JP2007024705A - Screening method of ryanodine receptor stabilizing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an evaluation method or the like of a ryanodine receptor function improving action that is useful for selecting a treating agent or a preventive agent for an abnormal disease related to muscular excitement or contraction or the like. <P>SOLUTION: This method for measuring bonding activity to a ryanodine receptor of a test substance includes following processes (1)-(4): (1) bringing a ryanodine receptor, the test substance, and a substance having a ryanodine receptor stabilizing action function in contact with each other; (2) measuring a bonding amount of the reference substance to the ryanodine receptor in step (1); (3) comparing the bonding amount measured in the step (2) with a bonding amount of the reference substance to the ryanodine receptor, in the absence of the test substance; and (4) a process for determining a bond inhibiting activity between the reference substance and the ryanodine receptor by the test substance based on the result in step (3). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a therapeutic or prophylactic agent for muscular excitement / contraction-related abnormal diseases, i.e., disorders involving stimulation conduction abnormalities, muscle contraction or relaxation dysfunction, specifically cardiac hypertrophy, heart failure, ischemic heart disease, Therapeutic or prophylactic agent for heart disease, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia, malignant hyperthermia, congenital myopathy, or impaired exercise tolerance associated with heart failure, or central Drugs for treating or preventing calcium handling disorders, specifically epilepsy, manic depression, anxiety, schizophrenia, ALS (amyotrophic lateral sclerosis), stroke, cerebral infarction, cerebral embolism, cerebrovascular disorder, Alzheimer Ryanodine receptor function improving agent useful as a therapeutic or preventive agent for Alzheimer's disease, Parkinson's disease, glaucoma, retinopathy, or macular degeneration, and a method for evaluating the ryanodine receptor function improving action of a test substance It is intended to.

The ryanodine receptor (RyR) is known as an intracellular calcium channel that controls the release of calcium from the endoplasmic reticulum (SR). The ryanodine receptor 2 (RyR2), an isoform that is abundant in the sarcoplasmic reticulum of the myocardium, is triggered by excessive phosphorylation by phosphokinase A (PKA) stimulation during arrhythmia and heart failure. , Protein bound on RyR2: FKBP12.6 dissociates, the RyR2 conformation is destabilized to increase the probability of opening, and calcium is released (leaked) from the sarcoplasmic reticulum, the calcium storehouse in the myocardium. Has been reported in clinical and animal models (see Non-Patent Documents 1 to 3). Recently, dissociation of RyR2 and FKBP12.6 is involved in calcium leak, and a substance that suppresses dissociation: 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy -2,3,4,5-Tetrahydro-1,4-benzothiazepine (hereinafter referred to as JTV-519) has been reported (see Patent Documents 1 to 3). JTV-519 is a 1,4-benzothiazepine derivative that has a myocardial protective effect and enhances the binding of FKBP12.6 to RyR2 hyperphosphorylated at a concentration of 100 nM to 1 μM (see Non-Patent Document 4). It is known that, in SR in which calcium leak is enhanced, suppression of calcium leak with IC ₅₀ = 30 nM (see Non-Patent Document 5). In addition, studies on changes in ryanodine receptor conformation using partial peptides suggest that JTV-519 stabilizes the ryanodine receptor conformation (see Non-Patent Document 6). On the other hand, 1,5-benzothiazepine derivative calcium antagonist: Diltiazem is known as a therapeutic agent for hypertension, paroxysmal supraventricular tachycardia, etc. 1 μM) has been reported to suppress calcium leak (see Non-Patent Document 5).
Ryanodine is an alkaloid known to bind to the ryanodine receptor, but ryanodine does not have an action of suppressing calcium leak from the ryanodine receptor, that is, improving the function of the ryanodine receptor.
As described above, JTV-519 was known to suppress the dissociation of RYR2 and FKBP and to suppress calcium leak from the sarcoplasmic reticulum. Diltiazem was also known to suppress calcium leaks from the sarcoplasmic reticulum. However, it was unclear whether JTV-519 or diltiazem interacted with the ryanodine receptor.

On the other hand, screening methods (evaluation methods) for drugs that suppress calcium leakage via RYR2 include (1) a method using FK506 or rapamycin as an index for the dissociation of FKBP from RYR2, and (2) labeled FKBP and phosphorylation. Methods such as immunoprecipitation, ultracentrifugation, etc. to recover the labeled RYR2 or complex with the endoplasmic reticulum and measuring the label activity, and (3) a method using the calcium leak from the sarcoplasmic reticulum by FK506 as an indicator. It was. However, these methods cannot simultaneously evaluate a large number of compounds. Therefore, an evaluation method capable of efficiently evaluating a large number of test compounds and supporting high-throughput screening has been demanded.
On the other hand, ryanodine receptor 1 (RyR1), an isoform abundant in the sarcoplasmic reticulum of skeletal muscle, is bound to FKBP12 by the same mechanism of action as RyR2, and skeletal muscle is abnormal in exercise tolerance. In RyR1, FKBP12 is dissociated. JTV-519 has been shown to increase the binding of RyR1 and FKBP12 and improve exercise tolerance. In addition, ryanodine receptor 3 (RyR3), which is an isoform abundant in brain and nerve cells, is known to bind FKBP12 like RyR1, and the dysfunction of RyR3 is caused by neuronal calcium overload. It is expected that neuronal cell death will be caused by the load, and that excitatory transmission abnormalities will be caused by a decrease in calcium content in the endoplasmic reticulum.

JP 2003-95977 A US Pat. No. 6,489,125 US Patent Application Publication No. 2004/0229781 S.O.Marx et al., Cell 101: 365-376 (2000) M.Yano et al., Circulation 102: 2131-2136 (2000) X.H.T.Wehrens et al., Cell 113: 829-840 (2003) X.H.T.Wehrens et al., Science 304: 292-296 (2004) M.Yano et al., Circulation 107: 477-484 (2003) Oda et al. Abstracts of the 8th Annual Meeting of the Japanese Heart Failure Society P135

  The problem to be solved by the present invention is to treat or prevent muscle excitement / contraction-related abnormal diseases, i.e., a disorder involving stimulation conduction abnormality, muscle contraction or relaxation dysfunction, specifically cardiac hypertrophy, heart failure, false Lyanodine receptor function improving agent for sarcoplasmic reticulum, useful as a therapeutic or preventive agent for blood heart disease, angina pectoris, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia, And providing a method for evaluating the ryanodine receptor function improving action of a test substance.

As described above, in the sarcoplasmic reticulum, a substance that suppresses calcium leak via a ryanodine receptor is effective as a therapeutic or prophylactic agent for heart failure and the like. Therefore, the present inventors have conducted intensive studies in order to construct a method for screening a compound that suppresses calcium leak via ryanodine receptor efficiently and simply.
That is, it was investigated whether compounds reported to be involved in calcium leak from sarcoplasmic reticulum, such as JTV-519, have binding activity for ryanodine receptors. As a result, JTV-519 was found to competitively inhibit the binding of diltiazem tritium labeled ([3H] diltiazem) and ryanodine receptor. The binding inhibition activity of [3H] diltiazem by JTV-519 was found to be concentration-dependent and excellent in quantification.
In other words, it was unclear whether JTV-519 or diltiazem, which suppresses calcium leak in the endoplasmic reticulum, interacts directly with the ryanodine receptor, but according to the present invention, the ryanodine receptor and the reference substance were directly bound. In addition, it was found that the binding between the reference substance and the ryanodine receptor is antagonized by another compound that suppresses calcium leak in the endoplasmic reticulum.
The present invention has been completed based on the above findings.

That is, the present invention
[1] A method for measuring the binding activity of a test substance to a ryanodine receptor, comprising the following steps (1) to (4):
(1) contacting a ryanodine receptor, a test substance, and a reference substance having a ryanodine receptor stabilizing action;
(2) a step of measuring the amount of the reference substance bound to the ryanodine receptor in (1),
(3) A step of comparing the binding amount measured in (2) with the binding amount of the reference substance to the ryanodine receptor in the absence of the test substance, and (4) based on the result of (3), depending on the test substance Determining the binding inhibitory activity of the reference substance and ryanodine receptor;
[2] A substance having a ryanodine receptor stabilizing action is diltiazem, 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy-2,3,4,5-tetrahydro-1,4 -The method according to [1], which is benzothiazepine or a pharmaceutically acceptable salt thereof;
[3] The method according to [1] or [2], wherein the ryanodine receptor is an endoplasmic reticulum ryanodine receptor;
[4] The method according to [3], wherein the endoplasmic reticulum is a sarcoplasmic reticulum;
[5] Evaluation method of ryanodine receptor function improving action using test substance binding activity to ryanodine receptor as an index;
[6] The evaluation method according to [5], wherein the binding activity to the ryanodine receptor is measured by the method according to any one of [1] to [4];
[7] The method further comprises selecting a test substance that binds to the ryanodine receptor and has no ryanodine receptor stabilizing action and a test substance that does not competitively inhibit the binding of the ryanodine receptor, [6] the evaluation method according to [6];
[8] The evaluation method according to [7], wherein the substance that binds to the ryanodine receptor and does not have a ryanodine receptor stabilizing action is ryanodine.
[9] Muscle excitement / contraction-related abnormal disease or central calcium handling abnormal disease using as an index the ryanodine receptor function improving action of the test substance evaluated by the method according to any one of [5] to [8] To select therapeutic or preventive drugs for
[10] Muscle excitement / contraction-related abnormal disease is cardiac hypertrophy, heart failure, ischemic heart disease, angina, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia, malignant hyperthermia [9] a screening method according to claim 9, wherein the exercise tolerance is reduced due to symptom, congenital myopathy or heart failure;
[11] Central calcium handling disorders include epilepsy, depression, anxiety, schizophrenia, ALS (Amyotrophic lateral sclerosis), stroke, cerebral infarction, cerebral embolism, cerebrovascular disorder, Alzheimer's disease, Parkinson's disease The screening method according to [9], which is glaucoma, retinopathy or macular degeneration;
[12] A substance having binding activity to a ryanodine receptor, comprising diltiazem or 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy-2,3,4,5-tetrahydro- A ryanodine receptor function improving agent comprising, as an active ingredient, a reference substance comprising 1,4-benzothiazepine and a substance that inhibits the binding of ryanodine receptor;
[13] The ryanodine receptor function improving agent according to [12], wherein the substance having binding activity to the ryanodine receptor is a substance that does not further inhibit the binding of ryanodine and the ryanodine receptor;
[14] The ryanodine receptor function improving agent according to [12] or [13], which is a therapeutic or prophylactic agent for a disorder of muscular excitement / contraction-related abnormality or central calcium handling abnormality;
[15] Muscle excitement / contraction-related abnormal disease is cardiac hypertrophy, heart failure, ischemic heart disease, angina, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia, malignant hyperthermia The ryanodine receptor function-improving agent according to [14], which is reduced exercise tolerance associated with symptom, congenital myopathy or heart failure;
[16] Central calcium handling disorder is epilepsy, depression, anxiety, schizophrenia, ALS (Amyotrophic lateral sclerosis), stroke, cerebral infarction, cerebral embolism, cerebrovascular disorder, Alzheimer's disease, Parkinson's disease A ryanodine receptor function improving agent according to [14], which is glaucoma, retinopathy or macular degeneration;
[17] Diltiazem, 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine or a pharmaceutically acceptable product thereof A reagent for measuring the binding activity to the ryanodine receptor, comprising a salt as an active ingredient;
About.

  According to the present invention, a therapeutic or prophylactic agent for a disorder associated with muscle excitability / contraction, that is, a disorder involving stimulation conduction abnormality, muscle contraction or relaxation dysfunction, specifically cardiac hypertrophy, heart failure, ischemic heart disease, angina Lyanodine receptor function-improving agent for sarcoplasmic reticulum, useful as a therapeutic or preventive agent for myopathy, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia, and ryanodine receptor as test substance It has become possible to provide a method for evaluating the function improving function.

In the present specification, the ryanodine receptor is a very large protein having one subunit of 565 kDa, and four subunits form a calcium release channel and are responsible for calcium release from the endoplasmic reticulum. Examples of ryanodine receptors include ryanodine receptor 1 (hereinafter referred to as RyR1), ryanodine receptor 2 (hereinafter referred to as RyR2) and ryanodine receptor 3 (hereinafter referred to as RyR3). ing.
RyR1 is mainly expressed in skeletal muscle, and the amino acid sequence or gene sequence of human RyR1 is described in Genbank ACCESSION No.P21817, and F Zorzato et al., J. Biol. Chem., 265: 2244-2256 (1990). It is disclosed. In this specification, RyR1 includes not only the human RyR1 but also homologs derived from other animal species.
RyR2 is mainly expressed in cardiac muscle, and the amino acid sequence or gene sequence of human RyR2 is disclosed in Genbank ACCESSION No.Q92736 and REA Tunwell et al., Biochem.J., 318: 477-487 (1996). Yes. In this specification, RyR2 includes not only the human RyR2 but also homologs derived from other animal species.
RyR3 is expressed in all cells but particularly highly expressed in the brain. The amino acid sequence or gene sequence of human RyR3 is Genbank ACCESSION No. Q15413, and G Marziali et al., FEBS Lett., 394: 76-82. (1996). In this specification, RyR3 includes not only the human RyR3 but also homologs derived from other animal species.
In the present invention, the ryanodine receptor is not particularly limited as long as it can take a three-dimensional structure to which a ligand can bind, but preferably, ryanodine derived from mammals such as humans, cows, pigs, monkeys, mice and rats. Provided in the cell fraction expressing the receptor. Specific examples include intact cells, cell homogenates, and cell fractions fractionated from the homogenates by centrifugation or the like. Preferably, the endoplasmic reticulum or a membrane fraction derived from the endoplasmic reticulum can be used.

Further, for example, an artificial lipid bilayer membrane prepared by a conventional method from a solution in which various lipids such as phosphatidylcholine, phosphatidylserine, and cholesterol are mixed at an appropriate ratio, preferably a ratio close to that in the cell membrane of a mammalian cell, is also used. It can be preferably used in one embodiment of the invention.
For example, skeletal muscle endoplasmic reticulum or RyR1-expressing gene-transferred cells can be preferably used as RyR1. RyR2 is preferably a myocardial endoplasmic reticulum or a RyR2-expressing gene-introduced cell. As RyR3, preferably, neuronal vesicles or RyR3-expressing gene-introduced cells can be used.

In the present specification, the reference substance having a ryanodine receptor stabilizing action is an action that suppresses calcium leak caused by the action of FK506 on the endoplasmic reticulum (SR) (hereinafter referred to as calcium leak inhibitory action), or ryanodine It represents a substance having an action (hereinafter referred to as an opening probability suppressing action) of suppressing an increase in opening probability of a calcium free channel formed by a receptor. Having a calcium leak inhibitory action means suppressing calcium outflow in the measurement of the amount of calcium that has been taken into the endoplasmic reticulum and the amount of natural outflow outside the endoplasmic reticulum. The method for measuring the calcium leak inhibitory action is well known to those skilled in the art and includes the method described in T Yamamoto et al., Cardiovasc. Res. 44: 146-155 (1999). Also, having an opening probability suppressing action means, for example, the generation of current caused by ryanodine receptor destabilization in the measurement of current between planar lipid bilayers reconstituted ryanodine receptor at resting calcium ion concentration It means to suppress the number of times. The method for measuring the opening probability suppressing action is well known to those skilled in the art, and examples include the method described in AR Marks et al., Science 281: 818-821 (1998).
The substance having a ryanodine receptor stabilizing action is not particularly limited as long as it has the above-described calcium leak inhibiting action and opening probability inhibiting action, and preferably includes a compound having a molecular weight of 500 or less. Specifically, diltiazem, JTV-519, ie 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy-2,3,4,5-tetrahydro-1,4-benzothi Azepine or a pharmaceutically acceptable salt thereof can be exemplified. Here, JTV-519 is the following formula (1):

で表される化合物を示す。式（１）で表される化合物の薬学上許容される塩（例えば塩酸塩などが挙げられる）もまたJTV-519の代わりに用いることができる。

The compound represented by these is shown. Pharmaceutically acceptable salts of the compound represented by the formula (1) (for example, hydrochloride and the like) can also be used in place of JTV-519.

  The compound used as the test substance is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, inorganic compounds, natural or synthetically prepared organic compounds, and the like. The test substance is specifically a peptide library of 3 to 50 amino acids, preferably 5 to 20 amino acids, or a molecular weight of 100 to 2000, preferably prepared using combinatorial chemistry techniques known to those skilled in the art. Can include 200 to 800 low molecular weight organic compound libraries.

Below, the aspect of this invention is demonstrated in detail.
I. Method for Measuring Ryanodine Receptor Binding Activity A first aspect of the present invention relates to a method for measuring binding activity for a ryanodine receptor. That is, by using a substance having a ryanodine receptor stabilizing action such as diltiazem or JTV-519 as a reference substance, and examining whether the test substance competitively inhibits the binding of the reference substance and the ryanodine receptor, The binding activity of the test substance to the ryanodine receptor can be evaluated.
The “method for measuring the binding activity to ryanodine receptor” of the present invention includes the steps described in the following (1) to (4). That is,
(1) contacting a ryanodine receptor, a test substance, and a reference substance having a ryanodine receptor stabilizing action;
(2) a step of measuring the amount of the reference substance bound to the ryanodine receptor in (1),
(3) A step of comparing the amount of binding measured in (2) with the amount of binding of the reference substance to the ryanodine receptor in the absence of the test substance,
(4) A step of determining the binding inhibitory activity of the reference substance and the ryanodine receptor by the test substance based on the result of (3).

In the above (1), as the ryanodine receptor, an aqueous solution containing RyR1, RyR2 and / or RyR3, a cell, or a cell fraction prepared from the cell can be used.
Here, RyR1 or RyR2 is preferably used as a ryanodine receptor in order to select a therapeutic or prophylactic agent for a disorder associated with muscle excitement and contraction, that is, a disorder involving stimulation conduction abnormality, muscle contraction or relaxation dysfunction. Used. The muscle excitement / contraction-related abnormal diseases specifically include cardiac hypertrophy, heart failure, ischemic heart disease, angina pectoris, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia , Malignant hyperthermia, congenital myopathy, or reduced exercise tolerance associated with heart failure. Especially when evaluating therapeutic or preventive drugs such as cardiac hypertrophy, heart failure, ischemic heart disease, angina pectoris, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, myasthenia, RyR1 is preferably used when evaluating malignant hyperthermia, congenital myopathy, or reduced exercise tolerance associated with heart failure.
In addition, RyR3 is preferably used as a ryanodine receptor in order to select a therapeutic or prophylactic agent for a central calcium handling disorder. Examples of the above-mentioned abnormal central calcium handling include epilepsy, depression, anxiety, schizophrenia, ALS (Amyotrophic lateral sclerosis), stroke, cerebral infarction, cerebral embolism, cerebrovascular disorder, Alzheimer's disease Parkinson's disease, glaucoma, retinopathy or macular degeneration.
The aqueous solution is not particularly limited as long as it contains a ryanodine receptor protein, and includes, for example, a normal aqueous solution, a cell lysate, or a suspension of a protein solid phase carrier by immunoprecipitation.
Examples of the cells include cells expressing a ryanodine receptor regardless of origins such as endogenous and exogenous.
The cell fraction means various fractions derived from such cells, and is not particularly limited as long as it is a fraction containing an endoplasmic reticulum, particularly an endoplasmic reticulum membrane. Specific examples include myocardial sarcoplasmic reticulum or a membrane fraction thereof.
Examples of the cell disruption method include a method of crushing cells with a Potter type homogenizer, a method of disrupting by ultrasonic waves, and the like. In order to obtain a fraction containing endoplasmic reticulum, a density gradient centrifugation method using sucrose can be used.
In the above, cells obtained by culturing transformants containing ryanodine receptor DNA, and cell fractions derived from the cells can also be used. Examples of host cells used for preparing a transformant include Escherichia coli, yeast, insect cells, and animal cells, and preferably CHO cells and the like. For example, RYR can be expressed in CHO cells and used [J. Biol. Chem. 278.28856- (2003)].
The ryanodine receptor is purified from recombinant cells into which an expression vector containing DNA encoding the ryanodine receptor has been introduced by affinity chromatography using an anti-ryanodine receptor antibody, His-tag, GST-tag, or the like. You can also

Furthermore, since the endoplasmic reticulum membrane has a lipid bilayer structure, a purified ryanodine receptor reconstituted in an artificial lipid bilayer membrane can be used.
Examples of the lipid constituting the artificial lipid bilayer membrane include phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol (Ch), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and the like. What mixed the seed | species or more by the appropriate ratio is used preferably.
For example, for artificial lipid bilayer membranes (proteoliposomes), a suitable amount of mixed lipid chloroform solution of PC: PI: Ch = 12: 12: 1 is dispensed into a glass tube, and the chloroform is evaporated with nitrogen gas vapor to obtain lipid. After drying into a film, add an appropriate buffer to suspend, then disperse uniformly by sonication, and add a buffer containing a surfactant such as sodium cholate to completely suspend the lipid. To do. Here, an appropriate amount of the purified ryanodine receptor is added, incubated for about 20 to 30 minutes with occasional stirring in ice, and then dialyzed against an appropriate buffer. Proteoliposomes can be prepared by centrifuging at about 100,000 × g for 30-60 minutes and collecting the sediment.

  The amount of the reference substance used above bound to the ryanodine receptor can be measured by methods well known to those skilled in the art. That is, it can be measured using a reference substance that has a ryanodine receptor stabilizing action, such as diltiazem or JTV-519, which is labeled for detection. Specifically, the amount of the labeled compound in the solution containing the labeled compound not bound to the ryanodine receptor, that is, the solution containing no ryanodine receptor in the above (1) is measured, and the difference from the total amount of the labeled compound is calculated. By doing so, the amount of the labeled compound bound to the ryanodine receptor in the above (2) or (3) can be measured. Alternatively, by separating the ryanodine receptor, the binding amount of the labeled compound that is bound to the ryanodine receptor can be directly measured.

  In addition, a homogeneous assay can be performed using a ryanodine receptor or a membrane fraction containing a ryanodine receptor. As the homogeneous assay method, a method well known to those skilled in the art such as SPA or α-screen can be used. That is, the ryanodine receptor or the membrane fraction containing the ryanodine receptor can be solid-phased on a carrier containing liquid scintillant, and chemiluminescence can be detected from the carrier by binding the labeling compound.

Examples of the labeled compound include a substance having a ryanodine receptor stabilizing action labeled with a radioisotope such as [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S]. Specifically, tritiated diltiazem: diltiazem, cis (+) [N-methyl-3H] (Perkin Elmer), or JTV-519 in which a hydrogen atom in a methoxy group is tritiated (US Patent Publication: For example, see US2004 / 229781).

Hereinafter, the procedure of the measuring method of the present invention will be described in detail.
In (1) above, the cell or cell fraction containing the ryanodine receptor is suspended in an appropriate buffer. Here, the buffer is usually adjusted to pH 6.5 to 8.3, preferably pH 7.0 to 7.8. As the buffer solution, for example, a phosphate buffer solution, Tris-hydrochloric acid buffer solution and the like can be used. Further, a surfactant such as Tween 20, NP-40, CHAPS, Triton X-100 may be added as appropriate. Furthermore, protease inhibitors such as aprotinin, leupeptin, and PMSF can be added for the purpose of suppressing degradation of the ryanodine receptor by protease. In addition, okadaic acid, caliculin A, sodium fluoride, sodium vanadylate and the like can be added for the purpose of suppressing the effects of phosphorylation and dephosphorylation of the ryanodine receptor.
The reaction is usually carried out at 0 to 50 ° C., preferably about 4 to 37 ° C., for about 30 minutes to 24 hours, more preferably about 1 to 2 hours.
After the reaction, the binding reaction is stopped by adding an excessive amount of ice-cold buffer or covalently binding the labeled compound by UV irradiation, and removing the unbound labeled compound by filtration, etc., and measuring the amount of the labeled compound . If the labeled compound is a compound labeled with a radioisotope, the radioactivity is measured with a liquid scintillation counter or a γ-counter.

Next, as described in (3) above, the binding amount of the reference substance (labeled compound) measured in (2) above in the presence of the test substance is determined using the reference substance (label) in the absence of the test substance. The amount of binding of the compound) to the ryanodine receptor is compared.
Here, if the binding amount of the reference substance in the presence of the test substance is smaller than the binding amount of the reference substance in the absence of the test substance, the test substance competitively inhibits the binding between the reference substance and the ryanodine receptor. Judgment can be made. And the following formula:

で、被験物質の阻害活性を定量的に評価することもできる。

Thus, the inhibitory activity of the test substance can be quantitatively evaluated.

II. TECHNICAL FIELD The present invention relates to 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy-2,3,4,5 having an action to improve ryanodine receptor function. -Elucidating the action mechanism that a compound such as tetrahydro-1,4-benzothiazepine (JTV-519) directly binds to a ryanodine receptor, and using the action mechanism as an index, a substance having an action to improve ryanodine receptor function Based on finding out that it can be evaluated and screened. That is, the present invention provides a method for evaluating a ryanodine receptor function improving action using the binding activity of a test substance to a ryanodine receptor as an index.
The binding activity of the test substance to the ryanodine receptor can be measured using as an index whether the test substance inhibits the binding of the reference substance already known to bind directly to the ryanodine receptor and the ryanodine receptor. .
That is, based on the ryanodine receptor binding activity of the test substance, which is measured by the above-described measurement method I, the ryanodine receptor function improving action of the test substance can be evaluated.
Specifically, when the test substance has an activity (binding inhibitory activity) that inhibits binding between a reference substance composed of a substance having a ryanodine receptor stabilizing action and a ryanodine receptor, the test substance has a ryanodine receptor function. It can be evaluated that it has an improving action. For example, a test substance having the inhibition rate of 50% or more, preferably 80% or more can be determined to be a substance having a ryanodine receptor function improving action, that is, a candidate compound for a ryanodine receptor function improving agent. .
Furthermore, in the evaluation method of the present invention, it is preferable to select a test substance that binds to the ryanodine receptor and does not competitively inhibit the binding of the ryanodine receptor and a substance that does not have a ryanodine receptor stabilizing action. An example of the substance is ryanodine.
That is, by using ryanodine instead of the reference substance in the same manner as described above, it can be determined whether or not the test substance inhibits the binding between the ryanodine receptor and ryanodine. The ryanodine used here is preferably labeled with tritium or the like. For example, a test substance having a binding inhibition rate of ryanodine receptor and ryanodine of a test substance of 50% or less, preferably 30% or less, more preferably 10% or less is determined to be a candidate compound for a ryanodine receptor function improving agent. can do.
The ryanodine receptor function improving action here is the action that stabilizes the conformation of the ryanodine receptor (the ryanodine receptor stabilizing action), that is, the action that normally functions the calcium concentration regulation function in the endoplasmic reticulum by the ryanodine receptor. Is mentioned. Stabilizing the conformation of the ryanodine receptor is considered to enhance the binding between the FK506 binding protein and the ryanodine receptor at the molecular level. Specifically, an action to prevent calcium leak (calcium leak inhibitory action) or an action to suppress an increase in opening probability of a calcium release channel formed by a ryanodine receptor (opening probability inhibitory action) can be mentioned.
Diltiazem or JTV-519 has the above-described ryanodine receptor function improving action (see Non-Patent Document 5). That is, the evaluation method of the present invention is characterized by evaluating whether or not the test substance has a ryanodine receptor function improving action equivalent to or higher than that of the reference substance.
In addition, the substance having the ryanodine receptor function improving action evaluated by the evaluation method of the present invention, that is, the ryanodine receptor function improving action of the candidate compound of the ryanodine function improving agent is the aforementioned calcium leak inhibitory action or opening probability inhibitory action. It can be confirmed by measuring the action.

III. Therapeutic or preventive drug and its screening method Based on the evaluation results of II described above, it is possible to select a therapeutic or prophylactic drug for various diseases involving ryanodine receptor function. That is, the third aspect of the present invention provides a therapeutic or prophylactic agent for a muscular excitement / contraction-related abnormal disease or a central calcium handling abnormal disease, and a screening method thereof.
Here, “muscular excitement / contraction-related abnormal diseases”, that is, diseases involving abnormal stimulation conduction, muscle contraction or relaxation dysfunction are skeletal muscle, myocardium, specifically limb muscles, left atrium, left ventricle, right There is no particular limitation as long as it is a disease accompanied by impaired contraction or relaxation function of the atrium and right ventricle. Specifically, cardiac hypertrophy, heart failure, ischemic heart disease, angina pectoris, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia, malignant hyperthermia (during specific anesthetic administration) Diseases such as hyperthermia), congenital myopathy, or reduced exercise tolerance associated with heart failure.
The “central calcium handling abnormal disease” is not limited as long as it is a neurological dysfunction in nerve cells and an organic disorder accompanied by cell death due to intracellular calcium overload. Specifically, epilepsy, depression, anxiety, schizophrenia, ALS (Amyotrophic lateral sclerosis), stroke, cerebral infarction, cerebral embolism, cerebrovascular disorder, Alzheimer's disease, Parkinson's disease, glaucoma, retinopathy, Or diseases such as macular degeneration can be mentioned.

The therapeutic or prophylactic agent obtained by the screening method of the present invention can be used as it is or a pharmaceutically acceptable carrier (including excipients, extenders, binders, lubricants, etc.), conventional additives, etc. It can mix and can prepare as a pharmaceutical composition. The pharmaceutical composition is prepared in a form (oral administration such as tablets, pills, capsules, powders, granules, syrups; parenteral administration such as injections, drops, external preparations, suppositories, etc.), etc. Depending on the dosage, it can be administered orally or parenterally. The dosage varies depending on the type of active ingredient, administration route, administration subject or patient's age, weight, symptoms, etc., and cannot be defined unconditionally, but the daily dosage is several mg to 2 g, preferably about several tens mg. It can be administered once to several times a day.
The present invention will be described in detail in the following examples, but the present invention is not limited thereto.

(Binding inhibitory effect)
Canine myocardial endoplasmic reticulum (sarcoplasmic reticulum, hereinafter referred to as SR) was isolated from the left ventricle. The binding affinity of diltiazem, cis (+) [N-methyl-3H] (Perkin Elmer) (hereinafter referred to as [ ³ H] -diltiazem) to 60 μg SR was examined. That is, the total amount of [ ³ H] -diltiazem bound to SR and the amount of [ ³ H] -diltiazem bound to SR in the presence of a large excess of diltiazem were measured, and the difference between them was determined to [ ³ H] to SR. -Calculate the specific binding amount of diltiazem (Fig. 1), and the Scatchard plot shown in Fig. 2 (the vertical axis shows the ratio of the binding compound concentration to the free labeled compound concentration, and the horizontal axis plots the specific binding amount) The dissociation constant was -1 / Slope, and the maximum amount of binding was the intersection with the X-axis). As a result, the dissociation constant was 248 nM, and the maximum binding amount was 785 fmol / mg SR.
In addition, Western Blot was performed to compare the amount of L-type calcium channel in the same amount of myocardial cell membrane (Sarcolemma or less SL) collected from the left ventricle of the dog at the same time. Although a band indicating a calcium channel was detected, it was hardly detected by SR. However, when binding to [3H] -diltiazem under the same conditions using SR and SL, the maximum amount of binding was detected to the same extent.
When [ ³ H] -diltiazem was bound to RYR2 isolated by immunoprecipitation from SR, specific binding was detected.
From the above, it was found that [ ³ H] -diltiazem binding to SR is not L-type calcium channel but binding to RYR2.
On the other hand, the competitive binding inhibition effect of [ ³ H] -diltiazem to the sarcoplasmic reticulum was evaluated. That is, the JTV-519, and calcium antagonists, ^[3 H] - binding was measured inhibition to 60μg dog myocardium er diltiazem. The results are shown in Table 1.
As a result, the inhibition constants (Ki) of JTV-519 and diltiazem were 850 nM and 910 nM, respectively. On the other hand, nifedipine, a dihydropyridine calcium antagonist, showed no binding inhibitory activity against [ ³ H] -diltiazem.
From the above, a compound having a ryanodine receptor stabilizing action similar to that of diltiazem and JTV-519 can be found by competitive assay.

表1の結果より、ジルチアゼム及びJTV-519はリアノジン受容体に対する結合活性を有することが確認された。一方、ジヒドロピリジン系のカルシウム拮抗剤であるニフェジピンは、リアノジン結合活性を示さなかった。

From the results in Table 1, it was confirmed that diltiazem and JTV-519 have a binding activity to the ryanodine receptor. On the other hand, nifedipine, which is a dihydropyridine calcium antagonist, did not show ryanodine binding activity.

  The ryanodine receptor function-improving agent of the present invention is a therapeutic or prophylactic agent for muscle excitement / contraction-related abnormal diseases, i.e., stimulation conduction abnormality, muscle contraction or relaxation dysfunction-related diseases, specifically cardiac hypertrophy, heart failure, It is useful as a therapeutic or prophylactic agent for ischemic heart disease, angina pectoris, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia. The evaluation method of the ryanodine receptor function improving action of the present invention is useful for screening for therapeutic or prophylactic agents for muscular excitement / contraction-related abnormal diseases or muscle contraction or relaxation dysfunction-related diseases.

The result of having investigated the coupling | bonding of a ligand, ie, diltiazem, cis (+) [N-methyl-3H] (Perkin Elmer Co., Ltd.) and canine myocardial sarcoplasmic reticulum at various ligand concentrations is shown. □ (total binding) indicates total binding to the sarcoplasmic reticulum, ● (nonspecific binding) indicates nonspecific binding to the sarcoplasmic reticulum, and Δ (specific binding) indicates specific binding. (Total binding) is the reaction between RI and SR, (Non-specific binding) is the count in the reaction in the presence of excess (100 μM) unlabeled diltiazem, and the number of moles from the specific activity of [ ³ H] -diltiazem And the value divided by the amount of SR added is the vertical axis. (Specific binding) represents the value calculated by (total binding)-(non-specific binding). FIG. 2 shows specific binding of the graph of FIG. 1 in a Scatchard plot.

Claims (17)

A method for measuring the binding activity of a test substance to a ryanodine receptor, comprising the following steps (1) to (4):
(1) contacting a ryanodine receptor, a test substance, and a reference substance having a ryanodine receptor stabilizing action;
(2) a step of measuring the amount of the reference substance bound to the ryanodine receptor in (1),
(3) A step of comparing the binding amount measured in (2) with the binding amount of the reference substance to the ryanodine receptor in the absence of the test substance, and (4) based on the result of (3), depending on the test substance Determining the binding inhibitory activity of the reference substance and ryanodine receptor.   The substance having a ryanodine receptor stabilizing action is diltiazem, 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy-2,3,4,5-tetrahydro-1,4-benzothi The method according to claim 1, which is azepine or a pharmaceutically acceptable salt thereof.   The method according to claim 1 or 2, wherein the ryanodine receptor is an endoplasmic reticulum ryanodine receptor.   The method of claim 3, wherein the endoplasmic reticulum is a sarcoplasmic reticulum.   A method for evaluating an effect of improving the function of a ryanodine receptor, using as an index the binding activity of a test substance to the ryanodine receptor.   6. The evaluation method according to claim 5, wherein the binding activity to the ryanodine receptor is measured by the method according to any one of claims 1 to 4.   Furthermore, the test substance which binds to a ryanodine receptor and does not have a ryanodine receptor stabilizing action and the test substance which does not competitively inhibit the binding of the ryanodine receptor is selected. Evaluation method.   The evaluation method according to claim 7, wherein the substance that binds to the ryanodine receptor and does not have a ryanodine receptor stabilizing action is ryanodine.   A therapeutic or preventive agent for muscular excitement / contraction-related abnormal diseases or central calcium handling abnormal diseases, wherein the ryanodine receptor function improving action of the test substance evaluated by the method according to any one of claims 5 to 8 is used as an index. Medicine selection method.   Abnormal muscle excitement / contraction-related disorders include cardiac hypertrophy, heart failure, ischemic heart disease, angina, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia, malignant hyperthermia, congenital The screening method according to claim 9, wherein exercise tolerance is reduced due to sexual muscular disorder or heart failure.   Central calcium handling disorders include epilepsy, depression, anxiety, schizophrenia, ALS (amyotrophic lateral sclerosis), stroke, cerebral infarction, cerebral embolism, cerebrovascular disorder, Alzheimer's disease, Parkinson's disease, glaucoma, The screening method according to claim 9, wherein the screening method is retinopathy or macular degeneration.   A substance having binding activity to a ryanodine receptor, comprising diltiazem or 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy-2,3,4,5-tetrahydro-1,4 -A ryanodine receptor function improving agent comprising a reference substance comprising benzothiazepine and a substance that inhibits binding of ryanodine receptor as active ingredients.   The ryanodine receptor function improving agent according to claim 12, wherein the substance having binding activity to the ryanodine receptor is a substance that does not further inhibit the binding of ryanodine and the ryanodine receptor.   The ryanodine receptor function-improving agent according to claim 12 or 13, which is a therapeutic or prophylactic agent for muscular excitement / contraction-related abnormal diseases or central calcium handling abnormal diseases.   Abnormal muscle excitement / contraction-related disorders include cardiac hypertrophy, heart failure, ischemic heart disease, angina, myocardial infarction, arrhythmia, atrial or ventricular tachycardia and fibrillation, or myasthenia, malignant hyperthermia, congenital The ryanodine receptor function-improving agent according to claim 14, which is reduced exercise tolerance due to sexual muscular disorder or heart failure.   Central calcium handling disorders include epilepsy, depression, anxiety, schizophrenia, ALS (amyotrophic lateral sclerosis), stroke, cerebral infarction, cerebral embolism, cerebrovascular disorder, Alzheimer's disease, Parkinson's disease, glaucoma, The ryanodine receptor function improving agent according to claim 14, which is retinopathy or macular degeneration. Diltiazem, 4- [3- (4-benzylpiperidin-1-yl) propionyl] -7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine or a pharmaceutically acceptable salt thereof A reagent for measuring the binding activity to ryanodine receptor, comprising as an active ingredient.

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