KR20210104528A - A method for screening a therapeutic agent of obesity - Google Patents

Abstract

The present invention relates to a method for screening a drug for preventing or treating obesity to use a substance that induces mitophagy of adipocyte mitochondria as an anti-obesity agent in the process of developing a drug for the treatment of obesity. More specifically, the present invention relates to a screening method using a substance stimulating the mitochondria of adipocytes to induce mitophagy and to observe fluorescence intensity due to the binding of autophagosomes and lysosomes formed in adipocytes while reducing the expression of a novel mitophagy protein called UCP1.

Description

Screening method for a drug for the treatment of obesity {A method for screening a therapeutic agent of obesity}

The present invention relates to a method for effectively screening a drug for preventing or treating obesity using a mechanism for inducing mitophagy in adipocytes.

Mitophagy is an intracellular molecular regeneration (or decomposition) mechanism that removes unnecessary intracellular mitochondria due to damaged or aged cells, and has the same meaning as damaged cells are eliminated by autophagy from cells. Mitophagy is surrounded by a membrane when mitochondrial damage occurs, autophagosome is formed, and it fuses with lysosome to selectively remove damaged mitochondria. It is a well-known fact that the activity of these mitophagy is very important in terms of regulating the function of cells or tissues as well as the function of mitochondria in various cells, including nerve cells. It is known that when the activity of mitophagy is lowered, the risk of neurological diseases, cancer, metabolic diseases, etc. increases. It is self-evident that many diseases occur when mitophagy does not occur in moderation, which is typically associated with neuronal mitophagy abnormalities such as Parkinson's and Alzheimer's, as well as metabolic diseases related to adipocytes or muscle cell mitophagy. occurrence is also expected. There is no new drug related to mitophagy regulation yet, but if it is possible to control mitophagy, it is expected that it will be possible to develop drugs to treat various diseases.

In the present invention, the properties of carbonylcyanide m-chlorophenylhydrazone (CCCP), a substance acting on mitochondria, for adipocyte mitophagy were identified, and based on this, correlation with changes in adipocyte morphology relationship was established. In addition, it confirmed the efficacy of drugs such as polyunsaturated phosphatidylcholine (PPC), which is well known as a fat reducing agent, on mitophagy of adipocytes. Identify connectivity. Polyene phosphatidylcholine and polyunsaturated phosphatidylcholine (PPC) have been approved for safety by the Ministry of Food and Drug Safety, and are well known for dissolving the fat cell wall into a liquid by breaking the binding of adipocytes and discharging them out of the body. Originally developed as an injection for liver function recovery (liver cell regeneration), it was known to be effective for cirrhosis and fatty liver, but now it is used as a fat-reducing agent. In addition, the present invention investigated how the PPC-induced expression change of UPC1 protein, which is important for maintaining the function of adipocyte mitochondria, is related to the generation of mitophagy caused by PPC [Lower fat - Change in the shape of adipocytes - Mitophagy occurrence of - UPC1 protein change], etc., were investigated.

Investigation of such mechanistic effects will suggest an effective method for developing innovative new drugs (first-in-class) weight control agents or obesity treatments using adipocyte mitophagy in the future.

Vecente Lahera, et al., Role of Mitochondrial Dysfunction in Hypertension and Obesity, Curr Hypertens Rep (2017) 19:11

An object of the present invention is to provide a method for effectively screening a drug for preventing or treating obesity using a mechanism that induces mitophagy in adipocytes.

The present invention provides an effective new drug screening technique for rapidly developing drugs for reducing body fat by searching for substances that induce mitophagy in adipocytes in the process of developing a body fat loss agent by inhibiting adipocytes. can

Accordingly, the present invention provides a screening method for a drug for preventing or treating obesity.

Specifically, the present invention

After contacting the adipocytes with the candidate substance outside the human body,

It provides a screening method for a drug for the prevention or treatment of obesity, comprising determining whether the candidate substance generates or does not generate mitophagy of adipocytes.

In the present invention, when the candidate substance generates mitophagy in adipocytes, the candidate substance can be determined as a drug for preventing or treating obesity. In this case, whether or not the adipocytes generate mitophagy can be confirmed through the fusion of autophagosome and lysosome of mitochondria of adipocytes or the ratio of LC3-I and LC3-II proteins. The fusion of the autophagosome and the lysosome is determined by measuring the fluorescence intensity when the autophagosome and the lysosome are fused or combined using a fluorescent dye. have.

The ratio of the LC3-I and LC3-II proteins may mean the degree to which the LC3-I protein is converted to the LC3-II protein. LC3 or Atg8 is a ubiquitin-type protein, where LC3-I undergoes post-translational modification. LC3 is cleaved at the C-terminus by protease Atg4 to become LC3-I, thereby exposing the C-terminal glycine residue. LC3-I is transformed to LC3-II by phosphatidylethanolamine (PE). The LC3 system plays an important role in the transport and maturation of autophagosomes, and LC3-II is attached to the membrane of the endoplasmic reticulum until degradation occurs in the autolysosome, so it is used as a marker of the autophagosome membrane. do. That is, as the ratio of LC3-II protein to LC3-I increased, it means that the number of generated autophagosomes increased, which means the generation of mitophagy. Therefore, by measuring the ratio of the LC3-II protein to the LC3-I, it can be confirmed that the autophagosome increases as the ratio of the LC3-II protein increases.

In the present invention, all methods known in the art may be applied as it is, in addition to the methods described above, to determine whether or not mitophagy occurs in adipocytes.

In addition, the present invention may further include the step of determining whether the candidate material enhances or inhibits UCP1 gene expression and/or UCP1 protein function or activity when the candidate material generates mitophagy in adipocytes. .

In this case, the candidate material inhibits the expression of the UCP1 gene; Alternatively, when inhibiting the function or activity of UCP1 protein, the candidate substance may be selected as a drug for preventing or treating obesity.

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 estimated to have potential as a substance that promotes or inhibits transcription or translation from the UCP1 gene nucleic acid sequence to mRNA or protein, or a drug that enhances or inhibits the function or activity of the UCP1 protein according to a conventional selection method, or It may be randomly selected individual nucleic acids, proteins, peptides, other extracts or natural products, compounds, and the like.

In the present invention, “UCP1 (uncoupling protein-1)” refers to a protein originating from a mammal, preferably a human, a mouse, a house mouse, a rabbit, an orangutan, a monkey, a hamster, a cat, a dolphin, or a gorilla. For example, the UCP1 protein is human (homo sapiens)-derived GenBank Accession No. AAH69556.1, NP_068605.1; GenBank Accession No. from rat (Rattus norvegicus). NP_036814.1, AAH88156.1; or GenBank Accession No. from mice (mus musculus). It may be a UCP1 protein having an amino acid sequence such as NP_033489.1 and AAH12701.1.

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 substance can be determined as a substance capable of preventing and treating obesity, reducing fat, or reducing body weight.

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 UCP1 gene expression or inhibits protein function obtained through the screening method of the present invention may be a candidate substance for the prevention and treatment of obesity, a fat reducing agent or a weight loss agent.

For example, the candidate substance that suppresses the UCP1 gene expression or exhibits the activity of inhibiting the function of a protein may be a UCP1 inhibitor. The “UCP1 inhibitor” refers to a drug that exhibits an effect of inducing mitophagy of adipocytes in obese patients. For example, the UCP1 inhibitor may include any agent that knocks down UCP1 gene mRNA expression or a protein thereof, or reduces function or activity. In the present invention, the UCP1 inhibitor comprises an antisense-oligonucleotide comprising a sequence complementary to the UCP1 gene, siRNA, shRNA, miRNA, or a vector comprising the same; or an antibody specific for UCP1 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 UCP1 gene or a fragment thereof, and binds to the mRNA to inhibit expression of the UCP1 gene or fragment.

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 UCP1 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)).

Such candidate substances for the prevention and treatment of obesity, fat reduction or weight control act as a leading compound in the subsequent development of a preventive and therapeutic agent for obesity, a fat reducer or a weight loss agent, By modifying and optimizing the structure to exhibit the effect of inhibiting the function of the UCP1 gene or a protein expressed therefrom, it is possible to develop a novel anti-obesity and therapeutic agent, a fat reducing agent or a weight loss agent.

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.

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 screening method for an effective drug for preventing or treating obesity that can dissolve adipocytes by generating mitophagy of adipocytes.

Figure 1 confirms the generation of mitophagy by m-chlorophenylhydrazone (CCCP), which is a conventional mitophagy-inducing substance. 3T3-L1 adipocytes were treated with 30 μM of CCCP for 3 hours to produce a mixture of Mtphagy dye and Lyso dye. It is the result of measuring the fluorescence intensity by binding. An increase in fluorescence intensity in FIG. 1 indicates the generation of mitophagy in adipocytes, and the bottom of FIG. 1 is an enlarged view of the results.
Figure 2 confirms the change of adipocytes by m-chlorophenylhydrazone (CCCP), which is a conventional mitophagy-inducing substance. is the result of observation. In FIG. 2, it was observed that the adipocytes were lysed by CCCP.
Figure 3 confirms the generation of mitophagy by polyunsaturated phosphatidylcholine (PPC), a conventional fat reducing agent. After 3T3-L1 adipocytes were treated with PPC at a concentration of 0.5 mg/ml for 3 hours, Mtphagy dye and Lyso It is the result of measuring the fluorescence by the binding of a dye. In FIG. 3 , an increase in fluorescence intensity indicates the generation of mitophagy in adipocytes.
4 is an observation of changes in adipocytes caused by polyunsaturated phosphatidylcholine (PPC), a mitophagy-inducing substance. After 3T3-L1 adipocytes were treated with various concentrations of PPC (0.1-50 mg/ml) for 3 hours. , the shape of fat cells was observed. In FIG. 4 , adipocyte lysis was observed in a concentration-dependent manner with respect to PPC.
5 is a graph showing the survival rate and body fat changes of adipocytes by polyunsaturated phosphatidylcholine (PPC), a mitophagy-inducing substance. (B) shows the change in the content of brown adipose tissue (BAT) in body fat after intramuscular injection of (10 mg/kg).
Figure 6 shows the change in the expression of UCP1 by polyunsaturated phosphatidylcholine (PPC), a mitophagy-inducing substance. After the adipocytes were treated with various concentrations of PPC (0.1-50 mg/ml), the expression of UCP1 was western blot. This is the result confirmed by the analysis method.
7 shows the mechanism of mitophagy by UCP1 siRNA. After UCP1 siRNA was treated in 3T3-L1 adipocytes, fluorescence intensity was measured by the binding of Mtphagy dye and Lyso dye.

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: Generation of mitophagy by m-chlorophenylhydrazone (CCCP)

1) Mitophagy generation and measurement by CCCP

Adipocyte mitophagy was measured using a mitophagy detection kit (MD01-10, DOJINDO, Germany) of 3T3-L1 adipocytes. Briefly, Mtphagy dye is loaded into adipocytes so that the dye is accumulated in the mitochondria of adipocytes, and then Lyso dye is loaded to transport the dye to lysosomes of cells. Mtphagy dye-labeled mitochondria then induce fusion of lysosomes. When mitophagy is generated by treatment with m-chlorophenylhydrazone (CCCP) and polyunsaturated phosphatidylcholine (PPC), autophagosomes are formed in the mitochondria of adipocytes, and autophagosomes thus formed bind to lysosomes. Then, Mtphagy dye and Lyso dye combine to show strong fluorescence. At this time, it is possible to determine whether or not mitophagy occurs in adipocytes by measuring the generated fluorescence intensity.

3T3-L1 adipocytes were treated with 30 μM of m-chlorophenylhydrazone (CCCP) for 3 hours to induce binding of Mtphagy dye and Lyso dye. As a result, it was confirmed that the mitophagy of adipocytes occurred through the increase in fluorescence intensity according to the combination of Mtphagy dye and Lyso dye (upper part of FIG. 1) . The same effect was obtained in the result of magnifying the microscope (lower part of FIG. 1) .

2) Confirmation of changes in the shape of adipocytes by CCCP

3T3-L1 adipocytes were treated with 30 μM of m-chlorophenylhydrazone (CCCP) for 3 hours, and the phenomenon of lysis and destruction of adipocytes was observed. According to the CCCP treatment of 30 μM, it was confirmed that the mitophagy of adipocytes was associated with the lysis of adipocytes ( FIG. 2 ) .

Example 2: Function of polyunsaturated phosphatidylcholine (PPC) on mitophagy

1) Generation and measurement of mitophagy by PPC

Polyunsaturated phosphatidylcholine (PPC) 0.5 mg/ml was treated in 3T3-L1 adipocytes for 3 hours. At this time, the generation of mitophagy was measured in the same manner as described in 1) of Example 1. As a result, it was found that mitophagy of adipocytes was generated, similar to the result in m-chlorophenylhydrazone (FIG. 3) .

2) Confirmation of changes in the shape of adipocytes by PPC

Polyunsaturated phosphatidylcholine (PPC) 0.5 mg/ml was treated in 3T3-L1 adipocytes for 3 hours, and a phenomenon in which adipocytes were lysed and destroyed was observed (FIG. 4) .

In addition, in the treatment group (0.1 mg/ml to 50 mg/ml) in which the concentration of polyunsaturated phosphatidylcholine (PPC) was different, the viability of adipocytes decreased in a concentration-dependent manner with respect to PPC, thereby reducing the number of adipocytes. It was found that there is (Fig. 5A) . On the other hand, 200 μl of PPC at a concentration of 10 mg/kg was divided into 4 times (50 μl/time) and administered subcutaneously around the area where there was a lot of brown adipose tissue (BAT) between the shoulder blades of ICR mice. Two days after PPC administration, mice were sacrificed, brown fat was reddish-lined, and the weight was measured. As a result, in the experimental group injected with PPC (10 mg/kg), it was confirmed that the content of brown adipose tissue (BAT) in body fat decreased (FIG. 5B) .

Example 3: Mechanism of action of UCP1 in adipocyte mitophagy development

In order to investigate the mechanism of mitophagy in adipocytes, the function of UCP1, a mitochondrial protein, was investigated. At this time, the method for knocking down UCP1 in adipocytes is as follows:

[Method of knocking down UCP1 in adipocytes]

(1) As Ucp1 siRNA for UCP1 knockdown, mouse Ucp1 siRNA commercially available from Bioneer Co., Ltd. was used.

(2) Ucp1 siRNA treatment time - 5 hours (media added 5 hours after transfection, cells harvested after 24 hours)

(3) Lipofectamine treatment - Lipofectamine®2000 Transfection Reagent

(4) Transfection medium - only high glucose

(5) Differentiation conditions

① Cell differentiation period - 8 days

② Differentiate with MDI (3-isobutyl-1-methylxanthine, dexamethasone and insulin)

③ 3 hours of starvation

④ Ucp1 siRNA treatment in differentiated cells (forward siRNA)

(6) Experimental schematic diagram

As a result of treating adipocytes with various concentrations of PPC (0.1 mg/ml to 50 mg/ml), it was confirmed that the expression of UCP1 was decreased in a concentration-dependent manner with respect to PPC (FIG. 6) .

3) Confirmation of mitophagy in UCP1 knockdown adipocytes

As a result of treating 3T3-L1 adipocytes with UCP1 siRNA, it was observed that mitophagy was significantly generated ( FIG. 7 ) .

Claims (7)

After contacting the adipocytes with the candidate substance outside the human body,
A screening method for a drug for the prevention or treatment of obesity, comprising determining whether the candidate substance generates or does not generate mitophagy of adipocytes. According to claim 1,
A method for screening a drug for preventing or treating obesity, wherein when the candidate substance generates mitophagy in adipocytes, the candidate substance is determined as a drug for preventing or treating obesity. According to claim 1,
Whether or not mitophagy occurs in adipocytes is determined by measuring fluorescence intensity according to the fusion of autophagosome and lysosome in mitochondria of adipocytes; Or a screening method of a drug for preventing or treating obesity, which is confirmed by measuring the LC3-I and LC3-II protein expression ratio. According to claim 1,
The screening method is a screening method of a drug for preventing or treating obesity, further comprising determining whether the candidate material enhances or inhibits the function or activity of the UCP1 protein. 5. The method of claim 4,
When the candidate substance inhibits the function or activity of the UCP1 protein, determining the candidate substance as a drug for preventing or treating obesity, a screening method for a drug for preventing or treating obesity. According to claim 1,
The screening method is a screening method of a drug for preventing or treating obesity, further comprising determining whether the candidate material promotes or suppresses the expression of the UCP1 gene. 7. The method of claim 6,
When the candidate substance suppresses the expression of the UCP1 gene, determining the candidate substance as a drug for preventing or treating obesity, a screening method for a drug for preventing or treating obesity.

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