JP7324656B2 - Method and kit for predicting drug cardiotoxicity - Google Patents

Description

The present invention relates to methods and kits for predicting drug cardiotoxicity.

The flow of drug discovery and development consists of (1) basic research to find efficacy, (2) search for compounds (50,000 to 200,000 types) that exhibit this efficacy, and the final candidate (3 to 5 types) after 4 to 6 screenings. and (3) confirming the safety (side effects) of the final candidate compound.

However, if side effects are confirmed in the safety test of (3), development will be discontinued or a pharmaceutical application will be filed due to the fear of side effects.

When narrowing down the number of final candidate compounds, electrocardiogram measurements using experimental animals are performed as a safety test to evaluate cardiotoxicity, and whether a symptom called QT prolongation, which is one of the side effects, is observed. Evaluate. The QT interval is the time from the onset of the QRS to the end of the T wave on an electrocardiogram and corresponds to the duration of the action potential in the ventricular muscle. If this time is extended, the myocardium becomes electrically unstable, and premature ventricular contractions and an arrhythmia called TdP are likely to occur, which may lead to sudden death.

QT prolongation is caused by inhibition of the function of hERG (human ether-a-go-go-related gene product) channel protein (which produces K+ current) on the cell membrane by drugs, and intracellular transport of hERG channel protein. is also known to occur when is inhibited (Non-Patent Documents 1 and 2).

Therefore, hERG channels are expressed in cells and electrophysiologically evaluated by the patch clamp method. However, this evaluation requires a difficult measurement technique and expensive equipment. In addition, it is difficult to process multiple specimens.

In addition, evaluation of the channel expression level by fluorescence immunostaining or the like is also performed, but it takes time until detection, and it is difficult to process multiple specimens.

Pharmacology and Experimental Therapeutics, 2006, Vol.312 Molecular Pharmacology, 2011, Vol.79

An object of the present invention is to provide a simple method for predicting drug cardiotoxicity.

As a result of diligent efforts to solve the above problems, the inventors expressed on the cell membrane surface a fusion protein in which one of the two fragments of the reporter protein was linked to the hERG channel protein and the transmembrane domain. and cultured for a certain period of time, after which the drug is removed, the other of the two fragments of the reporter protein is added to reconstitute the reporter protein, and the signal from this reporter protein is measured. , found that it is possible to predict the cardiotoxicity of drugs, and completed the present invention (Fig. 10).

The gist of the present invention is as follows.
(1) expressing on the cell membrane surface a protein obtained by fusing one of the two fragments of the hERG channel, the transmembrane region, and the full-length reporter protein; adding a test substance to a medium and culturing the cells; After removing the test substance, the other fragment obtained by dividing the full-length reporter protein into two is added to reconstitute the full-length reporter protein on the cell, and the signal from the reconstituted reporter protein is measured. A method of predicting cardiotoxicity of a test substance comprising:
(2) the reporter protein is luciferase, one fragment obtained by dividing the full-length reporter protein into two is the C-terminal fragment of luciferase, and the other fragment obtained by dividing the full-length reporter protein into two is the N-terminal fragment of luciferase; The method according to (1), wherein the signal from the reporter protein obtained is luminescence produced by the reaction between luciferin and luciferase.
(3) The method according to (1) or (2), wherein an epitope tag is added to a protein obtained by fusing one of two fragments obtained by dividing the hERG channel, transmembrane region, and full-length reporter protein.
(4) The method according to any one of (1) to (3), including replacing the medium with PBS after removing the test substance.
(5) The method according to any one of (1) to (4), wherein no treatment is performed to inactivate the fusion protein expressed on the cell membrane surface before adding the test substance to the medium.
(6) The method according to any one of (1) to (5), wherein the cardiotoxic potential of the test substance is evaluated from the intensity of the signal measured at zero elapsed time after removal of the test substance.
(7) The method according to any one of (1) to (5), wherein the degree of recovery from hERG channel transport inhibition by the test substance is evaluated from the intensity of the signal measured at multiple elapsed times after removal of the test substance. .
(8) Any of (1) to (5) to evaluate changes in cardiotoxicity due to the dosage of the test substance by measuring the signal from the reconstituted reporter protein while changing the amount of the test substance added. The method described in .
(9) The method according to any one of (1) to (8), wherein the cardiotoxicity is prolongation of ventricular muscle action potential duration.
(10) A DNA containing a sequence encoding one of the two fragments of the hERG channel, transmembrane region and full-length reporter protein.
(11) A vector into which the DNA of (10) has been inserted.
(12) A cell introduced with the vector described in (11).
(13) A kit comprising either the DNA of (10), the vector of (11) or the cell of (12).
(14) The kit according to (13), which further contains the other fragment obtained by dividing the full-length reporter protein into two.
(15) The kit according to (13) or (14), further comprising a luminescent substrate.
(16) The kit according to any one of (13) to (15), further comprising one fragment obtained by dividing the full-length reporter protein into two.
(17) The kit according to any one of (13) to (16), which is used for predicting cardiotoxicity of a test substance.

INDUSTRIAL APPLICABILITY According to the present invention, it has become possible to predict the degree and presence or absence of cardiotoxicity of drugs by a simple method.

Evaluation of channel membrane trafficking inhibition by luciferase reconstitution (for transient expression). Evaluation of channel membrane trafficking inhibition by luciferase reconstitution (for stable expression). Evaluation of channel membrane trafficking inhibition by luciferase reconstitution (for stable expression). Evaluation of channel membrane trafficking inhibition by fluorescence immunostaining. Evaluation of channel membrane trafficking inhibition by fluorescence immunostaining. Measurement of recovery from channel membrane trafficking inhibition by luciferase reconstitution. Measurement of recovery from channel membrane trafficking inhibition by luciferase reconstitution. Evaluation of channel membrane trafficking inhibition by fluorescence immunostaining. Evaluation of channel membrane trafficking inhibition by fluorescence immunostaining. Schematic diagram of the present invention.

Hereinafter, embodiments of the present invention will be described in more detail.

The present invention involves expressing on the cell membrane surface a protein obtained by fusing one of the two fragments of the hERG channel, the transmembrane region, and the full-length reporter protein, adding a test substance to the medium, and culturing the cells; After removing the test substance, the other fragment obtained by dividing the full-length reporter protein into two is added to reconstitute the full-length reporter protein on the cell, and the signal from the reconstituted reporter protein is Provided is a method of predicting cardiotoxicity of a test substance, comprising measuring.

The hERG channel is a protein expressed from hERG (human ether-a-go-go-related gene) and is a potassium ion channel responsible for repolarization of myocardial action potential.

The transmembrane region refers to a portion (domain) that penetrates the cell membrane in the structure of a membrane protein, and examples include transmembrane domains such as PDGFR, EGFR, IGFR, FGFR, NGFR, VEGFR, FGFR, and HGFR. can.

In the present invention, any reporter protein can be used as long as it can confirm hERG channel expression, and is typically a luminescent protein, a fluorescent protein, or the like.

Luciferases derived from bioluminescent organisms, such as luciferase, can be exemplified as photoproteins. Photoproteins may be naturally occurring or mutated.

Examples of luciferase include natural firefly luciferase (specifically, natural firefly luciferase from North America, luciferase from Heike firefly, luciferase from Genji firefly, luciferase from Himebotaru, luciferase from red click beetle, luciferase from railroad worm, luciferase from glowworm, luciferase from Cypridina luciferase. , sea cactus-derived luciferase), natural luciferases other than firefly luciferase (specifically, dinoflagellate (worm)-derived luciferase, Renilla-derived luciferase, euphorbia luciferase-derived luciferase, luminescent bacteria-derived luciferase, Latia-derived luciferase, luminescent lugworm-derived luciferase, deep sea natural luciferases such as shrimp-derived luciferase), variants thereof, and the like.

The luciferase is preferably heat-stable, and the heat-stable luciferase described in Patent No. 6349003 can be exemplified, and its amino acid sequence is shown in SEQ ID NO:13.

Fluorescent proteins include Green Fluorescent Protein (GFP), and proteins with similar properties such as TurboGFP, AcGFP, TagGFP, Azami-Green, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, Hyper, Blue Blue Fluorescent Protein (BFP), its related protein EBFP, Cyan Fluorescent Protein (CFP), ECFP, TagCFP, AmCyan, MidoriishiCyan, Red Fluorescent Protein (Red Fluorescent Protein, RFP), and proteins showing similar properties TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, Yellow Fluorescent Protein (YFP) and TagYFP showing similar properties, EYFP, Venus, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana, orange fluorescent protein KusabiraOrange, mOrange, infrared fluorescent protein TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed mRasberry, mPlum etc. can be exemplified. Fluorescent proteins may be naturally occurring or mutated.

In the present invention, one fragment obtained by dividing a full-length reporter protein into two and the other fragment preferably interact to reconstitute the full-length reporter protein and exhibit its function as a reporter protein. A C-terminal fragment and an N-terminal fragment obtained by dividing the reporter protein into two.

When the reporter protein is a luminescent or fluorescent protein, one fragment and the other fragment obtained by dividing the full-length reporter protein into two are, for example, the C-terminal fragment and the N-terminal fragment of the luminescent or fluorescent protein. The C-terminal fragment and the N-terminal fragment of the luminescent or fluorescent protein should be capable of producing luminescence or fluorescence when they interact with each other.

Although the ratio of the number of amino acids constituting one fragment obtained by dividing the full-length reporter protein into two and the number of amino acids constituting the other fragment is not limited, the fragment to be fused with the hERG channel and the transmembrane region is the same as the other fragment. It is preferable that the number of amino acids is smaller than (fragment added after removing the test substance). For example, when the reporter protein is luciferase consisting of the amino acid sequence of SEQ ID NO: 13, the C-terminal fragment of luciferase is a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and the N-terminal fragment of luciferase is the amino acid sequence of SEQ ID NO: 12. However, the peptide consisting of the amino acid sequence of SEQ ID NO: 7 or 12 may have 1 to 25 amino acids deleted from the N-terminus and / or C-terminus, or the full-length 1 to 30 amino acids before and after the corresponding amino acid sequence in the luciferase amino acid sequence may be added or substituted. Alternatively, the C-terminal fragment of luciferase may be a subfragment of 20 or fewer (eg, 12, 11, 10, 9, etc.) amino acids.

One fragment and the other fragment obtained by dividing the full-length reporter protein into two may contain overlapping amino acid sequences. For example, in the case of a C-terminal fragment and an N-terminal fragment of a luminescent or fluorescent protein, the number of overlapping amino acids may be 0, 1-200, or 5-30.

One fragment and the other fragment of the full-length reporter protein may be modified in their original sequence to alter expression efficiency, protein stability, degree of interaction, intensity of luminescence or fluorescence, etc. good.

When the reporter protein is luciferase, one fragment obtained by dividing the full-length reporter protein into two is a C-terminal fragment of luciferase, and the other fragment obtained by dividing the full-length reporter protein into two is an N-terminal fragment of luciferase, the rearrangement The signal from the reporter protein obtained may be luminescence produced by the reaction between luciferin and luciferase. Luciferin is a substance that emits light when oxidized by luciferase, and examples thereof include firefly luciferin, bacterial luciferin, dinoflagellate luciferin, vargrin, and coelenterazine.

An epitope tag may be added to a protein obtained by fusing one fragment obtained by dividing the hERG channel, the transmembrane region, and the full-length reporter protein into two. By adding an epitope tag, it can be used for detection of hERG channels in immunostaining and Western blot analysis. Although detection is possible without an epitope tag, multiple staining is possible in immunostaining by adding it. Examples of epitope tags include hemagglutinin tag (HA-tag), histidine tag (His-tag), c-Myc tag, FLAG tag and the like.

In the method of the present invention, a protein is expressed on the cell membrane surface by fusing the hERG channel, the transmembrane region, and one of the two fragments of the full-length reporter protein (for example, the C-terminal fragment of a luminescent or fluorescent protein). For example, the DNA encoding one of the two fragments of the full-length reporter protein, the DNA encoding the transmembrane region, and the hERG gene are incorporated into an appropriate expression vector, introduced into appropriate cells, and cultured for an appropriate time. allows the fusion protein to be expressed on the cell membrane surface. A leader sequence (eg, an Igκ chain leader sequence) may also be incorporated into the expression vector. A DNA encoding one fragment obtained by splitting a full-length reporter protein into two, a DNA encoding a transmembrane region, and the hERG gene can be prepared by known methods using PCR or the like. Commercially available expression vectors can be used, e.g. SV40, pcDNA3.1(-), bacterial pQE70, pQE60, pQE-9, pBluescriptII KS, pTrc99a, pKK223-3, pDR540, pRIT2T, pET-11a, etc. The hERG gene may be integrated upstream or downstream of the DNA encoding one of the two fragments of the full-length reporter protein, and between the hERG gene and the DNA encoding one of the two fragments of the full-length reporter protein, A DNA encoding a transmembrane region may be incorporated. A linker sequence consisting of 1 to 50 nucleotides may be inserted between the DNA encoding one of the two fragments of the full-length reporter protein, the DNA encoding the transmembrane region and the hERG gene. An epitope tag may be added to the fusion protein, and the insertion site does not matter. Expression may be either transient expression or stable expression.

Bacterial cells (e.g., Escherichia spp., Bacillus spp., Bacillus subtilis, etc.), fungal cells (e.g., yeast, Aspergillus, etc.), insect cells (e.g., S2 cells, Sf cells, etc.) can be used for expressing the fusion protein. , animal cells (eg, CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), plant cells, and the like.

To introduce into a host an expression vector containing a DNA encoding one of the two fragments of the full-length reporter protein, a DNA encoding the transmembrane region, and the hERG gene, the calcium phosphate method, Lipofectamine, Lipofectamine2000 (both of which are Invitrogen), GeneJuice (Merck), Xfect (Clontech), FuGENE 6 (Roche Applied), HilyMax (Dojindo Laboratories), and the like may be used.

The other fragment obtained by dividing the full-length reporter protein into two (e.g., N-terminal fragment of a luminescent or fluorescent protein) is, for example, a vector (e.g., pET28b vector) incorporating a DNA encoding the other fragment obtained by dividing the full-length reporter protein into two. is introduced into a protein expression host (eg, Escherichia coli) and cultured for an appropriate period of time, after which the target protein (the other fragment obtained by splitting the full-length reporter protein into two) can be collected from the culture. When the protein of interest is secreted into the medium, the medium may be collected, and the protein of interest may be separated from the medium and purified. If the protein of interest is produced intracellularly in the host, the cells may be lysed, and the protein of interest may be isolated and purified from the lysate. If a His-tag is added to the DNA encoding the other fragment of the full-length reporter protein, it can be purified using a Ni-immobilized carrier.

After expressing on the surface of the cell membrane a protein obtained by fusing one of the two fragments of the hERG channel, transmembrane region and full-length reporter protein (e.g., C-terminal fragment of a luminescent or fluorescent protein), the test substance is added to the medium. do. Addition of the test substance is preferably performed after confirmation of cell adhesion and proliferation. Moreover, it is preferable not to perform a treatment to inactivate the fusion protein expressed on the cell membrane surface before adding the test substance to the medium. Treatment to inactivate the fusion protein includes addition of an enzyme (eg, trypsin/EDTA) or compound (eg, Sulfo-NHS) that inactivates the protein. Therefore, a physical technique such as a cell scraper or pipetting may be used to detach adherent cells.

The test substance is not particularly limited, and may be any substance, and may be a synthetic compound or a compound extracted from a natural product. Also, the test substance may be a new lead compound or a compound derivatized from a lead compound. The test substance may be a single compound or a mixture of compounds.

After adding the test substance, the cells are cultured for an appropriate period of time. After that, the cells are collected in a container such as a tube by a physical method, and the medium is exchanged by centrifugation to remove the test substance. The detection sensitivity can be increased by concentrating the cell density by centrifugation or the like and simultaneously exchanging the cell suspension solution from the culture medium to PBS (phosphate buffered saline). After removing the test substance, the other fragment (for example, the N-terminal fragment of a luminescent or fluorescent protein) is added to reconstitute the full-length reporter protein on the cell, and the reconstituted reporter protein is added. Measure the signal from the protein. When the reporter protein is a luminescent or fluorescent protein, it is preferable to detect the presence or absence of luminescence, change in luminescence wavelength, or presence or absence of fluorescence or change in fluorescence wavelength as signal measurement. Specifically, an N-terminal fragment of a luminescent or fluorescent protein is added to a culture medium of cells expressing a protein fused with an hERG channel, a transmembrane region, and a C-terminal fragment of a luminescent or fluorescent protein, and allowed to stand for an appropriate time. (during which the luminescent or fluorescent protein is reconstituted), if necessary, after adding a luminescent substrate, the presence or absence of luminescence or a change in emission wavelength or the presence or absence of fluorescence or a change in fluorescence wavelength may be detected. A luminescent substrate may be appropriately selected according to the type of luminescent protein. For example, if the photoprotein is luciferase, luciferin may be selected as the luminescent substrate. Moreover, a person skilled in the art can appropriately determine the molar ratio of the luminescent protein and the luminescent substrate depending on the types of the luminescent protein and the luminescent substrate.

The emission wavelength may be appropriately selected according to the type of the photoprotein, but when the photoprotein is natural luciferase of North American firefly or its mutant, the emission wavelength is appropriately 562 nm.

The fluorescence wavelength may be appropriately selected depending on the type of fluorescent protein together with the excitation wavelength. For example, the following excitation wavelength and fluorescence wavelength may be selected. Excitation wavelength 380 nm - fluorescence wavelength 440 nm for EBFP, a blue fluorescent protein, excitation wavelength 433 nm - fluorescence wavelength 475 nm for CFP, a cyan fluorescent protein, and excitation wavelength 475 nm for EGFP, a green fluorescent protein. wavelength 488 nm-emission wavelength 509 nm, yellow fluorescent protein YFP excitation wavelength 514 nm-emission wavelength 527 nm, red fluorescent protein DsRed2 excitation wavelength 563 nm-emission wavelength 582 nm, infrared light In the case of mPlum, which is a fluorescent protein, an excitation wavelength of 590 nm and an emission wavelength of 649 nm are selected respectively.

Luminescence or fluorescence detection can be performed using commercially available equipment.

After adding the test substance to the cells, washing, fixing, blocking, staining of channels on the surface of the cell membrane with an anti-tag antibody, permeabilization, staining of intracellular channels with an anti-hERG antibody, and microscopic observation can be performed. When an epitope tag is added to the fusion protein, multiple staining can be performed by immunostaining.

The method of the present invention makes it possible to predict the cardiotoxicity of a test substance, in particular the possibility of prolonging the action potential duration of the ventricular muscle. The prolongation of action potential duration in ventricular muscle can be evaluated and judged by the presence or absence of prolongation of QT time in electrocardiogram measurement of animals.

In the method of the present invention, the cardiotoxicity of the test substance can be evaluated from the intensity of the signal measured at zero elapsed time after removal of the test substance.

For example, if the signal intensity measured with the test substance is lower than the signal intensity measured without the test substance, cardiotoxicity, i.e. prolongation of action potential duration in the ventricular muscle Probability is high. In other words, if no decrease in signal intensity is observed due to the addition of the test substance, it can be evaluated that the risk of developing long QT syndrome due to channel trafficking inhibition is low.

In addition, in the method of the present invention, the degree of recovery from hERG channel transport inhibition by the test substance can be evaluated from the intensity of the signal measured at a plurality of elapsed times after removal of the test substance.

For example, when the test substance was added, the intensity of the signal measured at time 0 after removal of the test substance was lower than when the test substance was not added, but the signal intensity increased rapidly over time and hERG If recovery from channel transport inhibition is rapid, it can be evaluated that the duration of side effects (cardiac toxicity, ie, long QT syndrome) and recovery time from side effects are short.

Furthermore, in the present invention, changes in cardiotoxicity depending on the dosage of the test substance can be evaluated by measuring the signal from the reconstituted reporter protein while varying the amount of the test substance added.

For example, if the intensity of the signal measured at zero elapsed time after removal of the test substance is lower than when the test substance is not added, the decrease in signal intensity can be reduced by reducing the amount of test substance added. Therefore, it can be evaluated that it can be used safely by adjusting the dosage to an appropriate range. Also, by comparing the degree of increase in signal intensity after removal of the test substance, differences in recovery time from side effects due to differences in dosage can be evaluated.

The present invention also provides a DNA comprising a sequence encoding one of the two fragments of the hERG channel, transmembrane region and full-length reporter protein. This DNA is incorporated into an expression vector, introduced into a host cell, and the protein expressed can be used in the method of the present invention. The DNA of the present invention can be prepared by inserting the DNA encoding one of the two fragments of the full-length reporter protein, the DNA encoding the transmembrane region and the hERG gene into an appropriate expression vector (described above). The present invention also provides a vector (described above) into which a DNA containing a sequence encoding one of the two fragments of the hERG channel, transmembrane region and full-length reporter protein is inserted. The vector of the present invention preferably has a restriction enzyme site upstream and/or downstream of the DNA containing the sequence encoding one of the two fragments of the hERG channel, transmembrane region and full-length reporter protein. The vectors may be further added with promoters, enhancers, splicing signals, poly A addition signals, selectable markers, SV40 replication origins, tags and the like. The present invention also provides a cell introduced with a vector into which a DNA containing a sequence encoding one of the hERG channel, transmembrane region, and one of the two fragments of the full-length reporter protein (described above) has been introduced. Such cells include bacterial cells (e.g., Escherichia spp., Bacillus spp., Bacillus subtilis, etc.), fungal cells (e.g., yeast, Aspergillus, etc.), insect cells (e.g., S2 cells, Sf cells, etc.), animal cells, etc. Examples include cells (eg, CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), plant cells, and the like.

A DNA, vector or cell of the invention may be included in a kit as a component of the kit. The kit further includes the other fragment obtained by splitting the full-length reporter protein into two pieces, a luminescent substrate (described above), ATP, a sensitizer (e.g., benzophenone, rose bengal, etc.), Mg ion, coenzyme A, a reducing agent (e.g., dithio threitol, ascorbic acid, organic sulfur-based reducing agents, etc.), an instruction manual describing how to use the kit and how to evaluate the test substance, one fragment of the full-length reporter protein used as a positive control in order to confirm the activity of the other divided fragment). The kit can be used to predict the cardiotoxicity of a test substance, in particular the potential of the test substance to prolong action potential duration in the ventricular muscle.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.
[Example 1] Evaluation of inhibition of channel translocation using luminescence reaction by reconstitution of luciferase
Preparation of plasmids
A pcDNA3.1(-) vector (manufactured by Thermo Fisher Scientific) was used as a plasmid vector for expression. Leader sequence (SEQ ID NO: 1), luciferase C-terminal fragment (SEQ ID NO: 2), transmembrane domain (SEQ ID NO: 4), hERG (KCNH2) at the multiple cloning site of pcDNA3.1(-) vector in order from the 5' side (SEQ ID NO: 5) were each incorporated. A synthetic gene was used as the DNA template for the hERG (human Ether-a-go-go-Related Gene, KCNH2, Kv11.1) channel (NM_000238) with a full-length sequence of 3480 bp, rather than a cell or tissue sample. . Also, thermostable luciferase (Patent No. 6347003) was used for the luciferase C-terminal fragment.

An Igκ chain leader sequence or the like can be used for the signal peptide, and a PDGFR domain or the like can be used for the transmembrane region. A transmembrane region is essential between the luciferase C-terminal fragment and the hERG channel in order to measure inhibition of hERG channel translocation by this detection system. Furthermore, an epitope tag can be added to this sequence, regardless of the insertion site. In this study, a hemagglutinin tag (HA- tag) (SEQ ID NO: 3) was added.

The amino acid sequences of the leader sequence, luciferase C-terminal fragment, transmembrane domain, hERG (KCNH2) and hemagglutinin tag (HA-tag) are shown in SEQ ID NOs: 6, 7, 8, 9 and 10, respectively.

Preparation of gene-introduced cells The prepared expression plasmid was introduced into HEK293 cells (Riken), a cell line derived from human embryonic kidney cells. Transfection was performed by the lipofection method, but any method such as electroporation, microinjection, or gene transfer using a viral vector may be used. Also, the type of expression plasmid is not limited. Furthermore, this assay method exemplified below can measure both transient expression and stable expression. In the production of stable expression strain transformants in this test, drug selection was performed using G418 (geneticin), an aminoglycoside antibiotic.

Methods for producing luciferase N-terminal fragments
A DNA fragment (SEQ ID NO: 11) of a luciferase N-terminal fragment amplified by PCR using thermostable luciferase (Patent No. 6347003) as a template was introduced into a pET28b vector (manufactured by Merck). This plasmid DNA was transformed into E. coli BL21(DE3) and cultured overnight in LB medium supplemented with kanamycin and IPTG. The cells were collected, lysed with a solution containing TritonX-100 and lysozyme, and then His-tag purified to obtain a luciferase N-terminal fragment (SEQ ID NO: 12).

Method for evaluating inhibition of channel translocation by luminescence reaction The prepared transfected cells were cultured in a well plate or dish (medium: MEM (manufactured by Sigma) + 10% FBS (manufactured by gelifesciences) + 1% NEAA (manufactured by Sigma).
). The pre-culture period should be about 1 to 3 days after the cells have adhered and started to proliferate. In addition, the culture environment is 37°C and 5% CO ₂ . After confirming the adhesion and growth of the cells, drug exposure treatment was performed by adding the drug pentamidine or probucol, which is a test compound, and culturing for a certain period of time. After drug treatment, the cells were collected in a tube and centrifuged to concentrate the cell density 10 to 100 times, and at the same time the cell suspension solution was exchanged from the culture medium to PBS. This operation can further increase the detection sensitivity. A physical method using a cell scraper or pipetting is preferable for detachment of cells, and an "enzymatic decomposition method" that inactivates proteins expressed on the cell membrane surface using trypsin/EDTA is not suitable. After reconstituting luciferase by adding luciferase N-terminal fragment to the prepared cell suspension and allowing it to stand at room temperature for 5 to 60 minutes, a luminescent reagent containing luciferin, ATP, etc. Luminescence was measured.

Detection of hERG Channels by Fluorescent Immunostaining Gene- introduced cells were seeded on well plates, dishes, or glass slides such as chambers, and cultured at 37° C. under 5% CO ₂ . The drug exposure treatment was performed by adding the drug pentamidine or probucol, which are test compounds, in the same manner as in the measurement by the luminescence reaction. After drug treatment, washing with PBS, fixation with 4% paraformaldehyde/phosphate buffer, blocking with BSA, staining of channels on the cell membrane surface with anti-HA-tag antibody, permeabilization with TritonX-100, cells with anti-hERG antibody The inner channel was stained and observed with a fluorescence microscope.

Results and Discussion The evaluation results of inhibition of channel translocation by luminescence reaction are shown in FIGS. 1 to 3. FIG. Each data is corrected based on the number of cells measured with the "cell" ATP measurement reagent Ver.

As reported in the literature, hERG channel trafficking inhibition was observed in cells exposed to pentamidine and probucol. In addition, since the inhibition rate was similar to the reported example and the results of fluorescent immunostaining shown in Figs. It can be said that it is useful for measuring transport inhibition.

Furthermore, the greatest advantage of this detection system is that it can be detected in a short period of time compared to fluorescent immunostaining methods, etc., and it is possible to measure a large number of samples. Therefore, even in a test requiring collection of samples at fixed time intervals, such as in Example 2 below, measurement can be performed easily. In addition, since this system includes a step of collecting cells, cell proliferation assays can be performed at the same time, and it is easy to make corrections based on the number of cells.

[Example 2] Evaluation of degree of recovery from channel membrane translocation inhibition using luminescence reaction by luciferase reconstitution Using the gene-introduced cells (stable expression strain) prepared in Example 1, channel membrane translocation inhibition by drug treatment was evaluated. was evaluated.
Measurement method of degree of recovery from inhibition of channel translocation by luminescence reaction
The generated transfected cells were cultured in well plates or dishes. The pre-culture period should be about 1 to 3 days after the cells have adhered and started to proliferate. In addition, the culture environment is 37°C and 5% CO ₂ . After confirming the adhesion and growth of the cells, drug exposure treatment was performed by adding the drug pentamidine or probucol, which is a test compound, and culturing for a certain period of time. After drug treatment, the medium was exchanged to remove the drug, and the cells were returned to the CO ₂ incubator. Cells were collected at regular time intervals after drug removal, and the cell density was concentrated 10- to 100-fold by centrifugation, while the cell suspension solution was replaced from the culture medium to PBS. This operation can further increase the detection sensitivity. A physical method using a cell scraper or pipetting is preferable for detachment of cells, and an "enzymatic decomposition method" that inactivates proteins expressed on the cell membrane surface using trypsin/EDTA is not suitable. After reconstituting luciferase by adding luciferase N-terminal fragment to the prepared cell suspension and allowing it to stand at room temperature for 5 to 60 minutes, a luminescent reagent containing luciferin, ATP, etc. Luminescence was measured.

Detection of hERG Channels by Fluorescent Immunostaining Gene- introduced cells were seeded on well plates, dishes, or glass slides such as chambers, and cultured at 37° C. under 5% CO ₂ . The drug exposure treatment was performed by adding the drug pentamidine or probucol, which are test compounds, in the same manner as in the measurement by the luminescence reaction. After drug treatment, the medium was exchanged to remove the drug, and the cells were returned to the CO ₂ incubator. At regular intervals after drug removal, washing with PBS, fixation with 4% paraformaldehyde/phosphate buffer, blocking with BSA, staining of channels on the cell membrane surface with anti-HA-tag antibody, permeabilization with TritonX-100, Intracellular channels were stained with an hERG antibody and observed with a fluorescence microscope.

Result and Discussion FIG. 6 and FIG. 7 show the measurement results of the degree of recovery from inhibition of channel membrane translocation by luminescence reaction. Each data is corrected based on the number of cells measured with the "cell" ATP measurement reagent Ver.

Figures 6 and 7 show that the hERG channel expression level on the cell membrane surface, which was inhibited by exposure to pentamidine and probucol, recovered with the lapse of time after drug removal. Moreover, the degree of recovery differs depending on the type of chemical and the treatment concentration. This result was similar to the data obtained by fluorescence immunostaining in FIGS. With this detection system, measurement can be performed in a short time of about 45 minutes from sample collection to detection.

INDUSTRIAL APPLICABILITY The present invention can be used as one of safety tests in drug discovery.

<SEQ ID NO: 1> Nucleotide sequence of leader sequence (mouse Igκ chain leader sequence).
gagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac
<SEQ ID NO: 2> Nucleotide sequence of luciferase C-terminal fragment.
tccggttatgtaaacaatccggaagcgaccaacgccttgattgacaaggatggatggctacattctggagacatagcttactgggacgaagacgaacacttcttcatcgttgaccgcctgaagtctctgattaagtacaaaggctatcaggtggctcccgctgaattggaatccatcttgctccaacaccccaacatcagagacgcaggtgtcgcaggtctt cccgacgatgacgccggtgaacttcccgccgccgttgttgttttggagcacggaaaagacgatgacggaaaaagagatcgtggattacgtcgccagtcaagtaacaaccgcgaaaaagttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggtcttaccggaaaaagagacgcaagaaaaatcagagagatcctcataaaggccaagaagggcggaaa gatcgccgtg
<SEQ ID NO: 3> Nucleotide sequence of HA-tag.
tatccatatgatgttccagattatgct
<SEQ ID NO: 4> Nucleotide sequence of transmembrane domain (PDGFR transmembrane region).
gctgtgggccaggacacgcaggaggtcatcgtggtgccacactccttgccctttaaggtggtggtgatctcagccatcctggccctggtggtgctcaccatcatctccccttatcatcctcatgctttggcagaagaagccacgt
<SEQ ID NO: 5> Nucleotide sequence of hERG.

<SEQ ID NO: 6> Amino acid sequence of leader sequence (mouse Igκ chain leader sequence).
ETDTLLLWVLLLWVPGSTGD
<SEQ ID NO: 7> Amino acid sequence of luciferase C-terminal fragment.
SGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIRDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKRDARKIREILIKAKKGGKIAV
<SEQ ID NO: 8> Amino acid sequence of HA-tag.
YPYDVPDYA
<SEQ ID NO: 9> Amino acid sequence of transmembrane domain (PDGFR transmembrane region).
AVGQDTQEVIVVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR
<SEQ ID NO: 10> Amino acid sequence of hERG.

<SEQ ID NO: 11> Nucleotide sequence of luciferase N-terminal fragment.

<SEQ ID NO: 12> Amino acid sequence of luciferase N-terminal fragment.
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTLCVRFSHARDP IFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLLSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPKGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDG
<SEQ ID NO: 13> Amino acid sequence of full-length luciferase.
MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTLCVRFSHARDP IFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLLSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPKGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKY KGYQVAPAELESILLQHPNIRDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKL

Claims (12)

expressing in cells a protein obtained by fusing one of the two fragments of the hERG channel, the transmembrane region, and the full-length reporter protein; adding a test substance to a medium and culturing the cells; removing the test substance; and then adding the other fragment obtained by dividing the full-length reporter protein into two to reconstitute the full-length reporter protein on the cell membrane surface, and measuring the signal from the reconstituted reporter protein. , a method for predicting cardiotoxicity of a test substance, wherein the transmembrane region is a PDGFR transmembrane region consisting of the amino acid sequence of SEQ ID NO: 9, and one fragment obtained by dividing the full-length reporter protein into two is SEQ ID NO: 7 A luciferase C-terminal fragment consisting of an amino acid sequence, the other fragment obtained by dividing the full-length reporter protein into two parts is a luciferase N-terminal fragment consisting of the amino acid sequence of SEQ ID NO: 12, and the signal from the reconstituted reporter protein is luciferin. and luciferase . 2. The method according to claim 1, wherein an epitope tag is added to a protein obtained by fusing one of two fragments of the hERG channel, transmembrane region and full-length reporter protein. 3. The method according to claim 1 or 2, comprising removing the test substance, concentrating the cell density, and simultaneously replacing the medium with PBS. The method according to any one of claims 1 to 3 , wherein no treatment for inactivating the fusion protein expressed on the cell membrane surface is performed before adding the test substance to the medium. The method according to any one of claims 1 to 4 , wherein the cardiotoxic potential of the test substance is evaluated from the intensity of the signal measured at zero elapsed time after removal of the test substance. The method according to any one of claims 1 to 4 , wherein the degree of recovery from inhibition of hERG channel transport by the test substance is evaluated from the signal intensity measured at a plurality of elapsed times after removal of the test substance. The method according to any one of claims 1 to 4 , wherein the change in cardiotoxicity due to the dose of the test substance is evaluated by measuring the signal from the reconstituted reporter protein while changing the amount of the test substance added. The method according to any one of claims 1 to 7 , wherein the cardiotoxicity is prolongation of action potential duration in ventricular muscle. A DNA containing a sequence encoding one of two fragments obtained by dividing an hERG channel, a transmembrane region, and a full-length reporter protein, a vector into which the DNA has been inserted, or a cell into which the vector has been introduced , and a full-length A kit containing the other fragment obtained by dividing a reporter protein into two , wherein the transmembrane region is a PDGFR transmembrane region consisting of the amino acid sequence of SEQ ID NO: 9, and one fragment obtained by dividing the full-length reporter protein into SEQ ID NO: The kit is a luciferase C-terminal fragment consisting of the amino acid sequence of SEQ ID NO: 12, wherein the other fragment obtained by dividing the full-length reporter protein into two is a luciferase N-terminal fragment consisting of the amino acid sequence of SEQ ID NO:12. 10. The kit of claim 9 , further comprising a luminescent substrate. 11. The kit according to claim 9 or 10, further comprising one fragment obtained by dividing the full-length reporter protein into two. The kit according to any one of claims 9 to 11, which is used for predicting cardiotoxicity of a test substance.