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
Method for assaying compounds or agents for ability to decrease the activity of microsomal prostaglandin E synthase or hematopoietic prostaglandin D synthase Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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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
Invention field
The present invention relates to novelty and useful method that a kind of detection compound or reagent reduce the ability of prostaglandin synthase activity.More particularly, the present invention relates to the method that a kind of detection compound or reagent reduce the ability of mPGES (mPGES) activity or hematopoietic prostaglandin D synthase (hPGDS) activity.
Background of invention
Prostaglandin is the eicosanoid (eicosanoids) that a class plays an important role in pain, fever and inflammatory process.They are synthetic from arachidonic acid in vivo, have five yuan of carbocyclic rings that constitute an arachidonic acid carbochain part.Prostaglandin is not a hormone, only plays local action near being synthesized the position.
PGE
2And PGD
2These two kinds of prostaglandins all play a part particular importance in fever, pain and inflammation.Especially, the PGD that mast cell produces under antigenic stimulus
2Level raises, and PGD
2It is a kind of main medium of respiratory tract anaphylaxis disease.Especially, except other symptom, it can cause bronchoconstriction, bronchus hyperfunction, nasal obstruction and eosinocyte and TH
2Cellular infiltration.PGE
2Be a kind of pain medium, be proved to be in inducing hyperalgia, fever, vasodilation and oedema, play a part certain.
In the building-up process, phospholipase A2 is converted into arachidonic acid (form with ester exists in vivo) with phosphatide in vivo.Subsequently, the prostaglandin endoperoxides synzyme is converted into arachidonic acid PGG2 (PGG again
2).But the prostaglandin endoperoxides synzyme is catalysis PGG also
2The reduction reaction of middle peroxide group, and form PGH
2(PGH
2), PGH wherein
2Be PGE
2And PGD
2Precursor.Producing PGE
2Situation under, Prostaglandin E synthase (PGES) co-factor glutathione (GSH) when existing with PGH
2Be converted into PGE
2At least there are two kinds of PGE
2Synzyme, i.e. tenuigenin PGES (cPGES) and microsome PGES (mPGES).These two kinds of PGES are expressed in many tissues and overlapping distribution widely.MPGES can by the inflammatory stimulus thing for example lipopolysaccharides (LPS), IL-1 and TNF-α induce, the expression of cPGES then is a composition.In addition, proved mPGES relevant with the COX-2 activity [Murakami etc., J.Biol.Chem.275:32783 (2000)].
PGD
2Generation be that PGD synzyme (PGDS) is with PGH
2Be converted into PGD
2The result.The known PGD that has two types
2Synthase.First kind is to carry fat type (lipocalin-type) PGDS (L-PGDS), is mainly seen in the central nervous system; Second kind is hematopoiesis type PGDS (hPGDS), is mainly seen in peripheral tissues.L-PGDS does not rely on glutathione (GSH), and hPGDS then depends on GSH, and has the GST activity.In addition, L-PGDS and hPGDS almost do not have structural homology between the two.
Because PGD
2And PGE
2Play an important role in fever, pain and inflammatory process, people have made many effort and have set up various detection methods, to detect the compound that those may reduce even suppress its generation.Especially, in order to measure the ability that certain compound or reagent reduce or suppress the prostaglandin synthase activity, many technology for example HPLC, ELISA or RIA etc. have been used to quantitative test PGD
2And PGE
2Generation.Yet these technology all have certain intrinsic limitation.For example, they need a plurality of washing steps, purification step and/or use radiomaterial.Also have, these methods are more time-consuming, and a day flux has only tens (HPLC) to the individual data point of hundreds of (ELISA and RIA).Therefore, they can not be used for high flux screening.
Fluorescence polarization technology is a kind of technology that is used to study intermolecular interaction.The principle of this technology depends on is identified bulk of molecule.Especially, when with plane polarization rayed fluorescence molecule the time, the electronic transition that is in ground state in the tested molecule is to excited state.After nanosecond, these excited state electronics transition are again got back to ground state at about 4-5.In this decay process, this testing molecule is launched fluorescence signal just.In the fluorescence polarization process, only remain under the static situation in whole excited state at tested molecule, could detect this fluorescent emission at grade.If this molecule moves during excited state or rotates, then institute's emitted fluorescence will be on the plane different with the polarized light that excites this electronics.As a result, will detect less than fluorescent emission.Generally accepted is that more little then its motility of molecule and rotatory are big more.Therefore, the signal that micromolecule produces will be more much smaller than big molecule, because big molecule will keep relative static during excited state.This character of the molecule just that fluorescence polarization utilizes.Especially, analyze in the process of certain part at fluorescence polarization method, this part, the tracer agent part of fluorescence labeling substance markers (promptly with) and the acceptor that combines with this part are placed in the solution.So, the competition mutually of this part and tracer agent in order to be attached on the acceptor.Then, with this solution of plane polarization rayed, carry out input again.If few of part in the solution, then existing most receptors will be in conjunction with tracer agent.Because acceptor is a kind of big molecule (for part), thus a kind of signal of the self-marker's of coming fluorescence can be obtained, and can obtain very strong fluorescence polarization signal.On the contrary, if there is plurality of ligand, then most of acceptor will combine with this part.As a result, if there is signal to produce, the fluorescence polarization signal that is produced separately by tracer agent will be significantly less than the aforementioned signal that tracer agent produced by bind receptor.Difference between these signals just makes in the art those skilled in the art can determine whether this part exists and can measure its concentration.Fluorescence polarization is by the milli fluorescence polarization degree, or mP is that unit is measured.
Therefore, fluorescence polarization uses more simple and effective than detection methods such as ELISA, HPLC and RIA.And it can easily be used for a large amount of compound or the reagent of high flux screening in a short period of time.
So, needed a kind of just fluorescence polarization method, be used for that assessing compound or reagent reduce or even suppress the ability of prostaglandin synthase activity, especially estimate certain reagent or compound and whether can reduce or suppress mPGES generation PGE
2Ability, and estimate certain reagent or compound and whether can reduce or suppress hPGDS and produce PGD
2Ability.
Needed also have a kind of high throughput system, is used for that assessing compound or reagent reduce or the activity that the suppresses prostaglandin synthase ability of the activity of mPGES or hPGDS especially.Reduce or even inhibition hematopoietic prostaglandin D
2Synthase (hPGDS) or derivable microsome PGE
2Perhaps, the active compound of synthase (mPGES) can be used for treating inflammation and allergic symptom, as arthritis, asthma and rhinitis etc., only gives some instances herein.In addition, pain and/or fever may also can be treated with such compound or reagent.
This paper should not be interpreted as being to recognize that to quoting of any list of references this class list of references is the application's " prior art ".
Summary of the invention
According to the present invention, we provide new effective ways, be used for that assessing compound or reagent reduce or even suppress the ability that mPGES or hPGDS produce the activity of prostaglandin product separately, this method is not used radioactive isotope, do not need various washing step, and can fail the interior mode of ex vivo behind the manipulation in vitro, in the mode of manipulation in vitro, or implement in the mode of cell, also the mode that can separate is implemented.In addition, method of the present invention can be implemented in high-throughout mode at an easy rate.
Broadly, the present invention can amplify and be a kind of method, is used for determining whether a kind of compound or reagent can reduce the activity of prostaglandin synthase and its substrate reactions formation prostaglandin product.Wherein this prostaglandin synthase is selected from mPGES (mPGES) and hematopoietic prostaglandin D synthase (hPGDS).This method of the present invention may further comprise the steps: with prostaglandin synthase and its substrate, co-factor and testing compound or reagent mix, make enzymatic reaction to take place; Then this potpourri is hatched the reagent that this stop buffer contains and stops unreacted substrate spontaneous nuclear transformation is the prostaglandin product with stop buffer; And then this potpourri hatched with a kind of such detectable, this detectable contains the prostaglandin product of fluorescent marker (being a kind of tracer agent) mark, and with the prostaglandin product as antibody that immunogene was produced; Subsequently, handle with this potpourri of plane polarization rayed with in same mode but do not contain the control mixture of testing compound or reagent, this plane polarization light wavelength is the wavelength of fluorescence that fluorescent marker sends.Measure and compare the fluorescence polarization value of testing mixture and control mixture.If the polarization value of testing mixture greater than the polarization value of control mixture, then show the compound of surveying or reagent can reduce the prostaglandin synthase activity.Therefore, such compound or reagent can easily be used for the treatment of suffer from inflammation, the experimenter of the combination in any of allergy, pain and fever or these illnesss, only be several examples herein.
In addition, the present invention may extend to aforesaid a kind of method, and wherein prostaglandin synthase is derivable mPGES (mPGES), and substrate is a PGH
2(PGH
2), co-factor is glutathione (GSH), the prostaglandin product is a PGE
2(PGE
2).The used mPGES of the present invention's method can derive from ox, sheep, rodent, horse, dog, people, cat or the like.In one embodiment, mPGES derives from the people, and its amino acid sequence is shown in Fig. 9 B and SEQ ID NO:2.In addition, the mPGES that is applied to the inventive method needn't be purified form.
The present invention can further extend to such method, as described in the application's book, is used for determining whether a kind of compound or reagent can reduce prostaglandin synthase and its substrate reactions and form a kind of activity of prostaglandin product.Wherein prostaglandin synthase is hematopoietic prostaglandin D synthase (hPGDS), and substrate is PGH
2, co-factor is GSH, the prostaglandin product is a PGD
2(PGD
2).As mPGES, the hPGDS that is applied to the inventive method can have numerous sources.In one embodiment, hPGDS is people hPGDS, and its amino acid sequence is shown in Figure 10 B and SEQ IDNO:4.And hPGDS needn't be purified form.
As mentioned above, in the methods of the invention, the reagent that stop buffer contains and stops unreacted substrate spontaneous nuclear transformation is the prostaglandin product.Especially, the substrate PGH of aforesaid two kinds of prostaglandin synthases
2Contain peroxide group.Do not explain its mechanism though there is obligation, do not want to be subjected to the restriction of any explanation certainly yet, but it is believed that mPGES and hPGDS catalysis PGH
2The oxygen bond rupture of peroxide group, and with PGH
2Be separately converted to PGE
2And PGD
2Yet, PGH
2Can spontaneous nuclear transformation be PGE also
2Or PGD
2The analysis result to the ability of compound or reagent reduction mPGES or hPGDS activity can be disturbed and change to this spontaneous nuclear transformation.Therefore in the methods of the invention, testing mixture is hatched with stop buffer, this stop buffer contains and stops PGH
2Spontaneous nuclear transformation is PGD
2Or PGE
2Reagent.An object lesson of this reagent is the FeCl that concentration is about 20mM
2Yet those skilled in the art just can easily be familiar with stoping other reagent of this synchronous conversion in the art, and this class reagent is also included within the inventive method.And this duration of hatching can change, and is enough to stop any remaining unreacted PGH but must look
2Be converted into the prostaglandin product.
In addition, the present invention may extend to such method, is used for determining whether a kind of compound or reagent can reduce prostaglandin synthase, and especially mPGES or hPGDS form a kind of activity of prostaglandin product with its substrate reactions.Wherein this testing mixture is hatched with detectable after stop buffer is hatched again, this detectable contain the prostaglandin product of useful fluorescent marker mark and with the prostaglandin product as immunogenic antibody.Numerous fluorescent markers of all knowing of those those of ordinary skill all can be applicable to the present invention's method in the art.The example of these fluorescent markers comprises fluorescein, phycoerythrin (PE), red (the Texas red of Texas, TR), rhodamine, free lanthanide series salt, lanthanide series salt, CyDye (Amersham Biotech company product), BODIPY (Molecular Probes company product) and the ALEXA (Molecular Probes company product) etc. of chelating, only be several examples herein.In one embodiment, fluorescent marker is that Texas is red.In addition, fluorescent marker can perhaps alternatively be attached to earlier on the linkers directly in conjunction with on the prostaglandin product, is attached on the prostaglandin product by this linkers again.The used concrete linkers of the present invention comprises, but be not limited to certainly, aminobutyric acid, aminocaproic acid, 7-aminoheptylic acid, 8-aminocaprylic acid, Fmoc-aminocaproic acid, one or more Beta-alanine, isothiocyanates group, succinimide ester, halo sulphonal or carbodiimide etc. only are several examples herein.In a specific embodiments of the present invention, this prostaglandin product is attached to earlier on the carbodiimide joint, by this joint be attached to again fluorescent marker such as Texas red on.
In addition, the present invention may extend to such method, is used for determining whether a kind of compound or reagent can reduce or suppress derivable mPGES (mPGES) and its substrate PGH
2(PGH
2) reaction generation PGE
2(PGE
2), the method may further comprise the steps:
(a) with mPGES and PGH
2, glutathione (GSH) and testing compound or reagent mix at least 30 seconds;
(b) potpourri of step (a) with contain FeCl
2Stop buffer hatch together;
(c) potpourri of step (b) is hatched with a kind of detectable, this detectable contains the PGE of useful Texas red marker
2With with PGE
2As immunogenic antibody;
(d) with wavelength be the potpourri and the control mixture of the linearly polarized light irradiating step (c) of 580 ± 20nm, and the potpourri of determination step (c) and the fluorescence polarization value of control mixture;
(e) measurement result of comparison step (d).
If the fluorescence polarization determination value of potpourri that contains test compound or reagent shows then that greater than the fluorescence polarization determination value of control mixture this compound or reagent can reduce the activity of mPGES.In one embodiment, mPGES is people mPGES, its amino acid sequence shown in Fig. 9 B and SEQID NO:2, PGE
2Be connected by the carbodiimide joint with Texas is red.The red excitation wavelength of Texas is 580nm.Yet this wavelength can be in 580nm ± 20nm scope.Similarly, the red fluorescent emission wavelength of Texas is considered to 620nm usually.Yet this wavelength can change in the scope of 620nm ± 20nm.This variation of excitation wavelength and emission wavelength is included among the present invention.And just as previously described, mPGES needn't be purified form.
In addition, the present invention may extend to such method, is used for determining whether a kind of compound or reagent can reduce hematopoietic prostaglandin D synthase (hPGDS) and its substrate PGH
2(PGH
2) reaction formation PGD
2(PGD
2), the method may further comprise the steps:
(a) with hPGDS and PGH
2, GSH and testing compound or reagent mix at least 30 seconds;
(b) with the potpourri of step (a) with contain FeCl
2Stop buffer hatch together;
(c) potpourri of step (b) is hatched with a kind of detectable, this detectable contains the PGD of useful Texas red marker
2With with PGD
2As immunogenic antibody;
(d) with wavelength be the potpourri and the control mixture of the linearly polarized light irradiating step (c) of 580 ± 20nm, and the potpourri of determination step (c) and the fluorescence polarization value of control mixture;
(e) measurement result of comparison step (d).
If the fluorescence polarization determination value of potpourri that contains test compound or reagent shows then that greater than the fluorescence polarization determination value of control mixture this compound or reagent can reduce the activity of hPGDS.In one embodiment, hPGDS is people hPGDS, its amino acid sequence shown in Figure 10 B and SEQID NO:4, the red and PGD of Texas
2Connect with chemical bond by the carbodiimide joint.
In addition, the present invention may extend to such method, is used for determining whether a kind of compound or reagent can reduce prostaglandin synthase such as mPGES or hPGDS, produce the activity of prostaglandin product, and this method are carried out in the high flux mode.
Therefore, one aspect of the present invention provides a kind of method, be used for that assessing compound or reagent reduce or even suppress prostaglandin synthase, as the ability of the activity of mPGES or hPGDS.So, compound or reagent that method of the present invention makes in the art those skilled in the art to identify to can be applicable to certain combination for the treatment of experimenter's pain, inflammation, fever or these illnesss.
Another aspect of the present invention provides a kind of method, be used for the ability that assessing compound or reagent reduce or suppress the activity of mPGES or hPGDS, and this method does not need washing step or utilizes radioactive isotope.
A further aspect of the invention provides a kind of method, be used for the ability that assessing compound or reagent reduce or suppress the activity of prostaglandin synthase such as mPGES or hPGDS, and this method can be failed the interior mode of ex vivo behind the manipulation in vitro, mode with manipulation in vitro, or realize that in the mode of cell also the mode that can separate is implemented.
The present invention also has another aspect to provide a kind of method, be used for the ability that assessing compound or reagent reduce or suppress the activity of prostaglandin synthase, and this method can the high flux mode be carried out.
By with reference to the following drawings with the present invention's detailed description, can understand these aspects of the present invention and other aspect better.
The accompanying drawing summary
Fig. 1 is substrate PGH
2Be converted into PGE
2Enzymic transformations and PGH
2Be converted into prostaglandin product P GD
2Or PGE
2Spontaneous nuclear transformation reaction between the competition synoptic diagram, wherein used enzyme is mPGES, prevents that the reagent of this spontaneous nuclear transformation reaction from being FeCl
2
Fig. 2 is the synoptic diagram of a kind of method of the present invention, and wherein prostaglandin synthase is mPGES, and the prostaglandin product is PGE
2
Fig. 3 is substrate PGH
2Be converted into PGD
2Enzymic transformations and PGH
2Be converted into prostaglandin product P GD
2Or PGE
2Spontaneous nuclear transformation reaction between the competition synoptic diagram, wherein used enzyme is hPGDS, prevents that the reagent of this spontaneous nuclear transformation reaction from being FeCl
2
Fig. 4 is the synoptic diagram of a kind of method of the present invention, and wherein prostaglandin synthase is hPGDS, and the prostaglandin product is PGD
2
Fig. 5 has shown the chemical constitution of MK-886.MK-886 is a kind of known mPGES inhibitor, is used to prove that whether method of the present invention makes us can determine a kind of compound or reagent can reduce or suppress the activity of prostaglandin synthase.
Fig. 6 utilizes prostaglandin synthase mPGES and known inhibitor MK-886 and the concentration-response curve synoptic diagram of the present invention's of drawing method.IC50＝27.5μM。These results show, method of the present invention makes in the art that those skilled in the art can determine whether a kind of compound or reagent can reduce the activity of prostaglandin synthase.Prostaglandin synthase in this example is mPGES.
Fig. 7 has shown the chemical constitution of HQL 79.
Fig. 8 is a column diagram, has shown by the active decline of HQL 79 caused hPGDS to detect by method of the present invention.
Fig. 9 A and 9B have shown and have been used for hereinafter nucleotide sequence and the amino acid sequence (being respectively SEQ ID NO:1 and 2) of the people mPGES of example I.
Figure 10 A and 10B have shown nucleotide sequence and the amino acid sequence (SEQ IDNO: be respectively 3 and 4) of people H-PGDS.
Detailed Description Of The Invention
The present invention is based on so unexpected surprising discovery: fluorescence polarization can be used for identifying that those can reduce prostaglandin synthase (such as mPGES or hPGDS) and produce prostaglandin (such as PGD2Or PGE2) compound or the reagent of activity. Therefore in a broad sense, the present invention may extend to such side Method is used for determining whether a kind of compound or reagent can reduce prostaglandin synthase and its substrate reactions Generate the activity of prostaglandin product, wherein said prostaglandin synthase is selected from mPGES or hPGDS, The method may further comprise the steps:
(a) prostaglandin synthase and its substrate, co-factor and testing compound or reagent mix;
(b) mixture of step (a) is hatched with stop buffer, this stop buffer contains prevention should The substrate spontaneous nuclear transformation is the reagent of prostaglandin product;
(c) mixture of step (b) is hatched with detecting reagent, this detection reagent contain with The prostaglandin product of fluorescence labeling substance markers, and with this prostaglandin product as immunogenic anti-Body;
(d) mixture and the control mixture of usefulness linearly polarized photon irradiating step (c), this polarised light Wavelength be the wavelength of fluorescence that fluorescent marker sends, and mixture and the contrast of determination step (c) The fluorescence polarization value of mixture; And
(e) measurement result of comparison step (d),
If the fluorescence polarization determination value of the mixture of step in the above-mentioned steps (d) is greater than control mixture The fluorescence polarization determination value, show that then this compound or reagent can reduce the activity of prostaglandin synthase.
Run through numerous terms of this specification and the appended claims and being defined as follows of phrase, that is:
Term used herein " compound " or " reagent " refer at present known or later sending out Existing any chemical analysis. The compound that the present invention is used or the example of reagent include organic compounds (example As artificial or naturally occurring), peptide (artificial or naturally occurring), carbohydrate, nucleic acid molecules Etc..
Term used herein " enzyme " refers to such biomolecule, but catalysis specialization for example Learn protein or the RNA of reaction. Enzyme does not affect the balance of catalytic reaction, but by reducing activation energy Mode improve reaction speed.
Term used herein " prostaglandin synthase " but refer to the catalysis PGH2(PGH
2) Be converted into the enzyme of prostaglandin product. The object lesson that is applied to prostaglandin synthase of the present invention comprises Derivable mPGES (mPGES), this enzyme is at co-factor glutathione (GSH) In the situation about existing with PGH2Be converted into PGE2(PGE
2). Another example is Before hematopoietic prostaglandin D synthase (hPGDS), this enzyme are incited somebody to action in the situation that co-factor GSH exists Row parathyrine H2Be converted into PGD2(PGD
2)。
Term used herein " substrate " refers under the effect of enzyme to produce the change of product Compound. The example of the used substrate of the present invention is PGH2。
Term used herein " co-factor " refers to the required organic molecule of enzymatic activity, inorganic Molecule, peptide or protein. In a specific embodiments of the present invention, prostaglandin synthase is mPGES Or hPGDS, co-factor is glutathione (GSH).
Term used herein " prostaglandin product " refers to because prostaglandin synthase acts on The substrate of prostaglandin synthase and the product that generates. Therefore, for mPGES, this prostate Plain product is PGE2 For hPGDS, this prostaglandin product is PGD2。
Term used herein " fluorescent marker " refers to can when using the irradiation of specific wavelength Send the material of fluorescence, this material can directly be attached on the purpose compound or be attached to earlier one and connect On the molecule, be attached to again on the purpose compound by this joint. Be applied to the fluorescence of the inventive method The example of label comprises, but certainly is not limited to fluorescein, phycoerythrin (PE), Texas The lanthanide series salt of red (TR), rhodamine, free lanthanide series salt, chelating, BODIPY, ALEXA, CyDye etc. A kind of specific fluorescent marker that is applied to the present invention's method is Ke Sasi is red.
Term used herein " joint " and " linkers " are used interchangeably, and refer to glimmering The chemical part of signal thing and the combination of prostaglandin product. Be applied to the concrete example of joint of the present invention Attached bag draw together aminobutyric acid, aminocaproic acid, 7-aminoheptylic acid, 8-aminocaprylic acid, Fmoc-aminocaproic acid, One or more Beta-alanines, isothiocyanates group, succinimide ester, halo sulphonal or Carbodiimides etc. only are several examples herein. Be applied to a concrete example of joint of the present invention Son is carbodiimide group.
Term used herein " control mixture " refers to and contains test compound or reagent Mixture is compared, and contains the mixture of the material such as same reagent, compound, cell of same quantity, And process in the mode the same with the mixture that contains test compound or reagent, difference is, This control mixture does not contain test compound or reagent.
Antibody
Just as explained above, method of the present invention has been used and has been had with prostaglandin product PGD for example2Or PGE2As immunogenic antibody. This antibody can be monoclonal antibody, polyclonal antibody, And even chimeric antibody. Many methods known in the art all can be used for preparing PGE2Or PGD2Polyclonal antibody. For Dispersal risk, can make multiple host animal by injection prostaglandin product Immunity, these host animals include but not limited to rabbit, mouse, rat, sheep, goat etc. One In the specific embodiments, PGE2Or PGD2Can be connected on the immunogenic carrier, for example cow's serum Albumin (BSA) or keyhole limpet hemocyanin (KLH). Can use multiple adjuvant to answer to improve immunity Answer, according to host's species, available adjuvant include but not limited to Freunds adjuvant (fully and not exclusively), Surface reactive material, the Pluronic polyols (Pluronic such as the mineral coagulants such as aluminium hydroxide, lysolecithin Polyols), polyanion, peptide, oil emu, keyhole limpet hemocyanin, dinitrophenol dinitrophenolate, BCG vaccine (bacille Calmette-Guerin, BCG) or spillikin bacillus (Corynebacterium parvum).
For the monoclonal antibody of preparation for the prostaglandin product, any passage cell by cultivating The technology that is the Dispersal risk molecule can be used. These technology include but not limited at first by Kohler Hybridoma cell technology [Nature 256:495-497 (1975)], trioma with the Milstein exploitation Technology, human B cell hybridoma cell technology [Kozbor etc., Immunology Today 4:72 (1983); Cote etc., Proc.Natl.Acad.Sci.U.S.A.80:2026-2030 (1983)], and system [Cole etc. are at Monoclonal for the EBV-hybridoma technology of standby human monoclonal antibody Antibodies and Cancer Therapy, Alan R.Liss, the 77-96 page or leaf in Inc. one book (1985)]. In addition, monoclonal antibody can also be utilized described in the patent PCT/US90/02545 Technology prepares in the germfree animal body. For the technology that preparation " chimeric antibody " is developed also can be utilized [Morrison etc., J.Bacteriol.159:870 (1984); Neuberger etc., Nature 312:604-608 (1984); Takeda etc., Nature 314:452-454 (1985)], this technology is with the prostatitis The special mouse antibodies molecular gene montage of parathyrine product is to the human antibody with suitable BA On the molecular gene.
Condition
Just as explained above, method of the present invention can be failed the interior mode of ex vivo behind the manipulation in vitro, with The mode of manipulation in vitro is implemented; Or implement wherein all reagent, enzyme, substrate in the mode of separating Deng separating earlier and be stored in the such buffering of TRIS, TRIS HCl, HEPEs or phosphate In the liquid; Also can implement for the mode on basis by cell. In addition, in the method for the invention, prostate Plain synthase needn't pass through purifying.
Take cell in the analysis on basis, method of the present invention be used for to be determined test compound or reagent Whether can prevent or reduce emiocytosis prostaglandin product, and in in-vitro method, cell can Cracking before using method of the present invention so that can be in born of the same parents testing compound or reagent in the medium.
The search compound library is sought candidate compound or the examination that can reduce or suppress the prostaglandin synthase activity
Agent
By convention, produce the noval chemical compound entity with useful quality and will pass through following steps: identify Have some required character or active compound (being called lead compound (lead compound)), Prepare the variant of this lead compound, and character and the activity of estimating these compound variants. Yet, Present trend is to shorten the required time in all stages of drug development. Because high flux screening (HTS) Method can fast and effeciently be tested in enormous quantities, so this method has replaced traditional guide's chemical combination The thing identification method.
In one embodiment, the high flux screening method relates to provides a collection of a large amount of potential treatments that contain The compound of property compound (candidate compound). Then, utilize this " going through of method screening of the present invention History (historic) compound group ", shown required feature activity in this batch compound to identify Those compounds (specific compounds category or subclass). The compound of so identifying can be used as Traditional " lead compound " or itself namely can be used as potential or actual therapeutic agent.
The combination of compounds storehouse
The combination of compounds storehouse is a kind of prefered method that helps to produce new lead compound.The combination of compounds storehouse is by making up for example reagent and with the set of the synthetic various compound of chemistry or biology mode of many chemistry " structural unit ".For example, linear combination compound library such as peptide library are to be called as amino acid whose chemical constitution unit by one group to combine in multiple possible mode according to predetermined compound length (being amino acid whose quantity in the polypeptide compound).Millions of kinds of compounds can synthesize by the mixing of this chemical constitution unit combination.For example, a commentator notices 100 interchangeable chemical constitutions of systematicness ground combined hybrid unit, can synthesize 100,000,000 kinds of tetramerization compounds or 10,000,000,000 kinds of pentamer compound (Gallop etc. in theory, the application combination technology is carried out the organic synthesis of drug discovery 2. combinations, library screening strategy and following direction, J.Med.Chem. (1994) 37 (9): 1233-1251).
The preparation in combination of compounds storehouse is well-known for those those of ordinary skill in the art.This combination of compounds storehouse include but not limited to the peptide storehouse (consult, as United States Patent (USP) 5,010,175, Furka (1991) Int.J.Pept.Prot.Res., 37:487-493, Houghton etc. (1991) Nature, 354:84-88).Unique method of the present invention is predicted and is intended for use in synthesizing of peptide anything but.Other chemical substance that produces chemical polynary storehouse also can be used.Such chemical substance includes but not limited to: class peptide (peptoids) (PCT publication number WO 91/19735, on Dec 26th, 1991), encoded peptide (encoded peptides) (the open WO 93/20242 of PCT, on October 14th, 1993), biological at random oligomer (the open WO 92/00091 of PCT, on January 9th, 1992), benzene phenodiazine class (benzodiazepines) (U.S. Patent number 5,288,514), various body class (diversomers) is hydantoins for example, (Hobbs etc. such as benzene phenodiazine class and dipeptides, (1993) Proc.Nat.Acad.Sci.USA 90:69096913), vinylogous polypeptide (vinylogouspolypeptides) (Hagihara etc. (1992) J.Amer.Chem.Soc.114:6568), non-peptide comparison peptide (Hirschmann etc. with β-D-glucose skeleton, (1992) J.Amer.Chem.Soc.114:92179218), the similar organic synthesis body of little compound library (Chen etc. (1994) J.Amer.Chem.Soc.116:2661), oligomerization carbamate (oligocarbamates) (Cho etc., (1993) Science 261:1303), and/or phosphonic acids peptide ester (Campbell etc., (1994) J.Org.Chem.59:658).This class combination of compounds storehouse comprises that synthetic storehouse (consults Gordon etc., (1994) J.Med.Chem.37:1385), United States Patent (USP) 5 (is consulted in nucleic acid library, peptide nucleic acid storehouse, 539,083 etc.), antibody library (is consulted (1996) Nature Biotechnology such as Vaughn, 14 (3): 309-314) and PCT/US96/10287), the carbohydrate storehouse (consults (1996) Science such as Liang, 274:1520-1522 and United States Patent (USP) 5,593,853), and little organic molecule library (for example can consult Baum (1993) C﹠amp about benzene phenodiazine class; EN, 33 pages, can consult United States Patent (USP) 5 about isoprenoid at January 18,569,588, can consult United States Patent (USP) 5,549 about a thiazolidine ketone (thiazolidinones) and a thiazine ketone (metathiazanones), 974, can consult United States Patent (USP) 5,525 about pyrrolidines, 735 and 5,519,134, can consult United States Patent (USP) 5,506,337 about the morpholino compounds class, can consult 5,288,514 etc. about benzene phenodiazine class).
The equipment of preparation combinatorial libraries can be buied through commercial approach and (consult as 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 robot systems of knowing that are used for the liquid phase chemical material had been developed already.These systems comprise the automatically working station, as Takeda Chemical Industries, and the automatic synthesizer device of LTD. (Osaka, Japan) exploitation, and many robot system (Zymate II that utilize mechanical hand, ZymarkCorporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), the manual synthetic operation that chemist carries out can be imitated in these automatically working stations.Above-mentioned any equipment all is suitable for the present invention.For these equipment can be turned round according to mode discussed in this article, can transform these equipment.And essence of these transformations (if any) and implementation method are conspicuous for those personnel that are proficient in technology in the relevant technologies field.In addition, many combinatorial libraries commercialization itself (is consulted ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St.Louis, MO, ChemStar, Ltd., Moscow, RU, 3DPharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).
The high throughput analysis of compound library
Nature adopts the inventive method of fluorescence polarization technology can adapt to high flux screening at an easy rate.High throughput screening system commercialization (is consulted the Corp. as Zymark, Hopkinton, MA; AirTechnical Industries, Mentor,, OH; Beckman Instruments, Fullerton, CA; Precision Systems Inc., Natick, MA etc.).These systems generally can make the whole procedure robotization, comprise that the suction of sample and reagent moves, the last reading etc. of microwell plate in the detecting device of hatching, be suitable for this analysis of the distribution of liquid, timing.These systems that can assemble flexibly can realize that high throughput analysis, operating flexibility capable of fast starting, that have height also can realize customizations highly.The manufacturer of this type systematic provides detailed scheme for various high throughput analysis.For example, Zymark company provides the technology communication for the screening system of purposes such as the regulation and control that detect genetic transcription, part combination.
By with reference to following non-limiting example, will have more thorough understanding to the present invention as the exemplary example of the present invention.It is in order to illustrate the preferred embodiment of the invention more fully that following embodiment is provided.Yet they never should be construed as restriction vast scope of the present invention.
Example I
The fluorescence polarization test of derivable mPGES (mPGES)
PGE
2(PGE
2) relate to a kind of main medium of inflammation and pain.MPGES (mPGES) but when glutathione exists catalysis PGH
2To PGE
2Conversion.It has induced the expression of mPGES in many inflammatory diseases.Therefore, the compound that can reduce or suppress the mPGES activity will be the active drug of treatment inflammation, pain, fever or the combination of these illnesss, only enumerate several diseases or unusual herein.
But developed a kind of already by induced microparticle body PGE
2Synthase is measured PGH
2To PGE
2The test method that transforms.This test method designs according to the fluorescence polarization principle.With this enzyme and PGH
2, glutathione and hatched together by compound or the reagent estimated.Through a short incubation period (at least 30 seconds), add and contain FeCl
2With the stop buffer of citric acid with any remaining PGH of cancellation
2, otherwise it is PGD with spontaneous nuclear transformation
2Or PGE
2, and therefore disturb PGH
2To PGE
2The quantitative test of Enzymatic transformation (Fig. 1).Then, add the tracer agent (PGE that contains fluorescence labeling (Texas is red)
2) and anti-PGE
2Detection of antibodies solution is to produce and PGE
2The distinctive signal (Fig. 2) that is inversely proportional to of generation.PGE from this enzymatic reaction generation
2With this antibody of special competition, and discharge the fluorescence labeling tracer agent.PGE
2The inhibition of synthase activity will cause the increase of FP value.
Material
Glutathione (GSH):
Available from Sigma (products catalogue #G-6529).
PGH
2
Available from Caymen Chemicals, Inc. (products catalogue #17020).
PGE
2Monoclonal antibody
Available from Assay Designs, Inc. (products catalogue #915-057)
PGE
2：
Available from Caymen Chemicals, Inc. (products catalogue #14010)
The expression of people mPGES enzyme
People mPGES enzyme is to adopt bacterial expression system described in the following document and step to express: [Jakobsson, (surveyor's Prostaglandin E synthase: be a kind of inducibility enzyme that relies on the microsome glutathione such as P., constituted potential novel drugs target, Proc.Natl.Acad.Sci.USA96:7220-7225 (June, 1999)).Therefore, used in this embodiment mPGES is not a form of taking purifying, and is included in the film fraction of taking from the bacterium of expressing mPGES.The dna sequence dna of coding people mPGES that is used for this embodiment is shown in Fig. 9 A and SEQ ID NO:1.The amino acid sequence of people mPGES that is used for this embodiment is shown in Fig. 9 B and SEQ ID NO:2.
Fluorescently-labeled prostaglandin product P GE
2
As mentioned above, many fluorescent markers are used for method of the present invention.In one embodiment, the red connection of the Texas of fluorescent marker is by a linkers and PGE
2(PGE
2) connect.In this specific embodiments, with the PGE of Texas's red marker
2Be that (SanMateo, California) company synthesizes Combinix.The mechanism that produces this a part is as described below:
PGE
2Tracer agent is synthetic
In this building-up process, with the PGE in the red cadaverine of Texas (Molecular Probes company product) the adding anhydrous methylene chloride
2(Cayman Chemicals) solution.Add dicyclohexylcarbodiimide (Sigma Aldrich), under nitrogen atmosphere in dark reaction stirred 24 hours.Carry out purifying by the reverse hplc chromatography, make water/acetonitrile gradient and with 0.05%TFA as modifier.The linkers that is used for this building-up process can change to some extent.
Method
At first, with containing K
2HPO
4And KH
2PO
4Reaction buffer dilute the film fraction that this contains enzyme, with the preparation phosphate-buffered enzyme solutions.Then compound to be measured or reagent are put into this enzyme solutions.Randomly, also can further hatch this solution.In this specific embodiments, hatched this solution about 30 minutes.Then with substrate PGH
2Acetone soln and co-factor GSH place in another container of 4 ℃.Again enzyme solutions is added and contain PGH
2Container with begin the reaction.Hatch this potpourri about 30 seconds.Then, will contain the FeCl that concentration is 20mM
2Stop buffer add this potpourri, in case any remaining PGH
2Spontaneous nuclear transformation is PGE
2Then, will contain anti-PGE
2Antibody and PGE
2The detection solution of the red tracer agent of-Texas adds this potpourri, and hatches whole potpourri.In this specific embodiments, this incubation period, be about 120 minutes.But, be familiar with this operator and can change the length of this embodiment described any incubation period and still can obtain useful results.Control mixture is not except containing this compound or reagent, and potpourri is identical therewith, and is subjected to same processing, that is to say, it contains same reagent, hatched the same time, or the like.Then, be whole potpourri of plane polarization rayed and the control mixture of 580nm with wavelength, and measure the fluorescence polarization value of this potpourri and control mixture with the fluorescence filter that excitation wavelength and emission wavelength are set at 580nm and 620nm respectively.Be at this surveying instrument under the situation of FP pattern and measure.Then this twice measured value compared, with its fluorescence polarization measurement value of potpourri of determining to contain this compound or reagent whether greater than the fluorescence measurement value of contrast solution.If the measured value of this potpourri shows then that greater than the measured value of control mixture this compound or reagent have reduced the activity of mPGES.
The result
Above-mentioned test has obtained checking by using a kind of known mPGES inhibitor.This inhibitor MK-886 can be available from Biomol Research Laboratories, Inc. (PlymouthMeeting, PA, Catalog #EI-266), and chemical abstracts registry no is CAS#118414-82-7.Its structure as shown in Figure 5.This result of experiment as shown in Figure 6.These results have shown the concentration-response curve of this mPGES test, and clearly illustrate that, the method can be used for checking certain compound or reagent to reduce even suppress the ability of mPGES activity.
Example II
The fluorescence polarization assay of hematopoietic prostaglandin D synthase (hPGDS)
The antigenicity thorn is goaded into action in the tracheae anaphylactia and is increased PGD
2Generation.Because hematopoietic prostaglandin D synthase (hPGDS) makes PGH
2Be converted into PGD
2And the PGD that produces
2, both combine with the chemokine receptors (CRTH2) of D type prostaglandin receptor (DP) and Th2 cell, and increase bronchiostenosis, vasodilation and schneiderian membrane expansion.Bronchus hyperfunction, rhinostegnosis and the eosinocyte and the Th2 cellular infiltration that cause therefrom cause allergic reaction.So the compound or the reagent of reduction or inhibition hPGDS activity can use easily as therapeutic agent.
Fluorescence polarization (FP) test (Fig. 3 and 4) to measuring h PGDS activity is also studied.This test designs according to the fluorescence polarization principle.With hPGDS and PGH
2, glutathione and hatched together by compound or the reagent estimated.Through the incubation period (about 30 seconds) of a weak point, add and contain FeCl
2Stop buffer (20mM) is with any remaining PGH of cancellation
2, otherwise it is PGD with spontaneous nuclear transformation
2And PGE
2Potpourri, therefore and disturb PGH
2To PGD
2The quantitative test of Enzymatic transformation (Fig. 3).Then, add the tracer agent (PGD that contains fluorescence labeling (Texas is red)
2) and anti-PGD
2Detection of antibodies solution is to produce and PGD
2The distinctive signal (Fig. 4) that is inversely proportional to of generation.PGD from this enzymatic reaction generation
2With this antibody of special competition, and discharge the fluorescence labeling tracer agent.For the above reasons, PGD
2The decline of synthase activity or inhibition will cause the increase of fluorescence polarization (FP) value.
Material
The expression of hPGDS
People's hematopoiesis PGD
2Synthase is to adopt to express according to bacterial expression system and step described in the following document: Kanaoka etc. (Structure and Chromosomal Localization ofHuman and Mouse Genes for Hematopoietic Prostaglandin D Synthase.Eur.J.Biochem.267:3315-3322 (2000)).Encode the nucleotide sequence of this enzyme shown in Figure 10 A and SEQ ID NO:3.Increase this coding nucleotide sequence and insert the pT7-7 carrier is used for the cell of transformed into escherichia coli FL21 (DE3) bacterial strain then.Sulfo--β-D-the galactoside that with ultimate density is 0.6mM then adds this cell transformed, to induce the production of people hPGDS enzyme.People hPGDS carries out purifying with GSH-agarose (Sepharose) 4B column chromatography.The amino acid sequence of used people hPGDS is shown in Figure 10 B and SEQ ID NO:4 among this embodiment.
GSH：
Available from Sigma (products catalogue #G-6529).
PGH
2：
Available from Caymen Chemicals, Inc. (Ann Arbor, Michigan) (products catalogue #17020).
Anti-PGD
2Antibody:
Monoclonal anti PGD
2Antibody is available from Institute Pasteur (catalog number (Cat.No.) 0465328).
PGD
2：
Available from Caymen Chemicals, Inc. (Ann Arbor, Michigan) (products catalogue #12010).
Fluorescently-labeled prostaglandin product P GD
2
As the foregoing description 1, use Texas red as fluorescent marker.PGD with Texas's red marker
2(PGD
2) be that (San Mateo, California) company generates Combinix.The mechanism that generates this a part is as described below:
PGD
2Tracer agent is synthetic
In this building-up process, with the PGD in the red cadaverine of Texas (Molecular Probes company product) the adding anhydrous methylene chloride
2(Cayman Chemicals) solution.Add dicyclohexylcarbodiimide (Sigma Aldrich), under nitrogen atmosphere in dark reaction stirred 24 hours.Carry out purifying by the reverse hplc chromatography, make water/acetonitrile gradient and with 0.05%TFA as modifier.Certainly, used linkers can change.
Method
Except used enzyme is hPGDS, used identical of the method and example I, the prostaglandin product is PGD
2Therefore, as mentioned above, with containing K
2HPO
4And KH
2PO
4Reaction buffer dilute this enzyme and GSH, with the preparation enzyme solutions.The enzyme solutions that then compound to be measured or reagent is added this phosphate-buffered.Randomly, also can further hatch this solution.This incubation period, can be from more than a few minutes to one hour.In this specific embodiment, hatched this enzyme solutions about 30 minutes.
Then with substrate PGH
2Acetone soln place in another container of 4 ℃.Again enzyme solutions is added and contain PGH
2Container with begin the reaction.Hatch this potpourri about 30 seconds.Then, will contain FeCl
2Add this potpourri with the stop buffer of citric acid, in case any remaining PGH
2Spontaneous nuclear transformation is PGE
2Or PGD
2Then, will contain with PGD
2For immunogenic antibody with the PGD of Texas's red marker
2The detection solution of (tracer agent) adds this potpourri, and hatches whole potpourri.In this embodiment, hatched this potpourri about 120 minutes.But other incubation period described in this incubation period and this example, all can change, and depends on the concentration of reagent.
Prepare the control mixture identical, and handle this control mixture, that is to say, hatched the same time with the same manner that contains enzymatic mixture with processing with containing enzymatic mixture, or the like.But this control mixture does not contain compound to be evaluated or reagent.Then, with wavelength is the whole potpourri of plane polarization rayed and the control mixture of 580nm (being the red wavelength that is excited of Texas), and measures the fluorescence polarization value of this potpourri and control mixture with the fluorescence filter that excitation wavelength and emission wavelength are set at 580nm and 620nm respectively.Be at this surveying instrument under the situation of FP pattern and measure.Nature, used wavelength will depend on the fluorescent marker that the method is used.Then this twice FP measured value compared, with its fluorescence polarization measurement value of potpourri of determining to contain this compound or reagent whether greater than the fluorescence measurement value of contrast solution.If the measured value of this potpourri shows then that greater than the measured value of control mixture this compound or reagent have reduced the activity of hPGDS.
The result
Above-mentioned test has obtained checking by using a kind of known hPGDS inhibitor HQL79.HQL79 is existing narration in WO 95/01350 patent.The structure of HQL79 as shown in Figure 7.This result of experiment as shown in Figure 8.These results clearly illustrate that method of the present invention finds that HQL79 has reduced the activity of hPGDS.
Conclusion
Example I and II show significantly, method of the present invention is simple, do not need numerous washing steps or radioactive isotope, and can easily be used for high-throughout mode, be used for determining whether a kind of compound or reagent can reduce or suppress the especially activity of hPGDS or mPGES of prostaglandin synthase.
Scope of the present invention will not be subjected to the restriction of specific specific embodiments described herein.Really, except content described herein, by above narration and accompanying drawing, various improvement of the present invention will become clearly for being familiar with this operator.Such improvement is intended to be included within the appended claim scope.
This paper has quoted various publications, and their disclosure as a reference and integral body is incorporated in this.
Sequence table
＜110〉Aventis Pharmaceuticals, Inc.
＜120〉detection compound or reagent reduce the method for the ability of mPGES or hematopoietic prostaglandin D synthase activity
<130>USAV2002/0098?WOPCT
＜140〉do not obtain
<141>2003-08-15
<150>US?60/404,008
<151>2002-08-16
<150>GB?0229244.9
<151>2002-12-16
<160>4
＜170〉PatentIn version 3 .2
<210>1
<211>459
<212>DNA
＜213〉people (Homo sapiens)
<400>1
atgcctgccc?acagcctggt?gatgagcagc?ccggccctcc?cggccttcct?gctctgcagc 60
acgctgctgg?tcatcaagat?gtacgtggtg?gccatcatca?cgggccaagt?gaggctgcgg 120
aagaaggcct?ttgccaaccc?cgaggatgcc?ctgagacacg?gaggccccca?gtattgcagg 180
agcgaccccg?acgtggaacg?ctgcctcagg?gcccaccgga?acgacatgga?gaccatctac 240
cccttccttt?tcctgggctt?cgtctactcc?tttctgggtc?ctaacccttt?tgtcgcctgg 300
atgcacttcc?tggtcttcct?cgtgggccgt?gtggcacaca?ccgtggccta?cctggggaag 360
ctgcgggcac?ccatccgctc?cgtgacctac?accctggccc?agctcccctg?cgcctccatg 420
gctctgcaga?tcctctggga?agcggcccgc?cacctgtga 459
<210>2
<211>152
<212>PRT
＜213〉people
<400>2
Met?Pro?Ala?His?Ser?Leu?Val?Met?Ser?Ser?Pro?Ala?Leu?Pro?Ala?Phe
1 5 10 15
Leu?Leu?Cys?Ser?Thr?Leu?Leu?Val?Ile?Lys?Met?Tyr?Val?Val?Ala?Ile
20 25 30
Ile?Thr?Gly?Gln?Val?Arg?Leu?Arg?Lys?Lys?Ala?Phe?Ala?Asn?Pro?Glu
35 40 45
Asp?Ala?Leu?Arg?His?Gly?Gly?Pro?Gln?Tyr?Cys?Arg?Ser?Asp?Pro?Asp
50 55 60
Val?Glu?Arg?Cys?Leu?Arg?Ala?His?Arg?Asn?Asp?Met?Glu?Thr?Ile?Tyr
65 70 75 80
Pro?Phe?Leu?Phe?Leu?Gly?Phe?Val?Tyr?Ser?Phe?Leu?Gly?Pro?Asn?Pro
85 90 95
Phe?Val?Ala?Trp?Met?His?Phe?Leu?Val?Phe?Leu?Val?Gly?Arg?Val?Ala
100 105 110
His?Thr?Val?Ala?Tyr?Leu?Gly?Lys?Leu?Arg?Ala?Pro?Ile?Arg?Ser?Val
115 120 125
Thr?Tyr?Thr?Leu?Ala?Gln?Leu?Pro?Cys?Ala?Ser?Met?Ala?Leu?Gln?Ile
130 135 140
Leu?Trp?Glu?Ala?Ala?Arg?His?Leu
145 150
<210>3
<211>600
<212>DNA
＜213〉people
<400>3
atgccaaact?acaaactcac?ttattttaat?atgaggggga?gagcagaaat?tattcgttac 60
atatttgctt?atttggacat?acagtatgaa?gaccacagaa?tagaacaagc?tgactggcct 120
gaaatcaaat?caactctccc?atttggaaaa?atccccattt?tggaagttga?tggacttact 180
cttcaccaga?gcctagcaat?agcaagatat?ttgaccaaaa?acacagattt?ggctggaaac 240
acagaaatgg?aacaatgtca?tgttgatgct?attgtggaca?ctctggatga?tttcatgtca 300
tgttttcctt?gggcagagaa?aaagcaagat?gtgaaagagc?agatgttcaa?tgagctgctc 360
acgtataatg?cgcctcatct?tatgcaagac?ttggacacat?atttaggggg?gagagaatgg 420
cttattggta?actctgtaac?ttgggcagac?ttctactggg?agatttgcag?taccacactt 480
ttggtcttta?agcctgacct?gttagacaac?catccaaggc?tggtgacttt?acggaagaaa 540
gtccaagcca?ttcctgccgt?cgctaactgg?ataaaacgaa?ggccccaaac?caaactctag 600
<210>4
<211>199
<212>PRT
＜213〉people
<400>4
Met?Pro?Asn?Tyr?Lys?Leu?Thr?Tyr?Phe?Asn?Met?Arg?Gly?Arg?Ala?Glu
1 5 10 15
Ile?Ile?Arg?Tyr?Ile?Phe?Ala?Tyr?Leu?Asp?Ile?Gln?Tyr?Glu?Asp?His
20 25 30
Arg?Ile?Glu?Gln?Ala?Asp?Trp?Pro?Glu?Ile?Lys?Ser?Thr?Leu?Pro?Phe
35 40 45
Gly?Lys?Ile?Pro?Ile?Leu?Glu?Val?Asp?Gly?Leu?Thr?Leu?His?Gln?Ser
50 55 60
Leu?Ala?Ile?Ala?Arg?Tyr?Leu?Thr?Lys?Asn?Thr?Asp?Leu?Ala?Gly?Asn
65 70 75 80
Thr?Glu?Met?Glu?Gln?Cys?His?Val?Asp?Ala?Ile?Val?Asp?Thr?Leu?Asp
85 90 95
Asp?Phe?Met?Ser?Cys?Phe?Pro?Trp?Ala?Glu?Lys?Lys?Gln?Asp?Val?Lys
100 105 110
Glu?Gln?Met?Phe?Asn?Glu?Leu?Leu?Thr?Tyr?Asn?Ala?Pro?His?Leu?Met
115 120 125
Gln?Asp?Leu?Asp?Thr?Tyr?Leu?Gly?Gly?Arg?Glu?Trp?Leu?Ile?Gly?Asn
130 135 140
Ser?Val?Thr?Trp?Ala?Asp?Phe?Tyr?Trp?Glu?Ile?Cys?Ser?Thr?Thr?Leu
145 150 155 160
Leu?Val?Phe?Lys?Pro?Asp?Leu?Leu?Asp?Asn?His?Pro?Arg?Leu?Val?Thr
165 170 175
Leu?Arg?Lys?Lys?Val?Gln?Ala?Ile?Pro?Ala?Val?Ala?Asn?Trp?Ile?Lys
180 185 190
Arg?Arg?Pro?Gln?Thr?Lys?Leu
195
Claims (28)
1. be used for determining whether compound or reagent can reduce prostaglandin synthase and substrate reactions forms this active method of prostaglandin product, wherein said prostaglandin synthase is selected from mPGES (mPGES) and hematopoietic prostaglandin D synthase (hPGDS), said method comprising the steps of:
(a) with prostaglandin synthase and substrate, co-factor and described compound or reagent mix;
(b) potpourri of step (a) is hatched the reagent that described stop buffer contains and stops described substrate spontaneous nuclear transformation is the prostaglandin product with stop buffer;
(c) potpourri of step (b) is hatched with detectable, described detectable contain with the prostaglandin product of fluorescence labeling substance markers and with this prostaglandin product as immunogenic antibody;
(d) with the potpourri and the control mixture of linearly polarized light irradiating step (c), wherein said plane polarization light wavelength can the fluorescence excitation label, and the potpourri of measuring process (c) and the fluorescence polarization value of control mixture; And
(e) measurement result of comparison step (d),
Wherein, show that then this compound or reagent have reduced the activity of prostaglandin synthase when the fluorescence polarization measurement value of the fluorescence polarization measurement value of finding potpourri (c) greater than control mixture.
2. according to the process of claim 1 wherein that described prostaglandin synthase is mPGES (mPGES), substrate is a PGH
2(PGH
2), co-factor is glutathione (GSH), the prostaglandin product is a PGE
2(PGE
2).
3. according to the method for claim 2, wherein said mPGES is people mPGES, and it contains the amino acid sequence of SEQ ID NO:2.
4. according to the process of claim 1 wherein that described prostaglandin synthase is hematopoietic prostaglandin D synthase (hPGDS), substrate is PGH
2, co-factor is glutathione (GSH), the prostaglandin product is a PGD
2(PGD
2).
5. according to the method for claim 4, wherein said hematopoietic prostaglandin D synthase is the artificial blood Prostaglandin D synthase, and it contains the amino acid sequence of SEQ ID NO:4.
6. according to the process of claim 1 wherein that the reagent of described stop buffer is FeCl
2
7. according to the process of claim 1 wherein that described fluorescent marker comprises fluorescein, phycoerythrin (PE), Texas red (TR), rhodamine, free group of the lanthanides salt, group of the lanthanides salt, BODIPY, ALEXA or the CyDye of chelating.
8. according to the method for claim 7, wherein said fluorescent marker is Texas red (TR).
9. according to the method for claim 2, the reagent of wherein said stop buffer is FeCl
2
10. according to the method for claim 9, the duration of wherein said incubation step (b) is at least 30 seconds, and the duration of incubation step (c) is at least 3 minutes.
11. according to the method for claim 10, wherein said fluorescent marker comprises fluorescein, phycoerythrin (PE), Texas red (TR), rhodamine, free group of the lanthanides salt, group of the lanthanides salt, BODIPY, ALEXA or the CyDye of chelating.
12. according to the method for claim 11, wherein said fluorescent marker is Texas red (TR), it is 580 ± 20nm that plane polarization excites light wavelength.
13. according to the method for claim 4, the reagent of wherein said stop buffer is FeCl
2
14. according to the method for claim 13, wherein said fluorescent marker comprises fluorescein, phycoerythrin (PE), Texas red (TR), rhodamine, free group of the lanthanides salt, group of the lanthanides salt, BODIPY, ALEXA or the CyDye of chelating.
15. according to the method for claim 14, wherein said fluorescent marker is Texas red (TR), and plane polarization to excite light wavelength be 580 ± 20nm.
16. be used for determining whether compound or reagent can reduce prostaglandin synthase and substrate reactions forms this active method of prostaglandin product, wherein said prostaglandin synthase is selected from hematopoietic prostaglandin D synthase (hPGDS) and mPGES (mPGES), said method comprising the steps of:
(a) with prostaglandin synthase and substrate, co-factor and described compound or reagent mix;
(b) potpourri of step (a) is hatched with stop buffer, wherein said stop buffer contains and prevents that unreacted substrate spontaneous nuclear transformation from being the reagent of prostaglandin product;
(c) potpourri of step (b) is hatched with detectable, wherein said detectable contain with the prostaglandin product of Texas's red marker and with the prostaglandin product as immunogenic antibody;
(d) be the potpourri and the control mixture of the linearly polarized light irradiating step (c) of 580 ± 20nm with wavelength, and at the potpourri of 620 ± 20nm measuring process (c) and the fluorescence polarization of control mixture; And
(e) measurement result of comparison step (d),
Wherein work as the fluorescence polarization measurement value of the fluorescence polarization measurement value of the potpourri of finding step (c), show that then this compound or reagent have reduced the activity of prostaglandin synthase greater than control mixture.
17. according to the method for claim 16, wherein said prostaglandin synthase is people's mPGES (mPGES), it contains the amino acid sequence of SEQ ID NO:2, and substrate is a PGH
2(PGH
2), co-factor is a glutathione, the prostaglandin product is a PGE
2(PGE
2).
18. according to the method for claim 17, the reagent of wherein said stop buffer is FeCl
2
19. according to the method for claim 18, wherein with the described prostaglandin product of Texas's red marker comprise one with this prostaglandin product and the red linkers that is connected of Texas.
20. according to the method for claim 19, wherein said linkers is selected from aminobutyric acid, aminocaproic acid, 7-aminoheptylic acid, 8-aminocaprylic acid, Fmoc-aminocaproic acid, one or more Beta-alanine, isothiocyanate group, succinimide ester, halo sulphonal and carbodiimide.
21. according to the method for claim 20, wherein said linkers is a carbodiimide.
22. according to the method for claim 16, wherein said prostaglandin synthase is artificial blood Prostaglandin D synthase (hPGDS), it contains the amino acid sequence of SEQ ID NO:4, and substrate is PGH
2, co-factor is a glutathione, the prostaglandin product is a PGD
2(PGD
2).
23. according to the method for claim 22, the reagent of wherein said stop buffer is FeCl
2
24. according to the method for claim 23, wherein with the described prostaglandin product of Texas's red marker comprise one with this prostaglandin product and the red linkers that is connected of Texas.
25. according to the method for claim 24, wherein said linkers is selected from aminobutyric acid, aminocaproic acid, 7-aminoheptylic acid, 8-aminocaprylic acid, Fmoc-aminocaproic acid, one or more Beta-alanine, isothiocyanate group, succinimide ester, halo sulphonal and carbodiimide.
26. according to the method for claim 25, wherein said linkers is a carbodiimide.
27. be used for determining whether compound or reagent can reduce people's mPGES (mPGES) and its PGH
2(PGH
2) substrate reactions and form PGE
2(PGE
2) this active method, wherein said people's mPGES contains the amino acid sequence of SEQ ID NO:2, said method comprising the steps of:
(a) with mPGES and PGH
2, glutathione and described compound or reagent mix;
(b) with the potpourri of step (a) with contain FeCl
2Stop buffer hatch together;
(c) potpourri of step (b) is hatched with detectable, described detectable contains the PGE with Texas's red marker
2And with PGE
2As immunogenic antibody;
(d) with wavelength be the potpourri and the control mixture of the linearly polarized light irradiating step (c) of 580 ± 20nm, and the potpourri of measuring process (c) and the fluorescence polarization value of control mixture; And
(e) measurement result of comparison step (d),
Wherein work as the fluorescence polarization measurement value of the fluorescence polarization measurement value of the potpourri of finding step (c), show that then this compound or reagent have reduced the activity of mPGES greater than control mixture.
28. be used for determining whether compound or reagent can reduce artificial blood Prostaglandin D synthase (hPGDS) and its PGH
2(PGH
2) substrate reactions formation PGD
2(PGD
2) this active method, wherein said artificial blood Prostaglandin D synthase contains the amino acid sequence of SEQ ID NO:4, said method comprising the steps of:
(a) with hPGDS and PGH
2, glutathione and described compound or reagent mix;
(b) with the potpourri of step (a) with contain FeCl
2Stop buffer hatch together;
(c) potpourri of step (b) is hatched with detectable, described detectable contains the PGD with Texas's red marker
2And with PGD
2As immunogenic antibody;
(d) with wavelength be the potpourri and the control mixture of the linearly polarized light irradiating step (c) of 580 ± 20nm, and the potpourri of measuring process (c) and the fluorescence polarization value of control mixture; And
(e) measurement result of comparison step (d),
Wherein work as the fluorescence polarization measurement value of the fluorescence polarization measurement value of the potpourri of finding step (c), show that then this compound or reagent have reduced the activity of hPGDS greater than control mixture.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40400802P | 2002-08-16 | 2002-08-16 | |
| US60/404,008 | 2002-08-16 | ||
| GB0229244.9 | 2002-12-16 | ||
| GBGB0229244.9A GB0229244D0 (en) | 2002-08-16 | 2002-12-16 | Method for assaying compounds for inhibition of the activity of prostaglandin synthase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1675543A true CN1675543A (en) | 2005-09-28 |
Family
ID=31889687
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA038193957A Pending CN1675543A (en) | 2002-08-16 | 2003-08-15 | Method for assaying compounds or agents for ability to decrease the activity of microsomal prostaglandin E synthase or hematopoietic prostaglandin D synthase |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP1543327A2 (en) |
| JP (1) | JP2006511789A (en) |
| KR (1) | KR20050035885A (en) |
| CN (1) | CN1675543A (en) |
| AU (1) | AU2003258278A1 (en) |
| CA (1) | CA2495391A1 (en) |
| IL (1) | IL166782A0 (en) |
| RU (1) | RU2005107329A (en) |
| WO (1) | WO2004016223A2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254655A (en) * | 2015-11-20 | 2016-01-20 | 江汉大学 | Fluorescent amino acid based on BODIPY as well as synthetic method and application thereof |
| CN109781990A (en) * | 2018-12-25 | 2019-05-21 | 无锡市人民医院 | A kind of β-trace protein detection kit and preparation method |
| CN113174424A (en) * | 2021-03-15 | 2021-07-27 | 合肥康诺生物制药有限公司 | Method for detecting enzyme activity in aerobic enzymatic reaction and method for judging fermentation end point of recombinant escherichia coli |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4746616B2 (en) | 2004-06-30 | 2011-08-10 | アベンティス・ファーマスーティカルズ・インコーポレイテツド | Methods involving microsomal prostaglandin E2 synthase |
| EP1882937B1 (en) | 2005-05-17 | 2012-02-22 | Taiho Pharmaceutical Co., Ltd. | Method for diagnosis of severity and prediction of recurrence in eosinophilic inflammatory disease |
| RU2464262C2 (en) | 2007-03-30 | 2012-10-20 | Санофи-Авентис | Pyrimidine hydrazide compounds as pgds inhibitors |
| KR100913148B1 (en) * | 2007-04-10 | 2009-08-19 | 이금필 | Magnetic force-based bio sensor comprising magnetic particles |
| CA2722420A1 (en) * | 2008-05-13 | 2009-11-19 | Cayman Chemical Company, Incorporated | Methods for assaying compounds or agents for ability to displace potent ligands of hematopoietic prostaglandin d synthase |
| CN106943407A (en) | 2010-07-26 | 2017-07-14 | 赛诺菲 | Purposes of the Ben oxadiazole derivatives in the medicine for the treatment of allergia or inflammatory disease is prepared |
-
2003
- 2003-08-15 JP JP2004529521A patent/JP2006511789A/en active Pending
- 2003-08-15 WO PCT/US2003/025766 patent/WO2004016223A2/en not_active Application Discontinuation
- 2003-08-15 CA CA002495391A patent/CA2495391A1/en not_active Abandoned
- 2003-08-15 IL IL16678203A patent/IL166782A0/en unknown
- 2003-08-15 AU AU2003258278A patent/AU2003258278A1/en not_active Abandoned
- 2003-08-15 RU RU2005107329/13A patent/RU2005107329A/en not_active Application Discontinuation
- 2003-08-15 CN CNA038193957A patent/CN1675543A/en active Pending
- 2003-08-15 EP EP03788590A patent/EP1543327A2/en not_active Withdrawn
- 2003-08-15 KR KR1020057002538A patent/KR20050035885A/en not_active Application Discontinuation
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254655A (en) * | 2015-11-20 | 2016-01-20 | 江汉大学 | Fluorescent amino acid based on BODIPY as well as synthetic method and application thereof |
| CN105254655B (en) * | 2015-11-20 | 2017-03-22 | 江汉大学 | Fluorescent amino acid based on BODIPY as well as synthetic method and application thereof |
| CN109781990A (en) * | 2018-12-25 | 2019-05-21 | 无锡市人民医院 | A kind of β-trace protein detection kit and preparation method |
| CN113174424A (en) * | 2021-03-15 | 2021-07-27 | 合肥康诺生物制药有限公司 | Method for detecting enzyme activity in aerobic enzymatic reaction and method for judging fermentation end point of recombinant escherichia coli |
| CN113174424B (en) * | 2021-03-15 | 2023-05-26 | 合肥康诺生物制药股份有限公司 | Method for detecting enzyme activity in aerobic enzymatic reaction and method for judging fermentation end point of recombinant escherichia coli |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003258278A1 (en) | 2004-03-03 |
| WO2004016223A3 (en) | 2004-10-21 |
| RU2005107329A (en) | 2005-08-27 |
| EP1543327A2 (en) | 2005-06-22 |
| WO2004016223A2 (en) | 2004-02-26 |
| JP2006511789A (en) | 2006-04-06 |
| IL166782A0 (en) | 2006-01-15 |
| CA2495391A1 (en) | 2004-02-26 |
| KR20050035885A (en) | 2005-04-19 |
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