WO2002052262A1

WO2002052262A1 - Method and kit for screening substrate of abc protein

Info

Publication number: WO2002052262A1
Application number: PCT/JP2001/011399
Authority: WO
Inventors: Toshihisa Ishikawa
Original assignee: The Circle For The Promotion Of Science And Engineering
Priority date: 2000-12-25
Filing date: 2001-12-25
Publication date: 2002-07-04
Also published as: JPWO2002052262A1

Abstract

A method for screening a substrate of an ATP-binding cassette (ABC) protein characterized by comprising mixing and incubating a cell membrane fraction with the expression of the ABC protein, a labeled ATP, vanadic acid and a test sample and adding the resultant mixture to a support having an antibody against the ABC protein immobilized thereon, or mixing and incubating the labeled reagent, the test sample, etc. on the support, and then assaying the immobilized label or the unimmobilized label; and a kit for screening a substrate or an inhibitor of the ABC protein which contains a cell membrane fraction with the expression of the ABC protein, a labeled ATP or its derivative, and a support having an antibody against the ABC protein immobilized thereon, or an antibody against the ABC protein and a support.

Description

Specification

FIELD OF THE INVENTION Field of the Invention Screening method and kit for substrates of ABC proteins

The present invention relates to a method for screening a substrate and an inhibitor for an ATP-binding cassette (ABC) protein. Background art

Along with drug metabolizing enzymes, ABC proteins (or ABC transporters) define the pharmacokinetic profile (absorption, distribution, metabolism, excretion, effective drug concentration at the target site) of the drug, and thus the overall drug. It also affects the pharmacological effect. For example, ABC transporters expressed in small intestinal epithelial cells and cerebrovascular endothelial cells have a significant effect on the bioavailability of orally administered drugs and drug transfer to the central nervous system. On the other hand, in the field of cancer chemotherapy, it is well known that overexpression of P-glycoprotein and MRP1 makes cancer cells resistant to anticancer drugs. Both P-glycoprotein and MRP1 belong to the ABC protein family. At present, the human ABC protein family consists of about 50 members, many of whose genes have been identified and their sequences clarified. In addition, ABC proteins are widely found in non-human animals, as well as in plants, yeast, and microorganisms. However, an automatic device for screening the substrate specificity of ABC protein has not yet been developed. Nucleotide sequence analysis of human and animal and plant genomes has progressed, and novel ABC proteins have been discovered based on sequence homology, but their functions are largely unknown.

ABC protein acts as a transporter and transports the drug through the cell membrane The sending mechanism is considered as follows.

(1) The ABC protein is present in the membrane across both sides (inside and outside of the cell) of the cell membrane.

(2) One molecule of ATP as an energy source and one molecule of a drug to be transported are bound to the ABC protein from the inside of the cell, and ATP is converted to ATP by the ATPase activity of the ABC protein. The drug is transported from the inside to the outside of the cell due to the hydrolysis of the ABC protein by the energy released at this time.

(3) The drug is released outside the cells, and the phosphoric acid generated in (2) is also released from the ABC protein.

(4) A new ATP molecule binds to the ABC protein from the inside of the cell, this ATP is decomposed into ADP and phosphoric acid, and the energy is released. This energy causes the ABC transformed in (2) above. The shape of the protein is restored.

(5) The ADP and phosphoric acid generated in the above (4) are released from the ABC protein, and the transport system returns to the state of the above (1).

In the above cycle, ATP binds to the ABC protein and is decomposed into ADP due to the presence of the drug to be transported in the above step (2), so that if ADP bound to the ABC protein is measured, Drugs recognized by ABC proteins can be detected. Therefore, by labeling ATP and measuring the labeled ABC protein (that is, the ABC protein to which the labeled ADP is bound), it is possible to detect the drug or inhibitor transported by the ABC protein. Can be.

However, it is practically difficult to measure labeled ABC protein because the above-mentioned transport system is in a dynamic state due to enzyme rotation. Therefore, if panadic acid is present in the transit system, the panadic acid is replaced with phosphoric acid in the step (2), and the rotation of the transit system stops, The labeled ADP remains bound to the ABC protein, and the label can be measured.

Conventionally, in this measurement, the membrane protein and ABC protein are released from the cell membrane, the released ABC protein is separated from other proteins by electrophoresis, and the label bound to the ABC protein (that is, labeled A DP) was measured. According to this method, it is possible to determine whether a test drug is a substrate or an inhibitor of ABC protein, but it is not possible to test a large amount of a test substance in a short time. Disclosure of the invention

Therefore, the present invention provides a method that allows a large number of test drugs to be tested in a short period of time as to whether or not they are substrates or inhibitors of these potent BC proteins.

The present inventors have conducted various studies to solve the above-mentioned problems, and as a result, have confirmed that it is possible to examine the presence or absence of labeling of ABC protein by introducing an immunological technique, and completed the present invention.

Accordingly, the present invention provides a method for screening a substrate or an inhibitor for an ATP-binding cassette (ABC) protein, comprising: a membrane fraction of cells expressing an ABC protein; labeled ATP or a derivative thereof; After mixing and incubating the test sample and performing a photoaffinity reaction, the mixture is added to a support on which an antibody against ABC protein is immobilized, and the immobilized label or non-immobilized label is immobilized. A method for measuring a mark.

The present invention further provides a method for screening a substrate or an inhibitor for ABC protein without using a photoaffinity reaction, wherein a membrane fraction of cells expressing ABC protein is immobilized with an antibody against ABC protein. The labeled ATP or its derivative and Mixing and incubating nadic acid and a test sample, and measuring the captured label or the non-captured label. The method of measuring without using the photoaffinity reaction has an advantage of reducing the measurement cost because ATP or a derivative thereof having no photoaffinity reactive group can be used.

The present invention further comprises a cell membrane fraction expressing ABC protein, labeled ATP or a derivative thereof, and a support on which an antibody against ABC protein is immobilized, or an antibody against ABC protein and a support. A kit for screening a substrate or an inhibitor for the ATP protein, comprising the kit. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bluff showing the appropriate amounts of doxorubidin and anamycin as substrates for P-glycoprotein according to the method of the present invention. Fig. 2 shows the immobilized monoclonal antibody specific to P-glycoprotein, and the iminotiter plate immobilized with a membrane protein, and the addition of membrane proteins to immobilize the P-glycoprotein on the iminotiter plate support. The results of verification by Western Plot method whether or not they were converted are shown. The P-glycoprotein was detected by enzymatic chemiluminescence (ECL). P-glycoprotein was detected only in the sample in which the membrane fraction was added to the immotiter plate on which the antibody was immobilized.

FIG. 3 shows the results of recognition of the substrate verapamil by the P-glycoprotein by the vanadate trapping method using an imnotiter plate on which the cell membrane expressing the P-glycoprotein is immobilized.

Figure 4 shows the effect of verapamil concentration on the level of radiolabel captured in the presence and absence of panadic acid in a single iminotiter plate immobilized cell membrane expressing P-glycoprotein. . FIG. 5 shows the levels of radiolabel captured with a panadate trap in the presence of 30 μM verapamil, using iminotiter plates immobilized with antibodies specific for P-glycoprotein and those without.

Figure 6 shows that 20 μΜ of the test compound (verapamil, mouth-damine 123, quinidine, doxorubicin, para-amino-hippuric acid, sulfobromophthalate, This is the result of performing a vanadic acid trap reaction in the presence of (lane). The amount of radiolabel captured in the well was measured using a liquid scintillation counter, and the difference between the amount of radiolabel obtained in the presence (20 μμΜ) and the amount in the absence (非 μΜ) of sodium panadate was determined. It was calculated. The result is the average of three experiments; : Shown by standard deviation. Embodiment of the Invention

The ABC protein transports substances and controls channels by using the energy obtained by hydrolysis of ATP as a driving force. The ABC protein has a characteristic ATP-binding site, and its primary structure is well maintained throughout the evolution across ABC protein types and organisms. In particular, the ATP binding site of the ABC protein has a conserved amino acid residue sequence compared to the primary structure of the transmembrane portion and other parts.

The Walker A and Walker B motifs (Walker, JE, Saraste M Runswick, MJ, Gay, NJ (1982) EMBO J 1 945-951) and the ABC signature motif (Higgins, CF (1992) Annu. Rev. Cell Biol 8 67-113) (http: //www.expasy. Ch / prosite /) Force S Almost without amino acid residue sequence common to its ATP binding site. Based on the amino acid sequence of a protein known or newly discovered from previous studies, or the sequence of the gene or cDNA encoding it Thus, a protein having the above motif and a protein similar thereto are defined as an ABC protein (http: // www. Gene. Ucl. Ac. Uk./nomenclature/genefamily/abc. Html).

The motif is Walker A (Gly-X-X-Gly-X-Gly_Lys-Ser- [Ser,

Thr, Gin]) (SEQ ID NO: 1) and Walker B ([Leu, He, Phe]-[lie, Leu, Val] -X-Asp- [Glu, Asp, Ser]) (SEQ ID NO: 2) Yes and the following ABC signature ([Leu, He, Val, Met, Phe, Tyr, Cys]-[Ser, Ala]-[Ser, Ala, Pro, Gly, Leu, Val, Phe, Tyr, Lys, Gin, His]-Gly- [Asp, Glu, Asn, Gin, Met, Trp]-[Lys, Arg, Gin,

Ala, Ser, Pro, Cys, Leu, lie, Met, Phe, Trp]-[Lys, Ar, Asn, Gin, Ser, Thr, Ala, Val, Met]-[Leu, He, Val, Met, Phe, Tyr, Pro, Ala, Asn]-{Pro, His, Tyr}-[Leu, He, Val, Met, Phe, Trp]-[Ser, Ala, Gly, Cys, Leu, lie, Val, Phe]- {Phe, Tyr, Trp, His, Pro}-{Lys, Arg, His, Pro}-[Leu, He, Val, Met, Phe, Tyr, Trp, Ser, Thr, Ala] (X is unspecified (SEQ ID NO: 3).

There are about 50 types of human ABC proteins known at present (Reference: http: //www.numanabc.org/ and http://gene.ucl.ac. UK / nomenclature / genefamily / abc. html), and it is expected to increase further with the publication of the results of human genome analysis in the future. P-glycoprotein, MRP1, CFTR, SUR1, ABC1, TAP1, etc. are typical human ABC proteins. ABC proteins are widely found in bacteria, yeast, plants and animals. (Literature: Special feature on ABC protein Biochim. Biophys. Acta (1999) 1461: 175-419). The method of the present invention can be applied in principle to any ABC protein.

Preparation of membrane fraction

In carrying out the method of the present invention, it is necessary to prepare a cell membrane fraction in which the target ABC protein is present. Can be prepared. '

(1) Preparation of cells expressing ABC protein

The cDNA encoding the ABC protein is incorporated into an expression vector, and transfected into culture cells (mammalian cells, insect cells, yeast, bacteria, etc.). Use an expression vector that has a gene that confers resistance to a particular antibiotic. By culturing the transfected cells in the presence of an antibiotic, only cells having the ABC protein expression vector are selected. Confirm the expression of ABC protein by RT-PCR (reverse transcription-polymerase chain reaction) or specific antibody. Cultivate the ABC protein-expressing cells thus obtained in large quantities

(2) Preparation of membrane fraction from cells expressing ABC protein

The cells expressing the ABC protein are collected by centrifugation of the culture medium. The cells are disrupted by sonication or osmotic treatment, and the membrane fraction is collected by centrifugation. The membrane fraction is suspended in a buffer, and the cell membrane fraction is isolated and purified by sucrose concentration gradient centrifugation. After measuring the protein concentration of the cell membrane fraction, flash freeze at liquid nitrogen temperature and store at -80 degrees Celsius.

Labeling of ATP or its derivatives

The label for labeling ATP or a derivative thereof is not particularly limited as long as it is detectable, and examples thereof include a radioactive label, ³² P, ^'33 P, ³ H, ¹⁴ C, ³⁵ S, and the like. it can. ATP derivatives include [ ³⁵ S] ATPγS and the like.

In addition, examples of the photoaffinity label include an azide group and benzophenone. A specific example is 8-azido.

In order to bind the labeled ΑΤΡ or a derivative thereof to the ABC protein, conventional ultraviolet irradiation may be used. antibody

The anti-ABC protein antibody for capturing the ABC protein may be a polyclonal antibody or a monoclonal antibody. Polyclonal and monoclonal antibodies against a specific ABC protein or a fragment thereof can be prepared according to a conventional method.

Support

The support for immobilizing the antibody for capturing the ABC protein is not particularly limited as long as it can immobilize the antibody, and commercially available immotiter plates, affinity beads, and magnetic beads , Ferrite beads and the like can be used. The shape is not particularly limited, but for testing a large number of test samples at once, for example, a 98 or 384 well microtiter plate is preferable.

Measuring method

In the present invention, the ABC protein-containing membrane fraction, labeled ATP or its derivative, panadic acid, and a test sample are reacted in a reaction solution. As a reaction medium, a buffer such as a phosphate buffer method, Tris / HCl, MOPS, or the like is used, and the pH is preferably 6 to 8, particularly preferably 7 to 7.5. The reaction time is about 10 seconds to 3 hours, for example, about 1 minute to 30 minutes. The reaction is stopped by, for example, adding an excess amount (20 to 100 times the amount of the reaction solution) of the buffer solution cooled to 0 ° C to the reaction solution and mixing.

Next, the membrane fraction is separated from the reaction solution by a conventional method, for example, by centrifugation. Alternatively, pass through a molecular sieve (eg, Sephadex G25) or anion exchange resin (eg, DEAE Sephadex) column. As a result, unreacted ATP and vanadic acid in the reaction solution are removed from the membrane fraction. ATP or its derivative non-specifically attached to the membrane fraction, panadic acid, and the like can be further removed by washing, centrifugation, or the like. next The ABC protein bound to the membrane fraction is released from the membrane fraction. Free this, i.e. solubilization of membrane fraction, Ri by a conventional method, for example, surfactants such as Bok di Bokun X - 100, Tw e en20, Tw e en80, NP - 40, C 1 2 E Medicine _{_{_{1 2 E 8, C 1 2}}} E 9, it digitonin, CHAPS, ZW I TTERGENT, cholic acid diisocyanato Li c arm, be performed Ri by the treatment with Dokishikoru acid isocyanatomethyl Li um like.

Next, the sample containing the ABC protein released as described above is added to a support on which an antibody against the λBC protein is immobilized, for example, a well of an imunotiter plate, and the mixture is added at a temperature of 20 ° C to 37 ° C. The ABC protein is captured on the support by binding to the antibody immobilized on the support by incubating for a period of minutes to 12 hours. Next, after washing the support, labeled ADP bound to the ABC protein captured on the support, that is, ADP or a derivative thereof captured on the support via the binding to the ABC protein. Measure the label.

The method of measuring the label depends on the type of label. For example, a radioactive label can be measured or detected by a liquid scintillation counter, a beta counter, a phosphor imager, or the like, and a fluorescent label can be, for example, a fluorometer, an immunonotiter reader for fluorescence Can be detected or measured.

The method of the present invention is preferably performed using very small amounts of sample and reagents, such as in a microtiter plate well. In this case, it is not always the case that a fixed amount of the sample is added to the well, and this may cause a measurement or detection error. Therefore, in a preferred embodiment of the present invention, in addition to the immobilized or non-immobilized label (that is, the label-bound ABC protein), the amount of total ABC protein added to the well is measured. It is preferable to determine the relative amount of the immobilized label to the amount of white matter It is preferable to measure the total ABC protein by labeling the ABC protein with a fluorescent label, which includes (1) a method of directly reacting the ABC protein with a fluorescent probe, and (2) a fluorescent antibody method. is there. As probes for labeling the thiol group for use in the method (1), monobromobimane ^ monobromobimane, 5-iod oacetamide, fluorescein, 7 -fluoro 2,1, (3) Benzoxaziazol-l-4-ammonium sulfonate (ammonium 7-fiuoro-2,1,3-benzoxadiazole-4-sulfonate, or sifenorei eroyo doacetaside, 丄 ucifer yellow iodoacetami de).

Further, as a probe for labeling an amino group, 2 ′, Τ-difluorofluorescein (2 ′, 1′-difluorofluorescein) and the like can be mentioned.

In the fluorescent antibody method (2), the ABC protein is sandwich-fixed with a specific antibody on an immunotiter plate, and then labeled with a secondary antibody having a fluorescent probe.

The present invention also includes a measurement method that does not use a photoaffinity reaction, and the method is performed as follows.

Immobilization of antibody on support

Antibodies to the ABC protein (either polyclonal or monoclonal antibodies) are diluted to an appropriate concentration with phosphate-buffered saline (PBS), added to the wells of an immi- notiter plate, and added at 4 ° C. Alternatively, the antibody is immobilized on the support by allowing to stand at room temperature for 1 hour to 12 hours. Thereafter, the PBS solution containing the antibody is removed from the wells, and a blocking solution (for example, PBS containing bovine serum albumin or skim milk) is added to the wells, and left at 4 ° C or room temperature for 1 to 12 hours to support the wells. Block the part of the body where the antibody is not immobilized. And then And remove the blocking solution.

Immobilization of cell membrane expressing ABC protein

After diluting the membrane fraction prepared from ABC protein-expressing cells with a buffer solution to an appropriate protein concentration, add the antibody to the immobilized plate of the imnotiter plate, and incubate at room temperature with shaking. To bind the antibody to the ABC protein. Then, the cell membrane is immobilized on the support by the bonding. The well is then washed to remove excess floating membrane fraction.

Measurement using a membrane fraction immobilized on a support

The cell membrane thus immobilized on the support is mixed with panadic acid, radiolabeled ATP or its derivative, and a test sample, and incubated at room temperature. Incubation time is about 1 minute to 1 hour. Then remove the reaction solution and wash the wells with PBS solution. Then measure the amount of radiolabel captured in the well. This measurement can be performed using any of a liquid scintillation counter, a beta counter, and a phosphor imager. Example

Next, the present invention will be described more specifically with reference to examples.

P-glycoprotein is known as one of the ABC proteins. This protein has the effect of transporting certain anticancer drugs extracellularly in tumor cells, which is one of the mechanisms of drug resistance of cancer cells to anticancer drugs. For example, doxorubicin, an anticancer drug 0H

Is a substrate for P-glycoprotein and is transported extracellularly by P-glycoprotein.

This makes the cancer cells resistant to doxorubicin. On the other hand, anamycin which modified the structure of doxorubicin

Does not become a substrate for P-glycoprotein, and therefore, cancer cells do not show resistance to anamycin (Priebe, W and Perez- Soler, R. (1993) Pharmac. Ther. 60: 215-234).

Example 1.

Thus, as a model experiment, substrate specificity of doxorubicin and anamycin for P-glycoprotein was examined using P-glycoprotein as ABC protein.

1) Preparation of cell membrane fraction

Multidrug-resistant KB-C2.5 cells overexpressing P-glycoprotein (Akiyama, S. et al. (1985) Somat. Cell Mol. Genet. 11: 117-126) or P-glycoprotein expression NIH3T3 cells (Currier, SJ et al. (1989) J. Biol. Chem. 264: 14376-14381) transfected into cells are disrupted by osmotic treatment, and the cell membrane fraction is subjected to a sucrose gradient. It was prepared by centrifugation (100,000 X g 4 ° C, 20 minutes).

2) ^[32 P] - 8- azido-ATP photoaffinity Raberi ring

The obtained cell membrane fraction (200 g protein) was combined with doxorubicin or anamycin (0-: LOO / zM concentration) as a test compound in 5 / xl of a vanadate-ATP trap solution (200 panatonate). Li um, 20 mu M ^[32 P] _{_8- azido - ATP, 3mM MgS0 4} 50mM Tr is / HCl (pH7.5), and incubated 100 M EGTA, in 2mM Wapai down) 37 ° C, 10 minutes . Thereafter, the reaction was stopped by adding 400 μl of a Tris-EGTA solution (50 mM Tris / HCl (pH 7.5), ΙΟΟμΜ EGTA) to the reaction medium at high speed and force (Takada, Y. et al. (1998) Biochim. Biophys. Acta 1373: 13: 1-136).

^[32 P] -8 was centrifuged (15,000 X g, 10 minutes) with a photoaffinity labeled membrane fraction was solubilized in PBS containing Triton X- 1Q0 in Azido_ATP.

The supernatant fraction was collected, and monobromobimane (50 mM DMS0 solution) was added to a final concentration of ImM. The sample was incubated at room temperature for 30 minutes to fluorescently label the membrane protein. monobromobimane is a derivative with fluorescence by covalently binding to the SH group of the protein (Kosower, N: S (1979) Proc. Natl. Acad. Sci. USA 76: 3382-3386).

After that, the cysteine dissolved in the PBS solution containing Triton X-100 was heated, and unreacted monobromobimane was blocked.

The sample thus obtained is placed in a well of an immunotiter plate on which the C219 antibody (an antibody specific for P-glycoprotein) is immobilized, and incubated at room temperature or at 37 ° C. The antigen-antibody reaction proceeded. Thereafter, the wells were washed three times with a washing solution (20 mM sodium phosphate buffer (pH 7.2), 140 mM NaCl, 0.05% Triton X-100).

In order to determine the relative amount of P-glycoprotein bound to the antibody in the wells of the immunotiter plate, the fluorescence intensity of each well was measured in a plate reader under 370-385 nm excitation light. 477-484 nm). Then, after drying in the air, cut off the well and measure the radioactivity with a counter. By calculating the ratio of the radioactivity / fluorescence, per unit P- glycoprotein ^[32 P] - 8-azido- ATP amount is photoaffinity label was obtained accurately.

Figure 1 shows the obtained results. As can be seen, doxorubicin was a substrate for P-glycoprotein, whereas anamycin was not. Therefore, the method of the present invention reflects the properties of doxorubicin, which is transported extracellularly as a substrate for P-glycoprotein, and anamycin, which is not a substrate for P-glycoprotein and is not transported out of the cell. Was.

Example 2.

The substrate specificity for P-glycoprotein was measured by a method using a cell membrane fixed on the surface of a support with an antibody and without using a photophilic reaction. .

1) Immobilization of antibody +

Monoclonal antibodies against human P-glycoprotein were phosphate-buffered The mixture was diluted 100 times with saline (PBS), added to a well of an immunotiter plate, and allowed to stand at 4 ° C for 12 hours. Thereafter, the PBS solution containing the antibody was removed from the well, a blocking solution (PBS containing 5% bovine serum albumin) was added to the well, and the plate was allowed to stand at 4 ° C for 12 hours. Thereafter, the booking solution was removed from the well to prepare an immobilized antibody-immobilized plate.

2) Preparation of cell membrane fraction

From the human liver cDNA library, a cDNA encoding a P-glycoprotein was cloned, incorporated into a paculovirus, and expressed in large amounts in insect cells. The cells were disrupted by osmotic treatment, and the cell membrane fraction was prepared by sucrose gradient centrifugation (100,000 X g, 4 ° C, 20 minutes).

3) Immobilization of cell membrane

After diluting the membrane fraction with a buffer solution, the antibody was added to a well of an immobilized immobilizer plate, and incubated for 3 hours while shaking at room temperature. Thereafter, the wells were washed to remove excess floating membrane fraction.

4) Confirmation of immobilization of P-glycoprotein on the support

A solution of sodium lauryl sulphate was added to the immobilized immobilized plate to immobilize the cell membrane, and the membrane protein was solubilized and subjected to polyatomic amide electrophoresis. After that, the membrane protein was electrophoretically transferred to a western blotting membrane, and the P-glycoprotein was detected with a specific antibody.

Fig. 2 shows the obtained results. P-glycoprotein was detected only in the sample in which the membrane fraction was added to the immobilized antibody titer plate. No P-glycoprotein was detected in samples in which the antibody was not immobilized or in which no membrane fraction was added. From this result On the other hand, it was confirmed that the P-glycoprotein was immobilized together with the cell membrane on the support on which the monoclonal antibody was immobilized.

Figure 2 shows immobilized P-glycoprotein-specific monoclonal antibodies and immobilized P-glycoprotein on the immobilized plate. The results of verification by Western Plot method whether or not they were converted are shown. The P-glycoprotein was detected by enzymatic chemiluminescence (ECL). P-glycoprotein was detected only in the sample in which the membrane fraction was added to the immobilized antibody titrant plate.

5) Detection of substrate recognition reaction of P-glycoprotein immobilized on support

Cell membranes expressing the P- glycoprotein in Ueru of immobilized I Takeno titer plates, 20 mu Micromax [shed ^{- 3 2 Ρ] ΑΤΡ, 200} μ Μ Panajin acid isocyanatomethyl Li um, 3 mM magnesium sulfate, 2 mM Wano Add 40 mM Tris / HCl buffer, pH 7.4 (reaction cocktail) containing 0.1 mM EGTA and 30 verapamil (蛋白 -glycoprotein substrate), and shake for 20 minutes at room temperature. While incubating. Thereafter, the reaction solution was removed, and the wells were washed five times with a PBS solution containing 0.05% Tween 20. The amount of radiolabel captured by the well was measured with a liquid scintillation counter. Fig. 3 shows the obtained results. High levels of radiolabel were observed only when both the above reaction cocktails contained both sodium panadate and verapamil (a substrate for P-glycoprotein). On the other hand, if any of them lacked the reactivity quter described above, the levels of captured radiolabel were significantly lower. From these results, it is sufficiently possible to detect P-glycoprotein substrate recognition by a panic acid trap in a system using immotiter plates in which cell membranes expressing P-glycoprotein are immobilized. It was proved that. In addition, this method enables measurement in a short time without using photoaffinity reaction and polyacrylamide electrophoresis. did.

6) Verapamil concentration dependence

Cell membranes expressing the P- glycoprotein in immobilized Imuno titer one plates of ^{Ueru, 20μΜ [o; - 32 P} ] ATP, 200 μ M Panajin acid isocyanatomethyl Li um, 3 mM magnesium sulfate, 3 mM Wapain, 0.1 A 40 mM Tris / HCl buffer (pH 7.4) containing mM EGTA and verapamil at a concentration of 0 to 15 μΜ was added, and the mixture was incubated at room temperature with shaking for 20 minutes. Thereafter, the reaction solution was removed, and the wells were washed five times with a PBS solution containing 0.05% Tween 20. The amount of radiolabel captured in the well was measured with a liquid scintillation counter.

The results obtained are shown in FIG. In the absence of sodium panadate, increasing the concentration of verapamil, a substrate for P-glycoprotein, hardly changed the level of captured radiolabel. On the other hand, when sodium panadate is present at a concentration of 200 M, increasing levels of verapamil increase the level of captured radiolabel. However, at concentrations of verapamil above 15 μΜ, the level of captured radiolabel did not change much. That is, in the presence of sodium panadate, the level of captured radiolabel plotted a saturation curve against verapamil concentration, and the apparent Michaelis-Menten constant was estimated to be 5.

7) Necessity of Antibody for Detection of 認識 -Glycoprotein Substrate Recognition Reaction

Panamate trapping reaction was performed in the presence of verapamil using immunotiter plates immobilized with a monoclonal antibody specific for Ρ-glycoprotein and those not immobilized. The reaction conditions were the following as 20 / ζΜ [shed - ³² Ρ] ΑΤΡ, 40 0 or, including 200 / X Micromax Panajin acid isocyanatomethyl Li um, 3 mM magnesium sulfate, 2mM Wapain, a 0.1 mM EGTA and 30μΜ concentrations of verapamil Add mM Tris / HCl buffer (pH 7.4) And incubated at room temperature with shaking for 20 minutes. Thereafter, the reaction solution was removed, and the wells were washed five times with a PBS solution containing 0.05% Tween 20. Then, the amount of radiolabel captured in the well was measured with a liquid scintillation counter.

Fig. 5 shows the obtained results. In iminotiter plates on which a quality-specific antibody is immobilized on P-glycoprotein, the level of radiolabel captured by the panadate trap in the presence of 30 μ パ verapamil indicates that the antibody is not immobilized. Was significantly higher than This result indicates that the Ρ-glycoprotein is efficiently immobilized on the support together with the cell membrane by the antibody, and that the Ρ-glycoprotein retains its activity.

8) Detection of Ρ-glycoprotein substrate by this method

Using immunotiter plates in which cell membranes were immobilized with a monoclonal antibody specific for Ρ-glycoprotein, 20 μΜ of test compounds (verapamil, rhodamine 123, quinidine, doxorubicin, paraamino hippuric acid, sulfo The reaction was carried out in the presence of (bromophthalein). The reaction conditions are as follows. 20 μΜ [ _α- 32 ² ] ΑΤΡ, 0 μΜ or 200Μ 40 mM Tris / HCl buffer containing sodium panadate, 3 mM magnesium sulfate, 2 mM papain, 0.1 mM EGTA (PH 7.4) was added, and the mixture was incubated with shaking at room temperature for 20 minutes. Thereafter, the reaction solution was removed, and the wells were washed five times with a PBS solution containing 0.05% Tween 20. The amount of radiolabel captured in the well was measured with a liquid scintillation counter to calculate the difference between the amount of radiolabel obtained in the presence and the absence of sodium panadate.

The results obtained are shown in FIG. In the presence of the P-glycoprotein substrates verapamil, rhodamine 123, quinidine, and doxorubicin, the level of radiolabel captured by the panadate trap was significantly higher than that in the absence of the panadate. Was expensive. On the other hand, P-glycoprotein substrate For non-paraaminohippuric acid and sulfobromophthalein, the levels of radiolabel captured by the vanadate trap were not different from those in the absence of vanadate. This result indicates that the method is effective for detecting a substrate of P-glycoprotein.

Figure 6 shows that 20 μΜ of the test compound (verapamil, mouth-123, quinidine, doxorubicin, para-aminopurpuric acid, etc.) was immobilized on the cell membrane immobilized with an antibody specific for P-glycoprotein. (Sulfobromophthalein) This is the result of performing a panadic acid trap reaction in the presence. The amount of radiolabel captured in the well was measured with a liquid scintillation counter, and the amount of radiolabel obtained in the presence (20 μΟ) and in the absence (Ο μΜ) of sodium panadate was measured. Was calculated. Results are shown as the mean ± standard deviation of three experiments. Industrial applicability

INDUSTRIAL APPLICABILITY According to the present invention, a substrate or an inhibitor for an ABC-binding cassette (ABC) protein can be screened very efficiently, and the invention can be applied to high-throughput screening.

Claims

The scope of the claims

1. A screening method for a substrate or an inhibitor of an ATP-binding cassette (ABC) protein, comprising the steps of: (a) membrane fraction of cells expressing ABC protein; (2) labeled ATP or a derivative thereof and panadic acid; The test sample is mixed and incubated.After performing the photoaffinity reaction, the mixture is added to the support on which the antibody against the ABC protein is immobilized, and the label or the captured on the support is captured. Measuring the missing label.

2. In a method for screening a substrate or an inhibitor for an ATP-binding cassette (ABC) protein, a membrane fraction of cells expressing ABC protein is added to a support on which an antibody against ABC protein is immobilized, In addition, a labeled ATP or a derivative thereof, panadic acid, and a test sample are mixed and incubated, and the label captured on the support or the label not captured is measured. .

3. The ABC protein contains Walker A (Gly-X-X-Gly-X-Gly-Lys-Ser- [Ser, Thr, Gin]) (SEQ ID NO: 1) and Walker B ([ Leu, lie, Phe]-[Ile, Leu, Val]-X-Asp- [Glu, Asp, Ser]) (SEQ ID NO: 2) and the following ABC signature ([Leu, He, Val , Met, Phe, Tyr, Cys]-[Ser, Ala]-[Ser, Ala, Pro, Gly, Leu, Val, Phe, Tyr, Lys, Gin, His] -Gly- [As, Glu, Asn, Gin , Met, Trp]-[Lys, Arg, in, Ala, Ser, Pro, Cys, Leu, He, Met, Phe, Trp]-[Lys, Arg, Asn, Gin, Ser, Thr, Ala, Val, Met ]-[Leu, lie, Val, Met, Phe, Tyr, Pro, Ala, Asn]-{Pro, His, Tyr}-[Leu, lie, Val, Met, Phe, Trp]-[Ser, Ala, Gly, Cys, Leu, 丄丄 e, al, Phe]-{Phe, Tyr, Tr, His, Pro}-{Lys, Ar, His, Pro}-[Leu, He, Val, Met, Phe, Tyr , Trp, Ser, Thr, Ala] (Where X represents an unspecified amino acid).) The method according to claim 1 or 2, wherein the protein is a protein having (SEQ ID NO: 3) or a protein similar thereto.

4. The method according to any one of claims 1 to 3, wherein the label is a radioactive label, a fluorescent label, or a photoaffinity group label.

5. The radiolabel is ^{^{^{32 P, 33 P, 35 S}}} , 14 C or ³ H, A process according to any one of claims 1-4.

6. A membrane fraction of cells expressing ABC protein, labeled ATP or a derivative thereof, and a support on which an antibody against ABC protein is immobilized, or an antibody and support against ABC protein, A kit for screening substrates or inhibitors of ATP protein.