CN1675543A - Method for assaying compounds or agents for ability to decrease the activity of microsomal prostaglandin E synthase or hematopoietic prostaglandin D synthase - Google Patents

Abstract

Provided herein is a novel and useful method for evaluating the ability of compounds or agents to decrease the activity of microsomal prostaglandin E synthase or hematopoietic prostaglandin D synthase to produce their respective prostaglandin products.

Description

Method for detecting the ability of a compound or reagent to reduce the activity of microsomal prostaglandin E synthase or hematopoietic prostaglandin D synthase

field of invention

The present invention relates to a novel and useful method for testing the ability of compounds or agents to reduce the activity of prostaglandin synthase. More specifically, the present invention relates to a method of testing the ability of a compound or agent to decrease microsomal prostaglandin E synthase (mPGES) activity or hematopoietic prostaglandin D synthase (hPGDS) activity.

Background of the invention

Prostaglandins are a class of eicosanoids that play an important role in pain, fever, and inflammatory processes. They are synthesized in vivo from arachidonic acid and have a five-membered carbon ring that forms part of the arachidonic acid carbon chain. Prostaglandins are not hormones and act only locally near the site where they are synthesized.

Two prostaglandins, _PGE2 and _PGD2, play a particularly important role in fever, pain and inflammation. In particular, the level _of PGD ₂ produced by mast cells, which is a major mediator of respiratory allergic diseases, is increased under antigenic stimulation. In particular, it can cause, among other symptoms, bronchoconstriction, hyperbronchitis, nasal congestion, and infiltration of eosinophils and _TH2 cells. PGE ₂ is a pain mediator that has been shown to play a role in the induction of hyperalgesia, fever, vasodilation, and edema.

During synthesis in vivo, phospholipase A2 converts phospholipids into arachidonic acid (which exists in vivo as an ester). Subsequently, prostaglandin endoperoxide synthase converts arachidonic acid to prostaglandin G2 (PGG ₂ ). Prostaglandin endoperoxide synthase can also catalyze the reduction reaction of the peroxide group in PGG ₂ to form prostaglandin H ₂ (PGH ₂ ), where PGH ₂ is the precursor of PGE ₂ and PGD ₂ . Where _PGE2 is produced, prostaglandin E synthase (PGES) converts _PGH2 to _PGE2 in the presence of the cofactor glutathione (GSH). There are at least two PGE ₂ synthetases, cytoplasmic PGES (cPGES) and microsomal PGES (mPGES). These two PGES are widely expressed and overlapped in many tissues. mPGES can be induced by inflammatory stimuli such as lipopolysaccharide (LPS), IL-1 and TNF-α, whereas the expression of cPGES is constitutive. In addition, mPGES has been shown to be linked to COX-2 activity [Murakami et al., J. Biol. Chem. 275:32783 (2000)].

_PGD2 production is the result of the conversion of _PGH2 to _PGD2 by prostaglandin D synthase (PGDS). Two types of PGD ₂ synthases are known. The first is lipocalin-type PGDS (L-PGDS), which is mainly found in the central nervous system; the second is hematopoietic PGDS (hPGDS), which is mainly found in peripheral tissues. L-PGDS is not dependent on glutathione (GSH), while hPGDS is dependent on GSH and has GST activity. Furthermore, there is little structural homology between L-PGDS and hPGDS.

Since _PGD2 and _PGE2 play important roles in fever, pain, and inflammatory processes, many efforts have been made to establish various assays for compounds that may reduce or even inhibit their production. In particular, to determine the ability of a compound or reagent to reduce or inhibit the activity of prostaglandin synthase, various techniques such as HPLC, ELISA or RIA have been used to quantify _PGD2 and _PGE2 production. However, these techniques have certain inherent limitations. For example, they require multiple washing steps, purification steps and/or use of radioactive materials. Also, these methods are time-consuming, with a daily throughput of only tens (HPLC) to hundreds (ELISA and RIA) of data points. Therefore, they cannot be used for high-throughput screening.

Fluorescence polarization is a technique used to study molecular interactions. The principle of this technique depends on the size of the molecule being identified. In particular, when a fluorescent molecule is irradiated with plane polarized light, the electrons in the ground state of the measured molecule transition to an excited state. After about 4-5 nanoseconds, these excited state electrons transition back to the ground state. It is during this decay that the analyte emits a fluorescent signal. During fluorescence polarization, this fluorescence emission can only be detected in the same plane if the analyte molecule remains stationary throughout the excited state. If the molecule moves or rotates during the excited state, the emitted fluorescence will be in a different plane of polarization than the one that excited the electron. As a result, no fluorescence emission will be detected. It is generally accepted that the smaller the molecule, the greater its mobility and rotation. Therefore, the signal produced by a small molecule will be much smaller than that of a large molecule, which will remain relatively stationary during the excited state. Fluorescence polarization takes advantage of this property of molecules. In particular, during the analysis of a ligand by fluorescence polarization, the ligand, a tracer (ie, a ligand labeled with a fluorescent marker), and a receptor bound to the ligand are placed in solution. The ligand and tracer then compete with each other for binding to the receptor. The solution is then irradiated with plane polarized light, followed by signal detection. If there is not much ligand in solution, most of the receptors present will bind the tracer. Since the receptor is a macromolecule (relative to the ligand), a signal from the fluorescence of the marker can be obtained, and a strong fluorescence polarization signal can be obtained. Conversely, if a large number of ligands are present, most of the receptors will bind to that ligand. As a result, the fluorescence polarization signal, if any, produced by the tracer alone will be significantly smaller than that previously produced by the receptor-bound tracer. It is the difference between these signals that allows one of ordinary skill in the art to determine the presence and concentration of the ligand. Fluorescence polarization is measured in units of millifluorescence polarization, or mP.

Therefore, fluorescence polarization is more convenient and effective than detection methods such as ELISA, HPLC and RIA. Moreover, it can be easily used for high-throughput screening of a large number of compounds or reagents in a short period of time.

What is needed, therefore, is a fluorescence polarization method for evaluating the ability of a compound or reagent to reduce or even inhibit the activity of prostaglandin synthase, and in particular to evaluate whether a certain reagent or compound is capable of reducing or inhibiting the ability of mPGES to produce PGE ₂ , and evaluating whether a certain reagent or compound can reduce or inhibit the ability of hPGDS to produce PGD ₂ .

Also needed is a high throughput system for evaluating the ability of compounds or agents to reduce or inhibit the activity of prostaglandin synthases, particularly mPGES or hPGDS. Compounds that reduce or even inhibit the activity of hematopoietic prostaglandin _D2 synthase (hPGDS) or inducible microsomal prostaglandin _E2 synthase (mPGES) may be useful in the treatment of inflammatory and allergic conditions such as arthritis, asthma, and rhinitis, etc. Here are just a few examples. Additionally, pain and/or fever may also be treatable with such compounds or agents.

Citation of any reference herein shall not be construed as an admission that such reference is "prior art" to the present application.

Summary of the invention

In accordance with the present invention, we provide a new efficient method for evaluating the ability of compounds or agents to reduce or even inhibit the activity of mPGES or hPGDS to produce the respective prostaglandin products, which method does not use radioactive isotopes and does not require extensive washing steps, Moreover, it can be performed in the form of extracorporeal manipulation and then infused back into the body, in the form of in vitro manipulation, or in a cell-based manner, or in a separate manner. Furthermore, the methods of the present invention can be easily implemented in a high-throughput manner.

Broadly, the invention can be extended to a method for determining whether a compound or agent is capable of reducing the activity of a prostaglandin synthase to react with its substrate to form a prostaglandin product. Wherein the prostaglandin synthase is selected from microsomal prostaglandin E synthase (mPGES) and hematopoietic prostaglandin D synthase (hPGDS). This method of the present invention comprises the steps of: mixing prostaglandin synthase with its substrate, cofactor and test compound or reagent so that the enzymatic reaction can take place; then incubating the mixture with a stop solution containing A reagent that prevents the spontaneous conversion of unreacted substrate to a prostaglandin product; the mixture is then incubated with a detection reagent that contains the prostaglandin product labeled with a fluorescent marker (i.e., a tracer) , and antibodies produced using prostaglandin products as immunogens; subsequently, this mixture and a control mixture treated in the same manner but without the test compound or reagent were irradiated with plane polarized light, the wavelength of which is the wavelength of the fluorescent marker The wavelength of the emitted fluorescence. Determine and compare the fluorescence polarization values of the test mixture and the control mixture. If the polarization value of the test mixture is greater than that of the control mixture, it indicates that the test compound or reagent can reduce the activity of prostaglandin synthase. Accordingly, such compounds or agents can readily be used to treat subjects suffering from inflammation, allergy, pain and fever, or any combination of these conditions, just to name a few.

Furthermore, the invention extends to a method as described above, wherein the prostaglandin synthase is inducible microsomal prostaglandin E synthase (mPGES), the substrate is prostaglandin _H2 ( _PGH2 ), and the cofactor is Glutathione (GSH), the prostaglandin product is prostaglandin E ₂ (PGE ₂ ). The mPGES used in the method of the present invention can be derived from cattle, sheep, rodents, horses, dogs, humans, cats and the like. In a specific embodiment, mPGES is derived from human, and its amino acid sequence is shown in FIG. 9B and SEQ ID NO:2. Furthermore, the mPGES used in the methods of the invention need not be in purified form.

The invention further extends to methods, as described herein, for determining whether a compound or agent is capable of reducing the activity of prostaglandin synthase to react with its substrate to form a prostaglandin product. The prostaglandin synthase is hematopoietic prostaglandin D synthase (hPGDS), the substrate is PGH ₂ , the cofactor is GSH, and the prostaglandin product is prostaglandin D ₂ (PGD ₂ ). As with mPGES, hPGDS for use in the methods of the invention can come from a variety of sources. In a specific embodiment, hPGDS is human hPGDS, the amino acid sequence of which is shown in Figure 10B and SEQ ID NO:4. Also, hPGDS need not be in purified form.

As noted above, in the method of the present invention, the stop solution contains an agent that prevents the spontaneous conversion of unreacted substrate to prostaglandin product. In particular, the substrate PGH ₂ of the two prostaglandin synthases described above contains a peroxide group. While under no obligation to explain their mechanism, and certainly do not wish to be bound by any explanation, it is believed that mPGES and hPGDS catalyze the breaking of the oxygen bond of the peroxide group of PGH ₂ and convert PGH ₂ to PGE ₂ and PGD ₂ , respectively. However, PGH ₂ is also converted spontaneously to PGE ₂ or PGD ₂ . This spontaneous conversion can interfere with and alter the results of assays for the ability of a compound or reagent to reduce mPGES or hPGDS activity. Therefore, in the method of the present invention, the mixture to be tested is incubated with a stop solution containing a reagent that prevents the spontaneous conversion of _PGH2 to _PGD2 or _PGE2 . A specific example of such an agent is _FeCl2 at a concentration of about 20 mM. However, one of ordinary skill in the art will readily be familiar with other reagents that prevent this simultaneous transformation, and such reagents are also encompassed by the methods of the present invention. Also, the duration of this incubation can vary, but must be long enough to prevent conversion of any remaining unreacted _PGH2 to prostaglandin products.

Furthermore, the invention extends to methods for determining whether a compound or agent is capable of reducing the activity of a prostaglandin synthase, particularly mPGES or hPGDS, from reacting with its substrate to form a prostaglandin product. Wherein the mixture to be tested is incubated with the stop solution, and then incubated with the detection reagent, the detection reagent contains a prostaglandin product labeled with a fluorescent marker and an antibody using the prostaglandin product as an immunogen. A wide variety of fluorescent labels known to those of ordinary skill in the art can be used in the methods of the present invention. Examples of these fluorescent labels include fluorescein, phycoerythrin (PE), Texas red (TR), rhodamine, free lanthanide salts, chelated lanthanide salts, CyDye (product of Amersham Biotech, Inc. ), BODIPY (product of Molecular Probes), and ALEXA (product of Molecular Probes), etc., are just a few examples here. In a specific embodiment, the fluorescent marker is Texas Red. In addition, the fluorescent label can be directly bound to the prostaglandin product, or alternatively first bound to a linker molecule through which it binds to the prostaglandin product. Specific linker molecules useful in the present invention include, but are of course not limited to, aminobutyric acid, aminocaproic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, Fmoc-aminocaproic acid, one or more β-alanines, Isothiocyanate groups, succinimidyl esters, halodiethylsulfone or carbodiimides, etc., are just a few examples. In a specific embodiment of the present invention, the prostaglandin product is first bound to a carbodiimide linker, and then bound to a fluorescent marker such as Texas Red through the linker.

Furthermore, the invention extends to methods for determining whether a compound or agent is capable of reducing or inhibiting the reaction of inducible microsomal prostaglandin E synthase (mPGES) with its substrate prostaglandin _H2 ( _PGH2 ) to produce Prostaglandin E ₂ (PGE ₂ ), the method includes the following steps:

(a) mPGES is mixed with PGH ₂ , glutathione (GSH), and the compound or reagent to be tested for at least 30 seconds;

(b) The mixture of step (a) is incubated with the stop solution containing FeCl ₂ ;

(c) incubating the mixture of step (b) with a detection reagent containing PGE ₂ labeled with Texas Red and an antibody using PGE ₂ as an immunogen;

(d) irradiating the mixture of step (c) and the control mixture with a plane polarized light having a wavelength of 580 ± 20nm, and measuring the fluorescence polarization values of the mixture of step (c) and the control mixture;

(e) comparing the measurement results of step (d).

If the measured fluorescence polarization of the mixture containing the test compound or agent is greater than the measured fluorescence polarization of the control mixture, it indicates that the compound or agent reduces the activity of mPGES. In a specific embodiment, mPGES is human mPGES, the amino acid sequence of which is shown in Figure 9B and SEQ ID NO: 2, and PGE ₂ and Texas Red are linked by a carbodiimide linker. The excitation wavelength of Texas Red is 580nm. However, this wavelength may be in the range of 580nm±20nm. Similarly, the fluorescence emission wavelength of Texas Red is generally considered to be 620 nm. However, this wavelength can vary within the range of 620nm±20nm. Such changes in excitation wavelength and emission wavelength are included in the present invention. Also, as stated previously, mPGES need not be in a purified form.

Furthermore, the invention extends to methods for determining whether a compound or agent is capable of reducing the reaction of hematopoietic prostaglandin D synthase (hPGDS) with its substrate prostaglandin H ₂ (PGH ₂ ) to form prostaglandin D ₂ (PGD ₂ ), this method includes the following steps:

(a) mixing hPGDS with _PGH2 , GSH and the compound or reagent to be tested for at least 30 seconds;

(b) incubating the mixture of step (a) with a stop solution containing _FeCl ;

(c) incubating the mixture of step (b) with a detection reagent comprising _PGD2 labeled with Texas Red and an antibody using _PGD2 as an immunogen;

(d) irradiating the mixture of step (c) and the control mixture with a plane polarized light having a wavelength of 580 ± 20nm, and measuring the fluorescence polarization values of the mixture of step (c) and the control mixture;

(e) comparing the measurement results of step (d).

If the measured fluorescence polarization of the mixture containing the test compound or agent is greater than the measured fluorescence polarization of the control mixture, it indicates that the compound or agent reduces the activity of hPGDS. In a specific embodiment, hPGDS is human hPGDS, the amino acid sequence of which is shown in Figure 10B and SEQ ID NO: 4, and Texas Red and PGD ₂ are chemically linked through a carbodiimide linker.

Furthermore, the present invention extends to methods for determining whether a compound or reagent is capable of reducing the activity of prostaglandin synthases, such as mPGES or hPGDS, to produce prostaglandin products, and this method is performed in a high-throughput manner .

Accordingly, one aspect of the present invention is to provide a method for evaluating the ability of a compound or agent to reduce or even inhibit the activity of a prostaglandin synthase, such as mPGES or hPGDS. Thus, the methods of the present invention enable one of ordinary skill in the art to identify compounds or agents useful for treating pain, inflammation, fever, or some combination of these conditions in a subject.

Another aspect of the invention is to provide a method for evaluating the ability of a compound or reagent to reduce or inhibit the activity of mPGES or hPGDS without the need for washing steps or the use of radioactive isotopes.

Still another aspect of the present invention is to provide a method for evaluating the ability of compounds or reagents to reduce or inhibit the activity of prostaglandin synthase such as mPGES or hPGDS, and this method can be operated in vitro and then infused back into the body. The manner in which the operation is performed, or performed in a cell-based manner, can also be performed in a discrete manner.

Yet another aspect of the present invention is to provide a method for evaluating the ability of a compound or reagent to reduce or inhibit the activity of prostaglandin synthase, which method can be performed in a high-throughput manner.

These and other aspects of the invention will be better understood with reference to the following drawings and detailed description of the invention.

Brief description of the drawings

Figure 1 is a schematic diagram of the competition between the enzymatic conversion reaction of the substrate _PGH2 to _PGE2 and the spontaneous conversion reaction of _PGH2 to the prostaglandin product _PGD2 or _PGE2 , where the enzyme used is mPGES, preventing this spontaneous conversion The reagent for the reaction is FeCl ₂ .

Figure 2 is a schematic diagram of a method of the present invention wherein the prostaglandin synthase is mPGES and the prostaglandin product is _PGE2 .

Figure 3 is a schematic diagram of the competition between the enzymatic conversion reaction of the substrate _PGH2 to _PGD2 and the spontaneous conversion reaction of _PGH2 to the prostaglandin product _PGD2 or _PGE2 , where the enzyme used is hPGDS, preventing this spontaneous conversion The reagent for the reaction is FeCl ₂ .

Figure 4 is a schematic diagram of a method of the present invention wherein the prostaglandin synthase is hPGDS and the prostaglandin product is _PGD2 .

Figure 5 shows the chemical structure of MK-886. MK-886, a known mPGES inhibitor, was used to demonstrate that the methods of the present invention allow us to determine whether a compound or agent is capable of reducing or inhibiting the activity of prostaglandin synthase.

Fig. 6 is a schematic diagram of the concentration-response curve of the method of the present invention drawn using prostaglandin synthase mPGES and the known inhibitor MK-886. IC50 = 27.5 μM. These results demonstrate that the methods of the present invention allow one of ordinary skill in the art to determine whether a compound or agent is capable of reducing prostaglandin synthase activity. The prostaglandin synthase in this example is mPGES.

Figure 7 shows the chemical structure of HQL 79.

Figure 8 is a bar graph showing that the decrease in hPGDS activity caused by HQL 79 can be detected by the method of the present invention.

Figures 9A and 9B show the nucleotide and amino acid sequences (SEQ ID NOs: 1 and 2, respectively) of the human mPGES used in Example 1 below.

Figures 10A and 10B show the nucleotide and amino acid sequences of human H-PGDS (SEQ ID NO: 3 and 4, respectively).

Detailed description of the invention

The present invention is based on the unexpected and surprising discovery that fluorescence polarization can be used to identify compounds or agents that reduce the activity of prostaglandin synthases such as mPGES or hPGDS to produce prostaglandins such as _PGD2 or _PGE2 . Broadly, therefore, the invention extends to a method for determining whether a compound or reagent is capable of reducing the activity of a prostaglandin synthase selected from the group consisting of mPGES from the reaction of a prostaglandin synthase with its substrate to form a prostaglandin product or hPGDS, this method includes the following steps:

(a) mixing prostaglandin synthase with its substrate, cofactor and test compound or reagent;

(b) incubating the mixture of step (a) with a stop solution containing a reagent that prevents the spontaneous conversion of the substrate to the prostaglandin product;

(c) incubating the mixture of step (b) with a detection reagent containing a prostaglandin product labeled with a fluorescent marker and an antibody using the prostaglandin product as an immunogen;

(d) irradiating the mixture of step (c) and the control mixture with linearly polarized light, the wavelength of which is the wavelength of fluorescence emitted by the fluorescent marker, and measuring the fluorescence polarization values of the mixture of step (c) and the control mixture; and

(e) comparing the assay results of step (d),

In the above step, if the fluorescence polarization measurement value of the mixture in step (d) is greater than the fluorescence polarization measurement value of the control mixture, it indicates that the compound or reagent can reduce the activity of prostaglandin synthase.

Definitions of various terms and phrases used throughout this specification and appended claims are as follows:

As used herein, the term "compound" or "agent" refers to any chemical moiety, now known or later discovered. Examples of compounds or agents useful in the present invention include organic compounds (eg, man-made or naturally occurring), peptides (man-made or naturally occurring), carbohydrates, nucleic acid molecules, and the like.

The term "enzyme" as used herein refers to a biomolecule such as a protein or RNA that catalyzes a specific chemical reaction. Enzymes do not affect the balance of the catalytic reaction, but increase the reaction rate by reducing the activation energy.

The term "prostaglandin synthase" as used herein refers to an enzyme that catalyzes the conversion of prostaglandin _H2 ( _PGH2 ) into prostaglandin products. Specific examples of prostaglandin synthases useful in the present invention include inducible microsomal prostaglandin E synthase (mPGES), which converts prostaglandin _H into Prostaglandin E ₂ (PGE ₂ ). Another example is hematopoietic prostaglandin D synthase (hPGDS), which converts prostaglandin _H2 to prostaglandin _D2 ( _PGD2 ) in the presence of the cofactor GSH.

The term "substrate" as used herein refers to a compound that is subjected to the action of an enzyme to produce a reaction product. An example of a substrate useful in the present invention is _PGH2 .

The term "cofactor" as used herein refers to an organic molecule, inorganic molecule, peptide or protein required for enzyme activity. In a specific embodiment of the invention, the prostaglandin synthase is mPGES or hPGDS and the cofactor is glutathione (GSH).

As used herein, the term "prostaglandin product" refers to the reaction product resulting from the action of prostaglandin synthase on a substrate of prostaglandin synthase. Thus, for mPGES, the prostaglandin product is _PGE2 ; for hPGDS, the prostaglandin product is _PGD2 .

The term "fluorescent marker" as used herein refers to a substance that fluoresces when irradiated with light of a specific wavelength, and which can be directly bound to a compound of interest or first bound to a linker molecule through which it binds to the target compound. Examples of fluorescent labels for use in the methods of the invention include, but are of course not limited to, fluorescein, phycoerythrin (PE), Texas Red (TR), rhodamine, free lanthanide salts, chelated lanthanide SALT, BODIPY, ALEXA, CyDye, etc. A particular fluorescent marker for use in the methods of the present invention is Texas Red.

As used herein, the terms "linker" and "linker molecule" are used interchangeably and refer to a chemical moiety that binds a fluorescent label and a prostaglandin product. Specific examples of linkers useful in the present invention include aminobutyric acid, aminocaproic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, Fmoc-aminocaproic acid, one or more β-alanines, isothiocyanates groups, succinimidyl esters, halodiethylsulfone or carbodiimides, etc., are just a few examples. A specific example of a linker applicable to the present invention is a carbodiimide group.

The term "control mixture" as used herein refers to a mixture containing the same amount of the same reagent, compound, cell, etc. Treated in the same manner, except that the control mixture does not contain the test compound or reagent.

Antibody

As explained above, the method of the present invention uses antibodies having as immunogens products of prostaglandins such as _PGD2 or _PGE2 . Such antibodies may be monoclonal, polyclonal, or even chimeric. A number of methods known in the art can be used to prepare polyclonal antibodies to _PGE2 or _PGD2 . To prepare antibodies, various host animals including, but not limited to, rabbits, mice, rats, sheep, goats, etc. can be immunized by injection of prostaglandin products. In a specific embodiment, _PGE2 or _PGD2 can be linked to an immunogenic carrier, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants can be used to enhance the immune response, depending on the species of the host, available adjuvants include but not limited to Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin , complex polyols (Pluronicpolyols), polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, bacille Calmette-Guerin (BCG) or Corynebacterium parvum.

For the production of monoclonal antibodies directed against prostaglandin products, any technique for producing antibody molecules from cultured subcultured cell lines can be used. These technologies include, but are not limited to, hybridoma cell technology originally developed by Kohler and Milstein [Nature 256:495-497 (1975)], trioma technology, human B cell hybridoma cell technology [Kozbor et al., Immunology Today 4: 72 (1983 ); Cote et al., Proc.Natl.Acad.Sci.U.S.A.80:2026-2030 (1983)], and EBV-hybridoma technology for preparing human monoclonal antibodies [Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. In addition, monoclonal antibodies can also be produced in germ-free animals using the techniques described in patent PCT/US90/02545. Techniques developed for the preparation of "chimeric antibodies" can also be utilized [Morrison et al., J.Bacteriol. (1985)], which splices the prostaglandin product-specific mouse antibody molecule gene into the human antibody molecule gene with appropriate biological activity.

condition

As explained above, the method of the present invention can be carried out in an in vitro operation mode after in vitro operation and then infused into the body; or in an isolated manner, wherein all reagents, enzymes, substrates, etc. are first separated and stored in In buffers such as TRIS, TRIS-HCl, HEPEs, or phosphate; it can also be performed in a cell-based manner. Furthermore, in the method of the present invention, the prostaglandin synthase need not be purified.

In cell-based assays, the methods of the invention are used to determine whether a test compound or reagent is capable of preventing or reducing the secretion of prostaglandin production by cells, while in in vitro methods, cells may be lysed prior to use of the methods of the invention such that The compound or reagent to be tested can be in the intracellular medium.

Search compound libraries for candidate compounds or assays that reduce or inhibit prostaglandin synthase activity agent

Conventionally, the generation of new chemical entities with useful properties goes through the following steps: identification of compounds with certain desired properties or activities (called lead compounds), preparation of variants of the lead compounds, and evaluation of these compounds Variant properties and activities. However, the current trend is to shorten the time required for all stages of drug development. High-throughput screening (HTS) methods have replaced traditional lead-identification methods due to their ability to rapidly and efficiently perform large-scale assays.

In a specific embodiment, high throughput screening methods involve providing a pool of compounds containing a large number of potential therapeutic compounds (candidate compounds). This "historic compound population" is then screened using the methods of the invention to identify those compounds (specific compound classes or subclasses) within the pool that exhibit the desired characteristic activity. Compounds so identified can be used as traditional "lead compounds" or as potential or actual therapeutic agents themselves.

Combinatorial Compound Library

Combinatorial compound libraries are a preferred method to help generate new leads. A combinatorial compound library is a collection of various compounds synthesized chemically or biologically by combining many chemical "building blocks" such as reagents. For example, a linear combinatorial compound library, such as a polypeptide library, is composed of a group of chemical structural units called amino acids combined in various possible ways according to a predetermined compound length (ie, the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized by combinatorial mixing of such chemical building blocks. For example, one reviewer noted that systematic combinatorial mixing of 100 interchangeable chemical building blocks could theoretically synthesize 100 million tetrameric compounds or 10 billion pentameric compounds (Gallop et al., Applied Combinatorial Techniques Conducting Drug Discovery 2. Combinatorial organic synthesis, library screening strategies and future directions, J. Med. Chem. (1994) 37(9): 1233-1251).

The preparation of combinatorial compound libraries is well known to those of ordinary skill in the art. Such combinatorial compound libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. -88). The synthesis of peptides is by no means the only method foreseen and contemplated for use in the present invention. Other chemistries that generate chemical multiplex libraries can also be used. Such chemicals include, but are not limited to: peptoids (PCT publication WO 91/19735, December 26, 1991), encoded peptides (PCT publication WO 93/20242, October 14, 1993), Japan), random bio-oligomers (PCT Publication WO 92/00091, January 9, 1992), benzodiazepines (U.S. Patent No. 5,288,514), diversomers such as hydantoins (Hobbs et al., (1993) Proc.Nat.Acad.Sci.USA 90:69096913), vinylogous polypeptides (vinylogouspolypeptides) (Hagihara et al. (1992) J.Amer.Chem. Soc.114:6568), non-peptidic peptidomimetics with β-D-glucose backbone (Hirschmann et al., (1992) J.Amer.Chem.Soc.114:92179218), organic synthetics similar to small compound libraries (Chen (1994) J.Amer.Chem.Soc.116:2661), oligomeric carbamates (oligocarbamates) (Cho et al., (1993) Science 261:1303), and/or phosphonic acid peptide esters (Campbell et al., (1994) J. Org. Chem. 59:658). Such combinatorial compound libraries include synthetic libraries (see Gordon et al., (1994) J. Med. Chem. 37:1385), nucleic acid libraries, peptide nucleic acid libraries (see U.S. Patent No. 5,539,083, etc.), antibody libraries (see Vaughn et al. (1996) Nature Biotechnology, 14(3):309-314) and PCT/US96/10287), carbohydrate libraries (see Liang et al. For benzodiazepines see Baum (1993) C&EN, Jan. 18, p. 33, for isoprenes see U.S. Patent 5,569,588, for thiazolidinones and metathiazanones ) can refer to US Patent No. 5,549,974, regarding pyrrolidines, referring to US Patents 5,525,735 and 5,519,134, regarding morpholino compounds, referring to US Patent 5,506,337, regarding benzodiazepines, referring to US Patent No. 5,288,514, etc.).

Equipment for preparing combinatorial libraries is commercially available (see e.g. 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford , MA).

A series of well-known robotic systems have been developed for liquid phase chemicals. These systems include automated workstations, such as the automated synthesis instrument developed by Takeda Chemical Industries, LTD. (Osaka, Japan), and many robotic systems utilizing robotic hands (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto , Calif.), these automated workstations mimic the manual synthesis performed by chemists. Any of the devices described above are suitable for use with the present invention. Modifications can be made to these devices in order for them to function as discussed herein. Rather, the nature and methods of making such modifications, if any, will be apparent to those skilled in the relevant art. In addition, many combinatorial libraries are themselves commercialized (see ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd., Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

High Throughput Analysis of Compound Libraries

Naturally, the method of the present invention employing fluorescence polarization technology can be easily adapted for high-throughput screening. High-throughput screening systems are commercially available (see, eg, Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Fullerton, CA; Precision Systems Inc., Natick, MA, etc.). These systems generally automate the entire procedure, including pipetting of samples and reagents, dispensing of liquids, timed incubations, final reading of microplates in detectors appropriate for the analysis, and the like. These flexibly assembleable systems enable high-throughput analysis, rapid start-up, high operational flexibility, and a high degree of specialization. Manufacturers of such systems provide detailed protocols for a wide variety of high-throughput analyses. For example, Zymark Corporation provides technical communications for screening systems for detection of regulation of gene transcription, ligand binding, and more.

A better understanding of the invention will be obtained by reference to the following non-limiting examples which serve as illustrative examples of the invention. The following examples are provided to more fully illustrate the preferred embodiments of the invention. However, they should in no way be construed as limiting the broad scope of the invention.

Example I

Fluorescence Polarization Assay of Inducible Microsomal Prostaglandin E Synthase (mPGES)

Prostaglandin E ₂ (PGE ₂ ) is a major mediator involved in inflammation and pain. Microsomal prostaglandin E synthase (mPGES) catalyzes the conversion of _PGH2 to _PGE2 in the presence of glutathione. It induces the expression of mPGES in many inflammatory diseases. Accordingly, compounds that reduce or inhibit the activity of mPGES would be useful agents for the treatment of inflammation, pain, fever, or combinations of these conditions, to name only a few diseases or disorders.

An assay has been developed to measure the conversion of _PGH2 to _PGE2 by inducible microsomal _PGE2 synthase. The test method is designed according to the principle of fluorescence polarization. The enzyme is incubated with _PGH2 , glutathione, and the compound or reagent being evaluated. After a short incubation (at least 30 seconds), stop solution containing FeCl ₂ and citric acid is added to quench any remaining PGH ₂ that would otherwise spontaneously convert to PGD ₂ or PGE ₂ and thus interfere with the conversion of PGH ₂ to PGE ₂ Quantitative analysis of enzymatic conversion (Figure 1). Then, a detection solution containing a fluorescently labeled (Texas Red) tracer (PGE ₂ ) and an anti-PGE ₂ antibody was added to generate a specific signal inversely proportional to the production of PGE ₂ ( FIG. 2 ). The PGE ₂ generated from this enzymatic reaction will specifically compete with the antibody and release the fluorescently labeled tracer. Inhibition of PGE ₂ synthase activity will result in an increase in FP values.

Material

Glutathione (GSH):

Available from Sigma (Catalog #G-6529).

PGH ₂

Available from Caymen Chemicals, Inc. (Catalog #17020).

PGE ₂ monoclonal antibody

Available from Assay Designs, Inc. (Catalog #915-057)

_PGE2 :

Available from Caymen Chemicals, Inc. (Catalog #14010)

Expression of human mPGES enzyme

The human mPGES enzyme is expressed by the bacterial expression system and steps described in the following documents: [Jakobsson, P. et al. Potential new drug target, Proc.Natl.Acad.Sci.USA96:7220-7225 (June, 1999)). Therefore, the mPGES used in this example was not in purified form, but was contained within a membrane fraction taken from mPGES-expressing bacteria. The DNA sequence encoding human mPGES used in this example is shown in Figure 9A and SEQ ID NO: 1. The amino acid sequence of human mPGES used in this example is shown in Figure 9B and SEQ ID NO:2.

Fluorescently labeled prostaglandin product PGE ₂

As noted above, a number of fluorescent labels are useful in the methods of the invention. In a specific embodiment, the Texas Red linkage of the fluorescent marker is linked to prostaglandin E ₂ (PGE ₂ ) via a linker molecule. In this particular embodiment, _PGE2 labeled with Texas Red was synthesized by Combinix (SanMateo, California). The mechanism for the production of this molecule is as follows:

PGE ₂ tracer synthesis

In this synthesis, Texas red cadaverine (Molecular Probes) was added to a solution of prostaglandin _E2 (Cayman Chemicals) in anhydrous dichloromethane. Dicyclohexylcarbodiimide (Sigma Aldrich) was added and the reaction was stirred in the dark under a nitrogen atmosphere for 24 hours. Purification was performed by reverse phase HPLC chromatography using a water/acetonitrile gradient with 0.05% TFA as modifier. The linker molecule used in this synthesis can vary.

method

First, the enzyme-containing membrane fraction was diluted with a reaction buffer containing K ₂ HPO ₄ and KH ₂ PO ₄ to prepare a phosphate-buffered enzyme solution. The compound or reagent to be tested is then placed in the enzyme solution. Optionally, this solution can also be further incubated. In this particular embodiment, the solution is incubated for about 30 minutes. The acetone solution of the substrate PGH ₂ and the cofactor GSH were then placed in another vessel at 4°C. The enzyme solution is then added to the vessel containing PGH ₂ to start the reaction. Incubate this mixture for about 30 seconds. Then, a stop solution containing _FeCl2 at a concentration of 20 mM was added to the mixture to prevent spontaneous conversion of any remaining _PGH2 to _PGE2 . Then, a detection solution containing anti-PGE ₂ antibody and PGE ₂ -Texas Red tracer was added to the mixture, and the whole mixture was incubated. In this particular embodiment, the incubation period is about 120 minutes. However, one skilled in the art can vary the length of any of the incubation periods described in this example and still obtain useful results. The control mixture is identical to the mixture, except that it does not contain the compound or reagent, and is treated in the same way, that is, it contains the same reagent, is incubated for the same period of time, etc. Then, the whole mixture and the control mixture were irradiated with plane polarized light at a wavelength of 580 nm, and the fluorescence polarization values of the mixture and the control mixture were measured with fluorescence filters whose excitation wavelength and emission wavelength were set to 580 nm and 620 nm, respectively. Measurements were performed with the measuring instrument in FP mode. These two measurements are then compared to determine whether the fluorescence polarization measurement of the mixture containing the compound or reagent is greater than the fluorescence measurement of the control solution. If the measured value of the mixture is greater than the measured value of the control mixture, it is indicated that the compound or agent reduces the activity of mPGES.

result

The assay described above was validated using a known mPGES inhibitor. This inhibitor, MK-886, is commercially available from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, Catalog #EI-266) under CAS #118414-82-7. Its structure is shown in Figure 5. The results of this experiment are shown in Figure 6. These results show the concentration response curve of this mPGES assay and clearly demonstrate that this method can be used to test the ability of a compound or reagent to decrease, or even inhibit, mPGES activity.

Example II

Fluorescence polarization analysis of hematopoietic prostaglandin D synthase (hPGDS)

Antigenic stimulation will increase PGD ₂ production in airway allergic diseases. PGD _{2 ,} produced as a result of the conversion of PGH ₂ to PGD ₂ by hematopoietic prostaglandin D synthase (hPGDS), binds to both the D-type prostaglandin receptor (DP) and the chemokine receptor (CRTH2) of Th2 cells, and Increased bronchoconstriction, vasodilation and dilation of the nasal mucosa. The resulting bronchial hyperactivity, nasal obstruction, and infiltration of eosinophils and Th2 cells lead to allergic reactions. Therefore, compounds or agents that reduce or inhibit the activity of hPGDS can readily be used as therapeutic agents.

Fluorescence polarization (FP) assays (Figures 3 and 4), which measure hPGDS activity, were also investigated. The assay is designed based on the principle of fluorescence polarization. hPGDS were incubated with _PGH2 , glutathione, and the compound or reagent being evaluated. After a short incubation period (about 30 seconds), stop solution containing FeCl ₂ (20 mM) is added to quench any remaining PGH ₂ , which would otherwise spontaneously convert to a mixture of PGD ₂ and PGE ₂ and thus interfere with PGH Quantitative analysis of the enzymatic conversion of ₂ to PGD ₂ (Figure 3). Then, a detection solution containing a fluorescently labeled (Texas Red) tracer (PGD ₂ ) and an anti-PGD ₂ antibody was added to generate a specific signal inversely proportional to the generation of PGD ₂ ( FIG. 4 ). PGD ₂ generated from this enzymatic reaction will specifically compete with the antibody and release the fluorescently labeled tracer. For the above reasons, a reduction or inhibition of PGD ₂ synthase activity will result in an increase in fluorescence polarization (FP) values.

Material

Expression of hPGDS

Human hematopoietic PGD ₂ synthase is expressed by the bacterial expression system and steps described in the following documents: Kanaoka et al. (Structure and Chromosomal Localization of Human and Mouse Genes for Hematopoietic Prostaglandin D Synthase. Eur. J. Biochem. 3315-3322 (2000)). The nucleotide sequence encoding the enzyme is shown in Figure 10A and SEQ ID NO:3. The coding nucleotide sequence was amplified and inserted into pT7-7 vector, which was then used to transform cells of E. coli FL21(DE3) strain. Thio-β-D-galactoside was then added to the transformed cells at a final concentration of 0.6 mM to induce the production of human hPGDS enzyme. Human hPGDS was purified by GSH-Sepharose (Sepharose) 4B column chromatography. The amino acid sequence of human hPGDS used in this example is shown in Figure 10B and SEQ ID NO:4.

GSH:

Available from Sigma (Catalog #G-6529).

_PGH2 :

Available from Caymen Chemicals, Inc. (Ann Arbor, Michigan) (Catalog #17020).

Anti-PGD ₂ antibody:

Monoclonal anti-PGD ₂ antibody was purchased from Institute Pasteur (cat. no. 0465328).

PGD ₂ :

Available from Caymen Chemicals, Inc. (Ann Arbor, Michigan) (Catalog #12010).

Fluorescently labeled prostaglandin product PGD ₂

As in Example 1 above, Texas Red was used as a fluorescent marker. Prostaglandin D ₂ (PGD ₂ ) labeled with Texas Red was produced by Combinix (San Mateo, California). The mechanism for generating this molecule is as follows:

PGD ₂ tracer synthesis

In this synthesis, Texas red cadaverine (Molecular Probes) was added to a solution of prostaglandin _D2 (Cayman Chemicals) in anhydrous dichloromethane. Dicyclohexylcarbodiimide (Sigma Aldrich) was added and the reaction was stirred in the dark under a nitrogen atmosphere for 24 hours. Purification was performed by reverse phase HPLC chromatography using a water/acetonitrile gradient with 0.05% TFA as modifier. Of course, the linker molecule used may vary.

method

The procedure was the same as that used in Example I, except that the enzyme used was hPGDS and the prostaglandin product was _PGD2 . Therefore, as _described above, the enzyme and GSH were diluted with a reaction buffer containing _K2HPO4 and _KH2PO4 to prepare _an enzyme solution. The compound or reagent to be tested is then added to the phosphate buffered enzyme solution. Optionally, this solution can also be further incubated. This incubation period can range from a few minutes to over an hour. In this particular example, the enzyme solution is incubated for about 30 minutes.

The acetone solution of the substrate PGH ₂ was then placed in another vessel at 4°C. The enzyme solution is then added to the vessel containing PGH ₂ to start the reaction. Incubate this mixture for about 30 seconds. Then, a stop solution containing _FeCl2 and citric acid was added to the mixture to prevent spontaneous conversion of any remaining _PGH2 to _PGE2 or _PGD2 . Then, a detection solution containing an antibody using PGD ₂ as an immunogen and PGD ₂ (tracer) labeled with Texas Red was added to the mixture, and the whole mixture was incubated. In this example, the mixture was incubated for about 120 minutes. However, this incubation period, as well as others described in this example, can vary depending on the concentration of the reagents.

A control mix identical to the enzyme-containing mix was prepared and treated in the same manner as the enzyme-containing mix, that is, incubated for the same period of time, etc. However, the control mixture does not contain the compound or reagent being evaluated. Then, the whole mixture and the control mixture were irradiated with plane polarized light with a wavelength of 580nm (the wavelength at which Texas Red was excited), and the fluorescence of this mixture and the control mixture was measured with fluorescence filters whose excitation wavelength and emission wavelength were set to 580nm and 620nm, respectively. Fluorescence polarization value. Measurements were performed with the measuring instrument in FP mode. Naturally, the wavelength used will depend on the fluorescent label used in the method. These two FP measurements are then compared to determine if the fluorescence polarization measurement of the mixture containing the compound or reagent is greater than the fluorescence measurement of the control solution. If the measured value of the mixture is greater than the measured value of the control mixture, it is indicated that the compound or agent reduces the activity of hPGDS.

result

The assay described above was validated using a known hPGDS inhibitor, HQL79. HQL79 has been described in WO 95/01350 patent. The structure of HQL79 is shown in Figure 7. The results of this experiment are shown in FIG. 8 . These results clearly show that the method of the present invention finds that HQL79 reduces the activity of hPGDS.

in conclusion

Examples I and II clearly show that the method of the present invention is simple, does not require numerous washing steps or radioactive isotopes, and can be readily used in a high-throughput format for determining whether a compound or reagent reduces Or inhibit the activity of prostaglandin synthase, especially hPGDS or mPGES.

It is intended that the scope of the invention not be limited by the particular embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

Various publications are cited herein, the disclosures of which are hereby incorporated by reference in their entirety.

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Claims (28)

1. For determining whether compound or reagent can reduce prostaglandin synthase and substrate reaction form the method for the activity of prostaglandin product, wherein said prostaglandin synthase is selected from microsomal prostaglandin E synthase (mPGES) and hematopoietic Prostaglandin D synthase (hPGDS), the method comprises the steps of: (a) mixing the prostaglandin synthase with the substrate, cofactor and said compound or reagent; (b) incubating the mixture of step (a) with a stop solution containing a reagent that prevents the spontaneous conversion of the substrate to the prostaglandin product; (c) incubating the mixture of step (b) together with a detection reagent containing a prostaglandin product labeled with a fluorescent marker and an antibody using the prostaglandin product as an immunogen; (d) irradiating the mixture of step (c) and the control mixture with plane polarized light, wherein the wavelength of the plane polarized light can excite the fluorescent marker, and measuring the fluorescence polarization value of the mixture of step (c) and the control mixture; and (e) comparing the assay results of step (d), Wherein, when the fluorescence polarization measurement value of the mixture (c) is found to be greater than the fluorescence polarization measurement value of the control mixture, it indicates that the compound or reagent reduces the activity of prostaglandin synthase. 2. The method according to claim 1, wherein the prostaglandin synthase is microsomal prostaglandin E synthase (mPGES), the substrate is prostaglandin H ₂ (PGH ₂ ), and the cofactor is glutathione (GSH) , the prostaglandin product is prostaglandin E ₂ (PGE ₂ ). 3. The method according to claim 2, wherein said microsomal prostaglandin E synthase is human mPGES, which contains the amino acid sequence of SEQ ID NO:2. 4. The method according to claim 1, wherein the prostaglandin synthase is hematopoietic prostaglandin D synthase (hPGDS), the substrate is PGH ₂ , the cofactor is glutathione (GSH), and the prostaglandin product is prostaglandin D ₂ (PGD ₂ ). 5. The method according to claim 4, wherein said hematopoietic prostaglandin D synthase is an artificial hematopoietic prostaglandin D synthase, which contains the amino acid sequence of SEQ ID NO:4. 6. The method according to claim 1, wherein the reagent of the stop solution is _FeCl2 . 7. The method according to claim 1, wherein said fluorescent marker comprises fluorescein, phycoerythrin (PE), Texas Red (TR), rhodamine, free lanthanide salts, chelated lanthanide salts, BODIPY, ALEXA or CyDye. 8. The method according to claim 7, wherein said fluorescent marker is Texas Red (TR). 9. The method according to claim 2, wherein the reagent of the stop solution is _FeCl2 . 10. The method according to claim 9, wherein the duration of the incubation step (b) is at least 30 seconds and the duration of the incubation step (c) is at least 3 minutes. 11. The method according to claim 10, wherein said fluorescent marker comprises fluorescein, phycoerythrin (PE), Texas Red (TR), rhodamine, free lanthanide salts, chelated lanthanide salts, BODIPY, ALEXA or CyDye. 12. The method according to claim 11, wherein the fluorescent marker is Texas Red (TR), and the wavelength of the plane polarized excitation light is 580±20 nm. 13. The method according to claim 4, wherein the reagent of the stop solution is _FeCl2 . 14. The method according to claim 13, wherein the fluorescent marker comprises fluorescein, phycoerythrin (PE), Texas Red (TR), rhodamine, free lanthanide salts, chelated lanthanide salts, BODIPY, ALEXA or CyDye. 15. The method according to claim 14, wherein the fluorescent marker is Texas Red (TR), and the wavelength of the plane polarized excitation light is 580±20 nm. 16. A method for determining whether a compound or reagent can reduce the activity of a prostaglandin synthase that reacts with a substrate to form a prostaglandin product, wherein the prostaglandin synthase is selected from the group consisting of hematopoietic prostaglandin D synthase (hPGDS) and microsomal Prostaglandin E synthase (mPGES), the method comprises the steps of: (a) mixing the prostaglandin synthase with the substrate, cofactor and said compound or reagent; (b) incubating the mixture of step (a) with a stop solution, wherein the stop solution contains a reagent that prevents the spontaneous conversion of unreacted substrates to prostaglandin products; (c) incubating the mixture of step (b) with a detection reagent, wherein the detection reagent contains a prostaglandin product labeled with Texas Red and an antibody using the prostaglandin product as an immunogen; (d) irradiating the mixture of step (c) and the control mixture with plane polarized light having a wavelength of 580 ± 20 nm, and measuring the fluorescence polarization of the mixture of step (c) and the control mixture at 620 ± 20 nm; and (e) comparing the assay results of step (d), Wherein, when the fluorescence polarization measurement value of the mixture of step (c) is found to be greater than the fluorescence polarization measurement value of the control mixture, it indicates that the compound or reagent reduces the activity of prostaglandin synthase. 17. The method according to claim 16, wherein said prostaglandin synthase is human microsomal prostaglandin E synthase (mPGES), which contains the amino acid sequence of SEQ ID NO: 2, and the substrate is prostaglandin H ₂ (PGH ₂ ), the cofactor is glutathione, and the prostaglandin product is prostaglandin E ₂ (PGE ₂ ). 18. The method according to claim 17, wherein the reagent of the stop solution is _FeCl2 . 19. The method according to claim 18, wherein said prostaglandin product labeled with Texas Red comprises a linker molecule linking said prostaglandin product and Texas Red. 20. The method according to claim 19, wherein the adapter molecule is selected from the group consisting of aminobutyric acid, aminocaproic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, Fmoc-aminocaproic acid, one or more beta-alanines , isothiocyanate groups, succinimidyl esters, halodiethylsulfones, and carbodiimides. 21. The method according to claim 20, wherein said linker molecule is a carbodiimide. 22. The method according to claim 16, wherein said prostaglandin synthase is human hematoprostaglandin D synthase (hPGDS), which contains the amino acid sequence of SEQ ID NO: 4, the substrate is PGH ₂ , and the cofactor is glutathione Glycerin, the prostaglandin product is prostaglandin D ₂ (PGD ₂ ). 23. The method according to claim 22, wherein the reagent of the stop solution is _FeCl2 . 24. The method according to claim 23, wherein said prostaglandin product labeled with Texas Red comprises a linker molecule linking said prostaglandin product and Texas Red. 25. The method according to claim 24, wherein said adapter molecule is selected from the group consisting of aminobutyric acid, aminocaproic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, Fmoc-aminocaproic acid, one or more beta-alanines , isothiocyanate groups, succinimidyl esters, halodiethylsulfones, and carbodiimides. 26. The method according to claim 25, wherein said linker molecule is a carbodiimide. 27. A method for determining whether a compound or an agent can reduce the activity of human microsomal prostaglandin E synthase (mPGES) by reacting with its prostaglandin H ₂ (PGH ₂ ) substrate to form prostaglandin E ₂ (PGE ₂ ), Wherein said human microsomal prostaglandin E synthase contains the amino acid sequence of SEQ ID NO: 2, said method comprises the following steps: (a) mixing mPGES with _PGH2 , glutathione and the compound or reagent; (b) incubating the mixture of step (a) with a stop solution containing _FeCl ; (c) incubating the mixture of step (b) with a detection reagent containing _PGE labeled with Texas Red and an antibody using _PGE as an immunogen; (d) irradiating the mixture of step (c) and the control mixture with a plane polarized light having a wavelength of 580 ± 20nm, and measuring the fluorescence polarization values of the mixture of step (c) and the control mixture; and (e) comparing the assay results of step (d), Wherein, when the fluorescence polarization measurement value of the mixture of step (c) is found to be greater than the fluorescence polarization measurement value of the control mixture, it indicates that the compound or reagent reduces the activity of mPGES. 28. A method for determining whether a compound or reagent can reduce the activity of human hematopoietic prostaglandin D synthase (hPGDS) and its prostaglandin H ₂ (PGH ₂ ) substrate reaction to form prostaglandin D ₂ (PGD ₂ ), wherein the Described human blood prostaglandin D synthase contains the amino acid sequence of SEQ ID NO: 4, and described method comprises the following steps: (a) mixing hPGDS with _PGH2 , glutathione and the compound or reagent; (b) incubating the mixture of step (a) with a stop solution containing _FeCl ; (c) incubating the mixture of step (b) with a detection reagent containing PGD ₂ labeled with Texas Red and an antibody using PGD ₂ as an immunogen; (d) irradiating the mixture of step (c) and the control mixture with a plane polarized light having a wavelength of 580 ± 20nm, and measuring the fluorescence polarization values of the mixture of step (c) and the control mixture; and (e) comparing the assay results of step (d), Wherein, when the fluorescence polarization measurement value of the mixture of step (c) is found to be greater than the fluorescence polarization measurement value of the control mixture, it indicates that the compound or reagent reduces the activity of hPGDS.

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