KR102052882B1 - Use of STIM2 inhibitor for preventing or treating Brody syndrome or Brody disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of Brody's disease or Brody syndrome comprising the STIM2 inhibitor as an active ingredient, the inhibition of SERCA1a by the binding of STIM2 protein and SERCA1a by the STIM2 inhibitor can relax skeletal muscle By promoting calcium re-accumulation from STIM2 inhibitors, thereby contributing to the prevention, alleviation or treatment of Brody syndrome or Brody's disease.

Description

Use of STIM2 inhibitor for preventing or treating Brody syndrome or Brody disease

The present invention relates to the use of a STIM2 inhibitor for the prevention or treatment of brody syndrome or brody disease.

Brody disease is an inherited skeletal myopathy caused by autosomal disease, which is caused by rhabdomyolysis of skeletal muscle tissue, which causes muscle stiffness in the movement or movement of the body. Accompanied by exercise-induced muscle stiffness and cramping (Non-Patent Document 1). Symptoms of Brody's disease are severe on the legs, arms, and face (especially the eyelids), causing mobility and facial problems. Recently, symptoms and signs of Brody's disease (in this case, Brody's syndrome) also occur in patients with no mutations in SERCA1a. The cause of SERCA1a is that despite no mutation in the gene of SERCA1a, This is because the activity is significantly reduced. In the case of brody syndrome, the cause is not known at all, and there are no diagnosis methods, but only symptoms and signs (muscle relaxation delay, muscle stiffness, cramps) and patients exist.

Accordingly, the present inventors completed the present invention by identifying the role of STIM2 involved in the relaxation of skeletal muscle in order to diagnose and treat a disorder in which the skeletal muscle relaxes.

N.C. Voermans et al. Brody syndrome: A clinically heterogeneous entity distinct from Brody disease. Neuromuscular Disorders, Vol. 22, Issue 11, p944-954.

An object of the present invention to provide a pharmaceutical composition for the prevention or treatment of Brody syndrome or Brody's disease comprising a STIM2 inhibitor.

As a result of knocking down the stromal interaction molecule 2 (STIM2) in differentiated skeletal muscle cells, the present inventors have found that SERCA1a activity is increased in the process of inducing skeletal muscle contraction and relaxation caused by cell membrane depolarization. Due to the significant increase in the concentration of calcium ions in the myocyte cytoplasmic reticulum, by confirming that it can contribute to the relaxation of skeletal muscle, the present invention was completed.

Accordingly, the present invention relates to a novel mechanism of action of STIM2 for the treatment of skeletal muscle relaxation disorder disease.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention provides a pharmaceutical composition for the prevention or treatment of Brody syndrome or Brody's disease, comprising STIM2 inhibitor as an active ingredient.

In the present invention, "stromal interaction molecule 2 (STIM2)" refers to proteins derived from mammals, preferably humans, mice, mice, rabbits, orangutans, monkeys, hamsters, cats, dolphins, gorillas and the like. it means. For example, the STIM2 protein is human-derived GenBank accession no. NM_001169117.1, NM_020860.3; GenBank accession no. From rat (Rattus norvegicus). NM_001105750.2; Or GenBank accession no. From mice (mus musculus). STIM2 protein having an amino acid sequence such as 001081103.2.

The term “STIM2 inhibitor” refers to a drug that has an effect of inhibiting the degradation of SERCA1a in patients with Brody syndrome or Brody's disease. For example, the STIM2 inhibitor may include any agent that knocks down the mRNA expression of the STIM2 gene or a protein thereof, or decreases function or activity. In the present invention, the STIM2 inhibitor is an antisense-oligonucleotide comprising a sequence complementary to the STIM2 gene, siRNA, shRNA, miRNA or a vector comprising the same; Or an antibody specific for the STIM2 protein.

In one embodiment, the STIM2 inhibitor comprises a sense sequence having a nucleotide sequence of SEQ ID NO: 1 and an antisense sequence having a nucleotide sequence of SEQ ID NO: 2;

A sense sequence having the nucleotide sequence of SEQ ID NO: 3 and an antisense sequence having the nucleotide sequence of SEQ ID NO: 4; And

It may be an siRNA selected from the group consisting of a sense sequence having a nucleotide sequence of SEQ ID NO: 5 and an antisense sequence having a nucleotide sequence of SEQ ID NO: 6.

In the present specification, “siRNA” refers to a double-chain RNA that induces RNA interference through cleavage of mRNA of a target gene, and has an RNA strand of a sense sequence having the same sequence as the mRNA of the target gene and a sequence complementary thereto. It consists of the RNA strand of the antisense sequence.

The siRNA may include a form expressed by inserting the siRNA itself or a base sequence encoding the siRNA synthesized in vitro into the expression vector.

In the present invention, the "vector" refers to a gene construct comprising an external DNA inserted into the genome encoding the polypeptide.

A vector related to the present invention is a vector in which the nucleic acid sequence that inhibits the gene is inserted into the genome, and examples of the vector include a DNA vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a viral vector.

In addition, the antisense has a sequence complementary to all or part of the mRNA sequence transcribed from the STIM2 gene or fragment thereof, and may bind to the mRNA to inhibit the expression of the STIM2 gene or fragment.

In addition, the shRNA (short hairpin RNA) can be used to target the shRNA common sequence region on a human or mouse prepared according to a conventional method.

In addition, the antibody may use polyclonal antibodies, monoclonal antibodies, human antibodies and humanized antibodies against STIM2 protein.

Examples of such antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabody; Linear antibodies (Zapata et al. Protein Eng. 8 (10): 1057-1062 (1995)); Single chain antibody molecules; And multispecific antibodies formed from antibody fragments and the like.

Digestion of the antibody with papain results in two identical antigen binding fragments, each "Fab" fragment with a single antigen binding site, and the remainder "Fc" fragment. Pepsin treatment results in an F (ab ') 2 fragment that has two antigen binding sites and still can cross-link antigen. Fv is a minimal antibody fragment that contains a complete antigen recognition and binding site. This site consists of a dimer of one heavy chain and one light chain variable region and is tightly coupled non-covalently.

Methods of making polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be prepared by injecting the mammal with one or more immunizing agents, if necessary, with an adjuvant. Typically, the immunizing agent and / or immunoadjuvant is injected into the mammal several times by subcutaneous injection or intraperitoneal injection. The immunizing agent may be a protein of the invention or a fusion protein thereof. Injecting an immunizing agent with a protein known to be immunogenic in the mammal being immunized can be effective.

Monoclonal antibodies according to the invention may be prepared by the hybridoma method described in Kohler et al. Nature, 256: 495 (1975) or by recombinant DNA methods (see, eg, US Pat. No. 4,816,576). Can be. Monoclonal antibodies are also phage antibodies using techniques described in Clackson et al. Nature, 352: 624-628 (1991) and Marks et al. J. Mol. Biol., 222: 581-597 (1991). It can be isolated from the library.

The monoclonal antibodies in the present invention are specifically identical or homologous to sequences corresponding to antibodies derived from a particular species or antibodies belonging to a particular antibody class or subclass, provided that a portion of the heavy and / or light chains exhibits the desired activity. However, the remainder of the chain (s) includes “chimeric” antibodies that are identical or homologous to antibodies from other species or antibodies belonging to different antibody classes or subclasses or fragments of such antibodies (Morrison et al. Proc). Natl Acad Sci USA, 81: 6851-6855 (1984)).

“Humanized” forms of non-human (eg, murine) antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′) containing minimal sequences derived from non-human immunoglobulins. , F (ab ') ₂ or other antigen binding sequence of the antibody. In most cases, humanized antibodies replace human immunoglobulins by replacing residues in the complementarity determination (CDR) of the recipient with CDR residues of a non-human species (donor antibody), such as mice, rats, or rabbits, with the desired specificity, affinity, and ability. Receptor antibody). In some cases the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are not found in the recipient antibody or in the CDR or framework sequences to be introduced. Humanized antibodies comprise substantially all of one or more, generally two or more variable domains, wherein all or substantially all CDR regions correspond to regions of non-human immunoglobulins, and all or substantially all FR regions are human immunoglobulins Corresponds to the region of the sequence. Humanized antibodies also include at least a portion of an immunoglobulin constant region (Fc), generally a portion of a human immunoglobulin region (Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992)).

The pharmaceutical composition may increase the activity of SERCA1a by knocking down STIM2 through an STIM2 inhibitor, thereby preventing, ameliorating, or treating Brody syndrome or Brody's disease caused by the deactivation of the SERCA1a. In the following examples, the differentiated skeletal muscle cells knocked down STIM2 significantly increased calcium ion in myocytes and inhibited direct binding of STIM2 protein and SERCA1a to increase the activity of SERCA1a. To control the recalculation pathway of calcium into key myocytes.

The SERCA1a is returned to the to the role that the consumption of ATP as a kind of Ca ^{2 +} -pump stored calcium in the cytoplasm in muscle cytoplasmic reticulum (sarcoplasmic reticulum), this process is then flexed back to normal condition the relaxed state Coming calcium is a key step in the process.

The inventors found that the binding of STIM2 and SERCA1a is mediated by the part (SEQ ID NO: 7; SERCA1a-binding region (STIM2-UI)) from the 453 th to 729 th amino acid sequences present at the C-terminus of STIM2. . In the samples obtained from rabbit skeletal muscle, full-length STIM2 and SERCA1a were identified. Also, in the differentiated skeletal muscle cells obtained from mouse, STIM2 and SERCA1a coexisted mainly around the nucleus. Irrespective of the species, the results were applicable to skeletal muscle in general.

In addition, it was confirmed that STIM2 knocked down differentiated skeletal muscle cells had significantly higher SERCA1a activity than normal skeletal muscle cells in the presence of micromolar calcium. That is, in skeletal muscle cells, SITM2 was found to play a role of directly binding to SERCA1a through the STIM2-UI moiety to lower the activity of SERCA1.

Thus, in patients with skeletal muscle relaxation disorders, such as Brody syndrome or Brody's disease, inhibition of expression of the STIM2 gene or protein, in particular the amount of expression of STIM2-UI (453-729 portion of STIM2), which is the binding site of SERCA1a of STIM2 When administered to the patient or the inhibitor thereof may increase the activity of SERCA1a, thereby promoting skeletal muscle relaxation can improve, alleviate or treat the symptoms of patients such as Brody syndrome or Brody disease.

In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical arts, and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg , Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloids Succinic silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates, polyethyleneglycol, sodium carboxymethylcellulose, polyarylates, waxes or wool, and the like.

In addition, the composition of the present invention may further comprise a lubricant, wetting agent, emulsifier, suspending agent or preservative, etc. in addition to the above components.

In one embodiment, the composition according to the invention may be prepared in an aqueous solution for parenteral administration, preferably in a buffered solution such as Hanks' solution, Ringer's solution or physically buffered saline. Can be used. Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

The compositions of the present invention can be administered systemically or topically and can be formulated into drip dosage forms by known techniques for such administration. For example, in oral administration, it may be mixed with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets. For oral administration, the active compounds can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.

Various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques. In addition, an effective amount of the STIM2 inhibitor may be administered in a form suitable for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like as a solution immediately before administration to saline or buffer.

As used herein, the term "administration" refers to introducing a composition of the present invention to a patient in any suitable manner, wherein the route of administration of the composition of the present invention is via oral or parenteral various routes as long as the target tissue can be reached. May be administered. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.

The present invention also provides a method of treating Brody syndrome or Brody's disease comprising administering a therapeutically effective amount of a STIM2 inhibitor to a subject in need thereof.

As used herein, a subject refers to a mammal that is the subject of treatment, observation or experiment, preferably human.

As used herein, "effective amount" means the amount necessary to delay or entirely stop the onset or progression of the particular disease to be treated, and the effective amount of the STIM2 inhibitor included in the pharmaceutical composition of the present invention is Brody syndrome or Brody's disease. It means the amount required to achieve the effect of inhibiting the SERCA1a activity decrease in. Thus, the effective amount is determined by the type of disease, the severity of the disease, the type and amount of other components contained in the composition, and the age, weight, general health, sex and diet of the patient, time of administration, route of administration, duration of treatment, simultaneous use. It can be adjusted according to various factors including the drug. It will be apparent to those skilled in the art that a suitable total daily dose may be determined by the practitioner within the correct medical judgment.

For the purposes of the present invention, the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved and whether other agents are used in some cases, the age, weight, general state of health, sex of the patient. And various factors and similar factors well known in the medicinal art, including diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or concurrent with the specific composition.

As used herein, “treatment” means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected. "Treatment" may also mean increasing survival compared to the expected survival rate without treatment. "Treatment" refers to both therapeutic treatment and preventive or preventative measures. Such treatments include the treatments required for the disorders that have already occurred as well as the disorders to be prevented. "Ameliorating" a disease means that the extent and / or undesirable clinical signs of the disease state are reduced and / or the time course of progression is slowed or lengthened, as compared with no treatment. do.

The present invention also provides a method of treating Brody syndrome or Brody's disease comprising administering to a subject a pharmaceutical composition for the prevention or treatment of Brody syndrome or Brody's disease comprising a pharmaceutically effective amount of a STIM2 inhibitor.

The pharmaceutical composition and the method of administration used in the method of treating Brody syndrome or Brody's disease have been described above, and the common content between the two is omitted to avoid excessive complexity of the present specification.

Individuals who can administer the composition for preventing or treating brody syndrome or brody disease include all animals except humans. For example, animals such as dogs, cats, and mice.

The present invention provides a composition for diagnosing Brody syndrome or Brody disease, which comprises an agent for measuring mRNA or protein level of a stromal interaction molecule 2 (STIM2) gene.

In the case of the brody syndrome or brody disease, the activity of SERCA1a is reduced. In other words, patients with Brody syndrome or Brody's disease have decreased activity of SERCA1a, and thus, the mRNA or protein level of STIM2 directly binding to SERCA1a is expected to be detectably increased compared to normal individuals. Therefore, Brody syndrome or Brody's disease can be diagnosed from this.

In the present invention, "diagnosis" refers to confirming the pathological state, and for the purpose of the present invention, the diagnosis is confirmed whether or not the onset, development and alleviation of these diseases by confirming the expression of the diagnostic marker of brody syndrome or Brody's disease. Means to check.

In the present invention, "diagnostic marker" means a substance capable of diagnosing cells of Brody syndrome or Brody's disease from normal cells, and a polypeptide showing an increase or a decrease in cells of Brody syndrome or Brody's disease compared to normal cells. Or organic biomolecules such as nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. The marker for diagnosing Brody syndrome or Brody's disease provided by the present invention may be a protein expressed from the STIM2 gene whose expression amount is increased or decreased in the cells of Brody syndrome or Brody's disease compared to normal cells.

The composition for diagnosing brody syndrome or brody disease of the present invention comprises an agent for measuring the mRNA expression level of STIM2 gene or the amount of protein expressed from the gene, and with such an agent oligonucleotide having a sequence complementary to STIM2 mRNA, such as STIM2 It may include primers or nucleic acid probes that specifically bind to mRNA or antibodies specific for STIM2 protein.

The present invention,

The stromal interaction molecule 2 (STIM2) gene is contacted with a candidate outside the body,

It provides a method for screening a drug for the prevention or treatment of Brody syndrome or Brody disease comprising determining whether the candidate substance promotes or inhibits the expression of the gene.

In addition, the present invention,

Contacting the substrate with a substrate of stromal interaction molecule 2 (STIM2),

It provides a method for screening a drug for the prevention or treatment of Brody syndrome or Brody's disease comprising determining whether the candidate substance enhances or inhibits the function or activity of the protein.

According to the screening method of the present invention, first, a candidate substance to be analyzed may be contacted with a biological sample collected from a Brody syndrome or Brody disease patient including the gene or protein.

In the present invention, the candidate material promotes or inhibits or knocks down the function or activity of the STIM2 protein or a substance that promotes or inhibits the transcription or translation of the STIM2 gene sequence into mRNA or a protein according to a conventional selection method. down substances), or may be randomly selected individual nucleic acids, proteins, peptides, other extracts or natural products, compounds, and the like.

Thereafter, the expression amount of the gene, the amount of the protein, and the activity of the protein may be measured or confirmed in the sample treated with the candidate, and as a result of the measurement, the amount of expression of the gene, the amount of the protein, or the activity of the protein is increased or decreased. The candidate substance may be determined as a substance capable of treating brody syndrome or brody disease.

The method for measuring the expression level or the amount of protein of the STIM2 gene may be carried out including a known process for separating mRNA or protein from a biological sample using known techniques.

The biological sample refers to a sample obtained from a living body in which the expression level of the gene or the protein level according to the development or progression of brody syndrome or brody disease is different from a normal control group, and the sample includes, for example, tissue, cells, and blood. , Serum, plasma, saliva and urine, and the like, but are not limited thereto.

The method of measuring the expression level of the gene, the amount of protein or the activity of the protein in the method can be carried out through a variety of methods known in the art, such as reverse transcripatase-polymerase chain reaction, real-time Real time-polymerase chain reaction, Western blot, Northern blot, Southern blot, electrophoric mobility shift assay (EMSA), immunofluorescence, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay RIA; radioimmuno assay, radioimmunodiffusion, or immunoprecipitation may be performed.

Candidates exhibiting activity that inhibits gene expression or decreases protein function obtained through the screening method of the present invention may be candidates for brody syndrome or brody disease treatment.

Such a candidate drug for brody syndrome or brody disease will act as a leading compound in the development of subsequent drug treatment related to brody syndrome or brody disease, and will promote or inhibit the function of the STIM2 gene or the protein expressed therefrom. By modifying and optimizing its structure to be effective, new brody syndrome or brody disease treatments can be developed.

For matters related to genetic engineering in the present invention, Sambrook et al. (Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001)) and Frederick et al. (Prederick M. Ausubel et al. Current protocols in molecular biology volumes 1, 2, 3, John Wiley & Sons, Inc. (1994)).

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

The present invention can inhibit the decrease of SERCA1a activity by binding STIM2 protein to SERCA1a by knocking down STIM2, thereby promoting skeletal muscle relaxation, thereby increasing calcium re-accumulation from STIM2 inhibitors, preventing, alleviating or treating brody syndrome or brody disease. Can contribute to

1 is STIM2 domain (S: signal peptide; EF: Ca 2 + binding domain; SAM: oligomerization domain; T: transmembrane domain; C: coiled-coil domain; P: Ser / Pro rich portion; K: Lys rich portion) It is shown.
FIG. 2A shows Coomassie blue staining results after 10% SDS-PAGE of recombinant GST-binding STIM2-UI protein successfully expressed in E. coli , B shows GST-binding STIM2-UI protein and triad sample Proteins obtained through the binding reaction were shown by Coomassie blue staining.
Figures 3-9 show the respective qTOF MS spectral results for bands 1-7 found in Figure 2B.
10 shows the results of coimmunoprecipitation of STIM2 and SERCA1a in rabbit skeletal muscle triad samples.
FIG. 11 shows the result of confirming that the siRNA for STIM2 was treated to differentiation skeletal muscle cells of mice by the immunooblot assay that the level of STIM2 protein was reduced by more than 90% by siRNA (# 3).
12 is a result of confirming the role of STIM2 in skeletal muscle by measuring oxalate assisted radiation ⁴⁵ calcium influx after knocking down STIM2 in mouse differentiated skeletal muscle cells.
FIG. 13 shows the results of measuring cytoplasmic calcium levels at resting phase in differentiated skeletal muscle cells knocked down by STIM2. FIG.
Figure 14 is the result of measuring the amount of calcium stored in myocyte cytoplasmic reticulum in mice differentiated skeletal muscle cells knocked down STIM2 by Topsigin (TG) treatment.

Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1. cDNA Construction and Expression for GST-Binding STIM2-UI Protein

Using mouse STIM2 cDNA (GenBank accession number: NM_AF328905.1) as a sample, PCR was performed using the PCR primers in Table 1. The PCR synthesized thus obtained was inserted into pGEX-4T-1 using the restriction enzymes EcoR1 and Sal1, and the nucleotide sequence of the completed cDNA was reconfirmed through the protein sequencer and transformed into E. coli (DH5α). It was converted and expressed with GST-STIM2-UI (see FIG. 1) protein, and purified using GST beads.

GST -connect STIM2 For cDNA production on UI part PCR primer  order SEQ ID NO: 8
Forward primer
(Using EcoR1 restriction enzyme) 5'-CGGAATTCATGAGCCTGACCTCTTCCCTTTATTC-3 ' SEQ ID NO: 9
Reverse primer
(Sal1 restriction enzyme used) 5'-CGTCGACTCACTCTCCATTATGACAAAGGTCATG-3 '

Triad  sample preparation

Triad vesicles were obtained from rabbit fast-twitch skeletal muscle (back and leg) and dissolved (1% triton X-100, 10 mM Tris-HCl, 1 mM Na ₃ VO ₄ , 10%). Triad sac was solubilized at 4 ° C. for 4 hours using glycerol, 150 mM NaCl, 5 mM EDTA, a protease inhibitor cocktail, and pH 7.4) to obtain a triad sample. After GST-linked STIM2 UI protein was purified with GST beads, 150 μg of triad samples were mixed and bound for 8 hours at 4 ° C. The proteins attached to the beads were separated on a 10% SDS-PAGE gel and coomassie. Visualization via Coomassie Blue staining.

Gel state proteolysis, A quadrupole  Time-of-flight mass spectrometry and database search

Proteins obtained in the binding reaction are protein digested using trypsin in a gel state and analyzed by quadrupole time-of-flight mass spectrometry (qTOF MS). The results were searched using Mascot database to identify the identity of the protein.

band# Protein name Mascot ID Monoisotopic  mass Bell Match figures Same Peptide One Fructose-bisphosphate aldolase class 2 ALF_ECOLI 39355 E. coli 112 AFQELNAIDVL (617.32)
IFDFVKPGVITGDDVQK (626.67)
VKAPVIVQFSNGGASFIAGK (664.37)
SKIFDFVKPGVITGDDVQK (698.37)
FTIAASFGNVHGVYKPGNVVLTPTILR (718.65) 2 3-oxoacyl- [acyl-carrier-protein] synthase 1 FABB_ECOLI 42934 E. coli 124 AVITGLGIVSSIGNNQQEVLASLR (813.78) 3 Peptidyl-prolyl cis-trans isomerase D PPID_ECOLI 68108 E. coli 90 ALDAYYALQQK (642.33)
LIDEALLDQYAR (710.37)
QAIFATPAFQVDGK (746.89) 4 GTP-binding protein TypA / BipA TYPA_ECOLI 67546 E. coli 200 EGFELAVSRPK (616.83)
INIVDTPGHADFGGEVER (642.65)
AVAFALFGLQDR (654.35)
ASGTDEAVVLVPPIR (762.42) 5 Chaperone protein ClpB CLPB_ECOLI 95700 E. coli 180 VIGQNEAVDAVSNAIRR (604.65)
ELVLGVVSHNFRPEFINR (709.38)
LVGAPPGYVGYEEGGYLTEAVR (766.39)
VIGQNEAVDAVSNAIR (828.44)
VFVAEPSVEDTIAILR (879.98) 6 Phenylalanine-tRNA ligase beta subunit SYFB_ECOLI 88077 E. coli 212 FVPDTQAPLGIR (657.36) SLAISLILQDTSR (708.90)
IGFVGVVHPELER (726.40)
VAVATIGAVLPGDFK (729.41)
VYGYNNIPDEPVQASLIMGTHR (830.74) 7 Sarcoplasmic Of
endoplasmic reticulum
Ca ^{2 +} - ATPase  1a (SERCA1a) AT2A1_MOUSE 110747 Mouse 121 VGEATETALTTLVEK (781.42) 8 No matching signal

Figure 1 shows a schematic for the STIM2 domain and STIM2-UI (453-729 amino acid sequence) used in the present invention. 2A shows GST-binding STIM2-UI protein expressed in E. coli , purified using GST beads and isolated on 10% SDS-PAGE gel and stained with Coomassie blue, B is GST-binding STIM2 The protein obtained through the coupling reaction between the UI protein and the triad sample was stained with Coomassie blue. GST alone is a negative control, and the proteins shown by binding to the GST-binding STIM2-UI protein are named for each band on the left and used for qTOF MS analysis. GST or GST-STIM2-UI proteins are marked separately on the gel with an asterisk.

As shown in FIG. 1 and FIG. 2, a protein binding to STIM2 was found through a binding reaction between a GST-STIM2-UI protein and a triad sample.

3-9 show the respective qTOF MS spectral results for bands 1-7 (# 1-7) shown in FIG. 2 (B).

The GST-STIM2-UI protein was found to bind SERCA1a (# 7) from the results.

Example 2.

Induction of Skeletal Muscle Cells and Differentiation

Skeletal muscle satellite cells were isolated from skeletal muscle of mice, and primary cultures were obtained to obtain skeletal muscle precursor cells (myoblasts). As a culture process, 5% of 37 ° C conditions were treated with cell culture (F-10 Nutrient Mixture composition: 20% FBS, 100 units / ml penicillin, 100 ㎍ / ml streptomycin, 2 mM L-glutamine, 20 nM basic fibroblast growth factor) Incubated in a CO ₂ incubator. At this time, a 10-cm or 96-well plate was used as a culture dish, and all dishes were coated with Matrigel. When skeletal muscle cells proliferated to about 70% of the culture dish, they were induced into differentiated skeletal muscle cells (cell differentiation composition: 5% heat-inactivated horse serum and 20% FBS and F-10 Nutrient Mixture use low-glucose DMEM, no bFGF, 18% CO ₂ incubator). Differentiated cells were called differentiated skeletal muscle cells (myotubes), and the degree of differentiation was observed by obtaining an image of the cells using an optical microscope.

Co-Immunoprecipitation and Immunoassay

Triad samples (500 μg total protein) were subjected to co-immunoprecipitation using anti-STIM2 antibody (10 μg / m), and the resultant was separated on a 10% SDS-PAGE gel. Proteins isolated on the gel were transferred to a poly-vinyldenefluoride (PVDF) membrane (100V for 2 hours) and treated with 5% non-fat milk for 1 hour, and the corresponding primary antibody (3-12 hours, anti- -STIM2 antibody (1: 1000) or anti-TRPC6 antibody (1: 1000), followed by 45 minutes of horseradish peroxidase-conjugated secondary antibodies followed by color reaction (SuperSignal ultrachemiliminescent substrate, Pierce) Visualization and analysis through.

In order to perform immunoassay using differentiated skeletal muscle cells (myotube), differentiated skeletal muscle cells were lysed in lysis buffer composition: 1% Triton X-100, 10 mM Tris-HCl (pH 7.4), 1 mM Na ₃ VO ₄ , 10% glycerol, 150mM NaCl, 5mM EDTA, protease inhibitors (1μM pepstatin, 1μM leupeptin, 1mM phenylmethysulfonyl fluoride, 20mg / ml aprotinin, 1μM trypsin inhibitor) were treated for 24 hours at 4 ° C. At this time, differentiated skeletal muscle cells differentiated in 10-cm culture dish was treated with 300 μl of lysis solution. The following antibody treatments and color reactions were carried out as mentioned above.

The results of FIG. 10 confirm the direct coupling of the full-length STIM2 and SERCA1a. Since the result was a sample obtained from rabbit skeletal muscle and mouse differentiated skeletal muscle cells, the experimental results of the full-length STIM2 protein originally present in the organism, the experimental results of Figure 1 using the STIM2 fragment is the original organism The same is true for the full-length STIM2, which is also found in all species.

STIM2 protein knockdown

SiRNA (GenBank accession number: NM_AF328905.1) for STIM2 was synthesized using the siRNA design program (Dharmacon RNAi Technologies) (Table 3). Skeletal muscle cells 3 days from the day of differentiation were treated with siRNAs shown in Table 3 for 4 hours.

SiRNA  Sequence SEQ  ID NO. Sense Sequence SEQ  ID NO. Antisense  order Mixed control siRNA 10 5'-ACGUGACACGUUCGGAGAAUU-3 ' 11 5'-UUCUCCGAACGUGUCACGUUU-3 ' # 1 siRNA One 5'-UGCCACAAUAUGAGAAGAAUU-3 ' 2 5'-UUCUUCUCAUAUUGUGGCAUU-3 ' # 2 siRNA 3 5'-AUCGGAACGAAGAGGAGGAUU-3 ' 4 5'-UCCUCCUCUUCGUUCCGAUUU-3 ' # 3 siRNA 5 5'-AAUGUUUCCAGAGUAAGCAUU-3 ' 6 5'-UGCUUACUCUGGAAACAUUUU-3 '

As shown in FIG. 11, as a result of treatment of the siRNA with mouse differentiated skeletal muscle cells, it was confirmed that the level of STIM2 protein was reduced by more than 90% by siRNA # 3. Alpha-actin and Coomassie blue staining results were used as the basis for the same sample application.

Differentiated cell homogenate preparation Oxalate Secondary radiation ⁴⁵ calcium Inflow measurement

STIM2 knockdown differentiated skeletal muscle cells are homogenized using homogenization solution (50 mM KH ₂ PO ₄ , 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, protease inhibitor cocktail, and 0.5 mM DTT, and pH 7.0) (250 μg) was mixed with the preparation solution (40 mM imidazole, 100 mM KCl, 5 mM MgCl ₂ , 5 mM NaN ₃ , 0.5 mM EGTA, and pH 7.0.), And ⁴⁵ calcium-influx measurements were performed using the reaction solution (5 mM MgATP, 5 mM K-oxalate). , and 70nM or 1μM of free ⁴⁵ Ca ²⁺ ), and the change in radioactivity was measured at 0, 1, 2, 3 and 4 minutes.

⁴⁵ Ca ^{2 +} -inflow
( nmole / mg / min) Uninfected  Control A mixed contrast siRNA STIM2  Knock-down 70 nM free ⁴⁵ Ca ^{2 +} condition 1.64 ± 0.15 1.70 ± 0.13 2.06 ± 0.11 One μM  freedom ⁴⁵ Ca ^{2 +}  condition 8.01 ± 1.30 8.10 ± 0.30 12.17 ± 0.61 ^*

Oxalate - the auxiliary radiation ⁴⁵ Ca-a result of executing the inlet (12), differentiated skeletal muscle cells STIM2 the knockdown as compared to the siRNA non-infected control cells or scrambled (scrambled) control siRNA treated cells, 1μM ⁴⁵ Ca ^{2 +} significantly while Increased radiation ⁴⁵ calcium-influx was observed. That is, it was confirmed that SERCA1a activity was significantly increased in skeletal muscle by the absence or almost no STIM2. Also, 1μM ⁴⁵ Ca ^{2 +} state of skeletal muscle is because up to going physiological cytosolic calcium levels of instantaneous return to relax after a contraction, and or contraction, STIM2 is by controlling the SERCA1a activity of contraction duration or relaxation of skeletal muscle in a steady state This suggests that you can control the start. * In Table 4 indicates a significant difference compared to the uninfected control (p <0.05).

Calcium Transport Measurements in Single Cells

When combined with calcium, 5 μM of fura-2 (a resting cytoplasmic calcium concentration) or fluo-4 (a cytoplasmic network from myoplasmic reticulum), a calcium fluorescence dye (Ca ^{2 +} dye) that fluoresces at a different wavelength than before calcium was combined The amount of calcium that can be liberated) was prepared and incubated into differentiated skeletal muscle cells at 37 ° C. for 45 minutes. The differentiated skeletal muscle cells were treated with imaging solution (125 mM NaCl, 5 mM KCl, 2 mM KH ₂ PO ₄ , 2 mM CaCl ₂ , 25 mM HEPES, 6 mM glucose, 1.2 mM MgSO ₄ , 0.05% BSA (fraction V), pH 7.4.) It is a state. Fluorescence microscopy (Nikon x40 oil-immersion objective, NA 1.30, ECLIPSE Ti, Nikon) was used to measure intracellular calcium migration. Fluorescence changes of calcium fluorescent dyes are transferred to a computer using a 75-watt Xenon lamp (FSM150Xe, Bentham Instruments, Ltd) and a 12-bit CCD camera (DVC-340M-OO-CL, Digital Video Camera Company) connected to a fluorescence microscope And analyzed using InCyt Im1 (fluo-4) or Im2 (fura-2) image acquisition and analysis software (v5.29, Intracellular Imaging Inc). In order to measure the amount of calcium in the experiment it can be liberated into the cytoplasm in a near cytoplasmic reticulum, after calcium is not present no calcium (Ca ^{2 +} -free) for 5 minutes a solution of the imaging cell, (thapsigargin (TG with tapsi )) Was treated to induce depletion of calcium in SR. TG was treated to the cells by manual dissolved in Me ₂ SO (<0.05%) changes in cell migration by calcium Me ₂ SO (<0.05%) itself was not viewed. The results for TG measurements with relatively long measurement times (more than 20 minutes) were statistically analyzed for the area of the calcium migration graph.

Uninfected  Control A mixed contrast siRNA STIM2  Knock-down Resting cytoplasmic calcium concentration (nM) 72.75 ± 9.16
(108) 72.50 ± 5.02
(116) 56.17 ± 6.05 ^*
(113) In myocytes  The amount of calcium available to the cytoplasm 1.00 ± 0.09 (83) 0.99 ±± 0.08
(88) 1.30 ± 0.14 ^*
(81)

As shown in Table 5, FIGS. 13 and 14, STIM2 was knocked down and measured the cytoplasmic calcium concentration of the resting phase in the differentiated skeletal muscle cells, it was confirmed that the concentration of resting cytoplasmic calcium by STIM2 knockdown. The results were analyzed compared to the uninfected control. As expected in the previous experiment, it was confirmed that the concentration of cytosolic calcium decreased as the activity of SERCA1a increased due to the absence of STIM2.

In addition, the amount of calcium stored in myocytes in STIM2-knockdown mouse differentiated skeletal muscle cells was measured by TG treatment, indicating that the amount of calcium stored in myocytes was increased by STIM2 knockdown. Confirmed. As described above, as expected, it was confirmed that the amount of calcium in myocytes was increased as the activity of SERCA1a was increased by the absence of STIM2.

* In Table 5 indicates a significant difference compared to the uninfected control (p <0.05).

Data analysis

All data were expressed as ± SE combining data obtained through a number of experiments. The value obtained in the control group was expressed as 1 and expressed in a normalized ratio method representing a relative change thereto. Absolute cytoplasmic calcium concentrations were expressed in absolute numbers. Significant differences were performed using unpaired t- test (GraphPad InStat, v2.04), and significant differences were marked with an asterisk when P <0.05. Graphing was done using the Origin v7 program.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Use of STIM2 inhibitor for preventing or treating Brody syndrome          or Brody disease <130> P17U11C11796 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA # 1_sense <400> 1 ugccacaaua ugagaagaau u 21 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA # 1_antisense <400> 2 uucuucucau auuguggcau u 21 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA # 2_sense <400> 3 aucggaacga agaggaggau u 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA # 2_antisense <400> 4 uccuccucuu cguuccgauu u 21 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA # 3_sense <400> 5 aauguuucca gaguaagcau u 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA # 3_antisense <400> 6 ugcuuacucu ggaaacauuu u 21 <210> 7 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> SERCA1a-binding region (STIM2-UI) <400> 7 Ser Leu Thr Ser Ser Leu Tyr Ser Asp His Ser Trp Val Val Met Pro   1 5 10 15 Arg Val Ser Ile Pro Pro Tyr Pro Ile Ala Gly Gly Val Asp Asp Leu              20 25 30 Asp Glu Asp Thr Pro Pro Ile Val Ser Gln Phe Pro Gly Thr Met Ala          35 40 45 Lys Pro Pro Gly Ser Leu Ala Arg Ser Ser Ser Leu Cys Arg Ser Arg      50 55 60 Arg Ser Ile Val Pro Ser Ser Pro Gln Pro Gln Arg Ala Gln Leu Ala  65 70 75 80 Pro His Ala Pro His Pro Ser His Pro Arg His Pro His His Pro Gln                  85 90 95 His Thr Pro His Ser Leu Pro Ser Pro Asp Pro Asp Ile Leu Ser Val             100 105 110 Ser Ser Cys Pro Ala Leu Tyr Arg Asn Glu Glu Glu Glu Glu Ala Ile         115 120 125 Tyr Phe Ser Ala Glu Lys Gln Trp Glu Val Pro Asp Thr Ala Ser Glu     130 135 140 Cys Asp Ser Leu Asn Ser Ser Ile Gly Arg Lys Gln Ser Pro Pro Leu 145 150 155 160 Ser Leu Glu Ile Tyr Gln Thr Leu Ser Pro Arg Lys Ile Ser Arg Asp                 165 170 175 Glu Val Ser Leu Glu Asp Ser Ser Arg Gly Asp Ser Pro Val Thr Val             180 185 190 Asp Val Ser Trp Gly Ser Pro Asp Cys Val Gly Leu Thr Glu Thr Lys         195 200 205 Ser Met Ile Phe Ser Pro Ala Ser Lys Val Tyr Asn Gly Ile Leu Glu     210 215 220 Lys Ser Cys Ser Met Asn Gln Leu Ser Ser Gly Ile Pro Val Pro Lys 225 230 235 240 Pro Arg His Thr Ser Cys Ser Ser Ala Gly Asn Asp Ser Lys Pro Val                 245 250 255 Gln Glu Ala Pro Ser Val Ala Arg Ile Ser Ser Ile Pro His Asp Leu             260 265 270 Cys His Asn Gly Glu Lys         275 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer_STIM2-UI_F <400> 8 cggaattcat gagcctgacc tcttcccttt attc 34 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer_STIM2-UI_R <400> 9 cgtcgactca ctctccatta tgacaaaggt catg 34 <210> 10 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> scrambled control siRNA_sense <400> 10 acgugacacg uucggagaau u 21 <210> 11 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> scrambled control siRNA_antisense <400> 11 uucuccgaac gugucacguu u 21

Claims (9)

Under conditions where the matrix interaction molecule 2 (STIM2) protein and SERCA1a form a complex, the STIM2 protein and SERCA1a are contacted with a candidate outside the body,
A method for screening a drug for preventing or treating brody syndrome, comprising determining whether the candidate substance promotes or inhibits formation of a complex of STIM2 protein and SERCA1a. The method of claim 1,
A method for screening a drug for the prophylaxis or treatment of brody syndrome, wherein the candidate substance inhibits the formation of the complex of STIM2 protein and SERCA1a and is determined as a therapeutic agent for brody syndrome. The method of claim 1,
Candidate is a method for screening a drug for the prevention or treatment of brody syndrome, which is to inhibit the decrease of SERCA1a activity by the binding of STIM2 protein and SERCA1a to promote skeletal muscle relaxation. The method of claim 1,
The STIM2 protein comprises a SERCA1a-binding region (STIM2-UI) of STIM2 represented by the amino acid sequence set forth in SEQ ID NO: 7 for screening a drug for preventing or treating brody syndrome. delete delete delete delete delete

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