WO2024035023A1 - Novel bax variant and pharmaceutical composition comprising same - Google Patents

Novel bax variant and pharmaceutical composition comprising same Download PDF

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WO2024035023A1
WO2024035023A1 PCT/KR2023/011541 KR2023011541W WO2024035023A1 WO 2024035023 A1 WO2024035023 A1 WO 2024035023A1 KR 2023011541 W KR2023011541 W KR 2023011541W WO 2024035023 A1 WO2024035023 A1 WO 2024035023A1
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bax
seq
bax protein
lysine
amino acid
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French (fr)
Korean (ko)
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백광현
최해슬
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차의과학대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity

Definitions

  • the present invention relates to variants of Bax protein, an apoptosis protein. More specifically, the present invention relates to a variant of the Bax protein in which one or more lysine residues of the Bax protein are replaced with arginine; A method of increasing the half-life of a Bax protein comprising substituting one or more lysine residues of the Bax protein with arginine; and a pharmaceutical composition containing the Bax protein variant and a screening method for anticancer substances.
  • Apoptosis is programmed cell death and relies on extrinsic and intrinsic signaling pathways.
  • the extrinsic pathway is initiated by death receptors on the cell surface, whereas the intrinsic pathway is initiated by stress signals such as UV damage, hypoxia, oxidative stress and DNA damage. Stress signals induce the apoptotic pathway initiated by pro-apoptotic proteins at the mitochondrial level.
  • Bax protein is a pro-apoptotic protein belonging to the Bcl-2 family and is involved in inducing apoptosis in mitochondria.
  • Bax which is in a non-apoptotic state in the cytoplasm, initiates translocation of the mitochondrial outer membrane after oligomerization with Bak, thereby increasing mitochondrial outer membrane permeability (MOMP) (Pena-Blanco, A. et al., Bax , Bak and beyond-mitochondrial performance in apoptosis.
  • MOMP mitochondrial outer membrane permeability
  • Cytochrome C plays an important role in the activation of pro-caspase-9 and induces activation of caspase-3, -6, and -7 and apoptosis (Slee, EA et al., Serial killers : ordering caspase activation events in apoptosis.
  • the Bax protein is an initiator of the intrinsic apoptotic pathway, and effective control of Bax may provide an effective approach to the control of apoptosis.
  • deubiquitination enzymes USP1, USP7, USP12, or USP49 specifically bind to Bax protein, which is known to be involved in apoptosis (Korean Patent Registration No. 10-2282107, etc.).
  • the present inventors conducted various studies to uncover the ubiquitination mechanism of Bax protein. As a result, it was found that not only was the ubiquitination of a specific amino acid residue of the Bax protein, that is, a protein variant in which lysine at positions 128 and/or 190 of the Bax protein replaced with arginine, suppressed, but the half-life was significantly increased. .
  • the purpose of the present invention is to provide Bax protein variants.
  • the present invention aims to provide a method for increasing the half-life of Bax protein.
  • the present invention aims to provide a pharmaceutical composition containing the Bax protein variant as an active ingredient.
  • the present invention aims to provide a screening method for substances with anticancer activity using the Bax protein variant.
  • a Bax protein variant in the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; Alternatively, a Bax protein variant is provided in which lysines at positions 128 and 190 are replaced with arginine.
  • the Bax protein variant may be a variant consisting of the amino acid sequence of SEQ ID NO: 3, 5, or 6.
  • the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; Alternatively, a method for increasing the half-life of the Bax protein is provided, comprising substituting arginine for lysines at positions 128 and 190.
  • a pharmaceutical composition for preventing or treating solid cancer comprising the Bax protein variant as an active ingredient.
  • the solid cancer may be stomach cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, etc.
  • the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at position 128; Lysine at position 190; or treating and culturing the candidate material in cells expressing a Bax protein variant in which lysines at positions 128 and 190 are substituted with arginine and the deubiquitination enzyme USP1, USP7, USP12, or USP49; and (ii) measuring whether the cells cultured in step (i) have died and selecting a substance that induces cell death.
  • a screening method for a substance with anticancer activity is provided.
  • the Bax protein variant according to the present invention can inhibit protein degradation through ubiquitination and exhibits a significantly increased half-life compared to wild type Bax. According to the method of the present invention, the half-life of the Bax protein can be increased when converting the wild-type Bax protein to the Bax protein variant according to the present invention.
  • the variant according to the present invention can be usefully applied as a targeted anticancer agent for the treatment of solid cancer through effective apoptosis of cancer cells.
  • the mutant according to the present invention can be usefully used as a target for promoting apoptosis of cancer cells and developing treatment. That is, the screening method of the present invention can be usefully used for screening substances with anticancer activity targeting the interaction between deubiquitinating enzymes USP1, USP7, USP12, and USP49 and Bax protein.
  • Figure 1 shows the protein structure including the predicted ubiquitination sites (K128, K189, K190) (indicated by arrows) of the Bax protein using Bioinformatics and PyMol.
  • Figure 2 shows the results of Western blotting after transfection of wild-type Bax or Bax mutants (Bax K128R, Bax K189R, or Bax K190R) labeled with HA-labeled ubiquitin and Myc, respectively, into HeLa cells ( Left panel) and the results of immunoprecipitation with Myc antibody (right panel) are shown.
  • Figure 3 shows Bax from 0 to 24 hours after transfection of wild-type Bax or Bax mutant (Bax K128R or Bax K190R) into HeLa cells and treatment with cycloheximide, a protein synthesis inhibitor. expression level The results measured through Western blotting are shown.
  • Figure 4 shows the results of calculating the relative Bax expression level from 0 to 24 hours from the results of Figure 3.
  • FIGS 5A to 5C show the results of transfection of Mock vector, wild-type Bax, Bax K128R, and Bax K190R into HeLa cells, and then observation of cells at 0, 24, and 48 hours.
  • BDM PUB http://bdmpub.biocuckoo.org/
  • jci-bioinfo http://www.jci-bioinfo.cn
  • RUBI http: //old.protein.bio.unipd.it/rubi/
  • Netchop http://www.cbs.dtu.dk/services/NetChop
  • Lysine residues on the Bax surface were finally selected using ://pymol.org/2/).
  • the present inventors performed a conservative amino acid substitution for the lysine sites, that is, substitution with arginine containing a basic side chain. That is, Bax variants in which lysines at positions 128, 189, and 190 were respectively replaced with arginine [i.e., Bax K128R (protein of SEQ ID NO: 3), Bax K189R (protein of SEQ ID NO: 4), and Bax K190R (SEQ ID NO: 5) protein)] was produced, and among these variants, it was confirmed that Bax K128R (protein of SEQ ID NO: 3) and Bax K190R (protein of SEQ ID NO: 5) were significantly inhibited in ubiquitination.
  • Bax K128R protein of SEQ ID NO: 3
  • Bax K190R protein of SEQ ID NO: 5
  • the variant can be usefully applied as a targeted anticancer agent for the treatment of solid tumors through effective apoptosis of cancer cells.
  • the present invention provides variants of the Bax protein (amino acid of SEQ ID NO: 1). That is, the present invention relates to the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; Alternatively, it provides a Bax protein variant in which lysines at positions 128 and 190 are replaced with arginine.
  • the amino acid sequence of the Bax protein and the base sequence encoding it are all known.
  • the amino acid sequence of the Bax protein is as shown in SEQ ID NO: 1, and the base sequence encoding it is as in SEQ ID NO: 2.
  • the Bax protein variant according to the present invention may be a variant in which lysine at position 128 is replaced with arginine in the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, that is, a variant consisting of the amino acid sequence of SEQ ID NO: 3.
  • the Bax protein variant according to the present invention may be a variant in which lysine at position 190 is replaced with arginine in the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, that is, a variant consisting of the amino acid sequence of SEQ ID NO: 5.
  • the Bax protein variant according to the present invention is a variant in which lysines at positions 128 and 190 of the Bax protein composed of the amino acid sequence of SEQ ID NO: 1 are replaced with arginine, that is, a variant composed of the amino acid sequence of SEQ ID NO: 6. You can.
  • the Bax protein variant according to the present invention has lysine at position 128; Lysine at position 190; Alternatively, it can be produced by substituting lysine at positions 128 and 190 with arginine.
  • a gene encoding the Bax protein e.g., a gene with the base sequence of SEQ ID NO: 2 as a template, 128 A mutant in which lysine is replaced with arginine can be obtained.
  • a polymerase chain reaction is performed using the gene encoding the Bax protein (e.g., a gene with the base sequence of SEQ ID NO: 2) as a template.
  • the gene encoding the Bax protein e.g., a gene with the base sequence of SEQ ID NO: 2
  • a mutant in which lysine at position 190 is replaced with arginine can be obtained.
  • the present invention relates to the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, including lysine at number 128; Lysine at position 190; Alternatively, a method for increasing the half-life of the Bax protein comprising substituting arginine for lysine at positions 128 and 190 is provided.
  • the method of the present invention replaces lysine at position 128 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with arginine (i.e., replacing the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of SEQ ID NO: 3) conversion to a Bax protein variant).
  • the method of the present invention replaces lysine at position 190 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with arginine (i.e., replacing the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of SEQ ID NO: 5) conversion to a Bax protein variant).
  • the method of the present invention replaces lysines at positions 128 and 190 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with arginine (i.e., replacing the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of SEQ ID NO: 6) It may include converting to a Bax protein variant consisting of an amino acid sequence.
  • the variant according to the present invention can be usefully applied as a targeted anticancer agent for the treatment of solid tumors through effective apoptosis of cancer cells.
  • the present invention provides a pharmaceutical composition for preventing or treating solid cancer, comprising the Bax protein variant as an active ingredient.
  • the solid cancer may be stomach cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, etc.
  • the pharmaceutical composition of the present invention contains the Bax protein variant as an active ingredient and may include a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be formulated in the form of injections, freeze-dried preparations for injection, etc. according to conventional pharmaceutical methods.
  • the pharmaceutically acceptable carrier may include a sterilized aqueous solution (eg, physiological saline, etc.), non-aqueous solvent, etc. Additionally, appropriate stabilizers, isotonic agents, preservatives, etc. may be included as needed.
  • the dosage of the Bax protein variant contained as an active ingredient in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, administration route and period, and can be appropriately selected by a person skilled in the art.
  • the Bax protein variant can be administered at a daily dose of 0.1 to 1,000 mg/kg, and the administration may be administered once a day or in divided doses.
  • the expression level of Bax is associated with malignant transformation, tumor progression, and metastasis, and therefore low expression of Bax is considered a negative factor in cancer diseases. Reduced levels of Bax degradation are seen in invasive prostate cancer. Therefore, the Bax protein variant according to the present invention can be usefully used in the screening of substances with anticancer activity targeting the interaction of Bax protein with deubiquitinating enzymes (e.g., USP1, USP7, USP12, and USP49). there is.
  • deubiquitinating enzymes e.g., USP1, USP7, USP12, and USP49.
  • the present invention provides (i) a Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; or treating and culturing the candidate material in cells expressing a Bax protein variant in which lysines at positions 128 and 190 are substituted with arginine and the deubiquitination enzyme USP1, USP7, USP12, or USP49; and (ii) measuring whether the cells cultured in step (i) have died and selecting a substance that induces cell death.
  • the Bax protein variant may be a variant consisting of the amino acid sequence of SEQ ID NO: 3, 5, or 6.
  • the Bax gene (gene of SEQ ID NO: 2) was used by cloning into the pcDNA3.1-6myc expression vector. Bax variants were created in which lysine positions 128, 189, or 190 of the Bax protein were replaced with arginine, respectively, using the primer set shown in Table 2 below. Specifically, each mutant uses the wild-type Bax gene (gene of SEQ ID NO: 2) as a template and uses each primer set. Polymerase chain reaction (PCR) was performed.
  • PCR Polymerase chain reaction
  • Bax K128R variant a total of 12 cycles were performed: 95°C for 30 seconds, 60°C for 30 seconds, and 68°C for 5 minutes.
  • Bax K189R variant and Bax K190R variant a total of 12 cycles were performed: 95°C for 30 seconds, 54.4°C for 30 seconds, and 68°C for 5 minutes.
  • HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Grand) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin (Gibco, Grand Island, NY, USA). Island, NY, USA) and cultured in a 5% CO 2 incubator.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • penicillin and streptomycin Gibco, Grand Island, NY, USA
  • Island, NY, USA 5% CO 2 incubator.
  • Transfected cells were lysed in lysis buffer (50mM Tris-HCl [pH 7.5], 1mM EDTA, 10% glycerol, 300mM NaCl, and 1% Triton Centrifuged at rpm for 20 minutes. The supernatant was taken, an antibody (Myc antibody) was added and reacted at 4°C overnight, then A/G PLUS agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added and reacted at 4°C in a rotator for 2 hours. Samples were washed twice with wash buffer (lysis buffer containing PMSF and PIC 1:100), boiled for 7 min with 2X SDS protein loading buffer, and then loaded on SDS-PAGE gel and subjected to Western blotting. proceeded.
  • lysis buffer 50mM Tris-HCl [pH 7.5], 1mM EDTA, 10% glycerol, 300mM NaCl, and 1% Triton Centrifuged at rpm for 20
  • Transfected cells were lysed in lysis buffer (50mM Tris-HCl [pH 7.5], 1mM EDTA, 10% glycerol, 300mM NaCl, and 1% Triton Centrifuged at rpm for 20 minutes.
  • lysis buffer 50mM Tris-HCl [pH 7.5], 1mM EDTA, 10% glycerol, 300mM NaCl, and 1% Triton Centrifuged at rpm for 20 minutes.
  • the supernatant was taken and boiled for 7 minutes with 2 moved to After blocking the membrane with bovine serum albumin (KE, AU) for 1 hour, primary antibodies (HA antibody and Myc antibody) were reacted with the membrane at 4°C overnight, and incubated with TBS-T ( Washed with 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1% (V/V) Tween 20), and incubated with mouse secondary antibody (SERACARE, Milford, MA, USA) (1:30,000, 1% Skim milk). was added and reacted at room temperature for 2 hours. Protein bands were detected using ECL reagent solution (Young In Frontier, Seoul, Korea).
  • Myc antibody to label wild-type Bax or Bax variants was used to culture 9E10 cells in Roswell Park with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Cultures cultured in Memorial Institute Medium (RPMI, Gibco, Grand Island, NY, USA) in a 5% CO 2 incubator were collected and filtered through a 0.22 ⁇ M filter before use. In addition, HA antibodies to label ubiquitination enzymes were used to culture 12CA5 cells in RPMI (RPMI) containing 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Gibco, Grand Island, NY, USA) cultured in a 5% CO 2 incubator was collected and filtered through a 0.22 ⁇ M filter before use.
  • wild-type and lysine-substituted Myc-Bax were transfected into HeLa cells, and 24 hours later, cycloheximide, a protein synthesis inhibitor, was added at a concentration of 100 ⁇ M. After treatment, cells were collected from 0 to 24 hours (0, 12, 18, 24 hours), and then Western blotting was performed.
  • Mock vector wild-type Myc-Bax, mutant Myc-Bax K128R, and Myc-Bax K190R were transfected into HeLa cells, 0, 24, 48. The state of the cells was observed through a microscope over time.
  • Densitometric analysis was performed with Image J (National Institutes of Health, Bethesda, MD, USA), and t -test was performed with GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA).
  • Bax protein structure including predicted ubiquitination sites (K128, K189, K190)
  • Figure 1 shows the protein structure including the predicted ubiquitination sites (K128, K189, K190) of Bax protein using Bioinformatics and PyMol as described above.
  • Wild-type Bax or Bax variants (Bax K128R, Bax K189R, or Bax K190R) labeled with HA-labeled ubiquitination enzyme and Myc, respectively, were transfected into HeLa cells, and Western blotting was performed. The results were is the same as in Figure 2 (left panel of Figure 2). Additionally, immunoprecipitation was performed using Myc antibody, and the results are shown in Figure 2 (right panel of Figure 2). From the results in Figure 2, it can be seen that the ubiquitination of Bax K128R and Bax K190R is significantly inhibited.
  • Wild-type Bax, Bax K128R, and Bax K190R were each transfected into HeLa cells and then treated with cycloheximide, a protein synthesis inhibitor.
  • Bax expression level from 0 to 24 hours It was measured through Western blotting, and the results are shown in Figure 3.
  • the results of calculating the relative Bax expression level from 0 to 24 hours from the results in FIG. 3 are shown in FIG. 4.
  • the expression level of wild-type Bax is considered 1 at 24 hours
  • the expression level of Bax K128R is 1.817264
  • the expression level of Bax K190R is 1.982131, which is about 2 times that of wild-type Bax. It showed a half-life that was twice as high. Therefore, it can be seen that Bax K128R and Bax K190R maintain a longer half-life.

Abstract

The present invention provides: a Bax protein variant in which lysine at position 128, lysine at position 190, or lysines at positions 128 and 190 in a Bax protein is(are) substituted with arginine; a method for increasing the half-life of a Bax protein, the method comprising substituting lysine at position 128, lysine at position 190, or lysines at positions 128 and 190 in the Bax protein with arginine; a pharmaceutical composition comprising the Bax protein variant; and a method for screening for an anticancer substance. The Bax protein variant according to the present invention can inhibit a mechanism of protein degradation through ubiquitination and exhibits a longer half-life than wild-type Bax.

Description

신규의 BAX 변이체 및 이를 포함한 약학 조성물Novel BAX variants and pharmaceutical compositions containing them
본 발명은 세포사멸 단백질인 Bax 단백질의 변이체에 관한 것이다. 더욱 상세하게는, 본 발명은 Bax 단백질의 하나 이상의 라이신 잔기를 아르기닌으로 치환한 Bax 단백질의 변이체; Bax 단백질의 하나 이상의 라이신 잔기를 아르기닌으로 치환하는 것을 포함하는 Bax 단백질의 반감기를 증가시키는 방법; 및 상기 Bax 단백질 변이체를 포함하는 약학 조성물 및 항암 물질의 스크리닝 방법에 관한 것이다.The present invention relates to variants of Bax protein, an apoptosis protein. More specifically, the present invention relates to a variant of the Bax protein in which one or more lysine residues of the Bax protein are replaced with arginine; A method of increasing the half-life of a Bax protein comprising substituting one or more lysine residues of the Bax protein with arginine; and a pharmaceutical composition containing the Bax protein variant and a screening method for anticancer substances.
세포사멸(apoptosis)은 프로그램된 세포사멸로서, 외부 및 내부 신호 전달 경로에 의존한다. 외부 경로는 세포 표면의 사멸 수용체에 의해 개시되는 반면, 내부 경로는 UV 손상, 저산소증, 산화 스트레스 및 DNA 손상과 같은 스트레스 신호에 의해 개시된다. 스트레스 신호는 미토콘드리아 수준에서 전구-세포사멸성 단백질(pro-apoptotic protein)에 의해 시작되는 사포사멸 경로를 유도한다. Apoptosis is programmed cell death and relies on extrinsic and intrinsic signaling pathways. The extrinsic pathway is initiated by death receptors on the cell surface, whereas the intrinsic pathway is initiated by stress signals such as UV damage, hypoxia, oxidative stress and DNA damage. Stress signals induce the apoptotic pathway initiated by pro-apoptotic proteins at the mitochondrial level.
Bax 단백질은 Bcl-2 계열에 속하는 전구-세포사멸성 단백질이며, 미토콘드리아에서 세포사멸 유도에 관여한다. 세포질에서 비활성(non-apoptosis) 상태인 Bax는 Bak과의 올리고머화 후에 미토콘드리아 외막의 전위(translocation)를 개시함으로써, 미토콘드리아 외막 투과도(MOMP)를 증가시킨다(Pena-Blanco, A. et al., Bax, Bak and beyond-mitochondrial performance in apoptosis. FEBS J 2018, 285, 416-431, doi:10.1111/febs.14186; Grosse, L. et al., Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis. EMBO J 2016, 35, 402-413, doi:10.15252/embj.201592789). 이후, 내막에 위치한 단백질 시토크롬 C가 세포질로 이동한다(Danial, N.N. et al., Cell death: critical control points. Cell 2004, 116, 205-219, doi:10.1016/s0092-8674(04)00046-7). 시토크롬 C는 프로카스파제 9(pro-caspase-9)의 활성화에 중요한 역할을 하며, 카스파제-3, -6, -7의 활성화 및 세포사멸을 유도한다(Slee, E.A. et al., Serial killers: ordering caspase activation events in apoptosis. Cell Death Differ 1999, 6, 1067-1074, doi:10.1038/sj.cdd.4400601; Kuribayashi, et al., What are caspases 3 and 7 doing upstream of the mitochondria? Cancer Biol Ther 2006, 5, 763-765, doi:10.4161/cbt.5.7.3228). Bax protein is a pro-apoptotic protein belonging to the Bcl-2 family and is involved in inducing apoptosis in mitochondria. Bax, which is in a non-apoptotic state in the cytoplasm, initiates translocation of the mitochondrial outer membrane after oligomerization with Bak, thereby increasing mitochondrial outer membrane permeability (MOMP) (Pena-Blanco, A. et al., Bax , Bak and beyond-mitochondrial performance in apoptosis. FEBS J 2018, 285, 416-431, doi:10.1111/febs.14186; Grosse, L. et al., Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis. EMBO J 2016, 35, 402-413, doi:10.15252/embj.201592789). Afterwards, the protein cytochrome C located in the inner membrane moves to the cytoplasm (Danial, NN et al., Cell death: critical control points. Cell 2004, 116, 205-219, doi:10.1016/s0092-8674(04)00046-7 ). Cytochrome C plays an important role in the activation of pro-caspase-9 and induces activation of caspase-3, -6, and -7 and apoptosis (Slee, EA et al., Serial killers : ordering caspase activation events in apoptosis. Cell Death Differ 1999, 6 , 1067-1074, doi:10.1038/sj.cdd.4400601; Kuribayashi, et al., What are caspases 3 and 7 doing upstream of the mitochondria? Cancer Biol Ther 2006, 5 , 763-765, doi:10.4161/cbt.5.7.3228).
따라서, Bax 단백질은 내인성(intrinsic) 세포사멸 경로의 개시자이며, Bax의 효과적인 제어는 세포사멸의 제어에 대한 효과적인 접근을 제공할 수 있다. 본 발명자들은 탈유비퀴틴화 효소 USP1, USP7, USP12, 또는 USP49가 세포사멸에 관여하는 것으로 알려져 있는 Bax 단백질에 특이적으로 결합한다는 것을 밝혀낸 바 있다(대한민국 특허등록 제10-2282107호 등).Therefore, the Bax protein is an initiator of the intrinsic apoptotic pathway, and effective control of Bax may provide an effective approach to the control of apoptosis. The present inventors have discovered that deubiquitination enzymes USP1, USP7, USP12, or USP49 specifically bind to Bax protein, which is known to be involved in apoptosis (Korean Patent Registration No. 10-2282107, etc.).
본 발명자들은 Bax 단백질의 유비퀴틴화 기전을 밝혀내기 위해 다양한 연구를 수행하였다. 그 결과, Bax 단백질의 특정 아미노산 잔기, 즉 Bax 단백질의 128번 및/또는 190번의 라이신을 아르기닌으로 치환한 단백질 변이체의 유비퀴틴화(ubiquitination)가 억제될 뿐만 아니라, 반감기가 유의성 있게 증가한다는 것을 발견하였다. The present inventors conducted various studies to uncover the ubiquitination mechanism of Bax protein. As a result, it was found that not only was the ubiquitination of a specific amino acid residue of the Bax protein, that is, a protein variant in which lysine at positions 128 and/or 190 of the Bax protein replaced with arginine, suppressed, but the half-life was significantly increased. .
따라서, 본 발명은 Bax 단백질 변이체를 제공하는 것을 목적으로 한다.Therefore, the purpose of the present invention is to provide Bax protein variants.
또한, 본 발명은 Bax 단백질의 반감기를 증가시키는 방법을 제공하는 것을 목적으로 한다.Additionally, the present invention aims to provide a method for increasing the half-life of Bax protein.
또한, 본 발명은 상기 Bax 단백질 변이체를 유효성분으로 포함하는 약학 조성물을 제공하는 것을 목적으로 한다.Additionally, the present invention aims to provide a pharmaceutical composition containing the Bax protein variant as an active ingredient.
또한, 본 발명은 상기 Bax 단백질 변이체를 사용한 항암 활성을 갖는 물질의 스크리닝 방법을 제공하는 것을 목적으로 한다.Additionally, the present invention aims to provide a screening method for substances with anticancer activity using the Bax protein variant.
본 발명의 일 태양에 따라, 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신이 아르기닌으로 치환된 Bax 단백질 변이체가 제공된다. 상기 Bax 단백질 변이체는 서열번호 3, 5, 또는 6의 아미노산 서열로 구성된 변이체일 수 있다.According to one aspect of the present invention, in the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; Alternatively, a Bax protein variant is provided in which lysines at positions 128 and 190 are replaced with arginine. The Bax protein variant may be a variant consisting of the amino acid sequence of SEQ ID NO: 3, 5, or 6.
본 발명의 다른 태양에 따라, 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신을 아르기닌으로 치환하는 것을 포함하는 Bax 단백질의 반감기를 증가시키는 방법이 제공된다. According to another aspect of the present invention, in the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; Alternatively, a method for increasing the half-life of the Bax protein is provided, comprising substituting arginine for lysines at positions 128 and 190.
본 발명의 또 다른 태양에 따라, 상기 Bax 단백질 변이체를 유효성분으로 포함하는 고형암의 예방 또는 치료용 약학 조성물이 제공된다. 상기 고형암은 위암, 간암, 폐암, 유방암, 난소암, 대장암 등일 수 있다.According to another aspect of the present invention, a pharmaceutical composition for preventing or treating solid cancer comprising the Bax protein variant as an active ingredient is provided. The solid cancer may be stomach cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, etc.
본 발명의 또 다른 태양에 따라, (i) 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신이 아르기닌으로 치환된 Bax 단백질 변이체 및 탈유비퀴틴화 효소 USP1, USP7, USP12, 또는 USP49을 발현하는 세포에 후보 물질을 처리하고 배양하는 단계; 및 (ii) 단계(i)에서 배양된 세포의 사멸 여부를 측정하고, 세포의 사멸을 유도하는 물질을 선별하는 단계를 포함하는 항암 활성을 갖는 물질의 스크리닝 방법이 제공된다.According to another aspect of the present invention, (i) in the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at position 128; Lysine at position 190; or treating and culturing the candidate material in cells expressing a Bax protein variant in which lysines at positions 128 and 190 are substituted with arginine and the deubiquitination enzyme USP1, USP7, USP12, or USP49; and (ii) measuring whether the cells cultured in step (i) have died and selecting a substance that induces cell death. A screening method for a substance with anticancer activity is provided.
본 발명에 따른 Bax 단백질 변이체는 유비퀴틴화를 통한 단백질 분해기작을 억제할 수 있으며, 야생형(wild type) Bax에 비해 유의성 있게 증가한 반감기를 나타낸다. 본 발명의 방법에 따라, 야생형 Bax 단백질을 본 발명에 따른 Bax 단백질 변이체로 전환할 경우 Bax 단백질의 반감기를 증가시킬 수 있다. 또한, 본 발명에 따른 변이체는, 암세포의 효과적인 세포사멸을 통하여, 고형암 치료를 위한 표적 항암제에 유용하게 적용될 수 있다. 또한, 본 발명에 따른 변이체는 암세포의 세포사멸 촉진 및 치료 개발을 위한 표적으로서 유용하게 사용될 수 있다. 즉, 본 발명의 스크리닝 방법은 탈유비퀴틴화 효소 USP1, USP7, USP12, 및 USP49와 Bax 단백질의 상호작용을 표적으로 하는 항암 활성을 갖는 물질의 스크리닝에 유용하게 사용될 수 있다.The Bax protein variant according to the present invention can inhibit protein degradation through ubiquitination and exhibits a significantly increased half-life compared to wild type Bax. According to the method of the present invention, the half-life of the Bax protein can be increased when converting the wild-type Bax protein to the Bax protein variant according to the present invention. In addition, the variant according to the present invention can be usefully applied as a targeted anticancer agent for the treatment of solid cancer through effective apoptosis of cancer cells. In addition, the mutant according to the present invention can be usefully used as a target for promoting apoptosis of cancer cells and developing treatment. That is, the screening method of the present invention can be usefully used for screening substances with anticancer activity targeting the interaction between deubiquitinating enzymes USP1, USP7, USP12, and USP49 and Bax protein.
도 1은 Bioinformatics 및 PyMol을 이용하여 Bax 단백질의 유비퀴틴화 예상 부위(K128, K189, K190)(화살표로 표기)를 포함한 단백질 구조를 나타낸다.Figure 1 shows the protein structure including the predicted ubiquitination sites (K128, K189, K190) (indicated by arrows) of the Bax protein using Bioinformatics and PyMol.
도 2은 HA-표지된 유비퀴틴 및 Myc로 각각 표지된 야생형 Bax 또는 Bax 변이체(Bax K128R, Bax K189R, 또는 Bax K190R)를 HeLa 세포에 형질감염(transfection)한 후, 웨스턴 블롯팅을 수행한 결과(좌측 패널)와 Myc 항체로 면역침강법을 수행한 결과(우측 패널)를 나타낸다.Figure 2 shows the results of Western blotting after transfection of wild-type Bax or Bax mutants (Bax K128R, Bax K189R, or Bax K190R) labeled with HA-labeled ubiquitin and Myc, respectively, into HeLa cells ( Left panel) and the results of immunoprecipitation with Myc antibody (right panel) are shown.
도 3은 야생형 Bax 또는 Bax 변이체(Bax K128R 또는 Bax K190R)를 HeLa 세포에 형질감염(transfection)한 뒤 단백질 합성 억제제인 시클로헥시미드(Cycloheximide)를 처리한 후, 0시간부터 24시간까지의 Bax 발현량을 웨스턴 블롯팅을 통하여 측정한 결과를 나타낸다.Figure 3 shows Bax from 0 to 24 hours after transfection of wild-type Bax or Bax mutant (Bax K128R or Bax K190R) into HeLa cells and treatment with cycloheximide, a protein synthesis inhibitor. expression level The results measured through Western blotting are shown.
도 4는 도 3의 결과로부터 0시간부터 24시간까지의 상대적인 Bax 발현량을 계산한 결과를 나타낸다.Figure 4 shows the results of calculating the relative Bax expression level from 0 to 24 hours from the results of Figure 3.
도 5a 내지 도 5c는 Mock 벡터, 야생형 Bax, Bax K128R 및 Bax K190R 각각을 HeLa 세포에 형질감염시킨 뒤, 0, 24, 48 시간에 세포를 관찰한 결과를 나타낸다.Figures 5A to 5C show the results of transfection of Mock vector, wild-type Bax, Bax K128R, and Bax K190R into HeLa cells, and then observation of cells at 0, 24, and 48 hours.
Bax에서 유비퀴틴화 가능성이 높은 것으로 추정되는 부위를 찾기 위해 BDM PUB(http://bdmpub.biocuckoo.org/), jci-bioinfo(http://www.jci-bioinfo.cn), RUBI(http://old.protein.bio.unipd.it/rubi/) 및 Netchop(http://www.cbs.dtu.dk/services/NetChop)을 참조하여 목록화(short-listed)하였으며, PyMol 프로그램(https://pymol.org/2/)을 사용하여 Bax 표면의 라이신 잔기들을 최종적으로 선택하였다. 이후, Protein Data Bank (PDB, https://www.rcsb.org/)로부터 Bax 단백질의 구조 분석을 수행하였으며, 선택된 라이신들이 Bax 단백질의 표면에 있음을 확인하여 128번 및 190번의 라이신 부위가 Bax 단백질의 유비퀴틴화와 관련될 수 있다는 것(즉, 유비퀴틴 (ubiquitin)의 C-말단의 글라이신과 결합할 수 있다는 것)을 밝혀냈다. To find sites in Bax that are likely to be highly ubiquitinated, BDM PUB (http://bdmpub.biocuckoo.org/), jci-bioinfo (http://www.jci-bioinfo.cn), and RUBI (http: //old.protein.bio.unipd.it/rubi/) and Netchop (http://www.cbs.dtu.dk/services/NetChop), and short-listed using the PyMol program (https://old.protein.bio.unipd.it/rubi/). Lysine residues on the Bax surface were finally selected using ://pymol.org/2/). Afterwards, structural analysis of the Bax protein was performed from the Protein Data Bank (PDB, https://www.rcsb.org/), and it was confirmed that the selected lysines were on the surface of the Bax protein, confirming that the lysine sites at positions 128 and 190 were found in Bax. It was discovered that it can be related to protein ubiquitination (i.e., it can bind to the glycine at the C-terminus of ubiquitin).
상기 결과로부터 본 발명자들은 상기 라이신 부위들에 대한 보존적 아미노산 치환, 즉 염기성 측쇄를 포함하는 아르기닌으로의 치환을 수행하였다. 즉, 128번, 189번, 및 190번 라이신을 각각 아르기닌으로 치환한 Bax 변이체[즉, Bax K128R(서열번호 3의 단백질), Bax K189R(서열번호 4의 단백질), 및 Bax K190R(서열번호 5의 단백질)]를 제작하였으며, 이들 변이체 중 Bax K128R(서열번호 3의 단백질) 및 Bax K190R (서열번호 5의 단백질)이 유비퀴틴화가 유의성 있게 억제되는 것을 확인하였다. 또한, Bax K128R(서열번호 3의 단백질) 및 Bax K190R(서열번호 5의 단백질)을 단백질 합성 억제제인 시클로헥시미드(Cycloheximide)로 처리하여 단백질의 반감기를 확인한 결과, 야생형(wild type) Bax에 비해 높은 반감기를 나타낸다는 것을 밝혀냈다. 따라서, 상기 변이체는, 암세포의 효과적인 세포사멸을 통하여, 고형암 치료를 위한 표적 항암제에 유용하게 적용될 수 있다. From the above results, the present inventors performed a conservative amino acid substitution for the lysine sites, that is, substitution with arginine containing a basic side chain. That is, Bax variants in which lysines at positions 128, 189, and 190 were respectively replaced with arginine [i.e., Bax K128R (protein of SEQ ID NO: 3), Bax K189R (protein of SEQ ID NO: 4), and Bax K190R (SEQ ID NO: 5) protein)] was produced, and among these variants, it was confirmed that Bax K128R (protein of SEQ ID NO: 3) and Bax K190R (protein of SEQ ID NO: 5) were significantly inhibited in ubiquitination. In addition, Bax K128R (protein of SEQ ID NO: 3) and Bax K190R (protein of SEQ ID NO: 5) were treated with cycloheximide, a protein synthesis inhibitor, and the half-life of the protein was confirmed. As a result, wild type Bax It was found that it exhibits a higher half-life compared to Therefore, the variant can be usefully applied as a targeted anticancer agent for the treatment of solid tumors through effective apoptosis of cancer cells.
본 발명은 Bax 단백질(서열번호 1의 아미노산)의 변이체를 제공한다. 즉, 본 발명은 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신이 아르기닌으로 치환된 Bax 단백질 변이체를 제공한다.The present invention provides variants of the Bax protein (amino acid of SEQ ID NO: 1). That is, the present invention relates to the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; Alternatively, it provides a Bax protein variant in which lysines at positions 128 and 190 are replaced with arginine.
Bax 단백질의 아미노산 서열 및 이를 코딩하는 염기서열은 모두 공지되어 있으며, 예를 들어, Bax 단백질의 아미노산 서열은 서열번호 1과 같고, 이를 코딩하는 염기서열은 서열번호 2와 같다.The amino acid sequence of the Bax protein and the base sequence encoding it are all known. For example, the amino acid sequence of the Bax protein is as shown in SEQ ID NO: 1, and the base sequence encoding it is as in SEQ ID NO: 2.
일 구현예에서, 본 발명에 따른 Bax 단백질 변이체는 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서 128번의 라이신이 아르기닌으로 치환된 변이체, 즉 서열번호 3의 아미노산 서열로 구성된 변이체일 수 있다. 다른 구현예에서, 본 발명에 따른 Bax 단백질 변이체는 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서 190번의 라이신이 아르기닌으로 치환된 변이체, 즉 서열번호 5의 아미노산 서열로 구성된 변이체일 수 있다. 또 다른 구현예에서, 본 발명에 따른 Bax 단백질 변이체는 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서 128번 및 190번의 라이신이 아르기닌으로 치환된 변이체, 즉 서열번호 6의 아미노산 서열로 구성된 변이체일 수 있다. In one embodiment, the Bax protein variant according to the present invention may be a variant in which lysine at position 128 is replaced with arginine in the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, that is, a variant consisting of the amino acid sequence of SEQ ID NO: 3. In another embodiment, the Bax protein variant according to the present invention may be a variant in which lysine at position 190 is replaced with arginine in the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, that is, a variant consisting of the amino acid sequence of SEQ ID NO: 5. In another embodiment, the Bax protein variant according to the present invention is a variant in which lysines at positions 128 and 190 of the Bax protein composed of the amino acid sequence of SEQ ID NO: 1 are replaced with arginine, that is, a variant composed of the amino acid sequence of SEQ ID NO: 6. You can.
본 발명에 따른 Bax 단백질 변이체는 생명공학 분야에서 통상적으로 사용되는 방법에 따라 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신을 아르기닌으로 치환함으로써 제작할 수 있다. 예를 들어, 하기 서열번호 7 및 8의 프라이머 세트를 사용하여, Bax 단백질을 코딩하는 유전자(예를 들어, 서열번호 2의 염기서열을 갖는 유전자)를 주형으로 하여 중합효소연쇄반응을 수행함으로써 128번의 라이신이 아르기닌으로 치환된 변이체를 얻을 수 있다. 또한, 예를 들어, 하기 서열번호 11 및 12의 프라이머 세트를 사용하여, Bax 단백질을 코딩하는 유전자(예를 들어, 서열번호 2의 염기서열을 갖는 유전자)를 주형으로 하여 중합효소연쇄반응을 수행함으로써 190번의 라이신이 아르기닌으로 치환된 변이체를 얻을 수 있다.The Bax protein variant according to the present invention has lysine at position 128; Lysine at position 190; Alternatively, it can be produced by substituting lysine at positions 128 and 190 with arginine. For example, by performing a polymerase chain reaction using the primer set of SEQ ID NO: 7 and 8 below and a gene encoding the Bax protein (e.g., a gene with the base sequence of SEQ ID NO: 2) as a template, 128 A mutant in which lysine is replaced with arginine can be obtained. In addition, for example, using the primer sets of SEQ ID NOs: 11 and 12 below, a polymerase chain reaction is performed using the gene encoding the Bax protein (e.g., a gene with the base sequence of SEQ ID NO: 2) as a template. By doing this, a mutant in which lysine at position 190 is replaced with arginine can be obtained.
따라서, 본 발명은 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신을 아르기닌으로 치환하는 것을 포함하는 Bax 단백질의 반감기를 증가시키는 방법을 제공한다.Therefore, the present invention relates to the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, including lysine at number 128; Lysine at position 190; Alternatively, a method for increasing the half-life of the Bax protein comprising substituting arginine for lysine at positions 128 and 190 is provided.
일 구현예에서, 본 발명의 방법은 서열번호 1의 아미노산 서열로 구성된 Bax 단백질의 128번의 라이신을 아르기닌으로 치환(즉, 서열번호 1의 아미노산 서열로 구성된 Bax 단백질을 서열번호 3의 아미노산 서열로 구성된 Bax 단백질 변이체로 전환)하는 것을 포함할 수 있다. 다른 구현예에서, 본 발명의 방법은 서열번호 1의 아미노산 서열로 구성된 Bax 단백질의 190번의 라이신을 아르기닌으로 치환(즉, 서열번호 1의 아미노산 서열로 구성된 Bax 단백질을 서열번호 5의 아미노산 서열로 구성된 Bax 단백질 변이체로 전환)하는 것을 포함할 수 있다. 또 다른 구현예에서, 본 발명의 방법은 서열번호 1의 아미노산 서열로 구성된 Bax 단백질의 128번 및 190번의 라이신을 아르기닌으로 치환(즉, 서열번호 1의 아미노산 서열로 구성된 Bax 단백질을 서열번호 6의 아미노산 서열로 구성된 Bax 단백질 변이체로 전환)하는 것을 포함할 수 있다.In one embodiment, the method of the present invention replaces lysine at position 128 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with arginine (i.e., replacing the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of SEQ ID NO: 3) conversion to a Bax protein variant). In another embodiment, the method of the present invention replaces lysine at position 190 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with arginine (i.e., replacing the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of SEQ ID NO: 5) conversion to a Bax protein variant). In another embodiment, the method of the present invention replaces lysines at positions 128 and 190 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with arginine (i.e., replacing the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1 with the amino acid sequence of SEQ ID NO: 6) It may include converting to a Bax protein variant consisting of an amino acid sequence.
본 발명에 따른 변이체는, 암세포의 효과적인 세포사멸을 통하여, 고형암 치료를 위한 표적 항암제에 유용하게 적용될 수 있다. 따라서, 본 발명은 상기 Bax 단백질 변이체를 유효성분으로 포함하는 고형암의 예방 또는 치료용 약학 조성물을 제공한다. 상기 고형암은 위암, 간암, 폐암, 유방암, 난소암, 대장암 등일 수 있다.The variant according to the present invention can be usefully applied as a targeted anticancer agent for the treatment of solid tumors through effective apoptosis of cancer cells. Accordingly, the present invention provides a pharmaceutical composition for preventing or treating solid cancer, comprising the Bax protein variant as an active ingredient. The solid cancer may be stomach cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, etc.
본 발명의 약학 조성물은 상기와 같이 상기 Bax 단백질 변이체를 유효성분으로 포함하고, 약학적으로 허용가능한 담체를 포함할 수 있다. 본 발명의 약학 조성물은 통상의 제제학적 방법에 따라 주사제, 주사용 동결건조제제 등의 형태로 제제화될 수 있다. 상기 약학적으로 허용가능한 담체는 멸균된 수용액(예를 들어, 생리식염수 등), 비수성용제 등을 포함할 수 있다. 또한, 필요에 따라 적절한 안정화제, 등장화제, 보존제 등을 포함할 수도 있다.As described above, the pharmaceutical composition of the present invention contains the Bax protein variant as an active ingredient and may include a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be formulated in the form of injections, freeze-dried preparations for injection, etc. according to conventional pharmaceutical methods. The pharmaceutically acceptable carrier may include a sterilized aqueous solution (eg, physiological saline, etc.), non-aqueous solvent, etc. Additionally, appropriate stabilizers, isotonic agents, preservatives, etc. may be included as needed.
본 발명의 약학조성물에 유효성분으로서 함유되는 상기 Bax 단백질 변이체의 투여량은 환자의 상태 및 체중, 질병의 정도, 투여경로 및 기간에 따라 상이하며, 당업자에 의해 적절하게 선택될 수 있다. 예를 들면, 상기 Bax 단백질 변이체는 1일 투여량으로서 0.1 ∼ 1,000 mg/kg의 투여량으로 투여할 수 있으며, 상기 투여는 하루에 한번 또는 수회 나누어 투여할 수도 있다. The dosage of the Bax protein variant contained as an active ingredient in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, administration route and period, and can be appropriately selected by a person skilled in the art. For example, the Bax protein variant can be administered at a daily dose of 0.1 to 1,000 mg/kg, and the administration may be administered once a day or in divided doses.
Bax의 발현 수준은 악성 전환(malignant transformation), 종양 생성(tumor progression), 및 전이와 관련되며, 따라서 Bax의 낮은 발현은 암 질환에 있어서 음성 인자(negative factor)로서 간주된다. 감소된 수준의 Bax 분해는 침습적인 전립선암에서 나타난다. 따라서, 본 발명에 따른 Bax 단백질 변이체는 탈유비퀴틴화 효소(예를 들어, USP1, USP7, USP12, 및 USP49)와 Bax 단백질의 상호작용을 표적으로 하는 항암 활성을 갖는 물질의 스크리닝에 유용하게 사용될 수 있다. The expression level of Bax is associated with malignant transformation, tumor progression, and metastasis, and therefore low expression of Bax is considered a negative factor in cancer diseases. Reduced levels of Bax degradation are seen in invasive prostate cancer. Therefore, the Bax protein variant according to the present invention can be usefully used in the screening of substances with anticancer activity targeting the interaction of Bax protein with deubiquitinating enzymes (e.g., USP1, USP7, USP12, and USP49). there is.
즉, 본 발명은 (i) 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신이 아르기닌으로 치환된 Bax 단백질 변이체 및 탈유비퀴틴화 효소 USP1, USP7, USP12, 또는 USP49을 발현하는 세포에 후보 물질을 처리하고 배양하는 단계; 및 (ii) 단계(i)에서 배양된 세포의 사멸 여부를 측정하고, 세포의 사멸을 유도하는 물질을 선별하는 단계를 포함하는 항암 활성을 갖는 물질의 스크리닝 방법을 제공한다.That is, the present invention provides (i) a Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; or treating and culturing the candidate material in cells expressing a Bax protein variant in which lysines at positions 128 and 190 are substituted with arginine and the deubiquitination enzyme USP1, USP7, USP12, or USP49; and (ii) measuring whether the cells cultured in step (i) have died and selecting a substance that induces cell death.
본 발명의 스크리닝 방법에 있어서, 상기 Bax 단백질 변이체는 서열번호 3, 5, 또는 6의 아미노산 서열로 구성된 변이체일 수 있다.In the screening method of the present invention, the Bax protein variant may be a variant consisting of the amino acid sequence of SEQ ID NO: 3, 5, or 6.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예는 본 발명을 예시하기 위한 것으로, 본 발명이 이들 실시예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the present invention is not limited by these examples.
실시예Example
1. 시험방법1. Test method
(1) 유비퀴틴화 부위의 선별 및 변이체의 제작(1) Selection of ubiquitination sites and production of variants
하기 3개의 웹사이트(표 1)를 통하여 bioinformatics 분석을 수행하여 Bax 단백질의 유비퀴틴화 부위 후보군을 분석하고, PyMol 프로그램(https://pymol.org/2/)을 사용하여 사용하여 Bax 표면의 라이신 잔기들을 최종적으로 분석하였다. 분석 결과, 128번, 189번, 및 190번 라이신이 가장 많이 반복적으로 나타나는 아미노산 잔기, 즉 효율이 가장 높은 아미노산 잔기로 나타났으며, 이를 유비퀴틴화 부위 후보군으로 하였다. We performed bioinformatics analysis through the following three websites (Table 1) to analyze candidates for ubiquitination sites on the Bax protein, and used the PyMol program (https://pymol.org/2/) to identify lysines on the Bax surface. The residues were finally analyzed. As a result of the analysis, lysines at positions 128, 189, and 190 were found to be the most repetitive amino acid residues, that is, the amino acid residues with the highest efficiency, and were selected as ubiquitination site candidates.
유비퀴틴화 예측 부위(Ubiquitination Prediction Sites)Ubiquitination Prediction Sites
http://bdmpub.biocuckoo.org/http://bdmpub.biocuckoo.org/
http://www.jci-bioinfo.cn, RUBI(http://old.protein.bio.unipd.it/rubi/http://www.jci-bioinfo.cn, RUBI (http://old.protein.bio.unipd.it/rubi/
http://old.protein.bio.unipd.it/rubi/http://old.protein.bio.unipd.it/rubi/
HeLa 세포주로부터 총 RNA를 추출하고, 이를 통해 cDNA를 합성하였다. 이후 하기 표 2의 프라이머 세트(서열번호 13 및 14)를 이용하여 Bax cDNA 밴드를 검출하였다. Bax 유전자(서열번호 2의 유전자)는 pcDNA3.1-6myc 발현벡터에 클로닝하여 사용하였다. 하기 표 2의 프라이머 세트를 사용하여 Bax 단백질의 128번, 189번, 또는 190번 라이신을 각각 아르기닌으로 치환한 Bax 변이체를 제작하였다. 구체적으로, 각각의 변이체는 야생형 Bax 유전자(서열번호 2의 유전자)를 주형으로 하고, 각각의 프라이머 세트를 이용하여 중합효소연쇄반응(polymerase chain reaction, PCR)을 수행하였다. Bax K128R 변이체의 경우, 95℃에서 30초, 60℃에서 30초, 및 68℃에서 5분으로 총 12 사이클을 수행하였다. Bax K189R 변이체 및 Bax K190R 변이체의 경우, 95℃에서 30초, 54.4℃에서 30초, 및 68℃에서 5분으로 총 12 사이클을 수행하였다.Total RNA was extracted from the HeLa cell line, and cDNA was synthesized from it. Afterwards, the Bax cDNA band was detected using the primer set (SEQ ID NOs: 13 and 14) shown in Table 2 below. The Bax gene (gene of SEQ ID NO: 2) was used by cloning into the pcDNA3.1-6myc expression vector. Bax variants were created in which lysine positions 128, 189, or 190 of the Bax protein were replaced with arginine, respectively, using the primer set shown in Table 2 below. Specifically, each mutant uses the wild-type Bax gene (gene of SEQ ID NO: 2) as a template and uses each primer set. Polymerase chain reaction (PCR) was performed. For the Bax K128R variant, a total of 12 cycles were performed: 95°C for 30 seconds, 60°C for 30 seconds, and 68°C for 5 minutes. For the Bax K189R variant and Bax K190R variant, a total of 12 cycles were performed: 95°C for 30 seconds, 54.4°C for 30 seconds, and 68°C for 5 minutes.
변이체variant 서열번호sequence number 서열order
Bax K128RBax K128R 77 정방향forward 5'- TGC ACC AGG GTG CCG GAA -3'5'- TGC ACC AGG GTG CCG GAA -3'
88 역방향reverse 5'- TTC CGG CAC CCT GGT GCC -3'5'- TTC CGG CAC CCT GGT GCC -3'
Bax K189RBax K189R 99 정방향forward 5'- ATC TGG AGG AAG ATG GGC -3' 5'- ATC TGG AGG AAG ATG GGC -3'
1010 역방향reverse 5'- GCC CAT CTT CCT CCA GAT -3'5'- GCC CAT CTT CCT CCA GAT -3'
Bax K190RBax K190R 1111 정방향forward 5'- ATC TGG AAG AGG ATG GGC -3' 5'- ATC TGG AAG AGG ATG GGC -3'
1212 역방향reverse 5'- GCC CAT CCT CTT CCA GAT -3'5'- GCC CAT CCT CTT CCA GAT -3'
Bax (야생형)Bax (wild type) 1313 정방향forward 5'- ATG GAC GGG TCC GGG GAG -3'5'- ATG GAC GGG TCC GGG GAG -3'
1414 역방향reverse 5'- TCA GCC CAT CTT CTT CCA -3'5'- TCA GCC CAT CTT CTT CCA -3'
(2) 형질감염(transfection) (2) Transfection
HeLa 세포를 10% 소태아혈청(FBS, Gibco, Grand Island, NY, USA)와 1% 페니실린 및 스트렙토마이신(Gibco, Grand Island, NY, USA)이 포함된 Dulbecco's Modified Eagle's Medium(DMEM, Gibco, Grand Island, NY, USA)에 5% CO2 인큐베이터에서 배양하였다. 형질감염을 위해, 600 μl의 NaCl과 폴리에틸렌이민 시약(PEI, Polysciences, Inc., Warrington, PA, USA) 14 μl, pcDNA3.1 6myc-Bax의 야생형 또는 변이체 DNA 2 μg을 혼합하여 상온에서 15분 반응시키고, HeLa 세포(1x106 세포)가 담긴 6 ml의 배양액에 혼합액을 넣어준 후, 37℃에서 48시간 동안 배양하였다.HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Grand) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin and streptomycin (Gibco, Grand Island, NY, USA). Island, NY, USA) and cultured in a 5% CO 2 incubator. For transfection, mix 600 μl of NaCl, 14 μl of polyethyleneimine reagent (PEI, Polysciences, Inc., Warrington, PA, USA), and 2 μg of wild-type or mutant DNA of pcDNA3.1 6myc-Bax and incubate for 15 min at room temperature. After reaction, the mixture was added to 6 ml of culture medium containing HeLa cells (1x10 6 cells), and then cultured at 37°C for 48 hours.
(3) 면역침강(3) Immunoprecipitation
형질감염된 세포를 용액 완충액(lysis buffer)(50 mM Tris-HCl[pH 7.5], 1 mM EDTA, 10% 글리세롤, 300 mM NaCl 및 1% Triton X-100)에 얼음에서 20분간 용해시킨 후, 13,000 rpm으로 20분 동안 원심분리하였다. 상등액을 취하여 항체(Myc 항체)를 넣고 4℃에서 밤새 반응시킨 다음, A/G PLUS 아가로스 비드(Santa Cruz Biotechnology, Santa Cruz, CA, USA)를 넣고 4℃ 로테이터에서 2시간 동안 반응시켰다. 샘플을 세척 완충액(PMSF 및 PIC 1:100이 포함된 용해 완충액)으로 2회 세척하고, 2X SDS 단백질 로딩 완충액과 함께 7분 동안 끓인 후, 샘플을 SDS-PAGE 겔에 로딩하고, 웨스턴 블롯팅을 진행하였다.Transfected cells were lysed in lysis buffer (50mM Tris-HCl [pH 7.5], 1mM EDTA, 10% glycerol, 300mM NaCl, and 1% Triton Centrifuged at rpm for 20 minutes. The supernatant was taken, an antibody (Myc antibody) was added and reacted at 4°C overnight, then A/G PLUS agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added and reacted at 4°C in a rotator for 2 hours. Samples were washed twice with wash buffer (lysis buffer containing PMSF and PIC 1:100), boiled for 7 min with 2X SDS protein loading buffer, and then loaded on SDS-PAGE gel and subjected to Western blotting. proceeded.
(4) 웨스턴 블롯팅(4) Western blotting
형질감염된 세포를 용액 완충액(lysis buffer)(50 mM Tris-HCl[pH 7.5], 1 mM EDTA, 10% 글리세롤, 300 mM NaCl 및 1% Triton X-100)에 얼음에서 20분간 용해시킨 후, 13,000 rpm으로 20분 동안 원심분리하였다. 상등액을 취하여 2X SDS 단백질 로딩 완충액과 함께 7분 동안 끓인 후, 샘플을 SDS-PAGE 겔에 로딩하고, 폴리비닐리덴 플루오라아드(polyvinylidene fluoride, PVDF) 미세다공성 막(Millipore, billerica, MA, USA)에 옮겼다. 상기 막을 소혈청 알부민(bovine seum albumin, KE, AU)을 이용하여 1시간 동안 블록킹한 후, 1차 항체(HA 항체 및 Myc 항체)를 상기 막과 함께 4℃에서 밤새 반응시키고, TBS-T(20 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1%(V/V) Tween 20)로 세척하고, 마우스 2차 항체(SERACARE, Milford, MA, USA)(1:30,000 , 1% Skim milk)를 가하고, 2시간 동안 실온에서 반응시켰다. ECL 시약 용액(Young In Frontier, Seoul, Korea)을 사용하여 단백질 밴드를 검출하였다.Transfected cells were lysed in lysis buffer (50mM Tris-HCl [pH 7.5], 1mM EDTA, 10% glycerol, 300mM NaCl, and 1% Triton Centrifuged at rpm for 20 minutes. The supernatant was taken and boiled for 7 minutes with 2 moved to After blocking the membrane with bovine serum albumin (KE, AU) for 1 hour, primary antibodies (HA antibody and Myc antibody) were reacted with the membrane at 4°C overnight, and incubated with TBS-T ( Washed with 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1% (V/V) Tween 20), and incubated with mouse secondary antibody (SERACARE, Milford, MA, USA) (1:30,000, 1% Skim milk). was added and reacted at room temperature for 2 hours. Protein bands were detected using ECL reagent solution (Young In Frontier, Seoul, Korea).
(5) 항체(5) Antibodies
야생형 Bax 또는 Bax 변이체를 표지하기 위한 Myc 항체는 9E10 세포를 10% FBS(Gibco, Grand Island, NY, USA)와 1% 페니실린/스트렙토마이신(Gibco, Grand Island, NY, USA)이 포함된 Roswell Park Memorial Institute Medium(RPMI, Gibco, Grand Island, NY, USA)에 5% CO2 인큐베이터에서 배양한 배양액을 모아 0.22 μM 필터에 필터링하여 사용하였다. 또한, 유비퀴틴화 효소를 표지하기 위한 HA 항체는 12CA5 세포를 10% FBS(Gibco, Grand Island, NY, USA)와 1% 페니실린/스트렙토마이신(Gibco, Grand Island, NY, USA)이 포함된 RPMI(Gibco, Grand Island, NY, USA)에 5% CO2 인큐베이터에서 배양한 배양액을 모아 0.22 μM 필터에 필터링하여 사용하였다.Myc antibody to label wild-type Bax or Bax variants was used to culture 9E10 cells in Roswell Park with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Cultures cultured in Memorial Institute Medium (RPMI, Gibco, Grand Island, NY, USA) in a 5% CO 2 incubator were collected and filtered through a 0.22 μM filter before use. In addition, HA antibodies to label ubiquitination enzymes were used to culture 12CA5 cells in RPMI (RPMI) containing 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Gibco, Grand Island, NY, USA) cultured in a 5% CO 2 incubator was collected and filtered through a 0.22 μM filter before use.
(6) 단백질 반감기 측정(6) Measurement of protein half-life
야생형 Bax 단백질과 라이신이 치환된 Bax 단백질들의 반감기를 비교, 측정하기 위해, 야생형과 라이신 치환 Myc-Bax를 HeLa 세포에 형질간염시키고, 24 시간 후에 단백질 합성 억제제인 시클로헥시미드를 100 μM 농도로 처리하여, 0 시간부터 24 시간까지(0, 12, 18, 24 시간) 세포를 모은 후, 웨스턴 블롯팅을 진행하였다.To compare and measure the half-life of wild-type Bax protein and lysine-substituted Bax protein, wild-type and lysine-substituted Myc-Bax were transfected into HeLa cells, and 24 hours later, cycloheximide, a protein synthesis inhibitor, was added at a concentration of 100 μM. After treatment, cells were collected from 0 to 24 hours (0, 12, 18, 24 hours), and then Western blotting was performed.
(7) 세포사멸능 확인(7) Confirmation of cell death function
반감기가 증가된 Bax 변이체가 야생형과 유사한 세포사멸 능력을 가졌는지 확인하기 위하여 Mock 벡터, 야생형 Myc-Bax, 변이체 Myc-Bax K128R, Myc-Bax K190R을 HeLa 세포에 형질감염시킨 후, 0, 24, 48 시간에 세포의 상태를 현미경을 통해 관측하였다.To confirm whether the Bax variant with an increased half-life had an apoptotic ability similar to the wild type, Mock vector, wild-type Myc-Bax, mutant Myc-Bax K128R, and Myc-Bax K190R were transfected into HeLa cells, 0, 24, 48. The state of the cells was observed through a microscope over time.
(8) 도출된 결과 확인 및 통계 분석(8) Confirmation of derived results and statistical analysis
덴시토메터 분석(Densitometric analysis)은 Image J (National Institutes of Health, Bethesda, MD, USA)로 수행하였고, t-test는 GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA)로 수행하였다.Densitometric analysis was performed with Image J (National Institutes of Health, Bethesda, MD, USA), and t -test was performed with GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA).
2. 시험결과 및 고찰2. Test results and considerations
(1) 유비퀴틴화 예상 부위(K128, K189, K190)를 포함한 Bax 단백질 구조(1) Bax protein structure including predicted ubiquitination sites (K128, K189, K190)
도 1은 상기와 같이 Bioinformatics 및 PyMol을 이용하여 Bax 단백질의 유비퀴틴화 예상 부위(K128, K189, K190)를 포함한 단백질 구조를 나타낸다. Figure 1 shows the protein structure including the predicted ubiquitination sites (K128, K189, K190) of Bax protein using Bioinformatics and PyMol as described above.
(2) 웨스턴 블롯팅/면역침강 분석을 통한 발현 분석(2) Expression analysis through Western blotting/immunoprecipitation analysis
HA-표지된 유비퀴틴화 효소 및 Myc로 각각 표지된 야생형 Bax 또는 Bax 변이체(Bax K128R, Bax K189R, 또는 Bax K190R)를 HeLa 세포에 형질감염(transfection)한 후, 웨스턴 블롯팅을 수행하였으며, 그 결과는 도 2와 같다(도 2의 좌측 패널). 또한, Myc 항체를 사용하여 면역침강법을 수행하였으며, 그 결과는 도 2와 같다(도 2의 우측 패널). 도 2의 결과로부터, Bax K128R 및 Bax K190R은 유비퀴틴화가 유의성 있게 억제되는 것을 확인할 수 있다. Wild-type Bax or Bax variants (Bax K128R, Bax K189R, or Bax K190R) labeled with HA-labeled ubiquitination enzyme and Myc, respectively, were transfected into HeLa cells, and Western blotting was performed. The results were is the same as in Figure 2 (left panel of Figure 2). Additionally, immunoprecipitation was performed using Myc antibody, and the results are shown in Figure 2 (right panel of Figure 2). From the results in Figure 2, it can be seen that the ubiquitination of Bax K128R and Bax K190R is significantly inhibited.
(3) 단백질 합성 억제제 처리에 따른 Bax 발현 분석(3) Bax expression analysis according to protein synthesis inhibitor treatment
야생형 Bax, Bax K128R 및 Bax K190R 각각을 HeLa 세포에 형질감염(transfection)한 뒤 단백질 합성 억제제인 시클로헥시미드(Cycloheximide)와 함께 처리하였다. 0시간부터 24시간까지의 Bax 발현량을 웨스턴 블롯팅을 통하여 측정하였으며, 그 결과는 도 3과 같다. 또한, 도 3의 결과로부터 0시간부터 24시간까지의 상대적인 Bax 발현량을 계산한 결과는 도 4와 같다. 도 3 및 도 4의 결과로부터 알 수 있는 바와 같이, 24시간 때 야생형 Bax의 발현량을 1로 보았을 때, Bax K128R의 발현량은 1.817264, Bax K190R의 발현량은 1.982131로 야생형 Bax에 비해 약 2배 높은 반감기를 나타내었다. 따라서, Bax K128R 및 Bax K190R는 반감기가 더 길게 유지되는 것을 확인할 수 있다.Wild-type Bax, Bax K128R, and Bax K190R were each transfected into HeLa cells and then treated with cycloheximide, a protein synthesis inhibitor. Bax expression level from 0 to 24 hours It was measured through Western blotting, and the results are shown in Figure 3. In addition, the results of calculating the relative Bax expression level from 0 to 24 hours from the results in FIG. 3 are shown in FIG. 4. As can be seen from the results in Figures 3 and 4, when the expression level of wild-type Bax is considered 1 at 24 hours, the expression level of Bax K128R is 1.817264 and the expression level of Bax K190R is 1.982131, which is about 2 times that of wild-type Bax. It showed a half-life that was twice as high. Therefore, it can be seen that Bax K128R and Bax K190R maintain a longer half-life.
(4) 야생형 Bax와 Bax 변이체의 세포사멸능 비교 분석(4) Comparative analysis of the apoptosis function of wild-type Bax and Bax mutants
Mock 벡터, 야생형 Bax, Bax K128R 및 Bax K190R 각각을 HeLa 세포에 형질감염(transfection)시킨 뒤, 0, 24, 48 시간에 세포를 관찰한 결과는 각각 도 5a 내지 도 5c와 같다. 세포사멸 기능을 가지는 Bax가 형질감염 되었을 때 시간이 지남에 따라서 Mock 벡터 대조군과 비교하였을 때 세포의 수가 적어지는 것을 확인할 수 있다. 또한, Bax의 라이신 부위를 치환시킨 Bax K128R 및 Bax K190R을 형질감염시킨 실험군에서도 세포사멸이 관측되었다는 것을 확인할 수 있다. 따라서, 본 발명에 따른 Bax의 변이체는 Bax 고유의 세포사멸능을 보유하고 있음을 알 수 있다.After transfecting HeLa cells with the mock vector, wild-type Bax, Bax K128R, and Bax K190R, the results of observing the cells at 0, 24, and 48 hours are shown in Figures 5A to 5C, respectively. When Bax, which has an apoptotic function, is transfected, it can be seen that the number of cells decreases over time compared to the mock vector control group. In addition, it can be confirmed that cell death was observed in the experimental group transfected with Bax K128R and Bax K190R in which the lysine region of Bax was replaced. Therefore, it can be seen that the Bax mutant according to the present invention possesses Bax's unique apoptosis ability.

Claims (11)

  1. 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신이 아르기닌으로 치환된 Bax 단백질 변이체.In the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; or a Bax protein variant in which lysines at positions 128 and 190 are replaced with arginine.
  2. 제1항에 있어서, 서열번호 3의 아미노산 서열로 구성된 Bax 단백질 변이체.The Bax protein variant according to claim 1, which consists of the amino acid sequence of SEQ ID NO: 3.
  3. 제1항에 있어서, 서열번호 5의 아미노산 서열로 구성된 Bax 단백질 변이체.The Bax protein variant according to claim 1, which consists of the amino acid sequence of SEQ ID NO: 5.
  4. 제1항에 있어서, 서열번호 6의 아미노산 서열로 구성된 Bax 단백질 변이체.The Bax protein variant according to claim 1, which consists of the amino acid sequence of SEQ ID NO: 6.
  5. 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신을 아르기닌으로 치환하는 것을 포함하는 Bax 단백질의 반감기를 증가시키는 방법.In the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; or a method for increasing the half-life of Bax protein comprising substituting arginine for lysines at positions 128 and 190.
  6. 제5항에 있어서, 서열번호 1의 아미노산 서열로 구성된 Bax 단백질의 128번의 라이신을 아르기닌으로 치환하는 것을 포함하는 Bax 단백질의 반감기를 증가시키는 방법.The method of claim 5, comprising substituting arginine for lysine at position 128 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1.
  7. 제5항에 있어서, 서열번호 1의 아미노산 서열로 구성된 Bax 단백질의 190번의 라이신을 아르기닌으로 치환하는 것을 포함하는 Bax 단백질의 반감기를 증가시키는 방법.The method of claim 5, comprising substituting arginine for lysine at position 190 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1.
  8. 제5항에 있어서, 서열번호 1의 아미노산 서열로 구성된 Bax 단백질의 128번 및 190번의 라이신을 아르기닌으로 치환하는 것을 포함하는 Bax 단백질의 반감기를 증가시키는 방법.The method of claim 5, comprising substituting arginine for lysines at positions 128 and 190 of the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1.
  9. 제1항 내지 제4항 중 어느 한 항에 따른 Bax 단백질 변이체를 유효성분으로 포함하는 고형암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating solid cancer, comprising the Bax protein variant according to any one of claims 1 to 4 as an active ingredient.
  10. 제9항에 있어서, 상기 고형암이 위암, 간암, 폐암, 유방암, 난소암, 또는 대장암인 것을 특징으로 하는 약학 조성물.The pharmaceutical composition according to claim 9, wherein the solid cancer is stomach cancer, liver cancer, lung cancer, breast cancer, ovarian cancer, or colon cancer.
  11. (i) 서열번호 1의 아미노산 서열로 구성된 Bax 단백질에 있어서, 128번의 라이신; 190번의 라이신; 또는 128번 및 190번의 라이신이 아르기닌으로 치환된 Bax 단백질 변이체 및 탈유비퀴틴화 효소 USP1, USP7, USP12, 또는 USP49을 발현하는 세포에 후보 물질을 처리하고 배양하는 단계; 및 (i) In the Bax protein consisting of the amino acid sequence of SEQ ID NO: 1, lysine at number 128; Lysine at position 190; or treating and culturing the candidate material in cells expressing a Bax protein variant in which lysines at positions 128 and 190 are substituted with arginine and the deubiquitination enzyme USP1, USP7, USP12, or USP49; and
    (ii) 단계(i)에서 배양된 세포의 사멸 여부를 측정하고, 세포의 사멸을 유도하는 물질을 선별하는 단계를 포함하는 항암 활성을 갖는 물질의 스크리닝 방법.(ii) A screening method for a substance with anticancer activity, comprising the steps of measuring whether the cells cultured in step (i) have died and selecting a substance that induces cell death.
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