KR20210104529A - A method for screening a therapeutic agent of disease associated with muscle - Google Patents

Abstract

The present invention can provide a novel method for screening a preventive or therapeutic drug for muscle diseases caused by muscle weakness, through confirming the decrease of an ATP production amount and the increase of expression of muscle degrading enzymes Atrogin-1 and MuRF1 protein in muscle disease caused by muscle weakness, in a process of developing a therapeutic drug for the muscle diseases caused by muscle weakness.

Description

Screening method of a drug for the treatment of muscle disease {A method for screening a therapeutic agent of disease associated with muscle}

The present invention relates to a method for effectively screening a drug for preventing or treating a muscle disease using ATP kinetics of muscle cells.

Skeletal muscle atrophy refers to a disease in which the ability to support or exercise the human body is reduced due to a decrease or complete loss of protein content resistance or mass of skeletal muscle.

In general, skeletal muscle atrophy is divided into skeletal muscle atrophy caused by denervation due to trauma, and skeletal muscle atrophy (pulmonary atrophy) caused by immobility such as resting supine or joint fixation. It may be caused by decreased blood supply to the muscles, immobilization, persistent weight loss, malnutrition (malnutrition or starvation), congestive heart failure, chronic obstructive pulmonary disease ), renal failure, severe burns, cancer, AIDS, etc. are known to be caused by various diseases.

In addition, there is sarcopenia, which is on the increase due to rapid aging, which shows symptoms similar to skeletal muscle sarcopenia.

Such skeletal sarcopenia has been highlighted as a socially important concern, and although active research is being conducted, a therapeutic agent that clearly shows efficacy in skeletal sarcopenia has not yet been found, so research for the development of effective drugs is continuously required. .

Therefore, the development of a more rapid and effective evaluation method for the development of these effective drugs is an important task along with research on the development of effective drugs.

Republic of Korea Patent No. 10-1985087

An object of the present invention is to provide a method for effectively screening a drug for preventing or treating a muscle loss-related disease by using a mechanism in which the ATP production level of muscle cells is changed.

The present invention confirmed that it is possible to screen the effect of a therapeutic candidate on muscle loss disease in an animal model of muscular atrophy by changing the level of adenosine triphosphate (ATP) production in muscle cells.

Accordingly, the present invention provides a screening method for a drug for preventing or treating a muscle loss disease due to muscle weakness.

Specifically, the present invention

After contacting the muscle cells with the candidate substance outside the human body,

It provides a screening method of a drug for the prevention or treatment of muscle loss-related diseases, comprising determining whether the candidate substance changes the ATP production level of muscle cells.

In the present invention, when the candidate substance reduces the ATP production level of muscle cells, the candidate substance may be determined as a drug for preventing or treating muscle loss-related diseases. At this time, whether the ATP production level of the muscle cells is changed can be confirmed through a luciferase/luciferin luminescence reaction or a change in the amount of glycerol-6-phosphate produced. The luciferase/luciferin luminescence reaction is a reaction that essentially requires ATP, and is well known as a method for confirming ATP changes in cells. In addition, the change in the amount of glycerol-6-phosphate produced indirectly by measuring the glycerol-6-phosphate produced in proportion to the concentration of ATP produced by inducing the phosphorylation reaction of glycerol by an absorption or fluorescence method to indirectly change the production concentration of ATP can be checked In addition to the method for measuring the change in the luciferase/luciferin reaction and the amount of glycerol-6-phosphate, any known method capable of confirming the change in ATP production in cells may be used.

In addition, the present invention provides that, when the candidate material reduces the ATP production level of muscle cells, the candidate material may include Atrogin-1 gene and/or MuRF1 gene expression; And/or it may further comprise the step of determining whether to enhance or inhibit the function or activity of the Atrogin-1 protein and/or the MuRF1 protein.

In this case, the candidate material inhibits the expression of the Atrogin-1 gene and/or the MuRF1 gene; Alternatively, when the function or activity of Atrogin-1 protein and/or MuRF1 protein is inhibited, the candidate substance may be selected as a drug for preventing or treating muscle loss-related diseases.

According to the screening method of the present invention, a candidate substance to be analyzed can be contacted with a cell containing the gene or protein first.

The candidate substance is a substance that promotes or inhibits transcription or translation from the Atrogin-1 gene and/or MuRF1 gene nucleic acid sequence to mRNA or protein according to a conventional selection method, or the function or activity of the Atrogin-1 protein and/or MuRF1 protein It may be an individual nucleic acid, protein, peptide, other extract or natural product, compound, which is estimated or randomly selected as having potential as a drug to enhance or inhibit .

In the present invention, “Atrogin-1 (uncoupling protein-1)” is a representative muscle-specific E3 ligase. Unlike other muscle diseases, the role of Atrogin-1 on muscle aging is relatively unknown. The Atrogin-1 protein refers to a protein derived from mammals, preferably humans, mice, rats, rabbits, orangutans, monkeys, hamsters, cats, dolphins, or gorillas. For example, the Atrogin-1 protein is human (homo sapiens)-derived GenBank Accession No. NP_478136.1, NP_680482.1; GenBank Accession No. from rat (Rattus norvegicus). NP_598205.1; or GenBank Accession No. from mice (mus musculus). It may be an Atrogin-1 protein having an amino acid sequence such as NP_080622.1 and AAH27211.1.

In the present invention, "MuRF1 (muscle RING-finger protein 1)" refers to a protein originating from mammals, preferably humans, mice, rats, rabbits, orangutans, monkeys, hamsters, cats, dolphins, or gorillas. . For example, the MuRF1 protein is GenBank Accession No. from rat (Rattus norvegicus). AAL16405.1; or GenBank Accession No. from mice (mus musculus). It may be a MuRF1 protein having an amino acid sequence such as ABB16283.1.

In general, ubiquitin ligase in the body is a protein that induces degradation of the protein by attaching ubiquitin as a kind of apoptosis signal to the surface of the protein to be annihilated. There is a ubiquitin ligase called MuRF1. However, the Atrogin-1 and MuRF1 are overexpressed in patients with myopathy mainly accompanied by pathological muscle loss or muscle atrophy, unlike the normal in vivo ubiquitin ligase that induces unnecessary protein death, and currently, sarcopenia (sarcopenia) ) is known as one of the direct molecular biological causes of When the Atrogin-1 and MuRF1 are overexpressed, the degradation pathway is excessively activated compared to the synthesis of muscle cells, which leads to the loss of normal muscle cells.

Thereafter, it is possible to measure the expression amount of the gene, the amount of the protein, or the activity of the protein in the cells treated with the candidate substance, and as a result of the measurement, the expression amount of the gene, the amount of the protein or the activity of the protein is increased or decreased When measured, the candidate material may be determined as a material capable of preventing and treating muscle diseases caused by muscle weakness.

In the above method, the method for measuring the expression amount of a gene, the amount of a protein, or the activity of a protein may be performed through various methods known in the art, for example, reverse transcriptase-polymerase chain reaction, real-time Real time-polymerase chain reaction, Western blot, Northern blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion and immunoprecipitation assay ), etc. can be used.

A candidate substance that inhibits Atrogin-1 gene and/or MuRF1 gene expression or inhibits protein function obtained through the screening method of the present invention is a candidate substance for the prevention and treatment of muscle diseases caused by muscle weakness. can

For example, the candidate substance that inhibits Atrogin-1 gene and/or MuRF1 gene expression or inhibits protein function may be an Atrogin-1 inhibitor or a MuRF1 inhibitor. The “Atrogin-1 inhibitor” or “MuRF1 inhibitor” refers to a drug that has an effect of increasing ATP production in muscle cells in patients with muscle disease due to muscle weakness. For example, the Atrogin-1 inhibitor or MuRF1 inhibitor may include any agent that knocks down the mRNA expression of the Atrogin-1 gene or the MuRF1 gene, or a protein thereof, or reduces the function or activity, respectively. In the present invention, the Atrogin-1 inhibitor or MuRF1 inhibitor is an antisense-oligonucleotide, siRNA, shRNA, miRNA, or a vector comprising the same, each comprising a sequence complementary to the Atrogin-1 gene or MuRF1 gene; Or it may be any one of antibodies specific for Atrogin-1 protein or MuRF1 protein.

As used herein, “siRNA” refers to double-stranded 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 a target gene and a complementary sequence thereto. It consists of an RNA strand of antisense sequence.

The siRNA may include a form in which siRNA synthesized in vitro or a nucleotide sequence encoding the siRNA is inserted into an expression vector to be expressed.

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

The vector related to the present invention is a vector in which a nucleic acid sequence inhibiting the gene is inserted into the genome, and these vectors 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 an mRNA sequence transcribed from the Atrogin-1 gene and/or MuRF1 gene or fragment thereof, and binds to the mRNA to form the Atrogin-1 gene and/or MuRF1 gene or fragment. expression can be inhibited.

In addition, the shRNA (short hairpin RNA) may be prepared according to a conventional method by targeting the shRNA common nucleotide sequence site on a human or mouse.

In addition, the antibody may be a polyclonal antibody, a monoclonal antibody, a human antibody, or a humanized antibody against Atrogin-1 protein and/or MuRF1 protein.

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

Papain digestion of the antibody yields two identical antigen-binding fragments, one “Fab” fragment with a single antigen-binding site, and the other “Fc” fragment. _{Pepsin treatment yields an F(ab') 2} fragment that has two antigen binding sites and is still capable of cross-linking antigen. Fvs are minimal antibody fragments that contain complete antigen recognition and binding sites. This region consists of a dimer of one heavy chain and one light chain variable region and is tightly bound non-covalently.

Methods for preparing polyclonal antibodies are known to those skilled in the art. The polyclonal antibody can be prepared by injecting an immunizing agent into a mammal at least once and, if necessary, by injecting it with an adjuvant. Typically, the immunizing agent and/or adjuvant is injected into the mammal several times by subcutaneous or intraperitoneal injection. The immunizing agent may be a protein of the invention or a fusion protein thereof. It may be effective to inject an immunizing agent with a protein known to be immunogenic in the mammal being immunized.

The monoclonal antibody according to the present invention can be prepared by the hybridoma method described in the literature (Kohler et al. Nature, 256:495 (1975)) or by the recombinant DNA method (see, e.g., U.S. Patent No. 4,816,576). can Monoclonal antibodies were also prepared using the techniques described in Clackson et al. Nature, 352:624-628 (1991) and Marks et al. J. Mol. Biol., 222:581-597 (1991) as phage antibodies. can be isolated from the library.

Specifically, the monoclonal antibody in the present invention is identical to or homologous to a sequence corresponding to an antibody derived from a specific species or an antibody belonging to a specific antibody class or subclass, provided that the monoclonal antibody exhibits the desired activity. However, the remainder of the chain(s) includes “chimeric” antibodies that are identical or homologous to antibodies derived 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 are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab') comprising minimal sequences derived from non-human immunoglobulins. , F(ab')2 or other antigen-binding sequence of the antibody). In most cases, humanized antibodies are human immunoglobulins in which residues from the recipient's complementarity determining (CDR) are substituted with CDR residues from a non-human species (donor antibody), such as mice, rats or rabbits, having the desired specificity, affinity, and ability. recipient antibody). In some cases, Fv framework residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may comprise residues that are not found in the recipient antibody, or in the CDR or framework sequences into which it is introduced. A humanized antibody comprises substantially all of one or more, generally two or more variable domains, wherein all or substantially all of the CDR regions correspond to regions of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin. It corresponds to the region of the sequence. In addition, humanized antibodies comprise 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)).

Candidate substances for the prevention and treatment of muscle diseases caused by muscle weakness act as a leading compound in the process of developing a treatment for and preventing muscle diseases caused by muscle weakness. By modifying and optimizing the structure of the MuRF1 gene or a protein expressed therefrom to exhibit the effect of inhibiting the function, it is possible to develop a new preventive and therapeutic agent for muscle disease caused by muscle weakness.

Matters related to genetic engineering in the present invention are Sambrook et al. (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and Frederick et al. M. Ausubel et al., Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)) makes it clearer.

In the present invention, "muscle disease" refers to any disease that can occur due to muscle weakness, for example, sarcopenia, muscular atrophy, muscle dystrophy or cardiac atrophy, but is limited thereto. it is not For example, the sarcopenia may be age-related sarcopenia.

The “weakened muscle strength” refers to a state in which the strength of one or more muscles is reduced. The muscle weakness may be limited to any one muscle, one side of the body, upper or lower extremities, or the like, or may appear throughout the body. In addition, subjective muscle weakness symptoms including muscle fatigue or muscle pain can be quantified in an objective way through medical examination. The causes of muscle weakness include, but are not limited to, decreased ATP production of muscle cells, muscle damage, decreased muscle mass due to decreased differentiation of muscle cells, and the like, muscle aging, and the like. For example, the muscle weakness may be a decrease in muscle mass due to a decrease in ATP production in muscle cells.

Age-related sarcopenia is a muscle disease in which the skeletal muscles that make up the arms and legs are greatly reduced.

Muscular atrophy is a disease in which muscles are atrophied, and there are various types of muscle atrophy in the arms and legs in an almost symmetrical manner. The most common are amyotrophic lateral sclerosis and spinal progressive muscular atrophy. All of the above diseases are caused by progressive degeneration of motor nerve fibers and cells in the spinal cord, but the cause is unknown. Specifically, amyotrophic lateral sclerosis, also called 'lou Gehrig's disease', is a disease in which motor cells of the spinal nerves or diencephalon are gradually destroyed, and the muscles controlled by these cells atrophy and cannot use their strength. Spinal progressive muscular atrophy has no cone degeneration and chronic degeneration of anterior horn cells in the spinal cord.

Muscle degenerative atrophy is a disease in which progressive muscle atrophy and muscle weakness appear, and pathologically means degenerative myopathy characterized by necrosis of muscle fibers. Damage to the muscle cell membrane leads to necrosis and degeneration of muscle fibers, resulting in muscle weakness and atrophy. Muscle degenerative atrophy can be divided into sub-diseases according to the extent and distribution of muscle weakness, age of onset, rate of progression, severity of symptoms, and family history. , limb-pelvic muscular dystrophy, Emery-Dreyfus muscular dystrophy, facial scapular dystrophy, myotonic muscular dystrophy, oropharyngeal muscular dystrophy, distal muscular dystrophy or congenital muscular dystrophy.

Cardiac atrophy is the atrophy of the heart due to external or internal factors, and it can cause brown atrophy of the heart that causes the reduction of fat tissue by drying out and thinning myocardial fibers when starvation, wasting disease, or old age.

Advantages and features of the present invention, and methods for achieving them, will become 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 a variety of different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the art to which the present invention pertains. It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims.

The present invention can provide a method for screening a therapeutic agent for a muscle disease caused by muscle weakness by checking whether or not the ATP production of muscle cells is changed.

Figure 1 confirms the decrease in the expression of the myolytic enzyme by the standard material (Exedin-4), after treating the differentiated myotube cells with the standard material (Exendin-4), the myolytic enzyme atrozin-1, a marker of muscle atrophy (Atrogin-1) and MuRF1 (Muscle RING-finger protein-1) mRNA expression levels were confirmed. In Figure 1, it was observed that the mRNA expression of the muscle degrading enzymes, which are markers of muscle atrophy, decreased compared to the untreated group (control).
Figure 2 confirms the increase in the expression of the muscle activator protein by the standard substance (Exendin-4), the standard substance (Exendin-4) treatment in differentiated myotube cells was treated with only 10 ng/ml of TNF-α. It was observed to suppress the level of decreased activity expression of factors related to muscle activity (AKT and mTOR) in atrophy-induced myotube cells.
Figure 3 confirms the ATP change according to the differentiation period of muscle cells and the treatment period of the muscle atrophy-inducing substance, using the luciferase / luciferin reaction method (left figure), which is widely used as a method to find out the ATP change in cells , It was observed that ATP activity increased as the differentiation period of myoblasts into myotubes and the treatment period of muscle atrophy-inducing factors increased by the differentiation medium (DM) containing horse serum (right graph).
4 is an observation of ATP changes in muscle cells using a standard substance (Exendin-4). ATP changes in cells were observed. In FIG. 4 , it was observed that myotube cells treated with the standard substance (Exendin-4) had decreased ATP activity compared to the untreated group (control), and the graph on the right shows the level of 100% Luminescence as a starting point.
5 shows the results of an experiment on ATP change using the candidate substance group, and the ATP change in myotube cells was observed after treating the differentiated candidate substance group using the luciferase/luciferin reaction method. In FIG. 5 , variously changed ATP activities were observed in myotube cells treated with candidate substances.

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

[Example]

Example 1: Confirmation of reduction in expression of myolytic enzymes by standard substances

It is known that the expression of genes of Atrogin-1 and MuRF1 (Muscle RING-finger protein 1), which are muscle proteases and muscle atrophy markers, are increased during the onset of muscular dystrophy. Therefore, it was attempted to confirm the decrease in the expression of muscle degrading enzyme by the standard material.

Specifically, C2C12 cells (myoblasts) were cultured for 5 days in a differentiation medium containing horse serum at 2% (v/v) to differentiate them into myotubes. Normal myotube cells untreated with differentiated myotube cells were used as a control group, and myotube cells treated with a standard substance (Exendin-4) 50 nM, a known treatment for sarcopenia, for 48 hours, were used as the experimental group. . Thereafter, total RNA was extracted from the cells of the control group and the experimental group, cDNA was synthesized, and the expression levels of the muscle proteolytic enzymes Atrogin-1 and MuRF1 (Muscle RING-finger protein-1) were compared using the PCR method. did.

As a result (FIG. 1), it was confirmed that the mRNA expression of the muscle degrading enzymes, a marker of muscle atrophy, in the standard substance (Exendin-4) treatment group (experimental group) was decreased compared to the control group.

Example 2: Confirmation of increase in muscle activator by standard material

The signaling mechanisms by which PI3K/AKT and mTOR are activated are known to be important factors for muscle activity. Therefore, based on the results of Example 1, a change in the expression of muscle activators was observed through treatment with a standard substance (Exendin-4) for the treatment of sarcopenia for muscle atrophy. Normal myotube cells (control) untreated to myotubule cells differentiated from the myoblast cell line C2C12 cells in the same manner as in Example 1, and myotube cells induced by muscular dystrophy by treatment with only 10 ng/mL of TNF-α (negative) control), myotube cells (experimental group) treated together with 10 ng/mL of TNF-α and 50 nM of standard (Exendin-4) were cultured for 48 hours. Then, the expression levels of muscle activity-related factors (AKT and mTOR) were compared in myotube cells of each experimental group.

As a result (FIG. 2), the standard substance (Exendin-4) treatment group showed a tendency to suppress the level of decrease in the activity of muscle activators caused by TNF-α compared to the control group in mRNA expression of muscle degrading enzymes, which are markers of muscle atrophy. .

Example 3: Confirmation of ATP change according to muscle cell differentiation period and muscular dystrophy inducing material treatment period

ATP is a hallmark of living cells and is responsible for transferring energy to three general types of cellular reactions: metabolic reactions, material transport, and muscle movement. Since the luciferase/luciferin luminescence reaction is known to require ATP, it is widely used as a method for examining changes in ATP in cells (left side of FIG. 3). Accordingly, C2C12 cells (myoblasts) were cultured for 5 days at 2% (v/v) in a differentiation medium containing horse serum to differentiate them into myotubes. After that, the growth medium was treated with a muscle atrophy inducer (TNF-α 10 ng/mL) and cultured for 6 days. At each observation time, 100 μl of ATP solution was treated for 10 minutes to measure luminescence intensity.

As a result (FIG. 3), as the differentiation period of myoblasts into myotube cells increased, and the duration of treatment with the muscle atrophy inducer increased, ATP activity increased (right side of FIG. 3).

Example 4: Confirmation of ATP Changes in Muscle Cells Using Standards

Based on the results of Example 3, the ATP change pattern in muscle cells by the sarcopenia treatment standard material (Exendin-4) was confirmed using the luciferase/luciferin reaction method as in Example 1. ATP solution 100 in the control group that was not treated with any myotube cells differentiated from the myoblast line C2C12 cells in the same manner as in Example 1 and the experimental group treated with 50 nM of the conventionally known sarcopenia treatment standard (Exendin-4) for 48 hours The luminescence intensity was measured by treating [mu]l for 10 minutes.

As a result (Fig. 4), the standard substance (Exendin-4) treated group showed an increase in ATP activity than the control group.

Example 5: Confirmation of ATP changes in muscle cells using candidate substances

Using the ATP change in the muscle cells identified by the standard material, the ATP change in the muscle cells was screened by treating the candidate material to select a candidate material for the treatment of sarcopenia. C2C12 cells (myoblasts) were differentiated into myotubes in a differentiation medium containing horse serum in the same manner as in Example 1. On the 5th day of differentiation, a candidate material was prepared and treated in the growth medium. On the second day of treatment, 100 μl of ATP solution was treated, and luminescence intensity was measured 10 minutes later.

As a result (FIG. 5), it was confirmed that the level of ATP activity was variously changed in the experimental group treated with the candidate substance.

Claims (8)

After contacting the muscle cells with the candidate substance outside the human body,
A method of screening for a drug for preventing or treating muscle disease due to muscle weakness, comprising determining whether the candidate substance increases or decreases ATP production in muscle cells. According to claim 1,
When the candidate substance reduces ATP production in muscle cells, determining the candidate substance as a drug for preventing or treating muscle disease due to muscle weakness, a method for screening a drug for preventing or treating muscle disease. According to claim 1,
A method of screening for a drug for preventing or treating muscle disease due to muscle weakness, wherein the change in ATP production in muscle cells is confirmed through a luciferase/luciferin luminescent reaction or a change in the amount of glycerol-6-phosphate. According to claim 1,
The screening method is a screening method of a drug for preventing or treating muscle disease due to muscle weakness, further comprising determining whether the candidate material enhances or inhibits the function or activity of the Atrogin-1 protein and/or MuRF1 protein. 5. The method of claim 4,
When the candidate substance inhibits the function or activity of Atrogin-1 protein and/or MuRF1 protein, a drug for preventing or treating muscle disease due to muscle weakness, which is determined as a drug for preventing or treating muscle disease due to muscle weakness screening method. According to claim 1,
The screening method is a screening method of a drug for preventing or treating muscle disease due to muscle weakness, further comprising determining whether the candidate material promotes or inhibits the expression of the Atrogin-1 gene and/or the MuRF1 gene. 7. The method of claim 6,
When the candidate material inhibits the expression of the Atrogin-1 gene and/or MuRF1 gene, it is determined as a drug for preventing or treating muscle disease due to muscle weakness, screening for a drug for preventing or treating muscle disease due to muscle weakness Way. According to claim 1,
A method of screening a drug for preventing or treating muscle disease due to muscle weakness, wherein the muscle disease caused by muscle weakness is sarcopenia, muscular atrophy, muscle dystrophy, or cardiac atrophy.

Images