# Patent

## Title
Polypeptide derived from the ryanodine receptor that inhibits calcium leakage

## Abstract

A polypeptide exhibiting the effect of inhibiting Ca2+ leakage in a target cell, the polypeptide having the amino acid sequence of any of SEQ ID NOs: 3 to 9 or an amino acid sequence obtained through deletion, insertion, substitution, or addition of one or more amino acids in the aforementioned amino acid sequence; a polynucleotide encoding the polypeptide; and a pharmaceutical composition containing, as an active ingredient, the polypeptide or an expression product of the polynucleotide.

## Background
TECHNICAL FIELD
The present invention relates to a polypeptide exhibiting the effect of inhibiting Ca2+ leakage (hereinafter may be referred to as the “Ca2+ leakage inhibitory effect”) in the endoplasmic reticulum of a target cell, and to a pharmaceutical composition containing the polypeptide as an active ingredient. The present invention also relates to a screening method for selecting a compound exhibiting an activity of enhancing or suppressing the Ca2+ leakage inhibitory effect against Ca2+ leakage induction in the endoplasmic reticulum of a target cell.
BACKGROUND ART
Ca2+ flux in myocardial cells is an important factor for myocardial contraction. In recent years, many reports have shown that abnormal Ca2+ regulation (abnormal calcium handling) in myocardial cells is closely related to onset of heart failure (see Non-Patent Documents 1 and 2). In general, Ca2+ flux in a myocardial cell is as follows. A myocardial cell contains sarcoplasmic reticulum serving as a Ca2+ pool, and influx of extracellular Ca2+ triggers Ca2+ release from the sarcoplasmic reticulum via ryanodine receptor (RyR) into the cytoplasm. This increases cytoplasmic Ca2+ concentration, resulting in myocardial contraction. Meanwhile, myocardial dilation requires a decrease in cytoplasmic Ca2+ concentration, which is attained through Ca2+ reuptake into the sarcoplasmic reticulum by means of sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Excess Ca2+ which has been initially taken up from the outside of the cell is discharged to the outside thereof by means of Na+—Ca2+ exchange pump (NCX). Recent studies have focused on the sarcoplasmic reticulum, which serves as a Ca2+ pool in a myocardial cell, in view that the sarcoplasmic reticulum is related to abnormal calcium handling, and depletion of Ca2+ in the sarcoplasmic reticulum has been shown in the case of heart failure. Therefore, it may be possible to develop an anti-heart failure drug or an antiarrhythmic drug on the basis of a new concept of targeting an increase in Ca2+ concentration in the sarcoplasmic reticulum to a normal level.
Conventionally, studies on a mechanism of abnormal calcium handling have focused on lowering SERCA activity. In practice, overexpression of SERCA has shown to improve cardiac contraction performance and cardiac relaxation performance (see Non-Patent Document 3). However, heart failure patients do not necessarily exhibit a change in SERCA amount or SERCA activity (see Non-Patent Document 4). Therefore, in recent years, studies have focused on involvement of RyR, in addition to SERCA, in the abnormal calcium handling mechanism.
RyR is a protein which binds specifically to ryanodine, which is an ergot alkaloid. RyR has a tetrameric structure containing subunits of about 5,000 amino acids, and is considered to form a foot structure on the N-terminal side and a channel on the C-terminal side. RyR is classified into three subtypes; i.e., RyR1, which is localized in skeletal muscle and the brain (in particular, the cerebellum); RyR2, which is localized in myocardium, smooth muscle, and the brain (entire brain); and RyR3, which is present in the brain (in particular, the hippocampus and thalamus), smooth muscle, and skeletal muscle. In the present invention, the ryanodine receptor encompasses all these subtypes, but is preferably RyR2. As described above, RyR2 is a homotetramer containing four subunits of about 5,000 amino acids, in which one subunit is bound to one molecule of FK506-binding protein 12.6 (FKBP12.6). Conceivably, when RyR2 is bound to FKBP12.6, the structure of RyR2 is stabilized, whereas when FKBP12.6 is dissociated from RyR2, Ca2+ is released from the sarcoplasmic reticulum to the cytoplasm.
As has been known, FKBP12.6 is dissociated from RyR2 through phosphorylation with protein kinase A (PKA). In heart failure patients, hyperphosphorylation with PKA causes dissociation of FKBP12.6 (60% or more), which results in enhancement of Ca2+ sensitivity of RyR2, leading to abnormal leakage of Ca2+ from the sarcoplasmic reticulum (see Non-Patent Document 5).
RyR2 mutation has been observed in several heart-related diseases [CPVT (catecholaminergic polymorphic ventricular tachycardia), FPVT (familial polymorphic ventricular tachycardia), and ARVD (arrhythmogenic right ventricular dysplasia)] (see Non-Patent Document 6). In practice, in vitro studies have shown that CPVT-related RyR2 mutation reduces the affinity of RyR2 to FKBP12.6, and enhances the sensitivity of RyR2 to phosphorylation (see Non-Patent Document 7).
In addition, experiments employing the sarcoplasmic reticulum of the left ventricular myocardium of a dog model with heart failure induced by high-frequency right ventricular pacing have shown that abnormal Ca2+ leakage via RyR2, which is observed in the heart failure model, is attributed to a change in RyR2 structure associated with reduction of the stoichiometric composition of FKBP12.6 (see Non-Patent Document 8).
Recent studies have shown that JTV519, which is a 1,4-benzothiazepine derivative, corrects abnormal Ca2+ leakage via RyR2 (see Non-Patent Document 9). JTV519 was developed as a therapeutic drug for ischemic disorders, and was subjected to clinical tests. According to Yano, M., et al., RyR2 is an effective target of JTV519, and a drug may be developed on the basis of a new concept of correcting calcium handling through the RyR2-stabilizing effect of JTV519.
In view of the foregoing, in the art, demand has arisen for development of a substance exhibiting the effect similar to that of JTV519 (i.e., RyR2-stabilizing effect), and exhibiting no side effects.

    Non-Patent Document 1: Beuckelmann, et al., Circulation, 85, 1046-1055 (1992)
    Non-Patent Document 2: Gwathmey, et al., Circ Res, 61, 70-76 (1987)
    Non-Patent Document 3: Baker, et al., Circ Res, 83, 1205-1214 (1998)
    Non-Patent Document 4: Munch, et al., J Mol Med, 76, 434-441 (1998)
    Non-Patent Document 5: Marx, et al., Cell, 101, 365-376 (2000)
    Non-Patent Document 6: Marks, Circulation, 106, 8-10 (2002)
    Non-Patent Document 7: Wehrens, et al., Cell, 113, 829-840 (2003)
    Non-Patent Document 8: Yano, et al., Circulation, 102, 2131-2136 (2000)
    Non-Patent Document 9: Yano, et al., Circulation, 107, 477-484 (2003)


DISCLOSURE OF THE INVENTION
As described above, if there was provided a new substance which improves myocardial performance through improvement or inhibition of abnormal calcium handling (e.g., Ca2+ leakage induction in myocardial sarcoplasmic reticulum), the substance would be useful for elucidation of calcium handling regulation mechanism in myocardial endoplasmic reticulum, elucidation of heart failure mechanism, or improvement of abnormal Ca2+ leakage regulation in endoplasmic reticulum.
If there was provided a polypeptide exhibiting Ca2+ leakage inhibitory effect, or an expression product of a polynucleotide (hereinafter may be referred to as a “polynucleotide expression product”) exhibiting Ca2+ leakage inhibitory effect, the polypeptide or the polynucleotide expression product would be expected as a therapeutic or preventive agent for, for example, diseases associated with abnormal intracellular calcium regulation (abnormal intracellular calcium handling), such as heart failure, cardiomyopathy, valvulopathy, ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, hypertension, peripheral circulatory disorder, diabetes, dysmnesia, cerebral infarction, and malignant hyperthermia. In addition, the polypeptide or the polynucleotide expression product could be employed for selecting, through screening, a candidate compound which promotes or suppresses the Ca2+ leakage inhibitory effect in the endoplasmic reticulum of a cell.
Therefore, the present invention contemplates provision of a polypeptide exhibiting the effect of inhibiting Ca2+ leakage in the endoplasmic reticulum of a target cell; a polynucleotide expression product exhibiting such Ca2+ leakage inhibitory effect; a pharmaceutical composition containing, as an active ingredient, the polypeptide or the polynucleotide expression product; and a screening method for selecting a compound exhibiting an activity of enhancing or suppressing the Ca2+ leakage inhibitory effect in the endoplasmic reticulum of a target cell, the method employing the polypeptide or the polynucleotide expression product.
In view of the foregoing, for the purpose of searching for a new substance exhibiting the Ca2+ leakage inhibitory effect in endoplasmic reticulum, firstly, the present inventors have searched for, as a therapeutic peptide which is considered to correct abnormality in RyR2 structure, a polypeptide exhibiting the Ca2+ leakage inhibitory effect in endoplasmic reticulum. As a result, the present inventors have newly found that a polypeptide having a sequence of amino acids corresponding to positions 2114 to 2149 of the amino acid sequence of RyR2 exhibits the Ca2+ leakage inhibitory effect in endoplasmic reticulum. Furthermore, on the basis of the aforementioned sequence of amino acids, the present inventors have prepared polypeptides having amino acid sequences of different lengths, and have found that some polypeptides of the thus-prepared polypeptides exhibit the Ca2+ leakage inhibitory effect. The present invention has been accomplished on the basis of these findings.
Accordingly, the present invention provides a polypeptide which is selected from the group consisting of polypeptides described below in (a) and (b):

    (a) a polypeptide having the amino acid sequence of any of SEQ ID NOs: 3 to 9; and
    (b) a polypeptide having an amino acid sequence obtained through deletion, insertion, substitution, or addition of one or more amino acids in the amino acid sequence of any of SEQ ID NOs: 3 to 9, and which exhibits the effect of inhibiting Ca2+ leakage in a target cell.


The present invention also provides a polynucleotide selected from the group consisting of polynucleotides described below in (c), (d), and (e):

    (c) a polynucleotide encoding a polypeptide having the amino acid sequence of any of SEQ ID NOs: 3 to 9;
    (d) a polynucleotide which can be hybridized with the polynucleotide described in (c) above under stringent conditions, and which can produce a corresponding expression product; and
    (e) a polynucleotide which has a DNA sequence 80% or more homologous to the polynucleotide described in (c) above, and which can produce a corresponding expression product, an expression product of the polynucleotide exhibiting the effect of inhibiting Ca2+ leakage in a target cell.


The present invention also provides a pharmaceutical composition containing, as an active ingredient, the aforementioned polypeptide or an expression product of the aforementioned polynucleotide.
The present invention also provides a screening method for selecting an agonist or an antagonist with respect to the Ca2+ leakage inhibitory effect in the endoplasmic reticulum of a target cell, which method is characterized by comprising measuring, in the presence or absence of a test substance, the level of the Ca2+ leakage inhibitory effect of the aforementioned polypeptide in the endoplasmic reticulum of a target cell; and comparing the level measured in the presence of the test substance with the level measured in the absence of the test substance, to thereby select the test substance as an agonist which enhances the inhibitory effect or as an antagonist which suppresses the inhibitory effect.
The present invention also provides a screening method for selecting an agonist or an antagonist with respect to the Ca2+ leakage inhibitory effect in the endoplasmic reticulum of a target cell, which method is characterized by comprising measuring, in the presence or absence of a test substance, the level of the Ca2+ leakage inhibitory effect of an expression product of the aforementioned polynucleotide in the endoplasmic reticulum of a target cell; and comparing the level measured in the presence of the test substance with the level measured in the absence of the test substance, to thereby select the test substance as an agonist which enhances the inhibitory effect or as an antagonist which suppresses the inhibitory effect.
The present invention also provides a screening method for selecting a candidate substance which enhances the Ca2+ leakage inhibitory effect of the aforementioned polypeptide in the endoplasmic reticulum of a target cell, which method comprises the following steps (1) to (5):

    (1) a step of providing a target cell transformed with an expression vector containing a polynucleotide encoding the aforementioned polypeptide;
    (2) a step of adding FK506 to the endoplasmic reticulum of the target cell so as to induce Ca2+ leakage in the presence or absence of a test substance;
    (3) a step of measuring, by means of a Ca2+ indicator, the amount of Ca2+ leaked from the endoplasmic reticulum of the target cell, to thereby determine the amount of change in Ca2+ leakage from the endoplasmic reticulum in the presence or absence of the test substance;
    (4) a step of adding FK506 to the endoplasmic reticulum of a non-transformed cell so as to induce Ca2+ leakage in the absence of the test substance, measuring by means of a Ca2+ indicator the amount of Ca2+ having leaked from the endoplasmic reticulum, and determining the difference between the amount of Ca2+ as measured outside the endoplasmic reticulum; i.e., the standard amount, and each of the amounts as determined in (3) above in the presence and absence of the test substance; and
    (5) a step of selecting the test substance as a candidate substance when the difference between the standard amount and the amount as determined in the presence of the test substance is smaller than the difference between the standard amount and the amount as determined in the absence of the test substance.


The present invention also provides a screening method for selecting a candidate substance which enhances the Ca2+ leakage inhibitory effect of an expression product of the aforementioned polynucleotide in the endoplasmic reticulum of a target cell, which method comprises the following steps (1) to (5):

    (1) a step of providing a target cell transformed with an expression vector containing the aforementioned polynucleotide;
    (2) a step of adding FK506 to the endoplasmic reticulum of the target cell so as to induce Ca2+ leakage in the presence or absence of a test substance;
    (3) a step of measuring, through RT-PCR, the amount of expression of the polynucleotide in the endoplasmic reticulum of the target cell in the presence or absence of the test substance;
    (4) a step of comparing the expression amount in the presence of the test substance with the expression amount in the absence of the test substance; and
    (5) a step of selecting the test substance as a candidate substance when the expression amount in the presence of the test substance is greater than the expression amount in the absence of the test substance.


The present invention also provides a screening method for selecting a candidate substance which enhances the Ca2+ leakage inhibitory effect of the aforementioned polypeptide in a target cell, which method comprises the following steps (1) to (5):

    (1) a step of providing a target cell transformed with an expression vector containing a polynucleotide encoding the aforementioned polypeptide;
    (2) a step of adding FK506 to the endoplasmic reticulum of the target cell so as to induce Ca2+ leakage in the presence or absence of a test substance;
    (3) a step of measuring, by means of an antibody against the polypeptide, the amount of the polypeptide produced in the target cell in the presence or absence of the test substance;
    (4) a step of comparing the production amount in the presence of the test substance with the production amount in the absence of the test substance; and
    (5) a step of selecting the test substance as a candidate substance when the production amount in the presence of the test substance is greater than the production amount in the absence of the test substance.


The present invention also provides a screening kit for selecting an agonist or an antagonist with respect to the Ca2+ leakage inhibitory effect of the aforementioned polypeptide or an expression product of the aforementioned polynucleotide in the endoplasmic reticulum of a target cell, which kit is characterized by comprising at least one species selected from the group consisting of the polypeptide and the expression product, which are contained in the target cell, and FK506.
The polypeptide of the present invention exhibits the effect of inhibiting Ca2+ leakage in the endoplasmic reticulum of a target cell, and therefore is useful as a therapeutic or preventive agent for, for example, diseases associated with abnormal intracellular calcium handling. The polypeptide can be employed for selecting, through screening, a compound exhibiting an activity of enhancing or suppressing the Ca2+ leakage inhibitory effect in the endoplasmic reticulum of a target cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the effects of synthesized polypeptides on FK506-induced Ca2+ leakage in the sarcoplasmic reticulum derived from canine myocardium.
FIG. 2 shows the effects of synthesized polypeptides on FK506-induced Ca2+ leakage in the sarcoplasmic reticulum derived from canine myocardium.
FIG. 3 shows the effects of synthesized polypeptides on FK506-induced Ca2+ leakage in the sarcoplasmic reticulum derived from canine myocardium.
FIG. 4 shows the effects of synthesized polypeptides on FK506-induced Ca2+ leakage in the sarcoplasmic reticulum derived from canine myocardium.
FIG. 5 shows the effects of synthesized polypeptides on FK506-induced Ca2+ leakage in the sarcoplasmic reticulum derived from canine myocardium.



BEST MODE FOR CARRYING OUT THE INVENTION
[Definition]
As used herein, abbreviations of amino acids, peptides, nucleotide sequences, nucleic acids, etc. are according to IUPAC-IUB nomenclature [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984)], “Guideline for preparation of a specification, etc. including nucleotide sequences or amino acid sequences” (edited by Japan Patent Office), and conventional symbols used in the art.
As used herein, the term “gene” encompasses double-stranded DNA, as well as single-stranded DNA (sense strand or antisense strand) constituting the double-stranded DNA. No limitation is imposed on the length of such DNA. Therefore, unless otherwise specified, the polynucleotide (DNA molecule) encoding the polypeptide of the present invention encompasses double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand) including cDNA, single-stranded DNA (antisense strand) having a sequence complementary to the sense strand, synthetic DNA, and fragments thereof.
As used herein, “gene (DNA molecule)” may include any functional region; for example, at least one of an expression inhibitory region, a coding region, a leader sequence, an exon, and an intron. The term “polynucleotide” encompasses RNA and DNA. The term “DNA molecule” encompasses cDNA, genomic DNA, and synthetic DNA. The polypeptide having a specific amino acid sequence, or the polynucleotide encoding the polypeptide encompasses its fragments, homologues, derivatives, and mutants.
Examples of mutants of the polynucleotide (mutant DNA) include naturally occurring allelic mutants; non-naturally occurring mutants; and mutants obtained through deletion, substitution, addition, or insertion. Note that such a mutant encodes a polypeptide having substantially the same function as the polypeptide encoded by the non-mutated polynucleotide.
Variation of the polypeptide (modification of amino acid sequence) does not necessarily naturally occur through, for example, mutation or post-translational modification, and may be artificially performed by use of a naturally occurring gene (e.g., a fragment of the ryanodine receptor gene according to the present invention). Examples of mutants of the aforementioned polypeptide include allelic mutants, homologues, and naturally occurring mutants, which are at least 80% (preferably 95%, more preferably 99%) homologous to the non-mutated polypeptide.
The mutant DNA is conservative, or silent with respect to the amino acid sequence encoded by the DNA (i.e., no change occurs in amino acid residues encoded by the mutated nucleic acid sequence). Examples of conservative amino acid substitutions are as follows.














Original amino
Conservatively substituted



acid residue
amino acid residue









Ala
Ser



Arg
Lys



Asn
Gln or His



Asp
Glu



Cys
Ser



Gln
Asn



Glu
Asp



Gly
Pro



His
Asn or Gln



Ile
Leu or Val



Leu
Ile or Val



Lys
Arg, Asn, or Glu



Met
Leu or Ile



Phe
Met, Leu, or Tyr



Ser
Thr



Thr
Ser



Trp
Tyr



Tyr
Trp or Phe



Val
Ile or Leu










In general, one or more codons encoding a Cys residue affect the disulfide bonds of a specific polypeptide.
Amino acid residue substitutions which are generally considered to change protein properties are, for example, the following substitutions:
a) substitution of a hydrophobic residue with a hydrophilic residue; for example, substitution of Leu, Ile, Phe, Val, or Ala with Ser or Thr;
b) substitution of an amino acid residue other than Cys and Pro with Cys or Pro;
c) substitution of a residue having an electrically positive side chain (e.g., Lys, Arg, or His) with an electrically negative residue (e.g., Glu or Asp); and
d) substitution of an amino acid residue having a very large side chain (e.g., Phe) with an amino acid residue having no side chain (e.g., Gly).
[1] Polypeptide of the Present Invention
The polypeptide of the present invention (and the polypeptide employed in the screening method of the present invention; hereinafter may be referred to simply as “the invention polypeptide”) is any of the following polypeptides (a) and (b):

    (a) a polypeptide having the amino acid sequence of any of SEQ ID NOs: 3 to 9; and
    (b) a homologue of the aforementioned polypeptide; i.e., a polypeptide having an amino acid sequence obtained through deletion, insertion, substitution, or addition of one or more amino acids in the amino acid sequence of any of SEQ ID NOs: 3 to 9. The invention polypeptide exhibits the effect of inhibiting Ca2+ leakage in a target cell.


The polypeptide represented by SEQ ID NO: 3, which is named “R2P,” has a sequence of 36 amino acids corresponding to positions 2114 to 2149 of the amino acid sequence of human ryanodine receptor type 2 (hRyR2). SEQ ID NO: 20 represents the DNA sequence encoding the amino acid sequence of R2P. As shown in SEQ ID NO: 2, the DNA encoding the aforementioned ryanodine receptor type 2 contains 14,901 bases. As shown in SEQ ID NO: 1, the amino acid sequence encoded by the DNA contains 4,967 amino acids (Genbank Accession No. NM 001035).
The polypeptide represented by SEQ ID NO: 4 is MSV-R2P (M+2112-2149), which is obtained through addition of three amino acids (methionine, serine, and valine) to the N-terminal of R2P (SEQ ID NO: 3). The polypeptide represented by SEQ ID NO: 5 is RP1-30 (2114-2143), which is obtained through deletion of six amino acids at the C-terminal of R2P. The polypeptide represented by SEQ ID NO: 6 is RP1-25 (2114-2138), which is obtained through deletion of 11 amino acids at the C-terminal of R2P. The polypeptide represented by SEQ ID NO: 7 is RP1-20 (2114-2133), which is obtained through deletion of 16 amino acids at the C-terminal of R2P. The polypeptide represented by SEQ ID NO: 8 is RP13-32 (2126-2145), which is obtained through deletion of 12 amino acids at the N-terminal of R2P and deletion of four amino acids at the C-terminal thereof. The polypeptide represented by SEQ ID NO: 9 is RP17-36 (2130-2149), which is obtained through deletion of 16 amino acids at the N-terminal of R2P. The DNA sequences encoding these polypeptides are represented by SEQ ID NOs: 21 to 26.
No particular limitation is imposed on the number and position of amino acids which undergo modification (i.e., deletion, insertion, substitution, or addition) in the amino acid sequence of any of SEQ ID NOs: 3 to 9 shown in (b) above, so long as the polypeptide having the thus-modified amino acid sequence exhibits substantially the same activity as the polypeptide having the amino acid sequence of any of SEQ ID NOs: 3 to 9 (in particular, R2P having the amino acid sequence of SEQ ID NO: 3); i.e., these polypeptides exhibit the same effect. Preferably, one to a plurality of amino acids undergo the aforementioned modification. However, as described below in Example, the polypeptides represented by SEQ ID NOs: 4 to 9 also exhibit Ca2+ leakage inhibitory activity. Therefore, as used herein, the expression “modification of a plurality of amino acids in R2P” refers to the case where 2 to 20 amino acids of the amino acid sequence of R2P undergo deletion, insertion, substitution, or addition. This amino acid modification is preferably performed such that homology in the corresponding amino acids between the thus-modified polypeptide and the polypeptide represented by SEQ ID NO: 3 is, for example, about 80% or more, more preferably about 95% or more, particularly preferably about 98% or more.
Polypeptide or polynucleotide homology can be analyzed through measurement employing sequence analysis software (e.g., FASTA program) (Clustal, V., Methods Mol. Biol., 25, 307-318 (1994)). In the most preferred and convenient method for homology analysis, for example, a sequence is stored in a computer-readable medium (e.g., flexible disk, CD-ROM, hard disk drive, external disk drive, or DVD), and subsequently, by use of the thus-stored sequence, known sequence databases are searched through well known search means. Specific examples of such known sequence databases include the following databases:

    
    
        DNA Database of Japan (DDBJ);
        Genebank; and
        the European Molecular Biology Laboratory Nucleic Acid Sequence Database (EMBL).
    
    


Homology analysis can employ numerous search algorithms. An example of such search algorithms is a program called “BLAST.” This BLAST program includes five BLAST means. Three of the five means (BLASTN, BLASTX, and TBLASTX) are designed for nucleotide sequence query, and the remaining two means (BLASTP and TBLASTN) are designed for protein sequence query (Coulson, Trends in Biotechnology, 12: 76-80 (1994); Birren, et al., Genome Analysis, 1: 543-559 (1997)).
Furthermore, an additional program (e.g., a sequence alignment program or a program for identifying a remote sequence) can be employed for analysis of an identified sequence in the art.
The invention polypeptide may be, for example, a polypeptide derived from human cell, in particular, human myocardial cell, smooth muscle cell, skeletal muscle cell, brain neuron, neuroblast, or pancreatic β cell; or a polypeptide derived from human tissue, in particular, human heart, arterial/venous vessel, skeletal muscle, smooth muscle, cerebrum (cerebral cortex), cerebellum, pancreas, or the like. Alternatively, the invention polypeptide may be prepared through chemical synthesis on the basis of the amino acid sequence data of such a human-derived polypeptide. Examples of the homologue of the invention polypeptide (a) described in (b) above include proteins which exhibit Ca2+ leakage inhibitory activity and are derived from yeast, nematode, plant tissue, or mammal other than human. Examples of the mammal include horse, sheep, cattle, dog, monkey, cat, bear, mouse, rat, and rabbit.
The aforementioned Ca2+ leakage inhibitory effect of the invention polypeptide in a target cell is more specifically the effect of inhibiting intracellular calcium increase induced by Ca2+ leakage in the endoplasmic reticulum of the cell, and increasing Ca2+ uptake into the endoplasmic reticulum, to thereby inhibit Ca2+ leakage and to improve abnormal intracellular calcium handling (regulation). Examples of the aforementioned cell endoplasmic reticulum include sarcoplasmic reticulum. Specific examples of the sarcoplasmic reticulum include sarcoplasmic reticulum prepared from normal or failing myocardium, sarcoplasmic reticulum prepared from vascular smooth muscle, and sarcoplasmic reticulum prepared from skeletal muscle. In, for example, myocardial tissue, the Ca2+ leakage inhibitory effect is expected to result in an increase in myocardial contractile force, leading to improvement of heart failure or arrhythmia.
The Ca2+ leakage inhibitory effect can also be regarded as the effect of stabilizing ryanodine receptor function. Specifically, the ryanodine receptor function stabilizing effect refers to the effect of maintaining ryanodine receptor function in a normal state (i.e., in a state of normal tissues or normal cells of a healthy individual). As used herein, the ryanodine receptor function can also be regarded as the function of inhibiting (suppressing) Ca2+ leakage and regulating the transmission of Ca2+ signals from the sarcoplasmic reticulum, so as to maintain the amount of Ca2+ stored in the sarcoplasmic reticulum. Therefore, “maintaining ryanodine receptor function” refers to maintaining the state where physiological stimulation (e.g., membrane potential change) induces opening/closing of the ryanodine receptor channel required for normal muscle contraction/relaxation, and Ca2+ is stored in the sarcoplasmic reticulum in an amount sufficient for the transmission of Ca2+ signals from the sarcoplasmic reticulum.
Stabilization or maintenance of the aforementioned ryanodine receptor function can be attained by, for example, inhibiting reduction of the bonding strength between RyR and FKBP12.6, inhibiting phosphorylation of the ryanodine receptor, or maintaining the structure of the ryanodine receptor.
No particular limitation is imposed on the aforementioned homologue, so long as the homologue exhibits substantially the same Ca2+ leakage inhibitory effect as the polypeptide having the amino acid sequence of any of SEQ ID NOs. 3 to 9. As used herein, the expression “substantially the same” refers to the case where the Ca2+ leakage inhibitory effect of the homologue is physiochemically or pharmacologically the same as that of the polypeptide. The homologue and the polypeptide may differ from each other in terms of the extent of Ca2+ leakage inhibitory effect, or quantitative factors (e.g., protein molecular weight).
Preferably, the invention polypeptide having a modified amino acid sequence described in (b) above is substantially the same as the polypeptide having the amino acid sequence of SEQ ID NO: 3 (i.e., non-modified amino acid sequence) in terms of, in addition to Ca2+ leakage inhibitory effect, for example, the intrinsic activity of the non-modified polypeptide (e.g., the effect of stabilizing or maintaining ryanodine receptor activity in the case where the polypeptide is a ryanodine receptor stabilizer), antigenicity, or immunogenic activity.
In the present specification, the activities of the polypeptides (a) and (b); for example, Ca2+ leakage inhibitory effect, effect of improving abnormal calcium handling (regulation), effect of stabilizing ryanodine receptor function, effect of maintaining (improving) ryanodine receptor function, effect of inhibiting reduction of the bonding strength between the ryanodine receptor and the FK506-binding protein, effect of inhibiting phosphorylation of the ryanodine receptor, and effect of maintaining the ryanodine receptor structure, may hereinafter be referred to simply as “Ca2+ leakage inhibitory activity.”
The Ca2+ leakage inhibitory effect of the invention polypeptide corresponds to the case where, when a substance which induces Ca2+ leakage is administered to a cell or tissue provided with the ability to express the invention polypeptide, in the cell or tissue, Ca2+ leakage is inhibited, or Ca2+ concentration in the endoplasmic reticulum is increased, as compared with the case of a cell or tissue which is not provided with such expression ability. More specifically, the Ca2+ leakage inhibitory effect of the invention polypeptide corresponds to the effect of inhibiting Ca2+ leakage in the endoplasmic reticulum of a cell or a tissue, the Ca2+ leakage being induced by, for example, a substance (e.g., FK506) which cuts the bond between ryanodine receptor and FKBP12.6, thereby releasing Ca2+ to cytoplasm, or a substance which cuts the bond between ryanodine receptor and FKBP12.6 through phosphorylation of the ryanodine receptor with protein kinase A (PKA).
As described above, the invention polypeptide has an amino acid sequence corresponding to a portion of the amino acid sequence of human ryanodine receptor type 2. Expression of the human ryanodine receptor has been found in various human tissues. The human ryanodine receptor has been found to have three subtypes; i.e., RyR1, which is localized in skeletal muscle and the brain (in particular, the cerebellum); RyR2, which is localized in myocardium, smooth muscle, and the brain (entire brain); and RyR3, which is present in the brain (in particular, the hippocampus and thalamus), smooth muscle, and skeletal muscle. Recent studies have reported dysfunction or reduced expression of RyR2 in a diabetic animal model [Bidasee, et al., Mol. Pharmacol., 60, 1356-1364 (2001); Okamoto, et al., Diabetologia, 40, 1485-1491 (1997)], which suggests involvement of RyR2 in diabetic conditions.
The invention polypeptide exhibits the effect of inhibiting Ca2+ leakage in the endoplasmic reticulum of a cell or a tissue, and thus the polypeptide can be employed as an active ingredient of a drug. As described below, studies have reported the relation between Ca2+ leakage and various diseases. According to these studies, a drug containing the invention polypeptide as an active ingredient may be employed for the treatment of such a disease.
Heart failure: Heart failure is a terminal event (syndrome) of various heart diseases. In heart failure, regardless of the original heart disease, an increase in calcium concentration in cytoplasm in diastole has been reported. Such calcium overload is closely related to calcium leakage via ryanodine receptor (Non-Patent Document 8), and thus use of a substance which inhibits such calcium leakage may provide a new promising strategy for the treatment of heart failure.
Cardiomyopathy: In hypertrophic cardiomyopathy, progression of hypertrophy has been shown to have a close relation to calcineurin, which is a calcium regulatory protein [Sussman M. A. et al., Science, 281: 1690-1693 (1998)]. Inhibition of intracellular calcium overload is considered effective for inhibiting progression of cardiac hypertrophy. Cardiomyopathy finally progresses, through reduction of myocardial contractile force, to heart failure. As described above, heart failure is closely related to calcium leakage (Non-Patent Document 8), and thus use of a substance which inhibits such calcium leakage may provide a new promising strategy for the treatment of cardiomyopathy.
Valvulopathy: Cardiac valvulopathy is a disease in which valvular insufficiency or valvular stenosis occurs, and load is chronically applied to the heart, finally leading to heart failure. Valvulopathy finally progresses, through reduction of myocardial contractile force, to heart failure. As described above, heart failure is closely related to calcium leakage (Non-Patent Document 8), and thus use of a substance which inhibits such calcium leakage may provide a new promising strategy for the treatment of valvulopathy.
Ischemic heart disease: As has been known, during ischemic reperfusion, many abnormalities occur in sarcoplasmic reticulum as well as in NCX, mitochondria, etc., which results in marked intracellular calcium overload. The extent of the role of ryanodine receptor in such calcium overload has not yet been elucidated, but JTV519, which has previously been shown by the present inventors to act on ryanodine receptor in the case of heart failure, is known to be effective for ischemic reperfusion injury [Hypertens. Res., 25: 303-9 (2002)]. Therefore, inhibition of calcium leakage is considered effective for reperfusion injury in ischemic heart disease, and a substance which inhibits calcium leakage may be employed as a new therapeutic agent for ischemic heart disease.
Myocardial infarction: Similar to the case of the aforementioned ischemic heart disease, inhibition of calcium leakage is considered effective for reperfusion injury in myocardial infarction. In addition, inhibition of calcium leakage is considered effective for prevention of heart failure in a post-myocardial infarction patient, and therefore a substance which inhibits calcium leakage may be employed as a new therapeutic agent for myocardial infarction.
Angina pectoris: Similar to the case of the aforementioned ischemic heart disease, inhibition of calcium leakage is considered effective for reperfusion injury in angina pectoris. Therefore, a substance which inhibits calcium leakage may be employed as a new therapeutic agent for angina pectoris.
Arrhythmia: Mutation of myocardial ryanodine receptor tends to cause calcium leakage (in the form of channel property), and fatal arrhythmia (in the form of disease) [Annu. Rev. Med., 54: 257-67 (2003)]. This fact implies that calcium leakage via ryanodine receptor is closely related to fatal arrhythmia. Therefore, use of a substance which inhibits such calcium leakage may provide a new strategy for the treatment of arrhythmia.
Hypertension and peripheral circulatory disorder: Onset of arteriosclerosis is related to an increase in intracellular Ca2+ concentration in vascular smooth muscle. Intracellular Ca2+ concentration in vascular smooth muscle is generally regulated by IP3 receptor. Recent studies have shown that ryanodine receptor is also distributed in vascular smooth muscle, and is involved in regulation of such Ca2+ concentration [Circulation Research, 89: 1051-1067 (2001)]. Inhibition of calcium leakage via ryanodine receptor may also inhibit arteriosclerosis in vessel walls. Therefore, a substance which inhibits calcium leakage may be employed as an ameliorating agent for hypertension and peripheral circulatory disorder.
Diabetes: Insulin secretion in β cells is closely related to calcium release via ryanodine receptor mediated by FKBP12.6 [J. Biol. Chem., 272: 3133-6 (1997)]. A substance which inhibits calcium leakage via ryanodine receptor may be employed for normalization of insulin secretion, and treatment of diabetes.
Dysmnesia: Abnormality in ryanodine receptor has been reported in Alzheimer's disease [Neuroscience, 92: 499-513 (1999)]. Therefore, a substance which corrects such abnormality may be employed for the treatment of Alzheimer's disease or dysmnesia.
Cerebral infarction: Cell death during cerebral ischemia is closely related to an increase in intracellular Ca2+ concentration. A large amount of ryanodine receptor is present in the endoplasmic reticulum of brain neuron, and dantrolene, which is a ryanodine receptor inhibitor, has been reported to inhibit neuron death during cerebral ischemia [Neurosci. Lett., 158: 105-108 (1993)]. Therefore, a substance which inhibits calcium leakage via ryanodine receptor may considerably reduce neuron death during cerebral ischemia, and the substance may be employed as a therapeutic agent for cerebral infarction.
Malignant hyperthermia: Malignant hyperthermia is a fatal genetic disease in which Ca2+ is released from skeletal muscle ryanodine receptor during anesthesia, causing high fever and muscle contracture, the disease resulting from enhancement of the sensitivity of skeletal muscle ryanodine receptor to an anesthetic (e.g., halothane) through mutation of the ryanodine receptor. The channel regulatory domain of skeletal muscle ryanodine receptor is involved in such abnormal enhancement of the sensitivity to halothane. Therefore, a substance which regulates abnormal calcium handling may promote stabilization of ryanodine receptor, and may be employed as a new therapeutic drug for malignant hyperthermia.
As described above, there have been reported the relation between inhibition of Ca2+ leakage in endoplasmic reticulum and onset of a disease such as heart failure, cardiomyopathy, valvulopathy, ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, hypertension, peripheral circulatory disorder, diabetes, dysmnesia, cerebral infarction, or malignant hyperthermia, and as well the effect of inhibiting Ca2+ leakage in endoplasmic reticulum associated with such a disease. The fact that the invention polypeptide exhibits the effect of inhibiting Ca2+ leakage in endoplasmic reticulum suggests that the polypeptide per se inhibits Ca2+ leakage in the sarcoplasmic reticulum of a target cell, and thus the polypeptide is useful for treating or preventing any of the aforementioned diseases.
[2] Polynucleotide (DNA Molecule) of the Present Invention
The polynucleotide of the present invention is any of the following polynucleotides (c), (d), and (e):

    (c) a polynucleotide encoding a polypeptide having the amino acid sequence of any of SEQ ID NOs: 3 to 9;
    (d) a polynucleotide which can be hybridized with the polynucleotide described in (c) above under stringent conditions, and which can produce a corresponding expression product; and
    (e) a polynucleotide which has a DNA sequence 80% or more homologous to the polynucleotide described in (c) above, and which can produce a corresponding expression product. An expression product of the polynucleotide of the present invention exhibits Ca2+ leakage inhibitory effect in a target cell.


Among the polynucleotides of the present invention (hereinafter may be referred to as the “invention DNA molecule(s)”), the aforementioned polynucleotide (c) may be a DNA molecule containing a polynucleotide having the DNA sequence of any of SEQ ID NOs: 20 to 26 or the complementary strand thereof.
The invention DNA molecules (polynucleotides) also include the polynucleotide (d); i.e., a polynucleotide which is hybridizable with the aforementioned polynucleotide (c) under stringent conditions, and can express a product exhibiting Ca2+ leakage inhibitory effect in a target cell. As used herein, the expression “hybridizable under stringent conditions” may refer to the case where, even when hybridization is performed in 0.2×SSC containing 0.1% SDS at 50° C., followed by washing in 1×SSC containing 0.1% SDS at 60° C., no dissociation occurs.
The invention DNA molecules (polynucleotides) also include the polynucleotide (e); i.e., a polynucleotide which has a DNA sequence 80% or more (preferably 95% or more, more preferably about 98% or more) homologous to the aforementioned polynucleotide (c), and which can express a product exhibiting Ca2+ leakage inhibitory effect in a target cell (in particular, Ca2+ leakage inhibitory effect in the sarcoplasmic reticulum of a target cell).
An essential requirement for such a polynucleotide (hereinafter may be referred to as a “modified polynucleotide”) is that the polynucleotide can expresses a product exhibiting Ca2+ leakage inhibitory effect in a target cell (in particular, Ca2+ leakage inhibitory effect in the sarcoplasmic reticulum of a target cell). In other words, the requirement is that a transformant obtained through transformation with a recombinant expression vector in which such a polynucleotide (modified polypeptide) has been inserted can express a protein exhibiting Ca2+ leakage inhibitory effect in a target cell (in particular, Ca2+ leakage inhibitory effect in the sarcoplasmic reticulum of a target cell).
The aforementioned modified polynucleotide encompasses a DNA molecule having a DNA sequence encoding an amino acid sequence (modified amino acid sequence) obtained through deletion, insertion, substitution, or addition of one or more amino acids in the amino acid sequence of any of SEQ ID NOs: 3 to 9 (in particular, SEQ ID NO: 3). Use of the modified polynucleotide may detect the non-modified invention DNA molecule.
The invention DNA molecule also encompasses homologues of a polynucleotide (RyR2 gene fragment) having the DNA sequence of any of SEQ ID NOs: 20 to 26. As used herein, the term “homologues of RyR2 gene fragment” refers to a series of related DNA molecules which have sequence homology to the DNA sequence of any of SEQ ID NOs: 20 to 26, and which are recognized as one DNA molecule family in terms of commonality in structural feature and polynucleotide expression pattern, and as well similarity in biological function. The homologues also include alleles of the RyR2 gene fragment.
Modification (variation) or the like of an amino acid sequence may naturally occur through, for example, mutation or post-translational modification. Such amino acid sequence modification may be artificially performed by use of a naturally occurring DNA molecule (e.g., the RyR2 gene fragment according to the present invention). The invention DNA molecule encompasses all the modified DNA molecules having the aforementioned properties, regardless of, for example, the cause and means of such modification or variation.
Examples of the aforementioned artificial means include genetic engineering techniques such as site-specific mutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987); Methods in Enzymology, 100, 468 (1983); Nucleic Acids Res., 12, 9441 (1984); and Zoku Seikagaku Jikken Koza 1 “Idenshi Kenkyuho II,” edited by The Japanese Biochemical Society, p. 105 (1986)]; chemical synthesis means such as the phosphotriester method and the phosphoramidite method [J. Am. Chem. Soc., 89, 4801 (1967); J. Am. Chem. Soc., 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981); and Tetrahedron Lett., 24, 245 (1983)]; and combinations of these techniques and means. More specifically, DNA synthesis may be performed through chemical synthesis employing the phosphoramidite method or the phosphotriester method, or may be performed by means of a commercially available automated oligonucleotide synthesizer. A double-stranded fragment can be obtained by annealing a synthesized complementary strand with a chemically synthesized single strand under appropriate conditions, or adding a synthesized complementary strand to a chemically synthesized single strand by use of an appropriate primer sequence and DNA polymerase.
The nucleotide sequence of a DNA molecule having the DNA sequence of any of SEQ ID NOs: 20 to 26, which molecule is one specific embodiment of the invention DNA molecule (e.g., human RyR2 gene fragment), is an example of a combination of codons encoding the amino acid residues of a polypeptide having the corresponding amino acid sequence represented by any of SEQ ID NOs: 3 to 9. The invention DNA molecule is not limited to a DNA molecule having such a specific DNA sequence, and may have a DNA sequence obtained through arbitrary combination and choice of codons for the amino acid residues of the invention polypeptide. Choice of codons can be performed through a customary method. Codon choice may be performed in consideration of, for example, the codon usage of a host to be employed [Nucleic Acids Res., 9, 43 (1981)].
[3] Production of the Invention DNA Molecule
The invention DNA molecule can be easily produced or obtained through a chemical DNA synthesis method for directly synthesizing a DNA molecule having a nucleic acid sequence encoding a portion of the RyR2 amino acid sequence, on the basis of the sequence data of a specific example of the nucleic acid sequence of the RyR2 gene fragment (i.e., the invention polypeptide) disclosed herein, or on the basis of the sequence data of a portion of the RyR2 amino acid sequence. Alternatively, the invention DNA molecule can be easily produced or obtained through a generally employed genetic engineering technique [see, for example, Molecular Cloning 2nd Ed, Cold Spring Harbor Lab. Press (1989); and Zoku Seikagaku Jikken Koza “Idenshi Kenkyuho I, II, III,” edited by The Japanese Biochemical Society (1986)].
In such a chemical DNA synthesis method for directly synthesizing a DNA molecule, for example, a DNA molecule can be easily synthesized through solid-phase synthesis employing the phosphoramidite method by means of an automated synthesizer.
Production of the invention DNA molecule through a genetic engineering technique can be specifically performed by preparing, through a customary method, a cDNA library from an appropriate source in which the DNA molecule is expressed, and selecting a desired clone from the library by use of an appropriate probe or antibody specific to the invention DNA molecule [e.g., Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); and Science, 222, 778 (1983)].
Examples of the aforementioned cDNA source include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom. Isolation of total RNA from such a source, isolation and purification of mRNA, obtainment of cDNA, cloning thereof, etc. can be performed through a customary method. Production of the gene of the present invention can also be performed by means of various commercially available mRNA or cDNA libraries (e.g., mRNA or cDNA libraries available from Clontech Lab. Inc.), in particular, human heart mRNA or cDNA libraries.
No particular limitation is imposed on the method for screening the invention DNA molecule from cDNA libraries, and such screening can be performed through a customary method. Specific examples of the screening method include a method in which a protein produced by cDNA is subjected to immunological screening by use of an antibody specific to the protein, thereby selecting the corresponding cDNA clone; plaque hybridization and colony hybridization employing a probe which is selectively bound to a target DNA sequence; and combinations of these methods.
The probe employed in the aforementioned method is generally DNA which is chemically synthesized on the basis of data of the DNA sequence of the invention DNA molecule. The gene of the present invention which has already been obtained or a fragment of the gene can be advantageously employed as the aforementioned probe. Furthermore, a sense primer or antisense primer designed on the basis of the DNA sequence data of the invention DNA molecule can be employed as a screening probe.
The polynucleotide employed as a probe is a partial polynucleotide corresponding to SEQ ID NO: 20, and has at least 15 continuous nucleotides, preferably at least 20 continuous nucleotides, more preferably at least 30 continuous nucleotides. The aforementioned positive clone per se for producing the invention DNA molecule can also be employed as a probe.
DNA/RNA amplification through PCR [Science, 230, 1350 (1985)] is preferably employed for obtaining the gene of the present invention. Particularly when full-length cDNA is difficult to obtain from a library, RACE [Rapid amplification of cDNA ends; Jikken Igaku, 12(6), 35 (1994)], in particular, 5′-RACE [M. A. Frohman, et al., Proc. Natl. Acad. Sci., USA., 8, 8998 (1988)], etc. is preferably employed.
The primer employed for performing PCR can be designed on the basis of the sequence data of the gene of the present invention as disclosed herein, and can be synthesized through a customary method. Isolation and purification of DNA/RNA fragments amplified by PCR can be performed through a customary method (e.g., gel electrophoresis).
The above-obtained invention DNA molecule or DNA fragments can be sequenced through a customary method; for example, the dideoxy method [Proc. Natl. Acad. Sci., U.S.A., 74, 5463 (1977)] or the Maxam-Gilbert method [Methods in Enzymology, 65, 499 (1980)], or can be conveniently sequenced by means of a commercially available sequencing kit.
[4] Genetic Engineering Production of the Invention Polypeptide
The invention polypeptide (expression product) can be easily and reliably mass-produced by use of the gene of the present invention through a generally employed genetic engineering technique (e.g., Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); or Proc. Natl. Acad. Sci., USA., 80, 5990 (1983)) in the form of an expression product of the gene or a protein containing the expression product. More specifically, the invention polypeptide (expression product) can be obtained through the following procedure: recombinant DNA (expression vector) which enables a gene encoding a desired protein to be expressed in a host cell is prepared; the thus-prepared vector is introduced into the host cell for transformation; the resultant transformant is cultured; and a target protein is collected from the resultant culture.
The present invention also provides, for example, a polypeptide encoded by the invention DNA molecule (polynucleotide); a vector containing, for example, the invention DNA molecule, and a host cell transformed with the vector, the vector and host cell being employed for producing the polypeptide; and a method for producing the invention polypeptide through culturing of the host cell.
The aforementioned host cell may be derived from prokaryote or eukaryote. For example, the prokaryotic host may be any of generally employed hosts such as Escherichia coli and Bacillus subtilis. Preferably, Escherichia coli, in particular, Escherichia coli K12 strain is employed. Examples of eukaryotic host cells include vertebrate cells and yeast cells. Preferred vertebrate cells to be employed include COS cell (i.e., simian cell) [Cell, 23: 175 (1981)], Chinese hamster ovary cell, and dihydrofolate reductase-deficient Chinese hamster ovary cell line [Proc. Natl. Acad. Sci., USA., 77: 4216 (1980)]; and preferred yeast cells to be employed include Saccharomyces yeast cell. Needless to say, the host cell to be employed is not limited to these examples.
In the case where the host is a prokaryotic cell, a vector which can be replicated in the host cell is employed. In such a case, preferably, there is employed an expression plasmid in which a promoter sequence, a Shine-Dalgarno (SD) sequence, and an initiation codon (e.g., ATG) required for protein synthesis initiation are provided upstream of the gene of the present invention so that the gene can be expressed in the vector. In general, an Escherichia coli-derived plasmid (e.g., pBR322, pBR325, pUC12, or pUC13) is often employed as the aforementioned vector. However, the vector to be employed is not limited to these examples, and may be any known vector. Examples of commercially available vectors to be used in an expression system employing Escherichia coli include pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2 and pMAL-P2 (New England Biolabs), pET21 and pET21/lacq (Invitrogen), and pBAD/His (Invitrogen).
In the case where the host is a vertebrate cell, an expression vector to be employed generally contains a promoter, a RNA splice site, a polyadenylation site, and a transcription termination sequence, which are located upstream of the gene of the present invention to be expressed. If necessary, the expression vector may further contain a replication origin. Specific examples of the expression vector include pSV2dhfr having an SV40 initial promoter [Mol. Cell. Biol., 1: 854 (1981)]. The expression vector may be any known commercially available vector other than the aforementioned ones. Examples of commercially available vectors to be used in an expression system employing an animal cell include vectors for animal cells, such as pEGFP-N and pEGFP-C (Clontech), pIND (Invitrogen), and pcDNA3.1/His (Invitrogen); and vectors for insect cells, such as pFastBac HT (GibcoBRL), pAcGHLT (PharMingen), and pAc5/V5-His, pMT/V5-His, and pMT/Bip/V5-his (Invitrogen).
Specific examples of expression vectors to be employed in the case where the host is a yeast cell include pAM82 having a promoter for acid phosphatase gene [Proc. Natl. Acad. Sci., USA., 80: 1 (1983)]. Commercially available expression vectors for yeast cell include pPICZ (Invitrogen) and pPICZα (Invitrogen).
No particular limitation is imposed on the promoter to be employed. In the case where the host is a bacterium belonging to the genus Escherichia, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, or PL/PR promoter is preferably employed. In the case where the host is a bacterium belonging to the genus Bacillus, SP01 promoter, SP02 promoter, penP promoter, or the like is preferably employed. In the case where the host is yeast, for example, pH05 promoter, PGK promoter, GAP promoter, or ADH promoter is preferably employed. In the case where the host is an animal cell, for example, SV40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, cytomegalovirus promoter, or SRα promoter is preferably employed.
A typical fusion protein expression vector is also preferably employed as the expression vector for the invention DNA molecule. Specific examples of the vector include pGEX (Promega) for expressing the DNA molecule in the form of a fusion protein with glutathione-S-transferase (GST).
When the sequence encoding a mature polypeptide corresponds to the sequence of a polynucleotide which promotes expression or secretion of the polypeptide from a host cell, the polynucleotide sequence is, for example, a secretion sequence or a leader sequence. When the host is a bacterium, such a sequence contains a marker sequence (hexahistidine tag or histidine tag) employed for purification of a fusion mature polypeptide, whereas when the host is a mammalian cell, such a sequence contains a hemagglutinin (HA) tag.
No particular limitation is imposed on the method for introducing a desired recombinant DNA (expression vector) into a host cell and on the transformation method employing the expression vector, and generally employed introduction and transformation methods can be employed.
The thus-transformed cell can be cultured through a customary method, and, through this culturing, a target protein encoded by a gene designed as desired is expressed and produced (accumulated or secreted) inside or outside the transformed cell, or on the membrane of the cell.
The medium employable for such culturing can be appropriately selected from among conventionally employed media in consideration of the type of a host cell to be employed, and the culturing can be performed under conditions suitable for growth of the host cell.
If desired, the thus-obtained recombinant protein (the invention polypeptide) can be subjected to isolation and purification through various separation methods utilizing, for example, physical and chemical properties of the protein [see, for example, “Biochemical Data Book II,” pp. 1175-1259, 1st Edition, 1st Impression, Jun. 23, 1980, published by Tokyo Kagaku Dojin Co., Ltd.; Biochemistry, 25 (25), 8274 (1986); and Eur. J. Biochem., 163, 313 (1987)].
Specific examples of such separation methods include common reconstitution treatment, treatment with a protein precipitant (salting out), centrifugation, the osmotic shock method, ultrasonic disruption, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, affinity chromatography, various liquid chromatography techniques such as high performance liquid chromatography (HPLC), dialysis, and combinations of these methods. Particularly preferred methods include affinity chromatography employing a column to which an antibody specific to the invention polypeptide is bound.
The polynucleotide encoding the invention polypeptide can be designed by use of the RyR2 gene DNA sequence of SEQ ID NO: 2. If desired, in the gene, codons corresponding to amino acid residues may be appropriately selected and changed.
In the amino acid sequence encoded by the RyR2 gene, when a portion of the amino acid residues (or amino acids) of the sequence is to be modified through substitution, insertion, deletion, addition, etc., any of the aforementioned methods (e.g., site-specific mutagenesis) can be employed.
[5] Expression Product of the Present Invention
An expression product of the invention polynucleotide, the expression product serving as an active ingredient of the pharmaceutical composition of the present invention and being employed in the screening method of the present invention (hereinafter the expression product may be referred to as the “invention expression product”), can be obtained through a genetic engineering technique described in [4] above.
Similar to the case of the invention polypeptide described in [1] above, the invention expression product exhibits Ca2+ leakage inhibitory effect in a target cell (in particular, Ca2+ leakage inhibitory effect in sarcoplasmic reticulum), and is useful as the aforementioned therapeutic or preventive agent.
[6] Chemical Synthesis of the Invention Polypeptide
The invention polypeptide can also be produced through a generally employed chemical synthesis method on the basis of data of the amino acid sequences of SEQ ID NOs: 3 to 9. The chemical synthesis method encompasses peptide synthesis through a generally employed liquid-phase method or solid-phase method.
More specifically, the peptide synthesis encompasses so-called stepwise elongation, in which amino acids are connected step-by-step in turn on the basis of amino acid sequence data, thereby elongating an amino acid chain; and fragment condensation, in which fragments, each containing several amino acids, are synthesized in advance, and then the fragments are subjected to coupling reaction. The invention polypeptide can be synthesized through any of these techniques.
Condensation employed in such peptide synthesis can be performed through a customary method. For example, such condensation can be performed through the azide method, the mixed-acid anhydride method, the DCC method, the active ester method, the redox method, the DPPA (diphenylphosphoryl azide) method, the DCC+additive (e.g., 1-hydroxybenzotriazole, N-hydroxysuccinamide, or N-hydroxy-5-norbornene-2,3-dicarboxyimide) method, or the Woodward method.
The solvent to be used in these methods can be appropriately selected from among solvents which are generally known to be employed in such peptide condensation. Examples of such a solvent include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate, and mixtures thereof.
Upon the aforementioned peptide synthesis, the carboxyl groups of amino acids or peptides which do not participate in reaction can be generally protected through esterification, for example, in the form of a lower alkyl ester (e.g., methyl ester, ethyl ester, or tert-butyl ester), or an aralkyl ester (e.g., benzyl ester, p-methoxybenzyl ester, or p-nitrobenzyl ester).
A functional group of the side chain of an amino acid (e.g., the hydroxyl group of a tyrosine residue) may be protected with, for example, an acetyl group, a benzyl group, a benzyloxycarbonyl group, or a tert-butyl group, but such protection is not necessarily performed. For example, the guanidino group of an arginine residue may be protected with an appropriate protective group such as a nitro group, a tosyl group, a p-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, or an adamantyloxycarbonyl group.
Reaction for removing such a protective group from the thus-protected amino acid, peptide, or finally produced invention polypeptide can also be performed through a conventionally employed method; for example, catalytic reduction, or a method employing liquid ammonia/sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, or the like.
The thus-obtained invention polypeptide can be appropriately purified through any of the aforementioned methods; for example, a method which is generally employed in the field of peptide chemistry, such as ion-exchange resin chromatography, partition chromatography, gel chromatography, or countercurrent distribution.
[7] Pharmaceutical Composition of the Present Invention
The pharmaceutical composition of the present invention contains, as an active ingredient, the invention polypeptide or the invention expression product. The pharmaceutical composition is useful as, for example, a pharmaceutical composition exhibiting Ca2+ leakage inhibitory effect in a target cell (in particular, Ca2+ leakage inhibitory effect in sarcoplasmic reticulum), or an agent for improving calcium handling (regulation). The pharmaceutical composition, which exhibits the aforementioned Ca2+ leakage inhibitory effect and calcium handling (regulation) improving effect, is also useful as a therapeutic or preventive agent for, for example, heart failure, cardiomyopathy, valvulopathy, ischemic heart disease, myocardial infarction, angina pectoris, arrhythmia, hypertension, peripheral circulatory disorder, diabetes, dysmnesia, cerebral infarction, or malignant hyperthermia.
More specifically, the invention polypeptide or the invention expression product (e.g., R2P), which serves as an active ingredient of the pharmaceutical composition of the present invention, exhibits the effect of inhibiting Ca2+ leakage induced by FK506 or the like in a sarcoplasmic reticulum specific thereto. This Ca2+ leakage inhibitory effect (or activity) can be employed for the treatment of diseases associated with Ca2+ leakage induction or with abnormal calcium handling in cells. Examples of target cells affected by such Ca2+ leakage induction or abnormal calcium handling include myocardial cell, smooth muscle cell, skeletal muscle cell, brain neuron, neuroblast, and pancreatic β cell. Examples of important tissues containing such cells include heart, arterial vessel, muscle tissue, cerebellum, cerebral cortex, midbrain, cranial nerve tissue (e.g., hypophysis), and pancreas.
Examples of the aforementioned sarcoplasmic reticulum include sarcoplasmic reticulum prepared from normal or failing myocardium, sarcoplasmic reticulum prepared from vascular smooth muscle, and sarcoplasmic reticulum prepared from skeletal muscle.
The invention polypeptide or the invention expression product, which is an active ingredient of the pharmaceutical composition of the present invention, also encompasses pharmaceutically acceptable salts thereof. Examples of such salts include nontoxic alkali metal salts, alkaline earth metal salts, and ammonium salts which are prepared through methods well known in the art, such as sodium, potassium, lithium, calcium, magnesium, barium, and ammonium salts. The aforementioned salts also encompass nontoxic acid addition salts prepared through reaction between the invention polypeptide or the invention expression product and an appropriate organic acid or inorganic acid. Typical examples of nontoxic acid addition salts include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate, ascorbate, benzenesulfonate, and napsylate.
The pharmaceutical composition of the present invention, which contains, as an active ingredient, the invention polypeptide, the invention expression product, or a salt thereof, is prepared into the form of a pharmaceutical product containing a pharmaceutically effective amount of the active ingredient and an appropriate pharmaceutical carrier or diluent.
Examples of the pharmaceutical carrier employed for preparation of such a pharmaceutical product include diluents and excipients, such as a filler, an extender, a binder, a humectant, a disintegrant, a surfactant, and a lubricant, which are generally employed in accordance with the pharmaceutical product form. Such a carrier is appropriately selected so as to fit the unit dosage form of the prepared pharmaceutical product. A particularly preferred pharmaceutical product is prepared by use of an appropriate ingredient which is commonly employed for protein preparations, etc., such as a stabilizer, a bactericide, a buffer, an isotonizing agent, a chelating agent, a pH-adjusting agent, or a surfactant.
Examples of the aforementioned stabilizer include human serum albumin, common L-amino acids, sugars, and cellulose derivatives. Such a stabilizer may be employed singly or in combination with, for example, a surfactant. Particularly, this combination may further enhance the stability of the active ingredient. No particular limitation is imposed on the L-amino acid to be employed, and the L-amino acid may be any of glycine, cysteine, glutamic acid, and the like. No particular limitation is imposed on the sugar to be employed, and examples of the sugar which may be employed include monosaccharides such as glucose, mannose, galactose, and fructose; sugar alcohols such as mannitol, inositol, and xylitol; disaccharides such as sucrose, maltose, and lactose; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, and hyaluronic acid; and derivatives thereof. No particular limitation is imposed on the surfactant to be employed, and the surfactant may be any of an ionic surfactant or a nonionic surfactant. Specific examples of the surfactant which may be employed include polyoxyethylene glycol sorbitan alkyl esters, polyoxyethylene alkyl ethers, sorbitan monoacyl esters, and fatty acid glycerides. No particular limitation is imposed on the cellulose derivative to be employed, and the cellulose derivative may be, for example, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose sodium.
The appropriate amount of a sugar to be added is about 0.0001 mg or more, preferably within a range of about 0.01 to about 10 mg, on the basis of 1 μg of the active ingredient. The appropriate amount of a surfactant to be added is about 0.00001 mg or more, preferably within a range of about 0.0001 to about 0.01 mg, on the basis of 1 μg of the active ingredient. The appropriate amount of human serum albumin to be added is about 0.0001 mg or more, preferably within a range of about 0.001 to about 0.1 mg, on the basis of 1 μg of the active ingredient. The appropriate amount of an L-amino acid to be added is about 0.001 to about 10 mg on the basis of 1 μg of the active ingredient. The appropriate amount of a cellulose derivative to be added is about 0.00001 mg or more, preferably within a range of about 0.001 to about 0.1 mg, on the basis of 1 μg of the active ingredient.
The amount of the active ingredient contained in the pharmaceutical composition of the present invention is appropriately determined within a wide range. The appropriate active ingredient content of the composition is generally about 0.00001 to about 70 wt. %, preferably about 0.0001 to about 5 wt. %.
The pharmaceutical composition of the present invention may also contain an additive such as a buffer, an isotonizing agent, or a chelating agent. Examples of the buffer include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid, and/or salts corresponding to these acids (e.g. alkali metal salts and alkaline earth metal salts of these acids, such as sodium, potassium, calcium, and magnesium salts). Examples of the isotonizing agent include sodium chloride, potassium chloride, sugars, and glycerin. Examples of the chelating agent include sodium edetate and citric acid.
The pharmaceutical composition of the present invention may be prepared in the form of a solution product. Alternatively, the composition may take the form of a storable lyophilized product which is prepared through lyophilization of the solution product so that the lyophilized product is dissolved in, for example, a buffer (including water or saline) upon use to prepare a solution of appropriate concentration.
The unit dosage form of the pharmaceutical composition of the present invention can be appropriately selected so as to meet the therapeutic purpose. Typical examples of the dosage form include solid forms such as a tablet, a pill, a dispersion, a powder, a granule, and a capsule; and liquid forms such as a solution, a suspension, an emulsion, a syrup, and an elixir. In accordance with administration routes, these dosage forms are further classified into, for example, an oral agent, a parenteral agent, a nasal agent, a vaginal agent, a suppository, a sublingual agent, and an ointment, which can be respectively formulated, molded, or prepared through a customary method. If necessary, the pharmaceutical composition of the present invention may contain, for example, a coloring agent, a preservative, a perfume, a flavoring agent, a sweetening agent, or another drug.
No particular limitation is imposed on the method for administration of the pharmaceutical composition of the present invention, and the administration method is determined on the basis of, for example, the product form, age, sex, and other conditions of the patient, and the severity of disease. For example, a tablet, a pill, a solution, a suspension, an emulsion, a granule, or a capsule is orally administered; an injection is intravenously administered singly or in combination with a common replacement fluid (e.g., glucose or amino acid), and is, if necessary, intramuscularly, intradermally, subcutaneously, or intraperitoneally administered singly; a suppository is intrarectally administered; a vaginal agent is intravaginally administered; a nasal agent is intranasally administered; a sublingual agent is intraorally administered; and an ointment is locally administered through a transdermal route.
No particular limitation is imposed on the dose of the active ingredient contained in the pharmaceutical composition of the present invention, and the dose is appropriately determined within a wide range in consideration of, for example, the desired therapeutic effect, the administration method, the treatment period, and the age, sex, and other conditions of a patient. The daily dose is generally about 0.01 μg to about 10 mg per kg body weight, preferably about 0.1 μg to about 1 mg per kg body weight. The composition may be administered once daily or in several divided doses per day.
[8] Screening of Substance (Agonist) which Promotes Ca2+ Leakage Inhibitory Effect and Substance (Antagonist) which Suppresses the Inhibitory Effect
The present invention also provides a screening method for selecting a candidate compound which promotes or suppresses the Ca2+ leakage inhibitory effect (or the effect of improving abnormal calcium handling (hereinafter may be referred to as the “abnormal calcium handling improving effect”)) in the endoplasmic reticulum of a target cell.
A characteristic feature of the screening method resides in that, in the presence or absence of a test substance, the level of the Ca2+ leakage inhibitory effect (or the abnormal calcium handling improving effect) of the invention polypeptide or an expression product of the invention polypeptide in the endoplasmic reticulum of a target cell is measured; and the level measured in the presence of the test substance is compared with the level measured in the absence of the test substance, to thereby select the test substance as an agonist which enhances the inhibitory effect or as an antagonist which suppresses the inhibitory effect.
The level of the Ca2+ leakage inhibitory effect or the abnormal calcium handling improving effect of the invention polypeptide in a target cell (particularly in the endoplasmic reticulum) is determined through, for example, the following procedure: firstly, in the presence or absence of the invention polypeptide and in the presence or absence of a test substance, the amount of Ca2+ taken into myocardium-derived endoplasmic reticulum is measured; subsequently, FK506 is added to the endoplasmic reticulum, to thereby cut the bond between ryanodine receptor and FKBP12.6 and to induce Ca2+ leakage; and then the inhibitory effect of the polypeptide on FK506-induced Ca2+ leakage in the endoplasmic reticulum of a cell or a tissue is determined on the basis of reduction of the amount of Ca2+ leaked from the sarcoplasmic reticulum as detected by means of a Ca2+ indicator.
The Ca2+ leakage inhibitory effect in a target cell (in particular, the Ca2+ leakage inhibitory effect in sarcoplasmic reticulum) or the abnormal calcium handling improving effect of the invention polypeptide or the invention expression product can be detected through a known method.
More specifically, Ca2+ leakage induction or Ca2+ leakage inhibitory effect can be detected, for example, as follow. (1) Ca2+ leakage from sarcoplasmic reticulum and the effect of inhibiting the Ca2+ leakage can be measured through, for example, the method described in M. Yano, et al., Circulation, 102, 2131-2136 (2000).
Specifically, for example, in the presence or absence of the invention polypeptide or the invention expression product, the amount of Ca2+ taken into myocardium-derived sarcoplasmic reticulum is measured, and subsequently, FK506 is added to the sarcoplasmic reticulum, to thereby cut the bond between ryanodine receptor and FKBP12.6 and to induce Ca2+ leakage. Thereafter, the inhibitory effect on FK506-induced Ca2+ leakage in the endoplasmic reticulum of a cell or a tissue can be detected by means of a Ca2+ indicator.
More specifically, sarcoplasmic reticulum is incubated with a calcium-containing buffer (free calcium concentration: 0.3 μmol/L), and ATP (0.5 mmol/L) is added to the resultant mixture, to thereby initiate Ca2+ uptake into the cell. The time course of Ca2+ uptake is measured by means of fluo 3 serving as a Ca2+ indicator, and absorbance at an excitation wavelength of 480 nm and an emission wavelength of 530 nm is measured by means of an absorbance meter. After Ca2+ uptake reaches a plateau, in the presence or absence of the invention polypeptide or the invention expression product, FK506 is added, and then Ca2+ leakage is measured by means of the Ca2+ indicator, whereby the Ca2+ leakage inhibitory effect can be detected. In the aforementioned method, instead of FK506, rapamycin may be employed as a Ca2+ leakage-inducing agent.
As used herein, “Ca2+ leakage inhibitory activity” encompasses the Ca2+ leakage inhibitory effect in endoplasmic reticulum, and as well the activity of the polypeptide exhibiting the abnormal calcium handling (regulation) improving effect, the effect of stabilizing ryanodine receptor function, the effect of maintaining (improving) ryanodine receptor function, the effect of inhibiting reduction of the bonding strength between ryanodine receptor and FK506-binding protein, the effect of inhibiting phosphorylation of ryanodine receptor, and/or the effect of maintaining the structure of ryanodine receptor. Therefore, furthermore, the below-exemplified detection method can be performed.
(2) For detection of the effect of inhibiting phosphorylation of ryanodine receptor, there can be detected the inhibitory effect on Ca2+ leakage in the endoplasmic reticulum of a cell or a tissue, the Ca2+ leakage being induced by a substance which cuts the bond between ryanodine receptor and FKBP12.6, and induces Ca2+ leakage through phosphorylation of the ryanodine receptor with protein kinase A (PKA).
Specifically, the aforementioned ryanodine receptor phosphorylation inhibitory effect of the invention polypeptide or the invention expression product can be detected by, for example, measuring the extent of phosphorylation mediated by protein kinase A in the presence or absence of the invention polypeptide or the invention expression product [see, for example, Steven O, Marx, et al., Cell, 101, 365-376 (2000), or Reiken, et al., J. Biol. Chem., 278, 444-453 (2003)].
(3) The ryanodine receptor structure maintaining effect of the invention polypeptide or the invention expression product can be detected by, for example, observing a change in MCA fluorescence intensity in endoplasmic reticulum through the method described in Yano. M., et al., Circulation, 102, 2131-2136 (2000).
(4) The effect of inhibiting reduction of the bonding strength between ryanodine receptor and FK506-binding protein (specifically, the effect of inhibiting reduction of the bonding strength between ryanodine receptor (RyR) and FK506-binding protein 12.6 (FKBP12.6) of the invention polypeptide or the invention expression product) can be detected by, for example, measuring the bonding strength between RyR and FKBP12.6 through surface plasmon analysis or a similar technique.
Specifically, inhibition of reduction of the bonding strength between RyR and FKBP12.6 can be measured through, for example, the following procedure: to a chip on which RyR or FKBP12.6 has been immobilized, correspondingly, an FKBP12.6- or RyR-containing solution which contains the invention polypeptide or the invention expression product, or contains neither the invention polypeptide nor the invention expression product is fed at a constant flow rate; a sensorgram representing formation of an RyR-FKBP12.6 complex is obtained on the basis of a change in surface plasmon resonance signal; and inhibition of reduction of the bonding strength is measured through comparison between binding rates obtained from sensorgrams corresponding to the respective conditions.
(5) For detection of the abnormal calcium handling (regulation) improving effect, a change in Ca2+ concentration in sarcoplasmic reticulum or the corresponding cell change, which is attributed to abnormal migration of Ca2+ into or from the sarcoplasmic reticulum via stabilization of ryanodine receptor, can be detected through at least one of the aforementioned detection methods (1) to (4), to thereby detect whether or not the abnormal calcium handling will be corrected.
The present invention also provides a screening method for selecting a candidate substance which enhances the Ca2+ leakage inhibitory effect (or the abnormal calcium handling improving effect) of the invention polypeptide in the endoplasmic reticulum of a target cell, which method comprises the following steps (1) to (5):

    (1) a step of providing a target cell transformed with an expression vector containing a polynucleotide encoding the invention polypeptide;
    (2) a step of adding FK506 to the endoplasmic reticulum of the target cell so as to induce Ca2+ leakage in the presence or absence of a test substance;
    (3) a step of measuring, by means of a Ca2+ indicator, the amount of Ca2+ leaked from the endoplasmic reticulum of the target cell, to thereby determine the amount of change in Ca2+ leakage from the endoplasmic reticulum in the presence or absence of the test substance;
    (4) a step of adding FK506 to the endoplasmic reticulum of a non-transformed cell so as to induce Ca2+ leakage in the absence of the test substance, measuring by means of a Ca2+ indicator the amount of Ca2+ having leaked from the endoplasmic reticulum, and determining the difference between the amount of Ca2+ as measured outside the endoplasmic reticulum; i.e., the standard amount, and each of the amounts as determined in (3) above in the presence and absence of the test substance; and
    (5) a step of selecting the test substance as a candidate substance when the difference between the standard amount and the amount as determined in the presence of the test substance is smaller than the difference between the standard amount and the amount as determined in the absence of the test substance.


The present invention also provides a screening method for selecting a candidate substance which enhances the Ca2+ leakage inhibitory effect (or the abnormal calcium handling improving effect) of the invention expression product in the endoplasmic reticulum of a target cell, which method comprises the following steps (1) to (5):

    (1) a step of providing a target cell transformed with an expression vector containing the invention polynucleotide;
    (2) a step of adding FK506 to the endoplasmic reticulum of the target cell so as to induce Ca2+ leakage in the presence or absence of a test substance;
    (3) a step of measuring, through RT-PCR, the amount of expression of the polynucleotide in the endoplasmic reticulum of the target cell in the presence or absence of the test substance;
    (4) a step of comparing the expression amount in the presence of the test substance with the expression amount in the absence of the test substance; and
    (5) a step of selecting the test substance as a candidate substance when the expression amount in the presence of the test substance is greater than the expression amount in the absence of the test substance.


The present invention also provides a screening method for selecting a candidate substance which enhances the Ca2+ leakage inhibitory effect (or the abnormal calcium handling improving effect) of the invention polypeptide in a target cell, which method comprises the following steps (1) to (5):

    (1) a step of providing a target cell transformed with an expression vector containing a polynucleotide encoding the invention polypeptide;
    (2) a step of adding FK506 to the endoplasmic reticulum of the target cell so as to induce Ca2+ leakage in the presence or absence of a test substance;
    (3) a step of measuring, by means of an antibody against the polypeptide, the amount of the polypeptide produced in the target cell in the presence or absence of the test substance;
    (4) a step of comparing the production amount in the presence of the test substance with the production amount in the absence of the test substance; and
    (5) a step of selecting the test substance as a candidate substance when the production amount in the presence of the test substance is greater than the production amount in the absence of the test substance.


In each of these screening methods, the amount of expression (or production) of the invention polypeptide represented by any of SEQ ID NOs: 3 to 9 (e.g., R2P represented by SEQ ID NO: 3 or an expression product of the polynucleotide encoding R2P; hereinafter these may be collectively referred to simply as “R2P”) can be determined by competitively reacting an antibody against the polypeptide with labeled R2P in a test liquid containing a test substance, and measuring the amount of the labeled R2P which has been bound to the antibody. Alternatively, the amount of R2P in the test liquid can be determined by reacting the test liquid with an antibody immobilized on a carrier and a different labeled anti-R2P antibody simultaneously or sequentially, and then measuring the activity of a labeling agent on the antibody-immobilized carrier.
According to the screening method of the present invention, binding between R2P and a ligand can be evaluated in, for example, a cell, a cell-free preparation, a chemical library, or a naturally occurring mixture.
An agent for promoting (or enhancing) or inhibiting (or suppressing) the effect of R2P in inhibiting Ca2+ leakage, thereby improving abnormal calcium handling (regulation) in cells may be a naturally occurring substrate or ligand, or a structural or functional mimic thereof.
In the screening method of the present invention, a high-throughput screening technique can be applied. With application of this technique, in the case of screening of a Ca2+ leakage inhibitor, a Ca2+ leakage promoter, or an agent for promoting or inhibiting improvement of abnormal calcium handling (regulation) in the sarcoplasmic reticulum of a cell, a synthesis reaction mixture, a cellular fraction (e.g., endoplasmic reticulum, membrane, cell envelope, or cell wall), or a preparation of any of these (including R2P and a labeled ligand for the polypeptide) is incubated in the presence or absence of a test substance to be screened. Whether the test substance is agonistic or antagonistic to the effects of R2P is determined through detection of reduction of the binding between R2P and the labeled ligand. A test substance which inhibits Ca2+ leakage or improves abnormal calcium handling (regulation) without inducing the effects of R2P is highly likely to be a good Ca2+ leakage inhibitor or abnormal calcium handling (regulation) improving agent. In contrast, a compound which binds to R2P and increases Ca2+ leakage is regarded as an inhibitor against R2P. The activity level of R2P can be detected by means of a reporter system (including, but not limited to, calorimetric labeling), integration of a reporter gene which responds to change in polynucleotide or polypeptide activity, or a binding assay known in the art.
A competitive assay employing R2P, an R2P mutant (MSV-R2P, RP1-20, RP13-32, RP17-36, RP1-30, or RP1-25), or another Ca2+ leakage inhibitor (e.g., JTV519, a calcium antagonist such as diltiazem, or a β-blocker such as propranolol) in combination with a compound which binds thereto can be employed for selecting, through screening, a candidate compound serving as a Ca2+ leakage inhibitor or promoter, or as an agent for improving or enhancing abnormal calcium handling (regulation).
Test substances (drug candidate compounds) to be screened through the screening method of the present invention include a substance which promotes Ca2+ leakage inhibitory activity (in particular, a substance which inhibits Ca2+ leakage or a substance which improves abnormal calcium handling (regulation); i.e., agonist), as well as a substance which suppresses Ca2+ leakage inhibitory activity (in particular, a substance which promotes Ca2+ leakage or a substance which enhances abnormal calcium handling (regulation); i.e., antagonist). In the screening system of the present invention, “agonist” corresponds to a substance which increases Ca2+ leakage inhibitory activity to be measured, whereas “antagonist” corresponds to a substance which reduces Ca2+ leakage inhibitory activity to be measured.
Specific examples of such a test substance include oligopeptide, protein, antibody, RNA molecule, siRNA, nonpeptidic compound (synthetic compound), fermented product, cell extract (e.g., plant extract or animal tissue extract), and plasma. Such a substance may be a newly developed or known substance.
Examples of Ca2+ leakage inhibiting substances include an inorganic or organic low-molecular-weight compound which binds to R2P and increases its activity; naturally occurring, synthetic, or genetically modified peptide and polypeptide; and a sense DNA molecule with respect to DNA encoding R2P, which is directly administered in vivo or which is inserted in a recombinant vector for administration.
Examples of Ca2+ leakage promoting substances include inorganic or organic low-molecular-weight compound, peptide, polypeptide, antibody, and antisense DNA molecule (see, for example, Okano, J. Neurochem, 56: 560 (1991); and Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)), each of which does not induce R2P-induced activity (Ca2+ leakage inhibitory activity), and can bind to a molecule site to which R2P binds, thereby inhibiting the activity of R2P by preventing binding of R2P to the molecule site, and RNA molecule exhibiting the RNA interference effect (see, for example, McCaffrey, A. P., et al., Nature, 418, 38-39 (2002)).
A candidate substance selected through the screening method of the present invention, which exhibits the effect of promoting or suppressing Ca2+ leakage inhibitory activity, can be evaluated as follow. Specifically, when Ca2+ leakage inhibitory activity is increased by about 20% or more (preferably about 30% or more, more preferably about 50% or more) as compared with the case of control (in the absence of a test substance), the candidate substance can be selected as a compound which promotes Ca2+ leakage inhibitory activity. In contrast, when, for example, Ca2+ leakage inhibitory activity is reduced by about 20% or more (preferably about 30% or more, more preferably about 50% or more) as compared with the case of control (in the absence of a test substance), the candidate substance can be selected as a compound which suppresses Ca2+ leakage inhibitory activity.
Similar to the case of the invention polypeptide or the invention expression product, a substance selected through the screening method of the present invention and promoting Ca2+ leakage inhibitory activity is useful as an active ingredient of a pharmaceutical composition for diseases associated with cellular abnormality resulting from Ca2+ leakage or abnormal calcium handling.
Conceivably, a substance selected through the screening method of the present invention and promoting Ca2+ leakage inhibitory activity reduces the amount of Ca2+ leaked from sarcoplasmic reticulum, to thereby increase Ca2+ concentration in the sarcoplasmic reticulum, and to improve abnormal muscle contraction/relaxation function or muscle contraction/relaxation dysfunction in cells. Therefore, a sarcoplasmic reticulum Ca2+ increasing agent containing such a substance as an active ingredient is useful in a variety of fields (including the field of pharmaceuticals).
The aforementioned sarcoplasmic reticulum Ca2+ increasing agent, an agent for promoting Ca2+ leakage inhibitory activity in endoplasmic reticulum, or an agent for improving abnormal calcium handling (regulation) in sarcoplasmic reticulum can be applied to any of the aforementioned diseases in the form of a pharmaceutical composition containing the agent (in an effective amount required for exhibiting its effect) and an appropriate pharmaceutical carrier.
The substance serving as an active ingredient of the pharmaceutical composition; i.e., a sarcoplasmic reticulum Ca2+ increasing substance, a substance for promoting Ca2+ leakage inhibitory activity in endoplasmic reticulum, or a substance for improving abnormal calcium handling (regulation) in sarcoplasmic reticulum, may be prepared in the form of a pharmaceutically acceptable salt. The type and preparation method of the salt, the preparation method of the pharmaceutical composition, the pharmaceutical carrier (e.g., diluent or excipient) employed in the pharmaceutical composition, the unit dosage form, administration route, and administration method of the pharmaceutical composition, and the like may be similar to those described in detail in the case of the aforementioned pharmaceutical composition containing, as an active ingredient, the invention polypeptide or the invention expression product.
No particular limitation is imposed on the amount of the active ingredient to be contained in the pharmaceutical composition and the dose of the active ingredient, and the amount and the dose are appropriately determined within a wide range in consideration of, for example, the desired therapeutic effect, the administration method, the treatment period, and the age, sex, and other conditions of a patient. The daily dose is generally about 0.01 μg to about 10 mg per kg body weight, preferably about 0.1 μg to about 1 mg per kg body weight. The composition may be administered once daily or in several divided doses per day.
[9] Screening Kit
The present invention also provides a screening kit for selecting an agonist or an antagonist with respect to the Ca2+ leakage inhibitory effect of the invention polypeptide or an expression product of the invention polynucleotide in the endoplasmic reticulum of a target cell, which kit is characterized by comprising at least one species selected from the group consisting of the polypeptide and the expression product, which are contained in the target cell, and FK506.
In any embodiment of the screening kit of the present invention, a critical point is to include, as essential components, the invention polypeptide (e.g., a polypeptide having the amino acid sequence of any of SEQ ID NOs: 3 to 9) or the invention expression product (e.g., an expression product of a polynucleotide having the DNA sequence of any of SEQ ID NOs: 20 to 26); and FK506, a substance which induces Ca2+ leakage in sarcoplasmic reticulum (i.e., a substance which cuts the bond between ryanodine receptor and FK506-binding protein or promotes phosphorylation of the ryanodine receptor, to thereby induce Ca2+ leakage in sarcoplasmic reticulum, to reduce the amount of Ca2+ taken into the sarcoplasmic reticulum, and to induce abnormal calcium handling (regulation), resulting in abnormal muscle contraction/relaxation function or muscle contraction/relaxation dysfunction in cells). Other components of the screening kit of the present invention may be similar to those included in a screening kit of this type. For example, the screening kit of the present invention may be a kit including a polypeptide (e.g., R2P) exhibiting the activity of inhibiting Ca2+ leakage (improving abnormal calcium handling) in endoplasmic reticulum, and a reagent serving as an optional component, such as a cell culture, a reaction diluent, a dye, a buffer, a fixative, or a detergent.
Specific examples of the screening kit of the present invention include a kit including the following components 1 to 4:
Component 1. Cell preparation obtained through culturing of a target cell (target R2P-expressing cell, which has been prepared through transformation of a cell to be measured with R2P-encoding polynucleotide (DNA molecule)) in a 60-mm dish (0.5×105 to 1×105 cells/well) at 5% CO2 and 37° C. by use of, for example, MOPS buffer containing EGTA-Ca buffer;
Component 2. Substance which induces Ca2+ leakage in endoplasmic reticulum (FK506);
Component 3. Substance which induces calcium uptake (e.g., ATP) and a Ca2+ indicator (e.g., fluo3); and
Component 4. Calcium ATPase inhibitor (e.g., thapsigargin).
In the screening method employing the screening kit of the present invention, significant difference test can be performed on the amount of change in Ca2+ leakage in a target cell as measured per unit visual field of a well to which a test compound (test substance or drug candidate compound) has been added, and on the amount of change in Ca2+ leakage in the same cell as measured in a control well to which a test compound has not been added.
The calcium release activity of ryanodine receptor can also be evaluated or detected on the basis of the number of change in channel opening frequency measured through a method other than the aforementioned methods (e.g., a method for measuring the opening frequency by means of single-channel recording) [Reiken, et al., J. Biol. Chem., 278, 444-453 (2003)].
The aforementioned measurement and evaluation can be performed through customary methods.
The present invention will next be described in more detail by way of an Example, which should not be construed as limiting the invention thereto.
EXAMPLE 1
In order to search for a novel substance exhibiting the effect of inhibiting Ca2+ leakage in endoplasmic reticulum, firstly, the present inventors synthesized a plurality of polypeptides as therapeutic peptides considered to correct abnormality in RyR2 structure, and searched for a polypeptide exhibiting the effect of inhibiting Ca2+ leakage in endoplasmic reticulum.
(a) Polypeptide Synthesis:
Synthesis of polypeptides was performed through solid-phase synthesis employing the Fmoc (9-fluorenylmethyloxycarbonyl) method.
TBTU [2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate] and HOBt [1-hydroxybenzotriazole hydrate] were employed as activating reagents, and reaction was performed through a continuous flow system.
The thus-synthesized polypeptides correspond to the following sequences, each having amino acids which are numbered from the N-terminal toward the C-terminal of the amino acid sequence of human ryanodine receptor 2 (hRyR2: GenBank Accession No. NM 001035).

    DR3-1: amino acid Nos. 1852-1871 SEQ ID NO: 17
    DR3-2: amino acid Nos. 1861-1880 SEQ ID NO: 18
    DR3-3: amino acid Nos. 1871-1890 SEQ ID. NO: 19
    R22068/87: amino acid Nos. 2068-2087 SEQ ID NO: 13
    R2JN28: amino acid Nos. 2086-2113 SEQ ID NO: 14
    R2JF: amino acid Nos. 2086-2133 SEQ ID NO: 15
    R2P: amino acid Nos. 2114-2149 SEQ ID NO: 3
    R22150/85: amino acid Nos. 2150-2185 SEQ ID NO: 16

(b) Measurement of Ca2+ Leakage Inhibitory Activity:



Ca2+ leakage inhibitory activity was measured through the method of Yano, M., et al. [Yano, et al., Circulation, 102, 2131-2136 (2000)].
Firstly, sarcoplasmic reticulum was prepared from canine myocardium through the method of Kranias, et al. [Kranias E., et al., Biochim. Biophys. Acta., 709, 28-37 (1982)].
Subsequently, the sarcoplasmic reticulum (0.2 mg/mL) was incubated in 0.5 mL of 20 mM MOPS buffer (pH 6.8) containing 0.15 mM potassium gluconate, 0.2 mM EGTA-calcium buffer (free calcium: Ca2+=0.3 μM), and 10 mM NaN3.
After the fluorescence value was maintained constant, 0.5 mM ATP (product of Sigma) and 0.5 mM magnesium chloride were added to the resultant mixture, to thereby initiate Ca2+ uptake. The time course of Ca2+ uptake was measured. Ca2+ uptake was measured by means of fluo 3 (product of Molecular Probes) serving as a Ca2+ indicator, and the fluorescence intensity at an excitation wavelength of 480 nm and an emission wavelength of 530 nm was measured by means of a fluorometer.
After Ca2+ uptake reached a plateau, 1 μM thapsigargin (product of Wako Pure Chemical Industries, Ltd.) was added to thereby inhibit sarco(endo)plasmic reticulum Ca-ATPase (SERCA) activity for elimination of the influence of SERCA. The difference between the fluorescence value before addition of ATP and the fluorescence value at the time of completion of ATP-induced active Ca2+ uptake (i.e., the fluorescence value at the time when Ca2+ uptake reached a plateau) was taken as 100%. Therefore, when the fluorescence value does not change even if thapsigargin is added, the amount of Ca2+ leakage is 0% [it has been reported that Ca2+ leakage (about 10 to about 20%) may occur in sarcoplasmic reticulum derived from normal myocardium; Doi, M., et al., Circulation, 105, 1374-1379 (2002)].
Ca2+ leakage induced by simultaneous addition of thapsigargin and 30 μM FK506 (product of Fujisawa Pharmaceutical Co., Ltd,), which was regarded as a control of Ca2+ leakage induction, was measured by means of a Ca2+ indicator.
Ca2+ leakage in the case where 0.3 μM JTV-519 (distributed by JT Inc.) exhibiting Ca2+ leakage inhibitory activity was added simultaneously with administration of thapsigargin and FK506, which was regarded as a control of Ca2+ leakage inhibitory activity, was measured by means of a Ca2+ indicator.
A polypeptide obtained in (a) above (concentration: 0.1 μM, 0.3 μM, or 1.0 μM) and FK506 (30 μM/L) were added simultaneously with administration of thapsigargin, and the effect of inhibiting the thus-induced Ca2+ leakage via ryanodine receptor was measured by means of a Ca2+ indicator in a manner similar to that described above.
The results are shown in FIGS. 1 to 3. In each of these figures, the vertical axis corresponds to the value as measured 20 seconds after addition of thapsigargin, FK506, and a polypeptide. The value is the average of data of measurement performed twice or three times.
As shown in FIGS. 1 to 3, among the above-synthesized polypeptides, R2P, which is represented by SEQ ID NO: 3 corresponding to amino acid Nos. 2114-2149 of human ryanodine receptor 2, inhibits Ca2+ leakage in a concentration-dependent manner. In addition, 1.0 μM R2P polypeptide was found to exhibit Ca2+ leakage inhibitory effect comparable to that of 0.3 μM JTV519, which is known to exhibit Ca2+ leakage inhibitory effect.
(c) Study on Ca2+ Leakage Inhibitory Effect of R2P-Related Peptides:
In the aforementioned test, R2P, which is represented by SEQ ID NO: 3 corresponding to amino acid Nos. 2114-2149 of human ryanodine receptor 2, was found to exhibit Ca2+ leakage inhibitory effect. Therefore, the present inventors further synthesized peptides having the below-described sequences, which are partial peptides and mutants of R2P. Each of the thus-synthesized peptides was examined whether or not it exhibits Ca2+ leakage inhibitory effect.
Specifically, there were synthesized the following peptides: a peptide (SEQ ID NO: 4) obtained through introduction of three amino acids to the N-terminal of R2P; partial peptides (SEQ ID NOs: 5 to 9 and 11) obtained through deletion of amino acids at the N-terminal, the C-terminal, and both the N- and C-terminals of R2P; a mutant (SEQ ID NO: 12) obtained through substitution of an amino acid of R2P with another amino acid; and a mutant (SEQ ID NO: 10) having a sequence reverse to that of RP1-20. Each of the thus-synthesized peptides was examined in terms of Ca2+ leakage inhibitory effect in a manner similar to that described in (b) above. The results are shown in FIGS. 4 and 5.
Synthesized Peptides:

    MSV-R2P: amino acid Nos. M+2112-2149 SEQ ID NO: 4
    RP1-30: amino acid Nos. 2114-2143 SEQ ID NO: 5
    RP1-25: amino acid Nos. 2114-2138 SEQ ID NO: 6
    RP1-20: amino acid Nos. 2114-2133 SEQ ID NO: 7
    RP13-32: amino acid Nos. 2126-2145 SEQ ID NO: 8
    RP17-36: amino acid Nos. 2130-2149 SEQ ID NO: 9
    RP1-20REV: amino acid Nos. 2133-2114 SEQ ID NO: 10
    RP1-10: amino acid Nos. 2114-2123 SEQ ID NO: 11
    R2MP: amino acid Nos. 2114-2149 (V2132M) SEQ ID NO: 12


As a result, the following polypeptides: MSV-RP2 represented by SEQ ID NO: 4, RP1-30 represented by SEQ ID NO: 5, RP1-25 represented by SEQ ID NO: 6, RP1-20 represented by SEQ ID NO: 7, RP13-32 represented by SEQ ID NO: 8, and RP17-36 represented by SEQ ID NO: 9 were found to exhibit Ca2+ leakage inhibitory effect in a concentration-dependent manner. However, the Ca2+ leakage inhibitory activity of each of the polypeptides was lower than that of R2P.
All the synthesized mutant peptides, the synthesized polypeptides having sequences located upstream and downstream of R2P, and the synthesized polypeptide derived from the DR3 region were found to exhibit no Ca2+ leakage inhibitory effect. In addition, the R2JF peptide represented by SEQ ID NO: 15, which had been obtained through introduction, to the N-terminal of R2P 1-20, of the peptide represented by SEQ ID NO: 14 and having no Ca2+ leakage inhibitory effect, was found to exhibit no Ca2+ leakage inhibitory effect (FIG. 2).
JTV-519 employed as a control is known to inhibit Ca2+ leakage from the sarcoplasmic reticulum of failing myocardium in a concentration-dependent manner, to exhibit the effect of improving cardiac function in a tachycardia-induced canine heart failure model, and to exhibit no effect on cardiac function in a normal canine model.
A drug containing, as an active ingredient, the R2P polypeptide of the present invention or a mutant thereof inhibits Ca2+ leakage in a concentration-dependent manner, and thus is considered to exhibit effects similar to those of JTV-519. Therefore, the drug can be expected as a therapeutic or preventive agent for a disease whose onset is caused by muscle contraction/relaxation dysfunction due to abnormal calcium handling or induction of Ca2+ leakage from sarcoplasmic reticulum.

## Claims

1. A purified polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.