WO2021029565A1 - Procédé de criblage d'une substance inhibant la liaison d'api5 et du fgf2 - Google Patents

Procédé de criblage d'une substance inhibant la liaison d'api5 et du fgf2 Download PDF

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Publication number
WO2021029565A1
WO2021029565A1 PCT/KR2020/009846 KR2020009846W WO2021029565A1 WO 2021029565 A1 WO2021029565 A1 WO 2021029565A1 KR 2020009846 W KR2020009846 W KR 2020009846W WO 2021029565 A1 WO2021029565 A1 WO 2021029565A1
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fgf2
binding
protein
api5
screening
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PCT/KR2020/009846
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English (en)
Korean (ko)
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이병일
봉승민
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국립암센터
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention comprises the steps of: i) introducing cysteine to the outside of FGF2;
  • step ii) labeling a fluorescent material on FGF2 into which the cysteine of step i) has been introduced;
  • Protein-protein, nucleic acid (DNA, RNA)-protein interactions, or appropriate nucleases and proteolytic enzymes activity in addition to normal physiological reactions such as cell development, growth, and homeostasis, can lead to the occurrence of various diseases including cancer. Even related abnormal physiological responses are involved in almost all cellular signal transductions. Therefore, materials such as organic or inorganic small molecules, aptamers, peptides, therapeutic proteins, and antibodies are rapidly being developed for diagnosis and treatment. Therefore, in order to verify the efficacy of these pharmacological candidates, quantitative analysis of small molecule compound-protein, nucleic acid-protein, peptide-protein, protein-protein avidity (interaction) or nuclease, protease activity is very important. I can.
  • heterogeneity methods include enzyme linked immunosorbent assay (ELISA), microarray, and surface plasmon resonance (SPR). These heterogeneous methods require a step of immobilizing a molecule on a substrate and a washing step for analysis of avidity (interaction) or enzyme activity. Furthermore, it is also essential to purely separate substances and reaction products bound to substances (low molecular weight compounds or nucleic acids or proteins) to be analyzed for final avidity (interaction) analysis or enzyme activity measurement.
  • ELISA enzyme linked immunosorbent assay
  • SPR surface plasmon resonance
  • homogeneous method does not require the immobilization, washing, or separation process required in the heterogeneous method, it has the advantage of being able to easily and quickly analyze the binding strength (interaction) or enzyme activity between molecules.
  • Representative examples of homogeneous methods include fluorescence resonance energy transfer (FRET) and fluorescence polarization (FP) technologies.
  • FRET fluorescence resonance energy transfer
  • FP fluorescence polarization
  • the fluorescence resonance energy transfer measurement method is widely used to measure the binding force (interaction) or enzyme activity between molecules, but due to the demands of a donor and an acceptor, a fluorescent substance is added to both molecules to be analyzed. There is a fatal drawback that must be labeled.
  • the fluorescence polarization measurement method which enables analysis of the binding strength (interaction) or enzyme activity between molecules even when a fluorescent substance is labeled on only one molecule, is a method of measuring the fluorescence polarization that occurs when the fluorescent substance in a solution is excitation. .
  • a fluorescent substance When a fluorescent substance is excited and is in a stable state, it emits fluorescence polarized light, and when the fluorescent molecule is excited, it rotates by Brownian motion to emit fluorescence to a plane different from the excitation plane, resulting in loss of fluorescent polarization. It is to use the principle.
  • low molecular weight compounds-protein, nucleic acid-protein, peptide-protein, protein -It can be said to be a powerful technology in the analysis of binding (interaction) and enzyme activity of proteins, receptors-ligands, enzymes-substrates, nucleic acids-nucleic acids, etc.
  • Republic of Korea Patent Registration No. 10-1576363 relates to a method of measuring fluorescence polarization in which a signal is amplified.
  • a signal is amplified.
  • biotin and streptavidin By amplifying the fluorescence polarization value using biotin and streptavidin, it is possible to analyze the binding force (interaction) between two molecules or measure enzyme activity. Is being disclosed.
  • the present inventors made diligent efforts to provide a method for effectively screening the binding (interaction) power of a low molecular weight compound and a protein, etc., particularly the binding power of FGF2 and API5 or inhibitors that inhibit it.As a result, by introducing cysteine into FGF2, API5 In the case of screening after reacting with, it was confirmed that the binding strength of FGF2 and API5 or inhibitors that inhibit it can be screened with high efficiency, and the present invention was completed.
  • an object of the present invention is the steps of i) introducing cysteine outside FGF2;
  • step ii) labeling a fluorescent material on FGF2 into which the cysteine of step i) has been introduced;
  • the present invention comprises the steps of: i) introducing cysteine to the outside of FGF2;
  • step ii) labeling a fluorescent material on FGF2 into which the cysteine of step i) has been introduced;
  • FGF2 binding inhibitor screening method comprising a.
  • the cysteine in step i) may be introduced at the C-terminus of FGF2.
  • the fluorescent material of step ii) is FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine), FAM (fluorescein amidite), Rhodamine, TAMRA (Carboxytetramethylrhodamine), CyTM, CFTM (Cyanine- based fluorescent dye) and Alexa Fluor may be any one or more selected from the group consisting of.
  • the protein capable of binding to FGF2 in step iii) may have a molecular weight greater than or equal to FGF2.
  • the protein capable of binding to FGF2 of iii) is any selected from the group consisting of FLAG, histidine, CBD, MBP, TRX and GST (Glutathione S-transferase). It may be a protein to which one or more tags are fused.
  • the protein capable of binding to FGF2 in step iii) may be API5.
  • measuring the fluorescence intensity in step iv) is FRET (Fluorescence Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer), FP (Fluorescence Polarization), HCS (High-content Screening). And it may be measured using one or more methods selected from the group consisting of HTS (High Throughput Screening).
  • the fluorescence intensity measured in step iv) decreases to a level of 50% to 100% compared to before mixing the candidate material of step iii), it is determined as an FGF2 binding inhibitor. It may be to further include the step of.
  • the present invention provides a FGF2 binding inhibitor selected by the screening method.
  • the binding may be a combination of FGF2 and API5.
  • the present invention provides a composition for inhibiting anticancer drug resistance comprising the FGF2 binding inhibitor.
  • FGF2 (protein) outside refers to a portion of the FGF2 protein that is directed to the outside of the FGF2 protein rather than the inside of the protein.
  • inside FGF2 (protein) refers to a portion of the FGF2 protein directed toward the inside of the FGF2 protein rather than outside the protein.
  • “Inhibition of anticancer drug resistance” of the present invention refers to all effects of reducing or alleviating the resistance or resistance of cancer cells to anticancer agents so that the anticancer effect of the anticancer agent against cancer cells can be fully exhibited.
  • API5 Apoptosis inhibitor 5
  • AAC-11 Apoptosis inhibitor 5
  • API5 has been reported to be involved in increasing cancer survival in several cancer cells. Through physical binding with Acinus (ACIN1), it inhibits the induction of apoptosis through Acinus-mediated DNA fragmentation and blocks the activity of Caspase-3, a apoptosis protein.
  • ACIN1 Acinus
  • the cytotoxicity of the anticancer drug was increased by effectively inhibiting E2F1-induced apoptosis and lowering the expression of API5.
  • it has been found that it is involved in the migration and invasion of cancer cells, which is deeply related to cancer metastasis, and plays an important role in immune evasion of cancer cells.
  • the "fibroblast growth factor 2 (FGF2)" of the present invention is one of a large family of fibroblast growth factors consisting of 23 FGF members, and is the most abundant among FGF members in the central nervous system. It plays an important role as a regulatory mediator of proliferation, differentiation, development and survival in various cell types and has mitogenic activity in the nervous system. In addition, FGF2 has a potential angiogenic effect, promotes the growth of smooth muscle cells, wound healing and tissue repair, as well as the pluripotent of human and mouse embryonic stem (ES) cells. It has been reported to remain in state. There are two types of FGF2: high molecular weight (22, 22.5, 24 or 34 kDa) and low molecular weight (18 kDa).
  • High molecular weight FGF2 has a nuclear localization signal (NLS) at the N-terminus, allowing high molecular weight FGF2 to be located in the nucleus, whereas low-molecular weight FGF2 does not have NLS at the N-terminus, whereas atypical dichotomy at the C-terminus NLS regulates some low molecular weight FGF2 to be located in the nucleus.
  • NLS nuclear localization signal
  • FGF2 binding inhibitor refers to a substance that inhibits FGF2 from binding to other proteins, and in the present invention, it may mean a substance that inhibits the binding of FGF2 and API5.
  • the fluorescence polarization value was very low after a certain period of time. This is because the existing Cys residues present inside FGF2 are not sufficiently exposed on the protein surface, so that the fluorescent substance is shared. It was determined that labeling through binding was not properly performed. On the other hand, in the case of FGF2 in which Cys was exposed to the outside of the protein by introducing Cys at the C-terminus, it was confirmed that the fluorescent label was maintained for a long period of time. In addition, the labeling efficiency values of FGF2 with Cys introduced at the C-terminus were mostly about 0.3 to 0.8, and it was determined that Alexa Fluor 488 was appropriately covalently bonded to the introduced Cys to be labeled.
  • the six candidate compounds are respectively API5 derived peptide (1), FGF2 derived peptide (2), API5 binding substance derived from thermal shift assay (3), a derivative of candidate 3 (4), and other candidate substances of 3 It is a derivative (5) and a negative control group (6).

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
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  • Physics & Mathematics (AREA)
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  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biophysics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de criblage d'une substance inhibant la liaison du FGF2 et son utilisation, le procédé comprenant les étapes consistant à : i) introduire une cystéine à l'extérieur du FGF2 ; ii) marquer le FGF2 avec introduction de cystéine de l'étape i) avec une substance fluorescente ; iii) mélanger le FGF2 marqué avec la substance fluorescente de l'étape ii), une protéine qui peut se lier au FGF2 et une substance candidate qui peut inhiber la liaison entre celles-ci ; et iv) mesurer l'intensité de la fluorescence des substances mélangées de l'étape iii). En utilisant le procédé de criblage de la présente invention, la cystéine est en outre introduite dans le FGF2 pour permettre un marquage plus efficace de la substance fluorescente et ainsi une valeur de polarisation de fluorescence mesurée au moyen d'un test de polarisation de fluorescence et similaire est amplifiée et la force de liaison (interaction) entre le FGF2 et l'API5 ou la substance inhibant celle-ci peut être criblée efficacement.
PCT/KR2020/009846 2019-08-09 2020-07-27 Procédé de criblage d'une substance inhibant la liaison d'api5 et du fgf2 WO2021029565A1 (fr)

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KR10-2019-0097467 2019-08-09
KR1020190097467A KR102210227B1 (ko) 2019-08-09 2019-08-09 Api5 및 fgf2의 결합 저해 물질 스크리닝 방법

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024196179A1 (fr) * 2023-03-21 2024-09-26 Nex-I, Inc. Épitope api5 et anticorps se liant de manière spécifique à celui-ci

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150123250A (ko) * 2013-03-06 2015-11-03 제넨테크, 인크. 암 약물 내성의 치료 및 예방 방법

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150123250A (ko) * 2013-03-06 2015-11-03 제넨테크, 인크. 암 약물 내성의 치료 및 예방 방법

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AHMED MENNATALLAH, LEGRAND CYRIL, YAGÜE RELIMPIO ANA, BERETTA CARLO A., MUSCHKO ALINA, WEGEHINGEL SABINE, MÜLLER HANS‐MICHAEL, SEH: "A time‐resolved live cell imaging assay to identify small molecule inhibitors of FGF2 signaling", FEBS LETTERS, vol. 593, no. 16, 28 May 2019 (2019-05-28), pages 2162 - 2176, XP055780641, ISSN: 0014-5793, DOI: 10.1002/1873-3468.13462 *
GIUSEPPE LA VENUTA, SABINE WEGEHINGEL, PETER SEHR, HANS-MICHAEL MULLER, ELENI DIMOU, JULIA P. STERINGER, MAREIKE GROTWINKEL, NIKOL: "Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 29, no. 34, 19 August 2016 (2016-08-19), pages 17787 - 17803, XP055353975, ISSN: 0021-9258, DOI: 10.1074/jbc.M116.729384 *
HAN BYEONG-GU, KIM KYOUNG HOON, LEE SANG JAE, JEONG KYUNG-CHAE, CHO JEA-WON, NOH KYUNG HEE, KIM TAE WOO, KIM SOON-JONG, YOON HYE-J: "Helical Repeat Structure of Apoptosis Inhibitor 5 Reveals Protein-Protein Interaction Modules", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 287, no. 14, 30 March 2012 (2012-03-30), pages 10727 - 10737, XP055780634, DOI: 10.1074/jbc.M111.317594 *
SEOUNG MIN BONG , BYUNG LL LEE: "Crystallization and preliminary X-ray analysis of API5-FGF2 complex", BIO DESIGN, vol. 6, no. 4, 30 December 2018 (2018-12-30), pages 92 - 93, XP009525786, ISSN: 2288-7105 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024196179A1 (fr) * 2023-03-21 2024-09-26 Nex-I, Inc. Épitope api5 et anticorps se liant de manière spécifique à celui-ci

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