a. culturing the host cells of the present invention under conditions suitable for the expression of the Prostaglandin g/H synthase 2 precursor polypeptide; and

b. recovering the Prostaglandin g/H synthase 2 precursor polypeptide from the host cell culture. Yet another embodiment of the present invention is a method for detecting a polynucleotide encoding a Prostaglandin g/H synthase 2 precursor polypeptide in a biological sample comprising the following steps:

a. hybridizing any polynucleotide of the present invention to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and

b. detecting said hybridization complex.

Still another embodiment of the present invention is a method for detecting a polynucleotide of the present invention or a Prostaglandin g/H synthase 2 precursor polypeptide of the present invention comprising the steps of:

a. contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the Prostaglandin g/H synthase 2 precursor polypeptide and

b. detecting the interaction

Even another embodiment of the present invention is a diagnostic kit for conducting any method of the present invention.

Yet another embodiment of the present invention is a method of screening for agents which decrease the activity of a Prostaglandin g/H synthase 2 precursor, comprising the steps of:

a. contacting a test compound with a Prostaglandin g/H synthase 2 precursor polypeptide encoded by any polynucleotide of the present invention;

b. detecting binding of the test compound to the Prostaglandin g/H synthase 2 precursor polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a Prostaglandin g H synthase 2 - precursor.

Still another embodiment of the present invention is a method of screening for agents which regulate the activity of a Prostaglandin g/H synthase 2 precursor, comprising the steps of:

a. contacting a test compound with a Prostaglandin g/H synthase 2 precursor polypeptide encoded by any polynucleotide of the present invention; and

b. detecting a Prostaglandin g H synthase 2 precursor activity of the polypeptide, wherein a test compound which increases the Prostaglandin g/H synthase 2 precursor activity is identified as a potential therapeutic agent for increasing the activity of the Prostaglandin g/H synthase 2 precursor, and wherein a test compound which decreases the Prostaglandin g/H synthase 2 precursor activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the Prostaglandin g/H synthase 2 precursor.

Even another embodiment of the present invention is a method of screening for agents which decrease the activity of a Prostaglandin g/H synthase 2 precursor, comprising the step of:

contacting a test compound with any polynucleotide of the present invention and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of Prostaglandin g/H synthase 2 precursor.

Yet another embodiment of the present invention is a method of reducing the activity of a Prostaglandin g/H synthase 2 precursor, comprising the step of:

contacting a cell with a reagent which specifically binds to any polynucleotide of the present invention or any Prostaglandin g/H synthase 2 precursor polypeptide of the present invention, whereby the activity of Prostaglandin g/H synthase 2 precursor is reduced.

Still another embodiment of the present invention is a reagent that modulates the activity of a Prostaglandin g/H synthase 2 precursor polypeptide or a polynucleotide wherein said reagent is identified by any methods of the present invention.

Even another embodiment of the present invention is a pharmaceutical composition, comprising:

an expression vector of the present invention or a reagent of the present invention and a pharmaceutically acceptable carrier.

Yet another embodiment of the present invention is the use of an expression vector of the present invention or a reagent of the present invention for modulating the activity of a Prostaglandin g/H synthase 2 precursor in a disease, preferably pain.

The invention thus provides a human prostaglandin G/H synthase 2 precursor that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site. Furthermore, human prostaglandin G/H synthase 2 precursor and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity. Polypeptides

Human prostaglandin G H synthase 2 precursor polypeptides according to the invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 550, or 572 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof, as defined below. A prostaglandin G/H synthase 2 precursor polypeptide of the invention therefore can be a portion of a prostaglandin G H synthase 2 precursor protein, a full-length prostaglandin G/H synthase 2 precursor protein, or a fusion protein comprising all or a portion of a prostaglandin G/H synthase 2 precursor protein.

Biologically active variants

Human prostaglandin G/H synthase 2 precursor polypeptide variants that are biologically active, e.g., retain enzymatic activity, also are human prostaglandin G/H synthase 2 precursor polypeptides. Preferably, naturally or non-naturally occurring human prostaglandin G/H synthase 2 precursor polypeptide variants have amino acid sequences which are at least about 95, 96, 97, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO:2 or a fragment thereof. Percent identity between a putative human prostaglandin G/H synthase 2 precursor polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff & Henikoff, 1992.

Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson & Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant. The FASTA algorithm is described by Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 55:2444(1988), and by Pearson, Meth. Enzymol. J 83:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch- Sellers algorithm (Needleman & Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAMJ. Appl. Math.26:l l (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l , and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRTX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a human prostaglandin G/H synthase 2 precursor polypeptide can be found using computer programs well known in the art, such as DNASTAR software.

The invention additionally, encompasses prostaglandin G/H synthase 2 precursor polypeptides that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc. Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The prostaglandin G/H synthase 2 precursor polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

The invention also provides chemically modified derivatives of prostaglandin G/H synthase 2 precursor polypeptides that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxy- methylcellulose, dextran, polyvinyl alcohol, and the like. The polypeptides can be modified at random or predetermined positions within the molecule and can include one, two, three, or more attached chemical moieties.

Whether an amino acid change or a polypeptide modification results in a biologically active prostaglandin G/H synthase 2 precursor polypeptide can readily be determined by assaying for enzymatic activity, as described in Example 4.

Fusion proteins

Fusion proteins are useful for generating antibodies against prostaglandin G/H synthase 2 precursor polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a human prostaglandin G/H synthase 2 precursor polypeptide. Protein affinity chromatography or library- based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

A human prostaglandin G/H synthase 2 precursor polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises a human prostaglandin G/H synthase 2 precursor polypeptide, such as those described above. The first polypeptide segment also can comprise full-length prostaglandin G/H synthase 2 precursor protein. The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β-gl cuτoιύdase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the prostaglandin G/H synthase 2 precursor polypeptide- encoding sequence and the heterologous protein sequence, so that the prostaglandin G/H synthase 2 precursor polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:l in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of species homologs

Species homologs of human prostaglandin G/H synthase 2 precursor polypeptide can be obtained using prostaglandin G/H synthase 2 precursor polypeptide polynuckotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of prostaglandin G/H synthase 2 precursor polypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides

A human prostaglandin G/H synthase 2 precursor polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a prostaglandin G/H synthase 2 precursor polypeptide. A coding sequence for human prostaglandin G/H synthase 2 precursor is shown in SEQ ID NO: 1.

Degenerate nucleotide sequences encoding human prostaglandin G/H synthase 2 precursor polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO: l or its complement also are prostaglandin G/H synthase 2 precursor polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of prostaglandin G H synthase 2 precursor polynucleotides that encode biologically active prostaglandin G/H synthase 2 precursor polypeptides also are prostaglandin G/H synthase 2 precursor polynucleotides. Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO: 1 or its complement also are prostaglandin G/H synthase 2 precursor polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.

Identification of polynucleotide variants and homologs

Variants and homologs of the prostaglandin G/H synthase 2 precursor polynucleotides described above also are prostaglandin G/H synthase 2 precursor polynucleotides. Typically, homologous prostaglandin G/H synthase 2 precursor polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known prostaglandin G/H synthase 2 precursor polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions-2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each-homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the prostaglandin G/H synthase 2 precursor polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of prostaglandin G/H synthase 2 precursor polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T_m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973). Variants of human prostaglandin G/H synthase 2 precursor polynucleotides or prostaglandin G/H synthase 2 precursor polynucleotides of other species can therefore be identified by hybridizing a putative homologous prostaglandin G/H synthase 2 precursor polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO: l or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to prostaglandin G/H synthase 2 precursor polynucleotides or their complements following stringent hybridization and/or wash conditions also are prostaglandin G/H synthase 2 precursor polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated T_m of the hybrid under study. The T_m of a hybrid between a prostaglandin G/H synthase 2 precursor polynucleotide having a nucleotide sequence shown in SEQ ID NO: l or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T_m = 81.5 °C - 16.6(log₁₀[Na⁺]) + 0.41(%G + C) - 0.63(%formamιde) - 600//), where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C. Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.

Preparation of polynucleotides

A human prostaglandin G/H synthase 2 precursor polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated prostaglandin G/H synthase 2 precursor polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise prostaglandin G/H synthase 2 precursor nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.

Human prostaglandin G H synthase 2 precursor cDNA molecules can be made with standard molecular biology techniques, using prostaglandin G/H synthase 2 precursor mRNA as a template. Human prostaglandin G/H synthase 2 precursor cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesize prostaglandin G/H synthase 2 precursor polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a human prostaglandin G/H synthase 2 precursor polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof.

Extending polynucleotides

Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to - detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Sarkar, PCR Methods Applic. 2, 318-322, 1993 ; Triglia et al., Nucleic Acids Res. 16, 8186, . 1988; Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991; Parker et al., Nucleic Acids Res.

19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto,

Calif). See WO 01/98340

Obtaining Polynucleotides

Human prostaglandin G/H synthase 2 precursor polypeptides can be obtained, for example, by purification from human cells, by expression of prostaglandin G/H synthase 2 precursor polynucleotides, or by direct chemical synthesis. Protein purification

Human prostaglandin G/H synthase 2 precursor polypeptides can be purified from any human cell which expresses the receptor, including host cells which have been transfected with prostaglandin G/H synthase 2 precursor polynucleotides. A purified prostaglandin G/H synthase 2 precursor polypeptide is separated from other compounds that normally associate with the prostaglandin G/H synthase 2 precursor polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

A preparation of purified prostaglandin G/H synthase 2 precursor polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.

Expression of polynucleotides

To express a human prostaglandin G/H synthase 2 precursor polynucleotide, the polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding prostaglandin G/H synthase 2 precursor polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding a human prostaglandin G/H synthase 2 precursor polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems. See WO 01/98340. Host cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed prostaglandin G/H synthase 2 precursor polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein. See WO 01/98340.

Alternatively, host cells which contain a human prostaglandin G/H synthase 2 precursor polynucleotide and which express a human prostaglandin G/H synthase 2 precursor polypeptide can be identified by a variety of procedures known to those of skill in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn, 1990) and Maddox et al, J. Exp. Med. 158, 1211-1216, 1983). See WO 01/98340.

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding prostaglandin G/H synthase 2 precursor polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a human prostaglandin G H synthase 2 precursor polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).

Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Expression and purification of polypeptides

Host cells transformed with nucleotide sequences encoding a human prostaglandin G/H synthase 2 precursor polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode prostaglandin G/H synthase 2 precursor polypeptides can be designed to contain signal sequences which direct secretion of soluble prostaglandin G H synthase 2 precursor polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane- bound prostaglandin G/H synthase 2 precursor polypeptide. See WO 01/98340.

Chemical synthesis

Sequences encoding a human prostaglandin G/H synthase 2 precursor polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a human prostaglandin G H synthase 2 precursor polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of prostaglandin G/H synthase 2 precursor polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule. See WO 01/98340.

As will be understood by those of skill in the art, it may be advantageous to produce prostaglandin G/H synthase 2 precursor polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter prostaglandin G/H synthase 2 precursor polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of a human prostaglandin G/H synthase 2 precursor polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of a human prostaglandin G/H synthase 2 precursor polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of a human prostaglandin G/H synthase 2 precursor polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.

Typically, an antibody that specifically binds to a human prostaglandin G/H synthase 2 precursor polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies that specifically bind to prostaglandin G/H synthase 2 precursor polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a human prostaglandin G/H synthase 2 precursor polypeptide from solution. See WO 01/98340.

Antisense oligonucleotides

Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of prostaglandin G/H synthase 2 precursor gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester intemucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.

Modifications of prostaglandin G/H synthase 2 precursor gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the prostaglandin G/H synthase 2 precursor gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base -pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co, Mt. Kisco, N.Y, 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. See WO 01/98340.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a human prostaglandin G/H synthase 2 precursor polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the prostaglandin G H synthase 2 precursor polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201). See WO 01/98340.

Differentially expressed genes

Described herein are methods for the identification of genes whose products interact with human prostaglandin G/H synthase 2 precursor. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, disorders involving pain. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human prostaglandin G/H synthase 2 precursor gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse franscriptase), PCR, and Northern analysis.

To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.

Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Patent 5,262,311).

The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human prostaglandin G/H synthase 2 precursor. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human prostaglandin G/H synthase 2 precursor. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human prostaglandin G/H synthase 2 precursor gene or gene product are up- regulated or down-regulated.

Screening methods

The invention provides assays for screening test compounds that bind to or modulate the activity of a human prostaglandin G H synthase 2 precursor polypeptide or a human prostaglandin G/H synthase 2 precursor polynucleotide. A test compound preferably binds to a human prostaglandin G/H synthase 2 precursor polypeptide or polynucleotide. More preferably, a test compound decreases or increases enzymatic activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.

Test compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl 33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).

High throughput screening

Test compounds can be screened for the ability to bind to prostaglandin G/H synthase 2 precursor polypeptides or polynucleotides or to affect prostaglandin G/H synthase 2 precursor activity or prostaglandin G/H synthase 2 precursor gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Yet another example is described by Salmon et al, Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

Binding assays

For binding assays, the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the prostaglandin G/H synthase 2 precursor polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the prostaglandin G/H synthase 2 precursor polypeptide can comprise a detectable label, such as . a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the prostaglandin G/H synthase 2 precursor polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a human prostaglandin G H synthase 2 precursor polypeptide can be determined without labeling either of the interactants. For example, a micro- physiometer can be used to detect binding of a test compound with a human prostaglandin G H synthase 2 precursor polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a human prostaglandin G/H synthase 2 precursor polypeptide (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a human prostaglandin G/H synthase 2 precursor polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opi . Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, a human prostaglandin G/H synthase 2 precursor polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Bartel et aϊ, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the prostaglandin G/H synthase 2 precursor polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding a human prostaglandin G/H synthase 2 precursor polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the prostaglandin G/H synthase 2 precursor polypeptide.

It may be desirable to immobilize either the prostaglandin G/H synthase 2 precursor polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the prostaglandin G/H synthase 2 precursor polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a human prostaglandin G/H synthase 2 precursor polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, the prostaglandin G/H synthase 2 precursor polypeptide is a fusion protein comprising a domain that allows the prostaglandin G/H synthase 2 precursor polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione deπvatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed prostaglandin G/H synthase 2 precursor polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a human prostaglandin G/H synthase 2 precursor polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated prostaglandin G/H synthase 2 precursor polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N- hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of sfreptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a prostaglandin G H synthase 2 precursor polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the prostaglandin G/H synthase 2 precursor polypeptide, can be deπvatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the prostaglandin G/H synthase 2 precursor polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the prostaglandin G/H synthase 2 precursor polypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to a human prostaglandin G/H synthase 2 precursor polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a prostaglandin G/H synthase 2 precursor polypeptide or polynucleotide can be used in a cell- based assay system. A prostaglandin G/H synthase 2 precursor polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a prostaglandin G/H synthase 2 precursor polypeptide or polynucleotide is determined as described above.

Enzymatic activity

Test compounds can be tested for the ability to increase or decrease the enzymatic activity of a human prostaglandin G/H synthase 2 precursor polypeptide. Enzymatic activity can be measured, for example, as described in Example 4.

Enzyme assays can be carried out after contacting either a purified prostaglandin G/H synthase 2 precursor polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases enzymatic activity of a human prostaglandin G/H synthase 2 precursor polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing prostaglandin G/H synthase 2 precursor activity. A test compound which increases enzymatic activity of a human prostaglandin G/H synthase 2 precursor polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human prostaglandin G/H synthase 2 precursor activity.

Gene expression

In another embodiment, test compounds that increase or decrease prostaglandin G/H synthase 2 precursor gene expression are identified. A prostaglandin G/H synthase 2 precursor polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the prostaglandin G/H synthase 2 precursor polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of prostaglandin G/H synthase 2 precursor mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a human prostaglandin G/H synthase 2 precursor polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a human prostaglandin G/H synthase 2 precursor polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a human prostaglandin G/H synthase 2 precursor polynucleotide can be used in a cell-based assay system. The prostaglandin G/H synthase 2 precursor polynucleotide can be naturally occurring in the cell or can be infroduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used. Pharmaceutical compositions

The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a human prostaglandin G/H synthase 2 precursor polypeptide, prostaglandin G/H synthase 2 precursor polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a prostaglandin G/H synthase 2 precursor polypeptide, or mimetics, activators, or inhibitors of a human prostaglandin G/H synthase 2 precursor polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, inframedullary, intrathecal, infraventricular, transdermal, subcutaneous, mtraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.

Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and fragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co, Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include^' amount, frequency, and method of administration.

Therapeutic indications and methods

Human prostaglandin G H synthase 2 precursor can be regulated to treat pain. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation). Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.

This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a human prostaglandin G H synthase 2 precursor polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

A reagent which affects prostaglandin G/H synthase 2 precursor activity can be administered to a human cell, either in vitro or in vivo, to reduce prostaglandin G/H synthase 2 precursor activity. The reagent preferably binds to an expression product of a human prostaglandin G H synthase 2 precursor gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the fransfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on- the outer surface of the liposome.

Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes. In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor- mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).

Determination of a therapeutical ly effective dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases enzymatic activity relative to the enzymatic activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for adminisfration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅o/ED₅o.

Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, fransfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, elecfroporation, "gene gun," and DEAE- or calcium phosphate-mediated fransfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding smgle- chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.

If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be infroduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of a human prostaglandin G/H synthase 2 precursor gene or the activity of a prostaglandin G/H synthase 2 precursor polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a human prostaglandin G/H synthase 2 precursor gene or the activity of a human prostaglandin G/H synthase 2 precursor polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to prostaglandin G/H synthase 2 precursor-specific mRNA, quantitative RT-PCR, immunologic detection of a human prostaglandin G/H synthase 2 precursor polypeptide, or measurement of enzymatic activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or. prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic methods

Human prostaglandin G H synthase 2 precursor also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding prostaglandin G/H synthase 2 precursor in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85, 4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-elecfrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of prostaglandin G/H synthase 2 precursor also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention. The invention thus provides a human prostaglandin G H synthase 2 precursor that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site. Human prostaglandin G/H synthase 2 precursor and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity.

EXAMPLE 1

Detection of prostaglandin g H synthase 2 precursor activity

The polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-prostaglandin g/H synthase 2 precursor polypeptide obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and prostaglandin g/H synthase 2 precursor activity is determined based on the conversion of arachidonic acid to PGE2 by radioimmunoassay. The cell extract is incubated for various times in 1-00 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 mM glutathione, 0.5 mM phenol, 100 μM hematin. The reaction is intitated with either 2 or 10 μm arachidonic acid and is terminated at 1 or 3 min by acidification with 0.1 N HC1 (final concentration). This mixture is neutralized with an equivalent amount of NaOH and analyzed for PGE2 formation using the methyl oximated PGE2 RIA kit (Amersham Corp.). It is shown that the polypeptide of SEQ ID NO: 2 has a prostaglandin g/H synthase 2 precursor activity.

EXAMPLE 2

Expression of recombinant human prostaglandin G/H synthase 2 precursor

The Pichia pastoris expression vector pPICZB (Invifrogen, San Diego, CA) is used to produce large quantities of recombinant human prostaglandin G/H synthase 2 precursor polypeptides in yeast. The prostaglandin G/H synthase 2 precursor-encoding DNA sequence is derived from SEQ ID NO: l. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human prostaglandin G/H synthase 2 precursor polypeptide is obtained. EXAMPLE 3

Identification of test compounds that bind to prostaglandin G/H synthase 2 precursor polypeptides

Purified prostaglandin G/H synthase 2 precursor polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human prostaglandin G/H synthase 2 precursor polypeptides comprise the amino acid sequence shown in SEQ ID NO:2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a human prostaglandin G/H synthase 2 precursor polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a human prostaglandin G/H synthase 2 precursor polypeptide.

EXAMPLE 4

Identification of a test compound which decreases prostaglandin G/H synthase 2 precursor gene expression

A test compound is administered to a culture of human cells fransfected with a prostaglandin G/H synthase 2 precursor expression construct and incubated at 37 °C for 10 to 45 minutes. A culture of the same type of cells that have not been fransfected is incubated for the same time without the test compound to provide a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P-labeled prostaglandin G/H synthase 2 precursor-specific probe at 65 ° C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: l. A test compound that decreases the prostaglandin G/H synthase 2 precursor-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of prostaglandin G/H synthase 2 precursor gene expression. EXAMPLE 5

Identification of a test compound which decreases prostaglandin G/H synthase 2 precursor activity

Enzymatic activity is measured using the method of U.S. Patent 5,681,842. A test compound dissolved in DMSO (3.3% v/v) is preincubated with microsomes from recombinant human prostaglandin G/H synthase 2 precursor expressed in the baculovirus/Sf9 cell system (Gierse et al, Biochem J. 305, 479, 1995), together with the cofactors phenol (2 mM) and hematin (1 μM) for 60 minutes prior to the addition of 10 μM arachidonic acid. The reaction is allowed to run for 2.5 minutes at room temperature prior to quenching with HC1 and neutralization with NaOH. PGE₂ production in the presence and absence of the test compound is determined by EIA analysis.

A test compound that decreases the enzymatic activity of the prostaglandin G/H synthase 2 precursor relative to the enzymatic activity in the absence of the test compound is identified as an inhibitor of prostaglandin G/H synthase 2 precursor activity.

EXAMPLE 6

Tissue-specific expression of prostaglandin G/H synthase 2 precursor

The qualitative expression pattern of prostaglandin G/H synthase 2 precursor in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

Quantitative expression profiling

To demonstrate that prostaglandin G/H synthase 2 precursor is involved in pain disorders, the following tissues are screened: fetal and adult brain, muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus, cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus,^" and spinal cord.

Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis" firstly described in Higuchi et al, BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies. If the amplification is performed in the presence of an internally quenched fluorescent oligonucleotide (TaqMan probe) complementary to the target sequence, the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996).

The amplification of an endogenous confrol can be performed to standardize the amount of sample RNA added to a reaction. In this kind of experiment, the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used. All "real time PCR" measurements of fluorescence are made in the ABI Prism 7700.

RNA extraction and cDNA preparation. Total RNA from the tissues listed above are used for expression quantification. RNAs labeled "from autopsy" were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.

Fifty μg of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix:

0.2 U/μl RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tπs-HCl pH 7.9; lOmM MgCl₂; 50 mM NaCl; and 1 mM DTT.

After incubation, RNA is extracted once with 1 volume of phenol:chloroform:isoamyl alcohol (24:24: 1) and once with chloroform, and precipitated with 1/10 volume of 3 M sodium acetate, pH5.2, and 2 volumes of ethanol.

Fifty μg of each RNA from the autoptic tissues are DNase treated with the DNA- free kit purchased from Ambion (Ambion, TX). After resuspension and spectrophotomefric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200ng/μL. Reverse transcription is carried out with 2.5μM of random hexamer primers. TaqMan quantitative analysis. Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-teframethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.

Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).

The assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 20X stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 μl.

Each of the following steps are carried out once: pre PCR, 2 minutes at 50° C, and 10 minutes at 95°C. The following steps are carried out 40 times: denaturation, 15 seconds at 95°C, annealing/extension, 1 minute at 60°C.

The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.

EXAMPLE 7

In vivo testing of compounds/target validation

Pain

Acute pain. Acute pain is measured on a hot plate mainly in rats. Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56°C) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking. The other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold. Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v, i.p, p.o, i.t, i.c.v, s.c, intradermal, transdermal) prior to pain testing.

Persistent pain. Persistent pain is measured with the formalin or capsaicin test, mainly in rats. A solution of 1 to 5% formalin or 10 to 100 μg capsaicin is injected into one hind paw of the experimental animal. After formalin or capsaicin application the animals show nocifensive reactions like flinching, licking and biting of the affected paw. The number of nocifensive reactions within a time frame of up to 90 minutes is a measure for intensity of pain.

Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v, i.p, p.o, i.t, i.c.v, s.c, intradermal, transdermal) prior to formalin or capsaicin administration.

Neuropathic pain. Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats. The operation is performed under anesthesia. The first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve. The second variant is the tight ligation of about the half of the diameter of the common sciatic nerve. In the next variant, a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L% spinal nerve only. The fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation.

Postoperatively, the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, 1ITC Inc. -Life Science Instruments, Woodland Hills, SA, USA; Electronic von Frey System, Somedic Sales AB, Hδrby, Sweden). Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10 °C where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity. A further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb. Chronic pain in general is assessed by registering the circadanian rhythms in activity (Surjo and Amdt, Universitat zu Kόln, Cologne, Germany), and by scoring differences in gait (foot print patterns; FOOTPRINTS program, Klapdor et al, 1997. A low cost method to analyze footprint patterns. J. Neurosci. Methods 75, 49-54).

Compounds are tested against sham operated and vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v, i.p, p.o, i.t, i.c.v, s.c, intradermal, transdermal) prior to pain testing.

Inflammatory Pain. Inflammatory pain is induced mainly in rats by injection of 0.75 mg carrageenan or complete Freund's adjuvant into one hind paw. The animals develop an edema with mechanical allodynia as well as thermal hyperalgesia. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc. -Life Science Instruments, Woodland Hills, SA, USA). Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy, Paw thermal stimulator, G. Ozaki, University of California, USA). For edema measurement two methods are being used. In the first method, the animals are sacrificed and the affected hindpaws sectioned and weighed. The second method comprises differences in paw volume by measuring water displacement in a plethysmometer (Ugo Basile, Comerio, Italy).

Compounds are tested against uninflamed as well as vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v, i.p, p.o, i.t, i.c.v, s.c, intradermal, transdermal) prior to pain testing.

Diabetic neuropathic pain. Rats treated with a single infraperitoneal injection of 50 to 80 mg/kg sfreptozotocin develop a profound hyperglycemia and mechanical allodynia within 1 to 3 weeks. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA).

Compounds are tested against diabetic and non-diabetic vehicle treated confrol groups. Substance application is performed at different time points via different application routes (i.v, i.p, p.o, i.t, i.c.v, s.c, intradermal, transdermal) prior to pain testing. REFERENCES

Jones DA, Carlton DP, Mclntyre TM, Zimmerman GA, Prescott SM. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J Biol Chem. 1993 Apr 25;268(12):9049-54.

Hla T, Neilson K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7384-8.

Kosaka T, Miyata A, Ihara H, Hara S, Sugimoto T, Takeda O, Takahashi E, Tanabe T. Characterization of the human gene (PTGS2) encoding prostaglandin-endoperoxide synthase 2. Eur J Biochem. 1994 May l;221(3):889-97.

Tazawa R, Xu XM, Wu KK, Wang LH. Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene. Biochem Biophys Res Commun. 1994 Aug 30;203(l):190-9.

Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J. 1994 Sep 15;302 ( Pt 3):723-7.

Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS, Simmons DL. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: Cloning, structure, and expression. Proc Natl Acad Sci U S A. 2002 Sep 19.

Claims

1. An isolated polynucleotide being selected from the group consisting of:

a. a polynucleotide encoding a prostaglandin G/H synthase 2 precursor polypeptide comprising an amino acid sequence selected form the group consisting of:

i. amino acid sequences which are at least about 95% identical to

the amino acid sequence shown in SEQ ID NO: 2; and

ii. the amino acid sequence shown in SEQ ID NO: 2.

b. a polynucleotide comprising the sequence of SEQ ID NO: 1 ;

c. a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a prostaglandin G H synthase 2 precursor polypeptide;

d. a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a prostaglandin G/H synthase 2 precursor polypeptide; and

e. a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a prostaglandin G/H synthase 2 precursor polypeptide.

2. An expression vector containing any polynucleotide of claim 1.

3. A host cell containing the expression vector of claim 2.

4. A substantially purified prostaglandin G/H synthase 2 precursor polypeptide encoded by a polynucleotide of claim 1.

5. A method for producing a prostaglandin G/H synthase 2 precursor polypeptide, wherein the method comprises the following steps:

a. culturing the host cell of claim 3 under conditions suitable for the expression of the prostaglandin G/H synthase 2 precursor polypeptide; and

b. recovering the prostaglandin G/H synthase 2 precursor polypeptide from the host cell culture.

6. A method for detection of a polynucleotide encoding a prostaglandin G/H synthase 2 precursor polypeptide in a biological sample comprising the following steps:

a. hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and

b. detecting said hybridization complex.

7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.

8. A method for the detection of a polynucleotide of claim 1 or a prostaglandin G/H synthase 2 precursor polypeptide of claim 4 comprising the steps of:

b. detecting the interaction

9. A diagnostic kit for conducting the method of any one of claims 6 to 8.

10. A method of screening for agents which decrease the activity of a prostaglandin G/H synthase 2 precursor, comprising the steps of:

a. contacting a test compound with any prostaglandin G/H synthase 2 precursor polypeptide encoded by any polynucleotide of claim 1 ;

b. detecting binding of the test compound to the prostaglandin G/H synthase 2 precursor polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a prostaglandin G/H synthase 2 precursor.

11. A method of screening for agents which regulate the activity of a prostaglandin G/H synthase 2 precursor, comprising the steps of:

a. contacting a test compound with a prostaglandin G/H synthase 2 precursor polypeptide encoded by any polynucleotide of claim 1 ; and

b. detecting a prostaglandin G/H synthase 2 precursor activity of the polypeptide, wherein a test compound which increases the prostaglandin G/H synthase 2 precursor activity is identified as a potential therapeutic agent for increasing the activity of the prostaglandin G/H synthase 2 precursor, and wherein a test compound which decreases the prostaglandin G/H synthase 2 precursor activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the prostaglandin G/H synthase 2 precursor.

12. A method of screening for agents which decrease the activity of a prostaglandin G/H synthase 2 precursor, comprising the step of:

contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of prostaglandin G H synthase 2 precursor.

13. A method of reducing the activity of prostaglandin G/H synthase 2 precursor, comprising the step of:

contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any prostaglandin G/H synthase 2 precursor polypeptide of claim 4, whereby the activity of prostaglandin G H synthase 2 precursor is reduced.

14. A reagent that modulates the activity of a prostaglandin G/H synthase 2 precursor polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.

15. A pharmaceutical composition, comprising:

the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.

16. Use of the expression vector of claim 2 or the reagent of claim 14 in the preparation of a medicament for modulating the activity of a prostaglandin G/H synthase 2 precursor in a disease.

17. Use of claim 16 wherein the disease is pain.