KR20200044705A - Method and Apparatus for Screening of Drugs Targeting Heterodimer of HER2 and HER3 and Method for Screening the Same - Google Patents

Abstract

The present invention relates to a method for screening a drug targeting a heterodimer of HER2 and HER3, the method comprising a step of analyzing the heterodimer of HER2 and HER3, and to a drug targeting a heterodimer of HER2 and HER3. The method of the present invention can be favorably applied to screen candidate drugs for the prevention or treatment of diseases associated with the heterodimer of HER2 and HER3.

Description

Methods and Apparatus for Screening of Drugs Targeting Heterodimer of HER2 and HER3 and Method for Screening the Same}

A drug screening method targeting a heterodimer of HER2 and HER3, comprising analyzing the heterodimer of HER2 and HER3, and a heterodimeric target drug of HER2 and HER3.

Human epidermal growth factor receptors (HER) are members of the growth factor receptor and receptor tyrosine kinase (RTK). HER family receptors are overexpressed and mutated in various types of cancer. Therefore, HER is a major therapeutic target in various cancers.

HER family receptors form homodimeric, heterodimeric, or dimeric or higher oligomers, activating their intracellular domains. In particular, the heterodimer composed of HER2 and HER3 has the highest proliferating capability among the possible combinations of HER family receptors. The detection of HER2-HER3 heterodimer in breast cancer patient samples is known to correlate with negative prognosis in HER2-positive breast cancer patients. Therefore, the development of a drug that specifically targets the HER2-HER3 heterodimer is continuing, but due to the lack of drug screening technology, successful development cannot be achieved.

One example includes measuring protein-protein interactions between a first protein and a second protein, wherein the first protein is HER2 and HER3, and the HER2 and HER3 form a heterodimer, and the second protein is a cell Or activation of signaling pathways involving HER2, HER3, or both (e.g., HER2-HER3 heterodimer forms) in cells or tissues, which are sub-proteins of HER2, HER3, or both of them in the tissue. Methods of measurement (or confirmation or determination or analysis) are provided. Another example provides a method of measuring (or identifying or determining or analyzing) the degree of activation of a HER2-HER3 heterodimer, comprising measuring the degree of phosphorylation of HER2 and / or HER3 in a HER2-HER3 heterodimer.

Another example includes measuring a protein-protein interaction between a first protein and a second protein, wherein the first protein is HER2 and HER3, and the HER2 and HER3 form a heterodimer, and the second protein is a cell Or HER2, HER3, or all of the HER2, HER3, or sub-proteins, or HER2, HER3, or both, which are suitable for application to the individual (individual patient) from which the cell or tissue is derived, among the signaling pathways within the tissue. For example, a method for screening a drug targeting HER2-HER3 heterodimer form) or a method for providing information on screening is provided. Another example is a cell or tissue comprising a HER2-HER3 heterodimer, or measuring the degree of activation of HER2 and / or HER3 (eg, phosphorylation of a tyrosine residue) in a HER2-HER3 heterodimer, or the cell or A method of selecting a drug targeting HER2, HER3, or both (e.g., HER2-HER3 heterodimer form) suitable for application to a tissue-derived individual (individual patient) or a method of providing information to the screening to provide.

Another example is a protein-protein interaction between a first protein and a second protein in an isolated cell or tissue treated with a candidate drug targeting the first protein, or in a cell or tissue isolated from an individual administered the drug. Measuring, the first protein is HER2 and HER3, the HER2 and HER3 form a heterodimer, and the second protein is a sub-protein of HER2, HER3, or both of them in a signaling pathway within a cell or tissue. Provided are methods of screening for drugs targeting phosphorus, HER2, HER3, or both (eg, HER2-HER3 heterodimer form). Another example is the degree of activation of HER2 and / or HER3 in the HER2-HER3 heterodimer in cells or tissues treated with a candidate substance targeting the HER2-HER3 heterodimer, or in cells or tissues isolated from the individual to whom the drug is administered. A method of screening for a drug targeting HER2, HER3, or both (eg, HER2-HER3 heterodimer form), comprising measuring (eg, phosphorylation of a tyrosine residue).

Another example includes measuring and / or comparing protein-protein interactions between a first protein and a second protein when treated with and without a candidate substance on a first protein or a substrate immobilized with the first protein, wherein the agent 1 protein is HER2 and HER3, the HER2 and HER3 form a heterodimer, and the second protein is HER2, HER3, or a sub-protein of HER2, HER3, or both of them in the signaling pathway in a cell or tissue A method of screening a drug targeting (eg, HER2-HER3 heterodimer form) is provided. Another example is to measure and / or compare the degree of activation of HER2 and / or HER3 (eg, phosphorylation of a tyrosine residue) with and without a candidate substance on a substrate where the HER2-HER3 heterodimer or HER2-HER3 heterodimer is immobilized. A method of screening for a drug targeting HER2, HER3, or both (eg, HER2-HER3 heterodimer form) comprising a step is provided. In the screening method, prior to the measuring and / or comparing step, a first protein (or HER2-HER3 heterodimer) or a first protein (or HER2-HER3 heterodimer) is immobilized on a substrate to which the second protein is immobilized. The step of reacting and the step of treating a candidate substance to the reactant may be further included. The drug targeting the HER2, HER3, or both (eg, HER2-HER3 heterodimer form) may be an anti-cancer agent. The anti-cancer agent may have an anti-cancer effect against cancer in which the HER2-HER3 heterodimer is detected, and / or may have an anti-cancer effect against cancer having resistance to a HER2 or HER3 targeted therapeutic agent.

Other examples are HER2 comprising at least one selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof, Provided is a composition for inhibiting the activity of HER3, and / or HER2-HER3 heterodimer. The HER2 and HER3 may be present alone or in a HER2-HER3 heterodimer. Other examples are one or more selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof, HER2, HER3, And / or inhibition of the activity of a HER2-HER3 heterodimer, or use of a composition for inhibiting the activity of a HER2, HER3, and / or HER2-HER3 heterodimer. Another example is HER2-HER3 hetero at least one selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof. Provided is a method for inhibiting the activity of a HER2, HER3, and / or HER2-HER3 heterodimer comprising administering to a subject in need thereof. The activity of the HER2, HER3, and / or HER2-HER3 heterodimer may be tyrosine kinase activity. Inhibition of activity of the HER2, HER3, and / or HER2-HER3 heterodimers may include HER2, HER3, and / or HER2-HER3 heterodimers (e.g., Y1289, Y1197, Y1222, Y1276, Y1328 of HER3, Y1196 of HER2, or these (Combination of two or more of them) may mean phosphorylation inhibition (reduction of phosphorylation level). The subject can be a cell, tissue, or individual (a mammal such as a human).

Another example is HER2, comprising one or more selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof. Provided is a composition for cell death comprising a -HER3 heterodimer. Other examples are one or more, HER2-HER3 hetero selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof. Provided for use in the preparation of a composition for cell death comprising a dimer, or cell death comprising a HER2-HER3 heterodimer. Another example is HER2-HER3 hetero at least one selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof. A method of killing a cell containing a HER2-HER3 heterodimer comprising administering to a subject in need thereof.

Another example is HER2- comprising one or more selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof. Provided is a pharmaceutical composition for the prevention and / or treatment of diseases associated with HER3 heterodimers. Other examples are one or more, HER2-HER3 hetero selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof. Provided for use in the prevention and / or treatment of dimer-related diseases, or in the manufacture of compositions for the prevention and / or treatment of HER2-HER3 heterodimer-related diseases. Another example is HER2-HER3 hetero at least one selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof. Provided is a method of preventing and / or treating HER2-HER3 heterodimer-related diseases, comprising administering to a subject in need thereof.

In another example, HER2-HER3 heterodimer is detected in a biological sample isolated from a subject in order to select a suitable subject for administration of a composition for preventing and / or treating a disease related to the HER2-HER3 heterodimer or applying treatment using the same. How to do. Another example is suitable for the administration of a composition for the prevention and / or treatment of the HER2-HER3 heterodimer-related disease or the treatment using the same, comprising detecting a HER2-HER3 heterodimer in a biological sample isolated from a subject. Provides a method for screening objects.

Another example provides an apparatus for use in the methods described above.

In the present specification, measurement of protein-protein interaction and / or enzymatic activity between sub-proteins of the HER2-HER3 heterodimer may be based on single-molecule pull-down and co-immunoprecipitation (co-IP).

One example provided herein includes measuring protein-protein interactions between a first protein and a second protein, wherein the first protein is HER2 and HER3, and the HER2 and HER3 form a heterodimer, and 2 proteins are signaling pathways involving HER2, HER3, or both HER2, HER3, or both (e.g., HER2-HER3 heterodimer forms) in cells or tissues, which are sub-proteins of HER2, HER3, or both. It relates to a method of measuring activation (or confirmation or determination or analysis) or a method of providing information on activation measurement. Another example relates to a method of measuring (or identifying or determining or analyzing) the degree of activation of a HER2-HER3 heterodimer, comprising measuring the degree of phosphorylation of HER2 and / or HER3 in a HER2-HER3 heterodimer.

Another example is a cell or tissue comprising a HER2-HER3 heterodimer, or measuring the degree of activation of HER2 and / or HER3 (eg, phosphorylation of a tyrosine residue) in a HER2-HER3 heterodimer, or the cell or Methods for selecting or targeting drugs that target HER2, HER3, or both (e.g., HER2-HER3 heterodimer forms) suitable for application to tissue-derived individuals (individual patients) It is about.

Another example includes measuring a protein-protein interaction between a first protein and a second protein in an isolated cell or tissue treated with a candidate drug targeting the first protein, wherein the first protein is HER2 and HER3, wherein HER2 and HER3 form a heterodimer, and the second protein is a sub-protein of HER2, HER3, or both, HER2, HER3, or both (e.g., HER2- HER3 heterodimer form). Another example is the degree of activation of HER2 and / or HER3 in the HER2-HER3 heterodimer in cells or tissues treated with a candidate substance targeting the HER2-HER3 heterodimer, or in cells or tissues isolated from the individual to whom the drug is administered. A method of screening for a drug targeting HER2, HER3, or both (eg, HER2-HER3 heterodimer form), comprising measuring (eg, phosphorylation of a tyrosine residue). The “drug targeting HER2, HER3, or both (eg, HER2-HER3 heterodimer form)” refers to the activity of HER2, HER3, or both (eg, HER2-HER3 heterodimer form) (eg tyrosine kinase activity) ).

Another example includes measuring and / or comparing a protein-protein interaction between a first protein and a second protein according to whether a first protein or a first protein is immobilized on a substrate, and the first protein Is HER2 and HER3, the HER2 and HER3 form a heterodimer, and the second protein is HER2, HER3, or a sub-protein of HER2, HER3, or both of them in a signaling pathway in a cell or tissue (e.g. , HER2-HER3 heterodimer form). Another example is measuring and / or comparing the degree of activation of HER2 and / or HER3 (eg, phosphorylation of a tyrosine residue) depending on whether a candidate substance is treated on a substrate on which the HER2-HER3 heterodimer or HER2-HER3 heterodimer is immobilized. It relates to a method of screening a drug targeting HER2, HER3, or both (eg, HER2-HER3 heterodimer form), including. In the screening method, prior to the measuring and / or comparing step, a first protein (or HER2-HER3 heterodimer) or a first protein (or HER2-HER3 heterodimer) is immobilized on a substrate to which the second protein is immobilized. The step of reacting and the step of treating a candidate substance to the reactant may be further included.

It will be described in more detail below.

As used herein, the term "protein-protein interaction (PPI)" may mean physical and / or chemical bonding or complex formation between a first protein and a second protein, eg, It may be measured by one or more of factors such as binding frequency, bonding strength (strength), and bonding time. In addition, the interaction (binding) between the first protein and the second protein means not only direct interaction (binding) between them, but also other proteins (proteins located between the first and second proteins in the signal transduction pathway). Intermediate interactions (binding) can also be included. The protein-protein interaction herein can be a single molecule reaction (a reaction between a first protein in one molecule and a second protein in one molecule).

In the present specification, the first proteins are HER2 (Human Epidermal growth factor Receptor 2 protein; ErbB2) and HER3 (Human Epidermal growth factor Receptor 3 protein; ErbB3), wherein HER2 and HER3 form a heterodimer (HER2-HER3 hetero Dimer).

HER2 and HER3 are members of the receptor tyrosine kinase (RTKs) of the HER family, respectively. The binding of a ligand to the extracellular domain of HER2 or HER3 induces receptor homo- or hetero-dimerization with other ErbB receptors, which causes intracellular autophosphorylation of specific tyrosine residues. Autophosphorylation of HER2 and / or HER3 leads to downstream signaling networks including MAPK and PI3K / Akt activation that affect cell proliferation, angiogenesis and metastasis. Overexpression and gene amplification of HER2 are frequently observed in breast cancer and gastric cancer, and are associated with poor prognosis and poor clinical outcome of cancer treatment. Meanwhile, HER3 is well known as a resistance mechanism for several targeted drug treatments. For this reason, these HER2 and / or HER3 are important targets in anticancer therapy. HER2 and HER3 may be derived from mammals such as humans. For example, HER2 is GenBank Accession No. It may be a polypeptide having an amino acid sequence provided in NP_001005862.1, NP_001276865.1, NP_001276867.1, NP_004439.2, but is not limited thereto. HER3 is GenBank Accession No. It may be a polypeptide having an amino acid sequence provided in NP_001005915.1, NP_001973.2, etc., but is not limited thereto.

The second protein is a sub-protein of HER2 and / or HER3 (e.g., HER2-HER3 heterodimer) in various biosignaling pathways, interacting with (binding to) HER2 and / or HER3 (e.g., HER2-HER3 heterodimer) ) It may be one or more selected from possible proteins. In another example, the sub-protein may be one or more selected from tyrosine kinases, such as tyrosine kinases capable of interacting (binding) with HER2 and / or HER3 (eg, HER2-HER3 heterodimer).

The step of measuring the protein-protein interaction between the first protein and the second protein performed in the method provided herein, and the step of measuring the degree of activation of the first protein or the first protein enzyme may be performed on isolated cells or tissues. It may be performed in vitro or in vitro .

The step of measuring the protein-protein interaction between the first protein and the second protein may include the following steps:

(1) preparing a substrate to which a first protein is immobilized by adding a sample containing a first protein or a first protein to a substrate containing a substance specifically binding to the first protein on a surface;

(2) reacting by adding the labeled second protein (bound material bound) to the substrate on which the prepared first protein is fixed; And

(3) Measuring a signal from the reactant obtained in step (2) (protein-protein interaction measurement).

In another example, measuring the degree of tyrosine kinase activation of HER2 and / or HER3 in a HER2-HER3 heterodimer (eg, phosphorylation of a tyrosine residue) can include the following steps:

(1-1) A sample containing HER2-HER3 heterodimer or HER2-HER3 heterodimer is added to a substrate containing a substance specifically binding to HER2 and / or HER3 on the surface, and the HER2-HER3 heterodimer is immobilized Preparing a substrate;

(2-1) phosphorylating the HER2-HER3 heterodimer and reacting by adding a labeled (labeled substance bound) phosphorylated Tyr specific binding substance (eg, an antibody); And

(3-1) Measuring a signal from the reactant obtained in step (2-1) (tyrosine kinase activation measurement).

In the step (2-1), the step of phosphorylating may include the step of treating a phosphorylating agent on the substrate or the HER2-HER3 heterodimer fixed to the substrate. The phosphorylating agent may be at least one selected from all substances capable of phosphorylating HER2 and / or HER3, and may be, for example, ATP and magnesium (eg, MgCl ₂ ), but is not limited thereto. In the drug screening method, the candidate substance can be treated before, after, or simultaneously with phosphorylation in step (2-1). Cholesterol-like detergent comprising DGTN, GDN, etc. in the HER2-HER3 heterodimer fixed to the substrate or substrate, before, after, or simultaneously with the phosphorylation and / or candidate substance treatment, and before the activation measurement in step (3-1) Structural and / or structural treatment of HER2-HER3 heterodimers by treating one or more detergents selected from (detergent, hereinafter, used interchangeably with surfactants) at concentrations above or above the critical micellar concentration (CMC) It may be characterized by maintaining functional activity.

The steps are described in more detail below:

Step (1) or (1-1): first protein immobilized substrate preparation step

The step (1) is a step of preparing a substrate in which the first protein is immobilized by adding a first protein or a test sample containing the first protein to a substrate containing a substance specifically binding to the first protein on the surface. .

The first proteins are HER2 and HER3, and specifically, they may be dimerized HER2-HER3 heterodimers. The sample containing the first protein comprises HER2 and / or HER3, or HER2-HER3 heterodimer, or cell, cell lysate, or cell lysate expressing HER2 and HER3 simultaneously. Isolated cells, recombinant cells engineered to simultaneously express HER2 and HER3 (e.g., HER2 in cells expressing HER2, recombinant cells introduced with a recombinant vector containing the HER3 gene, and cells expressing HER3) Or a recombinant cell into which a recombinant vector containing a gene has been introduced), or a lysate or lysate of the cell.

In one example, for heterodimer formation of HER2 and HER3, it may be characterized by treating a dimerization agent to cells that simultaneously contain or express HER2 and HER3. The dimerization agent is a ligand of HER3 (e.g., Neuregulin β1 (NRG1-b1); NP_039250, NP_039250.2, etc.), cholesterol-like detergent; for example, DGTN (digitonin; CAS Number: 11024-24 -1), GDN (glycol-diosgenin; CAS Number: 1402423-29-3), etc.) may be one or more selected from the group consisting of. In one embodiment, when NRG1-b1 is treated (injected) in cells expressing HER2 and HER3 simultaneously to form a HER2-HER3 heterodimer, the NRG1-b1 expresses HER2 and HER3 and then cells prior to lysis. Can be injected into. In another embodiment, when a cholesterol-like detergent is formed on cells expressing (including) HER2 and HER3 simultaneously to form a HER2-HER3 heterodimer, the cholesterol-like detergent is after cell lysis and of HER2 and / or HER3. Can be treated (added) before activation measurement (in one example, the cells are injected with an amount of NRG1-b1 of 10 ng / mL to 1000 ng / mL, 50 ng / mL to 200 ng / mL, or 10 ng / mL to 100 ng / mL). May be). At this time, the concentration of the cholesterol-like cleaning agent used may be a concentration that exceeds the critical micellar concentration (CMC) of the cleaning agent used, for example, based on the total cell lysate (cell lysis), 0.05% for DGTN ( w / v) or more, greater than 0.05% (w / v), or greater than 0.06% (w / v), such as 0.05 to 2% (w / v) or 0.06 to 2% (w / v), For GDN, 0.003% (w / v) or more, 0.003% (w / v) or more, 0.007% (w / v) or more, or 0.01% (w / v) or more, such as 0.003 to 2% (w / v) ), 0.005 to 2% (w / v), 0.007 to 2% (w / v), or 0.01 to 2% (w / v). When the concentration of the cleaning agent used is below CMC, it is difficult to maintain the micelle structure, protect the cell membrane permeation region of the protein, and maintain the tyrosine kinase activity, and the activity of the HER2-HER3 heterodimer (eg, the degree of phosphorylation) is lowered and not recovered. After confirmation, it is suggested that the detergent be used at the above concentration. Digitonin becomes quite unstable when dissolved, and precipitates form within a few hours. Therefore, when dissolved, adding about 10% (w / v) of glycerol (5 to 15% (w / v) or 8 to 12% (w / v)) keeps it more stable. GDN is soluble and stable for several days. All solutions can be maintained at pH 7 to 8. 7.2 to 7.6, or 7.4 using HEPES buffer. In addition, it is necessary to maintain an appropriate ion concentration by adding NaCl 150mM. A protease inhibitor cocktail can be added to prevent protein degradation by intracellular protease upon cell lysis (eg, Sigmaaldrich P8340, about 2% (w / v)).

The sample may be a cell, tissue, cell or tissue lysate, lysate, or extract isolated from an individual, body fluid (eg, blood (whole blood, plasma, or serum), saliva, etc.). The individual is any mammal (e.g., a primate such as human, monkey, primate, mouse, etc.) that wants to perform an activation test of a signal transduction pathway in a cell or tissue in which the first protein is involved, and / or to select an effective first protein targeted therapeutic agent, etc. Rats, etc.). In one example, the subject can be a patient with a disease associated with the first protein. The disease associated with the first protein may be a disease associated with overexpression of the first protein or activation of a signaling pathway in cells or tissues involved with the first protein, for example cancer.

The substrate may be of any material and / or all structures capable of immobilizing a first protein (HER2, HER3, or both) on the surface (both crystalline or amorphous can be used). In one example, the substrate may be made of a material having a refractive index of light (about 1.3) or higher, which accounts for a large portion of the biomaterial, in consideration of the ease of detection of the label signal. In one example, the substrate may have a thickness of about 0.1 to about 1 mm, about 0.1 to about 0.5 mm, 0.1 to about 0.25 mm, or about 0.13 to about 0.21 mm, and a refractive index of about 1.3 to about 2, about 1.3 To about 1.8, about 1.3 to about 1.6, or about 1.5 to about 1.54. The substrate may be any material that satisfies the refractive index range, and may be obtained from a material selected from the group consisting of glass (refractive index: about 1.52), quartz, etc., but is not limited thereto. The substrate may have any form that is commonly used for observation of biological samples, and may be, for example, a well type, a slide type, a channel type, an array form, a microfluidic chip, a microtube (capillary), etc., but is not limited thereto. It does not work. When observing a fluorescent microscope, the cover glass may be mounted on the substrate to which the sample is applied, and the material of the cover glass may be as described above, and the thickness may be less than or equal to the range described in the substrate (eg, refractive index 1.52, It may be 0.17 mm thick, but is not limited to this).

The substance that specifically binds to the first protein may be selected from all substances capable of specifically binding to the first protein, HER2, HER3, or both, for example, HER2, HER3, or both. Antibodies that specifically bind to or antigen-binding fragments thereof (eg, scFv, (scFv) 2, scFv-Fc, Fab, Fab 'and F (ab') 2 of antibodies, etc.), aptamers (protein or nucleic acid molecule) , Small molecule compounds, and the like. At this time, a substance that specifically binds to the first protein (HER2, HER3, or both) is a site that does not interfere with the interaction between the first protein and the second protein, that is, the first protein and the second protein It may be that the first protein binds at a site other than the site of interaction (binding).

In one example, the substrate is appropriately surface-modified to include (immobilize) a biological material (eg, antibody, etc.) that specifically binds to the first protein, or specifically to the first protein directly on the surface. The binding material may be fixed. When the substrate is surface-modified, the substrate can be treated (e.g., applied) with any compound having a functional group capable of immobilizing a biological material (e.g., antibody, etc.) that specifically binds to the first protein on one surface. And may be treated with a compound containing a functional group selected from the group consisting of aldehyde groups, carboxyl groups and amine groups. In one embodiment, the compound comprising a functional group selected from the group consisting of the aldehyde group, carboxyl group and amine group is biotin, bovine serum albumin (BSA), biotin-bound bovine serum albumin, polyethylene glycol (polyethylene) glycol; PEG), biotin-linked PEG (polyethylene glycol-biotin; PEG-biotin), polysorbate (eg, Tween20), or the like. The surface-treated substrate may be further treated (eg, applied) by one or more selected from the group consisting of neutravidin, streptavidin, avidin, and the like.

Step (2) or (2-1): Reacting the first protein and the second protein or HER2-HER3 Heterodimer  Phosphorylated, phosphorylated Tyrosine  Reacting with a specific binding agent

In step (2) or (2-1), the prepared first protein is immobilized on the substrate to which the labeled second protein is added, or the first protein is phosphorylated, and the labeled phosphorylated tyrosine specific binding substance is obtained. It is a step of adding and reacting. The labeled phosphorylated tyrosine-specific binding substance may be an antibody that specifically binds HER2 or HER3 phosphorylated tyrosine.

The second protein is a sub-protein of HER2 and / or HER3 (e.g., HER2-HER3 heterodimer) in various biosignaling pathways, as described above, HER2 and / or HER3 (e.g., HER2-HER3 heterodimer) ) And may be one or more selected from proteins capable of interacting (binding).

In the step (2-1), the step of phosphorylating may include the step of treating a phosphorylating agent on the substrate or the HER2-HER3 heterodimer fixed to the substrate. The phosphorylating agent may be at least one selected from all substances capable of phosphorylating HER2 and / or HER3, and may be, for example, ATP and magnesium (eg, MgCl ₂ ), but is not limited thereto. In the drug screening method, the candidate substance can be treated before, after, or simultaneously with phosphorylation in step (2-1). Before, after, or simultaneously with the phosphorylation and / or candidate treatment, and prior to the activation measurement in step (3-1), prior to step (1) or (1-) prior to HER2-HER3 heterodimer immobilized on the substrate or substrate. As described in 1), one or more detergents selected from cholesterol-like detergents including DGTN, GDN, etc. are treated with a concentration exceeding CMC (critical micellar concentration) to obtain structural and HER2-HER3 heterodimers. And / or maintaining functional activity.

The labeled second protein or labeled phosphorylated tyrosine specific binding substance is labeled with a labeling substance that generates a detectable signal by the second protein or labeled phosphorylated tyrosine specific binding substance (the labeling substance is, for example, a chemical) (Eg, covalent or non-covalent), recombinant, or physically, may be combined, or may mean a tag attached form to which a labeling material can be attached. The detectable signal can be selected from all signals (eg, light, radiation, etc.) that can be measured through conventional enzyme reaction, fluorescence, luminescence, and / or radiation detection. The labeling material may be at least one selected from the group consisting of all small molecule compounds, proteins, peptides, nucleic acid molecules, etc. capable of generating the label signal, for example, fluorescent dyes (small molecule compounds; Cyanine, Alex, Dylight, Fluoprobes, etc.) ), Fluorescent proteins (e.g., green fluorescent protein (GFP, enhanced GFP), yellow fluorescent protein (YFP), turquoise fluorescent protein (CFP), blue fluorescent protein (BPF), red fluorescent protein (RPF), mcherry, etc.) It may be one or more selected from the group consisting of. The tag may be at least one selected from all commonly used types such as His-tag / Ni-NTA. The concentration of use of the labeling material can be appropriately determined within a range of about 1 uM or less in order to enable accurate and easy detection without generating noise, for example, 1 nM to 1000 nM, 1 nM to 500 nM, 1 nM to 100 nM, It may be about 10nM to 1000nM, 10nM to 500nM, or 10nM to 100nM, but is not limited thereto. The signal generated from the labeling material can be measured by any signal detection means commonly used to detect or measure it (eg, a conventional fluorescence microscope, a fluorescence camera, a fluorescence intensity measurement (quantitative) device, etc.).

A method of screening for a drug targeting a first protein (HER2, HER3, or both (e.g., HER2-HER3 heterodimer form)), the method comprising: before the subsequent protein-protein interaction measurement step (step (3)), The method may further include a step of treating a candidate substance in the reactant of the first protein or the first protein and the second protein.

In order to more accurately measure the interaction between the first protein and the second protein, between the reacting step (step (2)) and the subsequent protein-protein interaction measuring step (step (3)), the substrate on which the reaction occurred It may further include the step of washing in a conventional manner.

Step (3) or (3-1): Protein-protein interaction measurement step or Tyrosine Residue  Phosphorylation measurement steps

Step (3) or (3-1) is a step of measuring a signal from the reactants obtained in step (2) or (2-1). The signal measurement detects (or measures or confirms) the label signal used in step (2) or (2-1) (for example, a signal measurable through conventional methods such as enzyme reaction, fluorescence, luminescence, or radiation detection). ).

In one example, the protein-protein interaction measurement in step (3) may be by real-time analysis.

In one example, when the label signal is a fluorescence signal, the signal detection is performed by supplying a light source absorbed by a label material that generates the fluorescence signal, for example, a fluorescence microscope, a fluorescence camera, and / or fluorescence The intensity measurement (quantitative) device or the like can be used to image and / or quantify.

In one embodiment, when the signal is a fluorescent signal, the fluorescent signal may be imaged and / or quantified using a fluorescent camera.

If the signal is a fluorescence signal, step (3) (step of measuring protein-protein interaction) is

(i) supplying a light source to the reactant in step (2); And

(ii) detecting a fluorescence signal generated by the supplied light source

It may include.

The step of supplying a light source in step (i) is a step of supplying a light source to the reactants of the first protein and the second protein obtained in step (2). If such a goal can be achieved, the supply time of the light source is There are no special restrictions. For example, the light source may be continuously supplied from before, simultaneously, or after step (1) to after step (2), or may be supplied for a predetermined time immediately before, simultaneously, or immediately after step (2), but is not limited thereto. It is not.

The light source may be any light source having a wavelength corresponding to a fluorescent signal, and may be, for example, a laser or a halogen lamp.

The wavelength of the light source can be adjusted according to the fluorescence signal used, for example, it can be selected from about 300nm to about 600nm or about 350nm to about 560nm. More specifically, the green fluorescent protein absorbs about 480 nm, the yellow fluorescent protein absorbs about 540 nm, the blue fluorescent protein absorbs about 375 nm, and the blue-green fluorescent protein absorbs about 425 nm, so the green fluorescent protein absorbs green fluorescence. When using, the wavelength of the light source is about 460 to about 500 nm, when using yellow fluorescence as the fluorescent signal, the wavelength of the light source is about 520 to about 560 nm, when using the blue fluorescence as the fluorescent signal, the wavelength of the light source is about 350 ~ About 400nm, when using a blue-green fluorescence as a fluorescent signal, the wavelength of the light source may be selected from about 400 to about 450nm.

In one embodiment, the protein-protein interaction measurement step of step (3) is performed by supplying a light source using a Total Internal Reflection Fluorescence (TIRF) microscope or a confocal microscope, and a fluorescent signal in a conventional manner. It can be performed by observing. In another example, a light source is supplied and a fluorescent signal is imaged by using a fluorescent camera for signal imaging, such as an EMCCD (Electron-multiplying charge-coupled device) camera or a Complementary metal oxide semiconductor (CMOS) camera. And / or quantitation.

In the following, the case where the protein-protein interaction measurement step of step (3) is performed using a total reflection microscope and a fluorescence camera will be described in more detail, for example:

A) The substrate of step (1) or step (2) is mounted on a total reflection microscope. In the total reflection microscope, the supply position of the light source is usually downward, and depending on the type of the total reflection microscope, the fluorescent signal is placed on the substrate (in this case, from bottom to top, the light source supply, the substrate, the lens, or the substrate, the light source supply, the lens Can be in order) or from below (in this case, from bottom to top, the lens, the light source supply, the substrate, or the light source supply, the lens, the substrate, or the lens, the substrate, the light source supply) Can be observed.

B) The light source may be a laser, and the intensity of the light source is about 0.5 mW to about 5 mW, about 0.5 mW to about 4.5 mW, about 0.5 mW to about 4 mW, about 0.5 mW to about 3.5 mW, about 0.5 mW to about 3 mW, about 0.5 mW to about 2.5 mW, about 0.5 mW to about 2 mW, about 1 mW to about 5 mW, about 1 mW to about 4.5 mW, about 1 mW to about 4 mW, about 1 mW to about 3.5 mW , About 1 mW to about 3 mW, about 1 mW to about 2.5 mW, about 1 mW to about 2 mW, about 1.5 mW to about 5 mW, about 1.5 mW to about 4.5 mW, about 1.5 mW to about 4 mW, about It may be from 1.5 mW to about 3.5 mW, from about 1.5 mW to about 3 mW, from about 1.5 mW to about 2.5 mW, or from about 1.5 mW to about 2 mW, such as about 2 mW, and the wavelength of the light source is fluorescence as described above. It may be appropriately selected according to the signal or the labeling material used, and / or the configuration of the equipment (for example, a strong light source may be used when an attenuation filter is used).

C) The fluorescence signal generated by the supply of the light source can be imaged and / or quantified by shooting with a fluorescence camera.

The photographing (or imaging) of the fluorescence signal may be performed simultaneously with the supply of the light source or within the signal generation maintenance time in consideration of the fluorescence signal generation maintenance time (light emission time, lifetime) of the labeling substance.

When shooting (or imaging) a fluorescent signal with a fluorescent camera (eg, an EMCCD camera), the exposure time per frame, laser power, camera gain value, and total shooting frame can be appropriately adjusted. For example, the shorter the exposure time per frame, the less signals are accumulated in one frame, and to compensate for this, the laser power may be increased or the sensitivity of the fluorescent camera may be increased. In one example, the exposure time per frame is about 0.001 seconds to about 5 seconds, about 0.001 seconds to about 3 seconds, about 0.001 seconds to about 2 seconds, about 0.001 seconds to about 1 second, about 0.001 seconds to about 0.5 seconds, About 0.001 seconds to about 0.3 seconds, about 0.001 seconds to about 0.1 seconds, about 0.01 seconds to about 5 seconds, about 0.01 seconds to about 3 seconds, about 0.01 seconds to about 2 seconds, about 0.01 seconds to about 1 second, about 0.01 Seconds to about 0.5 seconds, about 0.01 seconds to about 0.3 seconds, about 0.01 seconds to about 0.1 seconds, about 0.05 seconds to about 5 seconds, about 0.05 seconds to about 3 seconds, about 0.05 seconds to about 2 seconds, about 0.05 seconds to About 1 second, about 0.05 seconds to about 0.5 seconds, about 0.05 seconds to about 0.3 seconds, about 0.05 seconds to about 0.1 seconds, about 0.07 seconds to about 5 seconds, about 0. 07 seconds to about 3 seconds, about 0. 07 Seconds to about 2 seconds, about 0. 07 seconds to about 1 second, about 0. 07 seconds to about 0.5 seconds, about 0. 07 seconds to about 0.3 seconds, about 0.07 seconds to about 0.1 seconds, about 0.1 seconds to about 5 Seconds, about 0.1 seconds to about 3 seconds, about 0.1 seconds to about 2 seconds, about 0.1 seconds to about 1 second, about 0.1 second to about 0.5 second, or about 0.1 second to about 0.3 second, such as about 0.1 second, but is not limited thereto.

For example, when an EMCCD camera is used, photons generated from a labeling material (eg, eGFP) are converted into electrons through a device of the EMCCD and measured (photoelectric effect). At this time, the number of electrons generated per photon can be changed through a gain value. As the set gain value increases, the number of electrons generated per photon increases, so the sensitivity of the EMCCD increases and the background noise increases, so the signal-to-noise ratio is important. In one embodiment, to obtain a good signal-to-noise ratio, the gain value is about 10 to about 100, about 10 to about 80, about 10 to about 60, about 10 to about 50, about 20 to about 100, about 20 to about 80, about 20 to about 60, about 20 to about 50, about 30 to about 100, about 30 to about 80, about 30 to about 60, or about 30 to about 50, such as about 40 , It is not limited to this, and may be appropriately selected in consideration of camera sensitivity, life, equipment construction status, noise, test conditions, and the like.

Multiplying the total number of shooting frames by the exposure time gives the total shooting time (exposure time * total number of shooting frames = shooting time). Since the fluorescent signal disappears after the emission time of the fluorescent material, the total number of photographing frames and / or the exposure time can be adjusted so that the photographing time proceeds within the emission time.

In order to increase the accuracy of the protein-protein interaction measurement results, one or more of the imaging, such as two or more, three or more, four or more, five or more, seven or more, or ten or more (the upper limit is the Performed on a substrate of one or more channels (determined according to size and imageable area) or one or more channels (each channel includes two or more substrates), and the number of spots (also referred to as PPI complexes) on which signals are displayed is obtained. The resulting fluorescence signal can be quantified, which can be viewed as quantifying protein-protein interactions.

In another example, the protein-protein interaction may be quantified by quantifying the fluorescence intensity measured in step (3) using a conventional device. At this time, for the accuracy of the result, the result can be normalized based on the sample amount or the protein amount in the sample.

When two or more types of the first protein and / or the second protein are used, steps (1) to (3) may be performed for each first protein and second protein combination (ie, step (1)). (3) may be repeatedly performed as many times as the number of the first protein and the second protein combination).

The phosphorylation measurement step of the tyrosine residue in step (3-1) detects a signal arising from the reaction between a HER2-HER3 heterodimer and a substance (e.g., antibody) that specifically binds to a labeled HER2 or HER3 phosphorylated tyrosine residue. And / or quantifying. In one example, when a fluorescent material (such as a fluorescent protein) is used as a labeling material, phosphorylation of the tyrosine residue can be measured by measuring and quantifying the fluorescence intensity detected in the reactant, as described above.

The method provided herein, in addition to the steps (1) to (3) or (1-1) to (3-1) described above, further includes the following steps (4), and / or steps (5) May contain:

Step (4): comparing with the reference sample

Step (4) is a result obtained from the reference sample (protein-protein interaction level or tyrosine kinase activation level) obtained in step (3) or (3-1) above (protein-protein interaction level or tyrosine kinase activation level) ).

The reference sample may be appropriately selected according to the purpose of the invention.

For example, in the case of a method for measuring (or confirming or determining or analyzing) the activation of a signaling pathway in a cell or tissue, or a method for providing information for measuring the activation, the reference sample may include (1) normal cells, and / or (2) agents. Cells (e.g., normal cells or cancer cells) with known (identified) activation of signaling pathways involving 1 protein (HER2, HER3, or both (e.g., HER2-HER3 heterodimer form)), and / or ( 3) Cells isolated from individuals with known (identified) levels of activation of signaling pathways involved in the first protein (HER2, HER3, or both (e.g., HER2-HER3 heterodimer form)) (e.g., normal cells or Cancer cells), and / or (4) other cells capable of expressing HER2 and HER3 to form a HER2-HER3 heterodimer (eg, artificially (eg, recombinantly) expressing HER2 and HER3 to express HER2-HER3 hetero Cells forming dimers, etc.) It can be included. In one embodiment, when the test sample includes cancer cells isolated from an individual, it may be that cancer cells isolated from the individual include normal cells of the same tissue or organ.

As used herein, the term “normal cell” can mean any cell in a non-pathological state, wherein the “non-pathological state” is not a disease state, or a mutation, tumorigenesis, functional and / Or it may mean that it is not a condition that can cause a disease with morphological abnormalities. For example, normal cells may be cells that do not have a disease associated with a first protein (HER2, HER3, or both (eg, HER2-HER3 heterodimer form)) or a disease to be treated with a drug to be tested, from which the test sample is derived It may be derived from the same individual or tissue as an individual or tissue.

In addition, in the case of a screening method for a drug targeting a first protein (HER2, HER3, or both (eg, HER2-HER3 heterodimer form)), the reference sample is a sample in which a candidate substance is not treated or the candidate substance It may be a sample before treatment.

In order to perform step (4), the methods further comprise, prior to step (4), measuring the degree of protein-protein interaction or tyrosine kinase activation between the first protein and the second protein with respect to the reference sample. These steps may be performed with reference to steps (1), (2), and (3), or (1-1), (2-1), and (3-1) described above. .

Step (5)

The method provided in the present specification may further include, after the step (4), checking (determining) a desired item from the comparison result obtained in the step (4).

The details are as follows:

(i) within a cell or tissue HER2  And HER3 With heterodimer  Methods of measuring (or identifying or determining or analyzing) activation of the relevant signaling pathways, or methods of providing information for activation measurements

Step (5) may include the following steps:

If the protein-protein interaction level of the test sample measured in step (3) is higher than the protein-protein interaction level measured in the reference sample, a signal that the first protein is involved in the test sample or the individual from which the test sample originates Confirming (determining) that the level of activation of the delivery pathway is higher than the level of activation of normal cells or the level of activation known (verified) in the reference sample;

If the level of protein-protein interaction of the test sample measured in step (3) is equal to the level of protein-protein interaction measured in the reference sample, a signal in which the first protein is involved in the test sample or the individual from which the test sample is derived Confirming (determining) that the degree of activation of the delivery pathway is equivalent to the degree of activation of normal cells or the degree of activation known (verified) in the reference sample; And / or

If the protein-protein interaction level of the test sample measured in step (3) is lower than the protein-protein interaction level measured in the reference sample, a signal that the first protein is involved in the test sample or the individual from which the test sample originates Confirming (determining) that the level of activation of the delivery pathway is lower than the level of activation of normal cells or the level of known (confirmed) activation in the reference sample.

 Another example includes measuring a protein-protein interaction between a first protein and a second protein, wherein the first protein is HER2 and HER3, and the HER2 and HER3 form a heterodimer, and the second protein is a cell Or a sub-protein of HER2, HER3, or both in the signaling pathway within the tissue, targeting the first protein, wherein the step of measuring the protein-protein interaction is performed in a sample treated with a candidate substance and an untreated sample Method for screening candidate drugs to be targeted, method for confirming efficacy of candidate drugs targeting first protein, or method for screening candidate drugs for preventing or treating diseases associated with HER2-HER3 heterodimer formation, or preventing or treating such diseases It provides a method for confirming the efficacy of the candidate drug for. Another example includes measuring the degree of activation of HER2 and / or HER3 (eg, phosphorylation of a tyrosine residue) in a HER2-HER3 heterodimer, and measuring the activation comprises HER2-HER3 hetero treatment with a candidate substance. Selection of candidate drugs targeting HER2 and / or HER3 first proteins, performed on samples containing dimers or HER2-HER3 heterodimers and samples containing untreated HER2-HER3 heterodimers or HER2-HER3 heterodimers Method or method for confirming the efficacy of a candidate drug targeting a first protein, or a method for screening a candidate drug for the prevention or treatment of a disease associated with HER2-HER3 heterodimer formation, or the efficacy of a candidate drug for the prevention or treatment of the disease Provide a verification method.

The above method,

Treating (eg, contacting) the candidate substance with the test sample, and

The following steps (a), (b), (c), and (d) may include the steps of performing:

(a) Preparing a test sample comprising a HER2-HER3 heterodimer, or adding a test sample containing a HER2-HER3 heterodimer to a substrate containing a substance specifically binding to HER2 and / or HER3 on the surface, thereby HER2- Preparing a substrate on which the HER3 heterodimer is fixed;

(b) phosphorylating the HER2-HER3 heterodimer and reacting by adding a labeled phosphorylated tyrosine specific binding substance;

(c) measuring a signal from the reactant obtained in step (b) (measurement of protein-protein interaction or measuring the degree of tyrosine kinase activation of HER2 and / or HER3); And

(d) comparing each of the results obtained in step (c) with respect to the untreated test sample and the treated test sample.

As described above, the phosphorylated tyrosine-specific binding agent may be an antibody that specifically binds HER2 and / or HER3 phosphorylated tyrosine.

The candidate substance can be treated in step (b), for example, before, after, or simultaneously with the treatment of phosphorylated or phosphorylated tyrosine specific binding substance in step (b). Phosphorylation in step (b) may be performed by treating a phosphorylating agent. The phosphorylating agent may be at least one selected from all substances capable of phosphorylating HER2 and / or HER3, and may be, for example, ATP and magnesium (eg, MgCl ₂ ), but is not limited thereto.

In order to compare the results of the untreated test sample and the processed test sample in step (d), steps (a)-(c) are performed on the untreated test sample and the treated test sample. For each.

The test sample in which the candidate substance is not treated and the processed test sample respectively mean a test sample before treatment and a test sample after treatment, or a portion of the test sample and the candidate in which the candidate substance is processed by dividing the test sample It may mean some other test sample that has not been treated.

As a result of the comparison in step (d), when the level of protein-protein interaction of the test sample treated with the candidate substance or the degree of tyrosine kinase activation of HER2 and / or HER3 is lower than that of the test sample not treated with the candidate substance (i.e. When the candidate substance inhibits tyrosine kinase activation of HER2 and / or HER3), the candidate substance or candidate drug targeting HER2, HER3, or both (e.g., HER2-HER3 heterodimer form) or HER2-HER3 Selected as a candidate drug for the prevention or treatment of diseases associated with heterodimers (e.g., cancer), or the candidate substance targets HER2, HER3, or both (e.g., HER2-HER3 heterodimeric form) (i.e. It can be confirmed that it is effective as a drug (inhibiting tyrosine kinase activation of HER2 and / or HER3).

Accordingly, the method for selecting the candidate drug or the method for confirming the efficacy of the candidate drug, after the step (d), as a result of the comparison in the step (d), the protein-protein interaction level of the test sample treated with the candidate substance, or When the degree of tyrosine kinase activation of HER2 and / or HER3 is lower than in the test sample in which this candidate substance is untreated (i.e., the candidate substance interacts with the first protein and the second protein or the tyrosine kinase activation of HER2 and / or HER3 ), The candidate substance is selected as a candidate drug targeting HER2, HER3, or both (e.g., HER2-HER3 heterodimer form), or the candidate substance is HER2, HER3, or both (e.g. , Targeting the HER2-HER3 heterodimer form (i.e. inhibiting the interaction of the first and second proteins or tyrosine kinase of HER2 and / or HER3) It may further comprise the step of confirming that it is effective as a drug (inhibiting activation) (step (e)). The protein-protein interaction level of the test sample treated with the candidate drug or the degree of tyrosine kinase activation of HER2 and / or HER3 is lower than that of the test sample treated with the candidate drug- The protein interaction level or the degree of tyrosine kinase activation of HER2 and / or HER3 is not more than 75% of the protein-protein interaction level of the test sample in which the candidate substance is not treated or the degree of tyrosine kinase activation of HER2 and / or HER3 (i.e., 25 % Or more suppressed), 50% or less (ie, 50% or more suppressed), 45% or less, 40% or less, 35% or less, 30% or less, 25% or less (ie, 75% or more suppressed), 20% or less (ie 80% or more inhibition), 15% or less, or 10% or less (ie, 90% or more inhibition).

The candidate material can be selected from all biocompatible materials that can be used as target drugs for the first protein (HER2, HER3, or both (eg, HER2-HER3 heterodimer form)), such as small molecule chemical drugs (small molecular chemicals), proteins (eg, antibodies, antibody fragments, or analogs thereof), peptides, nucleic acid molecules (eg, DNA, RNA (eg, siRNA, microRNA, shRNA, etc.), PNA (peptide nucleic acid), aptamer, etc. ), Plant extracts, animal extracts, cell extracts, etc. may be selected from one or more, but is not limited thereto.

The test sample may be a cell (e.g., cell lysate, etc.) or tissue (e.g., cell lysate) overexpressed and / or activated with a first protein (HER2, HER3, or both (especially in the form of HER2-HER3 heterodimer)). Tissue lysate, etc.), or disease associated with the first protein or application of a drug targeting the first protein to be screened (treated), isolated cells (e.g., cell lysate, etc.) or tissue (e.g. Tissue lysate, etc.), and in one example, can be cells or tissues isolated from established cell lines or patients suffering from the disease (eg, cancer). For example, the test sample may be an established cancer cell line or a cell or tissue isolated from a cancer patient (the cancer may be related to overexpression and / or (activation) of the first protein).

The steps (a) to (e) may be performed in vitro .

The term description of each step is as described above.

The above-described method for selecting a candidate drug targeting the first protein may be usefully applied to an efficacy assay (or confirmation or test) of a drug developed as a candidate drug in the development of a new drug targeting the first protein.

If the level of protein-protein interaction of the test sample treated with the candidate substance is lower than that of the test sample treated with the candidate substance, further comprising selecting the candidate substance as a candidate drug targeting the first protein. You can.

The candidate substance can be selected from all biocompatible substances that can be used as target drugs for the first protein, such as small molecular chemicals, proteins (eg, antibodies, antibody fragments, or analogs thereof), peptides, etc. , Nucleic acid molecule (e.g., DNA, RNA (e.g., siRNA, microRNA, shRNA, etc.), PNA (peptide nucleic acid), aptamer, etc.), plant extract, animal extract, cell extract, etc. It can be, but is not limited to.

The test sample may be a cell (eg, cell lysate, etc.) or tissue (eg, tissue lysate, etc.) or tissue (e.g., cell lysate) in which the first protein is overexpressed and / or activated, or a disease related to the first protein or a first target for selection Application of a drug targeting a protein (treatment) can be isolated cells (e.g., cell lysate, etc.) or tissue (e.g., tissue lysate, etc.) associated with the disease of interest, in one example, an established cell line or the disease It may be a cell or tissue isolated from a patient suffering from (eg, cancer). For example, the test sample may be an established cancer cell line or a cell or tissue isolated from a cancer patient (the cancer may be related to overexpression and / or (activation) of the first protein).

The method of selecting a candidate drug targeting the first protein (HER2, HER3, or both (eg, HER2-HER3 heterodimer form)) described above is the first protein (HER2, HER3, or both (eg, HER2-) In the development of new drugs targeting HER3 heterodimer form)), it can be usefully applied to the efficacy assay (or confirmation or test) of drugs developed as candidate drugs.

As used herein, the term “targeting the first protein” refers to promoting or inhibiting the activity of the first protein (HER2, HER3, or both (eg, HER2-HER3 heterodimer form)). It may mean, for example, inhibiting the activity of the first protein (HER2, HER3, or both (eg, HER2-HER3 heterodimer form)) (tyrosine kinase activity). Inhibition of the activity of the first protein binds to the first protein, and / or degrades and / or structurally modifies the first protein, thereby reducing intrinsic function, such as intrinsic biosignaling function in cells and / or tissues. It can be ordered or removed.

As used herein, the term “drug” is any substance that exhibits a pharmacological effect, such as small molecule compounds, proteins (eg, antibodies, antibody fragments, or analogs thereof), peptides, nucleic acid molecules (eg, DNA, RNA (For example, siRNA, microRNA, shRNA, etc.), PNA (peptide nucleic acid), aptamer, etc., may be one or more selected from the group consisting of plant extracts, animal extracts, cell extracts, and the like.

 As used herein, the term “drug targeting the first protein” means any substance that inhibits the activity of the first protein, such as a small molecule, protein that inhibits the activity of the first protein ( For example, antibodies, antibody fragments, or analogs thereof), peptides, nucleic acid molecules (e.g., DNA, RNA (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA), miRNA (microRNA), etc.)), PNA ( peptide nucleic acid), aptamer, etc.), plant extracts, animal extracts, cell extracts, or the like. More specifically, a “drug targeting the first protein” binds to the first protein and / or degrades and / or structurally modifies the first protein to intrinsic function, such as intrinsic in cells and / or tissues. Any substance that reduces or eliminates the biosignaling function of, e.g., small molecule compounds that inhibit the activity of the first protein, proteins (e.g., antibodies, antibody fragments, or analogs thereof), peptides, nucleic acid molecules (e.g., DNA , RNA (eg, siRNA, microRNA, shRNA, etc.), PNA (peptide nucleic acid), aptamer, etc., may be one or more selected from the group consisting of plant extracts, animal extracts, cell extracts, and the like.

As used herein, the term “treatment targeting the first protein” can mean any medical and / or pharmaceutical action that inhibits the activity of the first protein, eg, the first as described above. The drug that inhibits the activity of the protein may be prescribed and / or administered to a subject in need of inhibiting the activity of the first protein. More specifically, “the treatment targeting the first protein” binds to the first protein, and / or degrades and / or structurally modifies the first protein to intrinsic function, such as intrinsic in cells and / or tissues. Drugs that reduce or eliminate the biosignaling function of may be prescribed and / or administered to a subject in need thereof.

The administration can be performed by oral or parenteral routes. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, local administration of the lesion site, intranasal administration, intrapulmonary administration, or rectal administration may be performed.

The individual, patient or subject may be any mammal, such as a primate such as a human or a monkey, a rodent such as a mouse or a rat, or may be a patient having a disease related to the first protein. The disease associated with the first protein may be a disease associated with overexpression of the first protein or activation of a signal transduction pathway in cells or tissues involved with the first protein, such as cancer, inflammation, and other immune diseases. In embodiments, the subject, patient or subject can be a cancer patient.

The cancer may be selected from all solid cancers and hematologic cancers, and may be, for example, cancer associated with overexpression of the first protein or activation of a signaling pathway in cells or tissues involved with the first protein. For example, the cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, melanoma of the skin or eye, rectal cancer, proximal cancer, esophageal cancer, small intestine cancer, endocrine Adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer , Kidney cancer, prostate cancer, vulva cancer, thyroid cancer, head and neck cancer, brain cancer, and the like, but may be one or more selected from the group consisting of, but not limited to. The cancer may include primary cancer as well as metastatic cancer. In addition, the cancer may be cancer having resistance to or obtained from existing anticancer treatment.

As used herein, the term "reactivity to a drug" may mean the degree to which the drug exerts an effect in an individual who has been administered the drug.

As used herein, the term “effect” refers to a medical and / or pharmaceutical effect that a drug or treatment seeks to achieve in a treatment subject, the prevention and / or treatment of a disease possessed by the treatment subject, and / or It may mean relief and / or improvement of symptoms. For example, when the drug is an anti-cancer agent and the treatment is an anti-cancer treatment, and the treatment target is a cancer patient, the effect may be an anti-cancer effect (prevention and / or treatment effect of cancer), and the anti-cancer effect inhibits the growth of cancer cells. In addition to the effect, it may include an effect of inhibiting migration, invasion, and / or metastasis, and / or suppressing exacerbation of cancer, and / or reducing or eliminating resistance, and the like. have.

In another example, an analytical device for use in a method of measuring activation of a signaling pathway in a cell or tissue described above, and / or a screening method of a drug is provided. The analysis device can be applied as a device for measuring the activation of a signal transduction pathway in a tissue, and / or a device for confirming the efficacy of a drug targeting a first protein. The first protein is HER2, HER3, or both (eg, HER2-HER3 heterodimer form).

The analysis device is applied to the method for measuring the activation of a signal transduction pathway in a cell or tissue described above, and / or a method for screening a drug, so that interaction between biomolecules in a small sample is accurately and efficiently observed, analyzed, detected, and And / or has the advantage of being measurable. An apparatus for measuring protein-protein interaction or an analytical method using the same can be usefully and effectively applied to a very small amount of a sample, such as a biopsy (eg, needle biopsy) sample. In addition, the analysis device or an analysis method using the same can accurately, efficiently observe, analyze, detect, and / or measure the degree of interaction or activation between various biomolecules (eg, proteins, nucleic acids, etc.) using a small sample. You can.

The analysis device,

The first protein specifically binds to a first protein capture material or a first protein containing a reaction portion, or

In addition to this, it may be to further include a signal detection means.

1 (NRG1-b1) 등), 콜레스테롤-유사 세정제 (Cholesterol-like detergent; 예컨대, DGTN(digitonin; CAS Number: 11024-24-1), GDN (glycol-diosgenin; CAS Number: 1402423-29-3) 등으로 이루어진 군에서 선택된 1종 이상)이 처리된 것일 수 있으며, 예컨대, HER2 및 HER3를 발현하고, NRG1-b1이 처리된 세포로서, 임의로 DGTN 및 GDN로 이루어진 군에서 선택된 1종 이상의 콜레스테롤-유사 세정제로 처리된 세포 또는 세포 용해물일 수 있다.The first protein is HER2 and HER3, especially HER2-HER3 heterodimer. In one example, the first protein may be used as a HER2-HER3 heterodimer, or a cell (expressing) containing HER2-HER3 heterodimer on the cell surface or a lysate of the cell, and the reaction unit (1) The HER2-HER3 heterodimer, or a cell containing (expressing) a HER2-HER3 heterodimer on the cell surface, or a lysate of the cell, or (2) they may include an immobilized substrate. Cells comprising (expressing) the HER2-HER3 heterodimer on the cell surface express HER2 and HER3, and the dimerization agents described below (e.g., ligands of HER3 (e.g. Neuregulin

1 (NRG1-b1), etc.), cholesterol-like detergent (Cholesterol-like detergent; for example, DGTN (digitonin; CAS Number: 11024-24-1), GDN (glycol-diosgenin; CAS Number: 1402423-29-3) Etc.) may be treated, for example, cells expressing HER2 and HER3, and treated with NRG1-b1, optionally one or more cholesterol-like selected from the group consisting of DGTN and GDN. Cells or cell lysates treated with a detergent.

The analytical device determines whether and / or the degree of interaction (binding) between HER2 and HER3, in particular, the HER2-HER3 heterodimer and sub-proteins on their signaling pathways, or the degree of activation of HER2 and / or HER3 (e.g., Tyr residues) Can be used to measure the degree of phosphorylation.

The reaction unit may include a substrate on which the first protein capture material is immobilized on the surface, or the first protein is immobilized with or without (directly) through the capture material. The first protein is HER2 and HER3, in particular, a HER2-HER3 heterodimer, and the first protein capture material is the first protein (HER2 and / or HER3) described in the step of preparing the substrate where the first protein is immobilized. It may be a substance that specifically binds, such as an antibody or antigen-binding fragment thereof. The reaction unit may be a well (eg, multi-well) type, slide type, channel type, array type, microfluidic chip, microtubule (capillary), but is not limited thereto.

The analysis device may be a device for measuring protein-protein interaction between the first protein and the second protein, and for this purpose, may further include a second protein that interacts with the first protein. In another example, the assay device may be a device that measures the degree of activation of HER2 and / or HER3 (e.g., phosphorylation of a Tyr residue), and for this purpose, a substance (e.g., an antibody) specifically binding to phosphorylated Tyr. It may further include. In one example, the second protein or a substance that specifically binds to phosphorylated Tyr is labeled with a labeling substance that generates a detectable signal (the labeling substance is, for example, chemically (e.g., covalent or non-covalent), or recombinant Red, or physically, may be combined), or may be in the form of a tag to which a labeling material is attached.

The first protein is HER2 and HER3, particularly HER2-HER3 heterodimer, and the second protein is as described above.

In addition, the apparatus for measuring protein-protein interaction, to normalize (normalize) the signal value measured by the signal detection means to the level of the first protein, a detection substance binding to the first protein and a binding substance to the detection substance It may further include a labeling material. The detection material binding to the first protein may be a biomolecule (eg, antibody) that binds to the first protein at a site different from the capture material of the first protein included in the multi-well described above, and the labeling material Is a biomolecule that is labeled with a labeling substance that generates a detectable signal (the labeling substance is, for example, chemically (e.g., covalently or non-covalently)) and is capable of binding to a substance for detection that binds the first protein (e.g., Antibody).

The signal detection means may be any signal detection means commonly used depending on the signal generated from the labeling substance used. For example, the signal detection means may include a signal stimulation unit and a signal detection unit, and in addition, may further include a signal analysis unit for analyzing (eg, quantitative or imaging) the measured signal. In one example, signal stimulation, signal detection, and signal analysis can each be performed at different sites, or at least two or more of them may be performed simultaneously or sequentially at one site. In one example, when the label is a fluorescent substance, the signal detection means may be selected from all means capable of generating and detecting a fluorescence signal, for example, a fluorescence signal stimulator (eg, light source), and a fluorescence signal It may include a detection unit, and / or a fluorescent signal analysis unit. In one embodiment, the signal detection means includes a total internal fluorescence microscope (Total Internal Reflection Fluorescence (TIRF) microscope) or a confocal microscope (for detecting light sources and fluorescence signals), or in addition, a fluorescence camera, such as EMCCD (Electron A multiplying charge-coupled device (CMOS) camera or a complementary metal oxide semiconductor (CMOS) camera may be additionally included to perform light source supply and imaging and / or quantification of fluorescent signals. The wavelength, intensity, and fluorescent camera measurement conditions of the light source (eg, exposure time per frame, laser power, camera gain value, total shooting frame, etc.) are as described above.

The device for measuring protein-protein interaction provided herein has the advantage of being able to accurately, efficiently observe, analyze, detect, and / or measure the interaction between the first protein and the second protein in a small sample. Therefore, the multi-well or analytical method using the same can be usefully and effectively applied to a very small amount of a sample, such as a biopsy (eg, needle biopsy) sample.

Another example provides a method for producing a heterodimer of HER2 and HER3. The method may include the step of treating a dimerization agent to cells that contain or express HER2 and HER3 simultaneously. The dimerization agent, as described above, a ligand of HER3 (eg, Neuregulin β1 (NRG1-b1)); NP_039250, NP_039250.2, etc.), Cholesterol-like detergents (e.g., DGTN (digitonin; CAS Number: 11024-24-1), GDN (glycol-diosgenin; CAS Number: 1402423-29-3, etc.) , May be one or more selected from the group consisting of, etc. In one embodiment, when the method includes the step of injecting (injecting) NRG1-b1 to cells simultaneously expressing HER2 and HER3, the NRG1-b1 is HER2 and HER3 may be expressed and then injected into cells prior to lysis In another embodiment, the cholesterol-like detergent is treated when the cells expressing HER2 and HER3 are treated with a cholesterol-like detergent. May be treated (added) to a washing step after cell lysis (in one example, the cells may be NRG1-b1 injected in an amount of 10 ng / mL to 1000 ng / mL or 50 ng / mL to 200 ng / mL). At this time, the concentration of cholesterol-like detergent used is used. The cleaning agent may have a concentration that exceeds the critical micellar concentration (CMC), for example, based on cell lysis, for DGTN, greater than 0.05% (w / v), greater than 0.05% (w / v), Or 0.06% (w / v) or more, such as 0.05 to 2% (w / v) or 0.06 to 2% (w / v), for GDN 0.003% (w / v) or more, 0.003% ( w / v) or more, 0.007% (w / v) or more, or 0.01% (w / v) or more, such as 0.003 to 2% (w / v), 0.005 to 2% (w / v), 0.007 to 2 % (w / v), or 0.01 to 2% (w / v).

Another example is sapitinib; 4- (3-Chloro-2-fluoroanilino) -7-methoxy-6-[[1- (N-methylcarbamoylmethyl) piperidin-4-yl] oxy] quinazoline; CAS No. 848942 -61-0), AEE788 (6- [4-[(4-Ethylpiperazin-1-yl) methyl] phenyl] -N-[(1R) -1-phenylethyl] -7H-pyrrolo [2,3-d] pyrimidin-4-amine; CAS No. 497839-62-0), Dacomitinib; ((E) -N- (4-((3-chloro-4-fluorophenyl) amino) -7-methoxyquinazolin-6 -yl) -4- (piperidin-1-yl) but-2-enamide; CAS No. 1110813-31-4), PD153035 (N- (3-Bromophenyl) -6,7-dimethoxy-4-quinazolinamine hydrochloride; CAS No. 183322-45-4), CUDC-101 (7-((4-((3-ethynylphenyl) amino) -7-methoxyquinazolin-6-yl) oxy) -N-hydroxyheptanamide; CAS No. 1012054-59 -9), PD158780 (4-N- (3-bromophenyl) -6-N-methylpyrido [3,4-d] pyrimidine-4,6-diaminel CAS No. 171179-06-9), Pelitinib ( (2E) -N- [4-[(3-Chloro-4-fluorophenyl) amino] -3-cyano-7-ethoxy-6-quinolinyl] -4- (dimethylamino) -2-butenamide; CAS No. 257933- 82-7), Saracatinib; (N- (5-Chloro-1,3-benzodioxol-4-yl) -7- [2- (4-methyl-1-pipera zinyl) ethoxy] -5-[(tetrahydro-2H-pyran-4-yl) oxy] -4-quinazolinamine; CAS No. 379231-04-6), and one or more selected from the group consisting of pharmaceutically acceptable salts thereof as an active ingredient, HER2-HER3 HER2 in cells containing a heterodimer (or included on the surface) and / Or HER3 activity (tyrosine kinase activity) inhibitory composition; A cell death composition comprising a HER2-HER3 heterodimer; Or it provides a pharmaceutical composition for the prevention or treatment of diseases associated with HER2-HER3 heterodimer (eg, cancer in which HER2-HER3 heterodimer is detected). Other examples include cells comprising HER2-HER3 heterodimer in at least one pharmaceutically effective amount selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, and saracatinib, Or comprising the step of administering to the individual containing the cell, HER2 and / or HER3 activity inhibition method; Cell death method comprising HER2-HER3 heterodimer; Or a disease associated with a HER2-HER3 heterodimer (eg, a cancer in which the HER2-HER3 heterodimer is detected).

The pharmaceutically acceptable salt may be selected and used without particular limitation on all salts commonly used in the pharmaceutical field. In one embodiment, the pharmaceutically acceptable salt is a non-toxic inorganic acid such as hydrochloric acid, bromic acid, sulfonate, sulfuric acid, phosphoric acid, nitric acid, etc .; Or it may be one or more selected from salts formed using organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, tartaric acid, citric acid, paratoluenesulfonic acid, methanesulfonic acid, etc., for example, hydrochloric acid, It is not limited thereto.

The HER2-HER3 heterodimer may be absent or inactive in normal cells or cancer cells that do not have resistance (eg, resistance to HER2 and / or target therapy (therapeutic agent) of HER3). Thus, a cell, disease, or subject to which a method and / or composition provided herein is applied or used in a method and / or composition is present in a HER2-HER3 heterodimer, normal cell or resistant (e.g., HER2 and / or It may be a cell or disease or subject associated with or present at a relatively high level compared to cancer cells that do not have a targeted treatment (resistance to the therapeutic agent) of HER3.

The HER2-HER3 heterodimer-containing cell may be a cell that includes a HER2-HER3 heterodimer (eg, included on the cell surface) or a higher level than normal cells, and in other words, HER2-HER3 heterodimer is conventional It may be detectable by a phosphorus protein detection means or cells detected at a higher level than normal cells. In one embodiment, the HER2-HER3 heterodimer-containing cells may be cancer cells, for example, cancer cells having the characteristics described above. In another example, the cancer cells may be cancer cells that are resistant to existing anti-cancer treatments, such as HER2 target treatment and / or HER3 target treatment. In another example, the cancer cell comprises a HER2-HER3 heterodimer (e.g., on the cell surface), or at a higher level than normal cells, and is used for conventional anti-cancer treatment, e.g., HER2 target treatment and / or HER3 target treatment. It may be a cancer cell resistant to.

In one example, the HER2-HER3 heterodimer-related disease may be a disease associated with a cell containing the HER2-HER3 heterodimer described above, and the presence (e.g., a higher level than normal cells) or detection of HER2-HER3 heterodimer ( For example, a disease characterized by a higher level than normal cells).

In one example, the cancer may be an existing anti-cancer agent, such as a cancer resistant to HER2 target treatment and / or HER3 target treatment. In one example, the subject may be a cell comprising a HER2-HER3 heterodimer, or an individual (a mammal such as a human) containing the cell. More specifically, the subject can be a cancer patient, for example, where HER2-HER3 heterodimers are present (eg, present at a higher level than normal individuals or individuals resistant to HER2 target treatment and / or HER3 target treatment). May be a cancer patient. In another example, the subject may be a cancer patient resistant to existing anti-cancer treatments, such as HER2 target treatment and / or HER3 target treatment. Thus, the method comprises cancer patients with HER2-HER3 heterodimer present (e.g., at a higher level than normal individuals or individuals resistant to HER2 target treatment and / or HER3 target treatment) prior to the administration step. Alternatively, the method may further include identifying a cancer patient resistant to existing HER2 target treatment and / or HER3 target treatment. The subject or cancer patient may be a cell, tissue, or individual (a mammal such as a human). The existing anti-cancer treatment may be a treatment using a conventional anti-cancer agent. The HER2 target treatment and / or HER3 target treatment may be treatment using a conventional HER2 target treatment and / or HER3 target treatment. The conventional anti-cancer agent or conventional HER2 target therapeutic agent and / or HER3 target therapeutic agent may be, for example, one or more selected from the group consisting of neratinib, lapatinib, and the like.

In one example, the pharmaceutical composition, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient, diluent, filler, extender, wetting agent, disintegrant, emulsifier (surfactant), lubricant, sweetener, flavoring agent, suspension agent , Preservatives and the like may further include one or more adjuvants selected from the group consisting of. The adjuvant may be appropriately adjusted according to the formulation to which the pharmaceutical composition is applied, and may be used by selecting one or more of all adjuvants that can be commonly used in the pharmaceutical field. In one embodiment, the pharmaceutically acceptable carrier is conventionally used for the formulation of drugs, lactose, dextrose, sucrose, trehalose, alginy, histidine, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, With alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It may be one or more selected from the group consisting of, but is not limited thereto.

The effective amount of the active ingredient, or the pharmaceutical composition can be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, intranasal administration, intrapulmonary administration, rectal administration, or local administration of a lesion site may be administered. When administered orally, the pharmaceutical composition may be formulated in a dosage form that can coat the active ingredient or protect it from degradation in the stomach so that the active ingredient does not degrade in the stomach.

As used herein, "pharmaceutical effective amount" may mean a content or dosage in a pharmaceutical composition of an active ingredient capable of exhibiting a desired pharmacological effect, a method of formulation, a mode of administration, age, weight of a patient, Factors such as sex, morbidity, food, time of administration, interval of administration, route of administration, rate of excretion, and sensitivity to response may be appropriately determined. For example, the daily or single dose of the active ingredient is 0.0001 to 1000 mg / kg (weight), 0.001 to 500 mg / kg, 0.01 to 100 mg / kg, 0.1 to 50 mg / kg, or 0.5 to 20 mg / kg range, but is not limited thereto. The daily or single dose may be formulated as a single dosage form in unit dose form, or appropriately divided into dosage forms, or may be prepared by incorporating into a multi-dose container.

The pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an aqueous or oily medium, or may be formulated in the form of a powder, granule, tablet or capsule, and additionally a dispersant or stabilizer for formulation. It can contain.

The pharmaceutical composition is administered to humans, primates including humans, monkeys, etc., rodents including mice, rats, etc., mammals including dogs, cats, cows, pigs, sheep, goats, horses, etc., or isolated from the mammals. Cells, tissues, or cultures thereof.

In another example, HER2-HER3 heterodimer is detected in a biological sample isolated from a subject in order to select a suitable subject for administration of a composition for preventing and / or treating a disease related to the HER2-HER3 heterodimer or applying treatment using the same. How to do. Another example is suitable for the administration of a composition for the prevention and / or treatment of the HER2-HER3 heterodimer-related disease or the treatment using the same, comprising detecting a HER2-HER3 heterodimer in a biological sample isolated from a subject. Provides a method for screening objects. When the method detects a HER2-HER3 heterodimer in the biological sample or is detected at a higher level than the normal sample, the subject may be administered or administered with a composition for preventing and / or treating a HER2-HER3 heterodimer-related disease. The method may further include identifying (selecting or deciding) an object suitable for application. The method may further include detecting a HER2-HER3 heterodimer in a normal sample, for comparison, prior to the step of confirming (selecting or determining). The normal sample may be cells without pathological symptoms, such as cells that are not cancer cells. The subject may be a mammal such as a human, and the biological sample may be cells isolated from the subject, for example, cancer cells.

The methods provided herein provide a technique for screening drugs using HER2-HER3 heterodimers, an effective inhibitor of HER2-HER3 heterodimers or diseases associated with HER2-HER3 heterodimers (e.g., HER2-HER3 heterodimers) It can be usefully used for screening candidate drugs for the prevention or treatment of diseases (cancer) associated with cells (cancer cells) expressing.

1A is a schematic diagram showing the process of forming HER2-HER3-eGFP heterodimer by treating NRG1-b1 after expressing HER3-eGFP in SKBR3.
1B is a graph showing that the amount of HER2-HER3-eGFP heterodimer is proportional to the amount of HER3-eGFP present in NRG1-b1 treated SKBR3 cells.
1C is a graph showing the amount of HER2-HER3 heterodimer detected when NRG1-b1 was treated before cell lysis and after cell lysis.
Figure 1d is a schematic diagram showing the process of inducing, extracting, and detecting heterodimers of HER2 and HER3 present in cells by treating NBR1-b1 on SKBR3 cells.
1E is a graph showing the amount of HER2-HER3 heterodimer detected according to the protein concentration added to the substrate.
Figure 1f is a graph showing the amount of HER2-HER3 heterodimer generated when treating ligands (NRG1-b1) to three breast cancer cell lines (SKBR3, BT474, and HCC1419) expressing HER2 and HER3.
Figure 1g is a graph measuring the presence of a dimer using an EGFR antibody, HER2 antibody, or HER3 antibody to confirm that a different type of dimer other than HER2-HER3 exists, such as EGFR-HER2, when NRG1-b1 is treated. to be.
Figure 1h shows the results of measuring the degree of RTK phosphorylation of SKBR3 cells before and after ligand treatment through a phospho-RTK array.
Figure 1i is a schematic diagram showing the process of overexpressing HER2-eGFP in HEK293T cells to form a HER2-eGFP-HER2-eGFP homodimer and attaching it to a substrate (surface treated with PEG, neutravidin, biotin, and anti-HER2 antibody) to be.
Figure 1j shows the results of measuring the fluorescence signal from HER2-eGFP-HER2-eGFP homodimer or HER2-eGFP monomer attached to the substrate in Figure 1i.
1K is a graph showing the ratio of a single molecule fluorescence signal off in a fluorescent signal obtained from a homodimer or monomer in FIG. 1j, and a ratio of off in two steps (dimer) and off (oligomer) in two or more steps.
Figure 1l is a graph showing the results of measuring the degree of dimer production of HER2 and HER3-eGFP according to detergent type.
Figure 1m is a graph showing the results of measuring the phosphorylation degree according to the presence or absence of the labeled surfactant and ligand (EGF) treatment by interaction with eGFP-labeled p85a.
Figure 2a is a schematic diagram showing an exemplary process for measuring the Tyr phosphorylation level of HER2-HER3 heterodimer.
Figure 2b is a graph showing the result of measuring the degree of phosphorylation (pTyr HER3 counts) of HER3 Tyr residue after treatment of ATP and Mg ^{+ 2} to the substrate on which the immobilized HER2-HER3 heterodimer.
2C is a graph showing the results of measuring phosphorylated HER3 Y1289 by adding ATP and Mg2 + after washing HER2-HER3 heterodimer attached to a substrate from cell lysate using digitonin or GDN at various concentrations, respectively.
Figure 2d is a graph showing the results of measuring the phosphorylated HER3 Y1289 after adding ATP and Mg2 + after washing the HER2-HER3 heterodimer attached to the substrate from the cell lysate using a surfactant of the indicated type and concentration, respectively.
Figure 2e is a schematic diagram showing the process of introducing a single position mutation corresponding to EGFR into HER2 and HER3, respectively.
Figure 2f is a graph measuring the degree of activation of HER2-HER3 heterodimer when the single-position mutations described in Figure 2e are HER2 and HER3, respectively.
Figure 2g is a schematic diagram showing an exemplary process for measuring the Tyr phosphorylation level of the HER2-HER2 homodimer.
Figure 2h is a graph showing the result of measuring the degree of phosphorylation (HER2 pTyr counts) of HER2 Tyr residue after treatment of ATP and Mg ^{+ 2} to the substrate on which the immobilized HER2-HER2 homodimer.
Figure 2i is a graph showing the degree of activation of HER2 according to the type of detergent used for washing the HER2-HER2 homodimer-forming cell lysate through the phosphorylation of HER2 Y1196.
3A is a schematic diagram showing that the HER2-HER3 heterodimer attached to the substrate induces interaction with the eGFP-labeled sub-signaling protein when undergoing phosphorylation.
3B is a graph showing the results of measuring the fluorescence signal of a lower signaling protein (ePCGFP-labeled PLCgSH2) induced by one HER2-HER3 heterodimer as actual data of the experiment described in FIG. 3A.
FIG. 3C is a graph showing how much a sub-signaling protein (PLC gamma 1) interacts with a single HER2-HER3 heterodimer in terms of the size of the fluorescent signal and the number of single molecule off events obtained from the fluorescent signal.
FIG. 3D is a graph showing how much a sub-signaling protein (p85a) interacts with a single HER2-HER3 heterodimer in terms of the size of the fluorescent signal and the number of single molecule off events obtained from the fluorescent signal.
Figure 3e is a graph showing how much of the sub-signaling protein (PI3K: p85a-p110a complex) interacts with one HER2-HER3 heterodimer as the size of the fluorescence signal.
Figure 3f is a schematic diagram showing that the HER2 homodimer attached to the substrate induces an interaction with the eGFP labeled sub-signaling protein when undergoing phosphorylation.
FIG. 3G is a graph showing how much a single HER2 homodimer interacts with a low-level signaling protein (PLC gamma 1) as the size of a fluorescence signal and the number of single molecule off events obtained from the fluorescence signal.
3H is a graph showing how much a single HER2 homodimer interacts with a low-level signaling protein (p85 a) as a fluorescence signal and the number of single molecule off events obtained from the fluorescence signal.
Figure 3i is a graph showing how much a single HER2 homodimer interacts with the amount of sub-signaling proteins (PI3K: p85a-p110a complex) as the size of the fluorescence signal and the number of single molecule off events obtained from the fluorescence signal, respectively.
Figure 3j is a schematic diagram showing the dephosphorylation of phosphorylated tyrosine of HER2-HER3 heterodimer attached to the surface by treatment with PTPN1.
The graph on the left of FIG. 3K is a graph showing the results of measuring the degree of phosphorylation according to the time of ATP and Mg2 + treatment on the immunoprecipitated HER2-HER3 heterodimer (measurement of the amount of pY1289 of HER3), and the graph on the right shows the immunoprecipitated HER2 -HER3 heterodimer is treated with ATP and Mg2 + to phosphorylate tyrosine of HER3 and shows the result of measuring the degree of phosphorylation with PTPN1 treatment time (measure the amount of pY1289 of HER3).
FIG. 3L is a schematic diagram expressing simulating the tyrosine signaling system in cells by processing ATP, Mg2 +, PTPN1, and eGFP-labeled p85a at a time on a HER2-HER3 heterodimer attached to a surface.
3M is a graph showing the result of measuring the fluorescent signal obtained from the experiment simulated in FIG. 3 over time.
FIG. 3N is a graph showing the time taken to bind the new p85a to the same HER2-HER3 heterodimer after the first p85a binding obtained in FIG. 3m.
Figure 4a is a graph measuring the phosphorylation of HER3 tyrosine residues over time when treated with ATP and Mg2 + marked on the HER2-HER3 heterodimer.
Figure 4b is a graph showing by measuring the phosphorylation rate of the HER3 Tyr residue while increasing the concentration of ATP treatment to HER2-HER3 heterodimer.
Figure 4c is a graph showing the measurement of the phosphorylation rate of the HER2 Tyr residue while increasing the concentration of ATP treatment using HER2-HER2 homodimer.
Figure 4d is a graph showing the degree of activation of HER2 obtained by pre-treatment of Lapatinib at various concentrations in HER2-HER3 heterodimer and HER2 homodimer, followed by ATP and magnesium chloride according to Lapatinib concentration.
Figure 4e is a graph showing the degree of activation of HER2 obtained by simultaneously treating Lapatinib, ATP, and magnesium chloride on HER2-HER3 heterodimer and HER2 homodimer according to Lapatinib concentration.
Figure 4f is a graph showing the HER3 Y1289 phosphorylation level obtained by treating Lapatinib, ATP, and magnesium chloride with various concentrations of PTPN1 (Protein Tyrosine Phosphatase, Non-receptor type 1) in HER2-HER3 heterodimer according to Lapatinib concentration.
Figure 5a is a schematic diagram showing an exemplary screening process (HER2-HER3 heterodimer (in vitro) assay) of a candidate drug that inhibits phosphorylation of HER2-HER3 heterodimer.
Figure 5b is a graph showing the degree of HER3 Tyr1289 phosphorylation obtained by treating 280 candidate substances from the Tyrosine kinase inhibitor library provided by MCE (MedChemExpress) to HER2-HER3 heterodimer in an amount of 10 uM.
5C is a graph showing the degree of HER3 Tyr1289 phosphorylation obtained by treating 33 compounds selected as having excellent HER3 Tyr1289 phosphorylation inhibitory effect in the results of FIG. 5B to HER2-HER3 heterodimer in an amount of 1 uM.
Figure 5d is a result of Figure 5c in the results of HER3 Tyr1289 phosphorylation inhibitory effect selected with 8 compounds and 12 drugs HER2-HER3 heterodimer positive cell lines HCC-1419, SK-BR-3, and BT-474 with no inhibitory effect , HER2-HER3 heterodimer negative cell lines MCF-7, MDA-MB-231, and shows the results of cell proliferation obtained by treatment with HCC-1954.
5E shows the results of FIG. 5C, after treating the three best drugs (Sapitinib, AEE788, Dacomitinib) with ligands (NRG1-b1) for 1 hour or 18 hours in SKBR3, the degree of activation of cells was varied with various antibodies ( pHER3, pAkt, pErk antibody).
Figure 5f is a graph showing the results of measuring the degree of inhibition of HER3 phosphorylation of the three drugs (Sapitinib, AEE788, Dacomitinib) with the best effect confirmed in the results of Figure 5c.
5g is a graph showing cell viability when lapatinib is treated with SKBR3 according to the presence or absence of a ligand.
5h is a graph showing cell viability in the case of treating dacomitinib with SKBR3 depending on the presence or absence of a ligand.
5i is a graph showing cell viability when CUDC-101 is treated with SKBR3 according to the presence or absence of a ligand.
Figure 5j is a graph showing the cell viability when treated with Sapitinib in SKBR3 depending on the presence or absence of a ligand.
5K is a schematic diagram of an experiment confirming the anti-tumor effect of dacomitinib in SKBR3 xenograft mice.
FIG. 5L is a graph showing the size of tumor tissue measured on the date indicated in FIG. 5K.
Figure 5m is a graph showing the final weight of cancer tissue extracted from mice 20 days after the start of the test when lapatinib and dacomitinib were treated alone (with vehicle) or when lapatinib and dacomitinib were treated with NRG1-b1 to be.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited to the following examples.

Example  One: HER2 - HER3 heterodimer  formation

On the substrate HER2  Attach

HER3-eGFP recombinant gene (HER3: NM_001982.3 BC082992.1, eGFP: MH087225 linker: PTFLYKVVDPVPVAT or GGGSGGGT, vector: pEGFP-N1 addgene, pEGFP-N1-FLAG) linked to eGFP at the end of the intracellular domain of HER3 -Overexpressing SKBR3 breast cancer cells (Korea Cell Line Bank or ATCC) are injected through electroporation to induce overexpression of HER3. 30ug of HER3-eGFP plasmid is transfected into 5X10 ^ 6 SKBR3 cells using electroporation (electroporation conditions: 950V, 35ms, 2 pulses). Incubated in 90 mm culture dish for 1 day (RPMI1640, FBS 10%) to allow SKBR3 to express HER3-eGFP. The existing medium was removed and cultured with FBS-free medium (RPMI1640) for 12-18 hours to remove the influence of unspecified growth factors.

NRG1-b1 (100 ng / ml) was added to the prepared SKBR3 and incubated for 10 minutes to induce heterodimer formation of HER2 protein and HER3-eGFP protein on the cell surface (see FIG. 1A).

The level of HER2-HER3-eGFP heterodimer production according to the expression level of HER3-eGFP was measured by a single-molecule Total Internal Reflection Fluorescence Microscope, and the results are shown in FIG. 1B. As shown in FIG. 1B, HER2-HER3 heterodimer was rarely generated when NRG1-b1 was not treated after introducing HER3-eGFP plasmid into SKBR3, whereas HER2-HER3 heterodimer was treated when NRG1-b1 was treated. It can be seen that it increases in proportion to the expression level of HER3-eGFP. The above results show that NRG1-b1 should be added to generate HER2-HER3 heterodimer.

In addition, when NRG1-b1 was treated before cell lysis and after cell lysis, the level of HER2-HER3 heterodimer generation was measured by a single-molecule Total Internal Reflection Fluorescence Microscope, and the results were measured. It is shown in Figure 1c. Cell lysis was performed using lysis buffer (1% Digitonin or 1% GDN, 10% glycerol, 50mM HEPES-NaOH [pH 7.4], 150mM NaCl, 2% protease inhibitor cocktail (Sigmaaldrich P8340)% concentration all w / v). Was carried out. 1C, when NRG1-b1 was treated after cell lysis (ie, cell lysate was treated with NRG1-b1; Extract + NRG1-b1), and when HER2-HER3 heterodimer was not treated with NRG1-b1 On the other hand, when NRG1-b1 was treated before cell lysis, the level of HER2-HER3 heterodimer production was remarkably high, while little was produced to a similar extent. These results indicate that NRG1-b1 must be administered prior to cell lysis to generate HER2-HER3 heterodimer, and when NRG1-b1 is administered simultaneously with or after cell lysis, HER2-HER3 heterodimer cannot be generated at a significant level. Shows

On the substrate HER3  Attach

1d is a schematic diagram showing the process of inducing, extracting, and detecting heterodimers of HER2 and HER3 present in cells by treating NBR1-b1 on SKBR3 cells (HER3 Ab: Novus Biologicals; Cat # BAM348, HER2 Ab) : Thermo Fisher; Cat # MA5-15050, Cy3-labeled Ab: Jackson ImmunoResearch Labs; Cat # 111-165-046). The amount of HER2-HER3 heterodimer production can be measured by measuring the HER2 immunolabeling count (the number of fluorescence spots measured by a total reflection single-molecule fluorescence microscope) using an anti-HER2 antibody.

Referring to FIG. 1D, the amount of HER2-HER3 heterodimer in NRG1-b1 treatment and no treatment was measured, and the results are shown in FIG. 1E. As shown in FIG. 1E, the amount of HER2-HER3 heterodimer increases as the protein concentration added to the substrate increases when NRG1-b1 is treated, whereas HER2-HER3 heterodimer is irrespective of the added protein concentration when NRG1-b1 is not treated. It can be seen that it is hardly generated.

Three types of breast cancer cell lines expressing HER2 and HER3 (SKBR3, BT474, and HCC1419, obtained from the Korea Cell Line Bank above) were treated with a ligand (NRG1-b1) to induce HER2-HER3 heterodimer production, and HER2-HER3 heterodimer production was generated. After the induced cell lysate was treated with a substrate surface-treated with an anti-HER3 antibody to immunoprecipitate the HER2-HER3 heterodimer, the HER2 immunolabeling count was measured using an anti-HER2 antibody to measure the amount of HER2-HER3 heterodimer production. . The results are shown in Figure 1f. The cell lines SKBR3, BT474, and HCC1419 are HER2 overexpressing cell lines and express HER3 together.

When the cell lysate is attached to a substrate using various types of antibodies, the degree of HER2-HER3 heterodimer production is measured by differently treating the ligand (NRG1-b1) and the type of antibody attached to the substrate according to the process of FIG. 1D. One result is shown in Figure 1g (IP: target molecule of the antibody treated on the substrate; Detection: the target of the antibody used in the detection process of Figure 1d). As shown in Fig. 1g, the amount of EGFR-HER3, EGFR-HER2, HER2-HER3 heterodimer and EGFR-HER3 heterodimer when the ligand is not treated is HER2-HER3 heterodimer when the ligand is treated. Very small compared to the production amount. These results show that HER2-HER3 heterodimer is specifically produced by ligand treatment.

The phospho-RTK array (Phospho-RTK Array Kit; Cat # ARY001B, R & D systems) was used to measure the degree of RTK phosphorylation of SKBR3 cells before and after ligand treatment, and the results are shown in FIG. 1H. The phospho-RTK array is coated with an antibody targeting the substance at each marked position. Each of NRG1-b1 untreated (-) or treated (+) SKBR3 cell extracts is sprinkled on a phospho-RTK array to immunoprecipitate the protein in the array and then treated with phospho-tyrosine antibody to measure the phosphorylation of the protein. have. In FIG. 1H, the darker the spot color, the higher the phosphorylation degree. As shown in FIG. 1h, it can be seen that when NRG1-b1 was treated with SKBR3, the degree of phosphorylation of HER3 increased significantly.

Detergent treatment

Surfactant treatment during cell lysis

The degree of dimer formation of HER2 and HER3-eGFP upon treatment with various types of surfactant was measured by eGFP count, and the results are shown in FIG. 1L. After expressing HER3-eGFP in the HER2 overexpressing cell line SKBR3, cell lysates were obtained using various surfactants (1% (w / v); Digitonin, GDN, CHAPSO, DDM, or TX100) shown in the figure. After the immunoprecipitation of the HER2-HER3 heterodimer to the substrate using an anti-HER2 antibody, the eGFP signal was measured and quantified. Lysis proceeded at a concentration of 1% (w / v). Other surfactants may be used to detect HER2-HER3 heterodimer.

To confirm the phosphorylation activity of the EGFR dimer (homodimer), the degree of phosphorylation of the immunoprecipitated EGFR was measured by interaction with eGFP-labeled p85a (eGFP-p85a), and the results are shown in FIG. 1m. After EGFR-expressing cell line HCC1666 (Korea Cell Line Bank) was treated with EGF (Life Technologies PHG0311L), cell lysates were obtained using the surfactant (1% (w / v); Digitonin or TX100) shown in the figure. After dimerization of the dimer with a substrate using EGFR antibody (Thermo scientific MS-378-B1), ATP (200 uM) and EDTA (1 mM) or Mg2 ⁺ (10 mM) were additionally added to phosphorylation. These results show that the use of Digitonin is not applicable to maintaining the activity of all proteins. When considering with the results of Figure 2d below, it can be seen that the activity of EGFR is maintained even when using different types of detergent, while the activity of HER2-HER3 heterodimer is maintained when using digitonin or GDN. HER2-HER3 heterodimer detection may be performed using other detergents.

cell From lysate  Attached to the substrate HER2 - HER3 heterodimer  Surfactant treatment (washing)

-D-glucoside) 1.00%(w/v), DDM(n-Dodecyl

-D-maltoside) 0.01%(w/v), 및 TX100 0.10%(w/v)를 각각 사용하여 세척한 후, HER3의 Y1289의 인산화 정도를 전반사 단분자 형광현미경 (single-molecule Total Internal Reflection Fluorescence Microscope)으로 측정하여, 그 결과를 도 2d에 나타내었다. After the cell lysis as described above, the cell lysate was treated on a substrate to attach a HER2-HER3 heterodimer to the substrate and washed with detergent. To test the degree of HER3 activation according to detergent type, DGTN (digitonin) 0.10% (w / v), GDN 0.01% (w / v), CHAPSO (3-([3-Cholamidopropyl] dimethylammonio) -2 -hydroxy-1-propanesulfonate) 1.00% (w / v), OG (n-octyl-

-D-glucoside) 1.00% (w / v), DDM (n-Dodecyl)

-D-maltoside) After washing with 0.01% (w / v) and TX100 0.10% (w / v), the phosphorylation degree of HER3 Y1289 is totally reflected single-molecule Total Internal Reflection Fluorescence. Microscope), the results are shown in Figure 2d.

As shown in Figure 2d, when washing the HER2-HER3 heterodimer attached to the substrate, when using digitonin or GDN as a detergent, the phosphorylation level of Y1289 of HER3 appeared to be high, whereas when using CHAPSO, OG, DDM, and TX100 It can be seen that appears low.

In addition, in order to test the degree of HER3 activation according to the concentration of detergent, typically, digitonin and GDN are used in various concentrations to determine the phosphorylation level of Y1289 in HER3 when washing cell lysates, total reflection single-molecule fluorescence microscopy Internal Reflection Fluorescence Microscope), the results are shown in Figure 2c. In FIG. 2c, it can be seen that the magnitude of phosphorylation occurring after the immunoprecipitation rapidly changes before and after the concentrations of the CMC (Critical Micelle Concentration) of Digitonin and GDN, respectively, indicated by dotted lines. From these results, it can be said that the activity of the immunoprecipitated dimer is associated with the cell membrane permeation region protected by the surfactant (because, at a concentration lower than CMC, the surfactant does not form micelles, so that the hydrophobic cell membrane is not formed). This is because it cannot cover the transmission area). The substrate described on the right side of FIG. 2C shows that SKBR3 cells were lysed with Digitonin or GDN at a concentration of 1% (w / v), followed by immunoprecipitation; Washing the immunoprecipitated HER2-HER3 heterodimer with various concentrations of surfactant (one point in the graph is an independent experiment with washing with the corresponding concentration of surfactant); It means that phosphorylation is induced by adding ATP and Mg2 + to a solution in which a surfactant having a CMC or higher (Digitonin is 0.1% (w / v), GDN is 0.01% (w / v)) is added. As shown in Figure 2c, in the case of digitonin, when the cell cleaning concentration is about 0.05% (w / v) or more, the degree of HER3 activation is excellent, and when it is lower, the degree of activation decreases. have.

In addition, ATP and Mg2 + were added after washing the HER2-HER3 heterodimer attached to the substrate from cell lysates with surfactants of various types and concentrations (see FIG. 2D), respectively, and phosphorylated HER3 Y1289 was measured. The results are shown in Figure 2d. In all cases, it was carried out above the CMC of the surfactant. The phosphorylation was measured with reference to Example 3 and Figure 2a below. It can be seen from the results shown in FIG. 2D that the activity of the immunoprecipitated HER2-HER3 heterodimer can be maintained only when using a specific surfactant (Digitonin, GDN). The right substrate of FIG. 2D shows that after the SKBR3 cells were lysed with each surfactant at a concentration of 1% (w / v), immunoprecipitation was performed; Washing the immunoprecipitated HER2-HER3 heterodimer with various types of surfactants (one point in the graph is an independent experiment with washing at the corresponding concentration); It means that phosphorylation is induced by adding ATP and Mg2 + to a solution added with Digitonin above CMC.

HER2 - HER3 heterodimer  Substrate attachment

In this example, the process of attaching the HER2-HER3 heterodimer to the substrate is as follows: After surface treatment of PEG, neutravidin, biotin, and anti-HER3 antibody on the substrate (see below), cell lysate is injected. After attaching the HER2-HER3 heterodimer to the substrate surface, wash with digitonin (0.1% (w / v)).

(1) PEG surface treatment

a. The coverslip and quartz slide were reacted with sulfuric acid and hydrogen peroxide 2: 1 solution (piranha solution) for 30 minutes. When the reaction was over, neutralized in 18 MΩ distilled water for 10 minutes.

b. The neutralized coverslip and slide were washed at least 5 times in 18 MΩ distilled water.

c. The coverslip and slide were reacted with aminosilane solution (3ml N- [3- (Trimethoxysilyl) propyl] ethylenediamine, 5ml acetic acid, 92ml methanol) for 20 minutes, and when the reaction was completed, it was washed twice with methanol and completely submerged in methanol.

d. The prepared coverslip and slide were dried using compressed nitrogen gas. A PEG buffer (42 mg sodium bicarbonate, 347.5 mg potassium sulfate was dissolved in 5 ml 18 MΩ distilled water) was prepared.

e. After applying a PEG solution (3 mg biotin-PEG, 100 mg mPEG, at 1 ml of PEG buffer) to the slide, cover it with a coverslip and evenly bury the PEG solution between the slide and coverslip, and react for 2 hours.

f. After the reaction, the coverslip and slide were washed with 18 MΩ distilled water and dried with compressed nitrogen gas. The completed coverslip and slide were placed in a 50ml tube to prevent contact with the PEG solution treated surface, and then vacuum packed and frozen (-20 ℃). There was no contact with the surface treated with the PEG solution on the coverslip and slide.

(2) Assembly and use

a. The frozen coverslip and slide were melted in a 37 ℃ incubator to prevent water droplets from forming.

b. A reaction chamber was created between the coverslip and the slide using double-sided tape and epoxy, so that the sides of the PEG-treated coverslip and slide face each other.

c. When the epoxy was sufficiently hardened, a neutravidin solution (100 ug / ml neutravidin, PBS or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) was added to the chamber and reacted for 5 minutes.

d. The neutravidin solution remaining in the chamber was washed with PBS and biotin conjugated anti-HER2 antibody (eBioscience, Cat.No.BMS120BT, clone 2G11) or anti-HER3 antibody (R & D systems, Cat.No.BAM348, clone: # 66201) (2 ~ 5ug / ml, PBS or 0.1% Triton-X-100, 50mM HEPES [pH 7.4], 150mM NaCl) was added and reacted for 5 minutes.

e. The antibody remaining in the chamber was washed with PBS, and the cell lysate was injected, followed by reaction for 15 minutes.

f. The lysate remaining in the chamber was washed with washing buffer (0.1% digitonin, or 0.01% GDN, 50mM HEPES [pH 7.4], 150mM NaCl, 1% glycerol).

Example  2. HER2 - HER2 homodimer  formation

Referring to the method of Example 1, the HER2-eGFP recombinant gene linked to eGFP at the end of the intracellular domain of HER2 (HER2: NM_004448, linker PTFLYKVVDPVPVAT or GGGSGGGT, eGFP: MH087225, vector: pEGFP-N1 addgene pEGFP-N1-FLAG) Was injected into the HEK293T cell line (Korea Cell Line Bank, or ATCC) through electroporation to induce overexpression of HER2, and induced the formation of HER2-HER2 homodimer (also referred to as HER2 homodimer) on the cell surface. After lysis of the prepared HER2 homodimer-forming cells, the substrate was treated and attached to the HER2 homodimer. The process of generating the HER2-HER2 homodimer and attaching the substrate is schematically shown in FIG. 1I.

With reference to the procedure of Example 1, after surface treatment of PEG, neutravidin, biotin, and anti-HER2 antibody on a substrate, cell lysate was injected, and after attaching the HER2 homodimer to the substrate surface, the DGTN (digitonin) 0.1% (w / v).

Fluorescence signals from the HER2-eGFP-HER2-eGFP homodimer or HER2-eGFP monomer attached to the substrate were measured by total reflection fluorescence microscopy (TIRF), and the results are shown in FIG.

In addition, in the fluorescent signal obtained from the homodimer or monomer in FIG. 1j, a single molecule fluorescent signal off phenomenon was observed, and the ratio of the dimer in two stages and the oligomer in two or more stages was obtained, and is shown in FIG. 1k. . When a single molecule fluorescent signal is turned off, the fluorescent molecule is denatured and the fluorescent signal does not appear when the fluorescent signal is continuously obtained by continuously shining an excitation laser on the fluorescent molecule. If present (for example, a dimer or more multimer), a fluorescence signal that decreases in a staircase form at one position can be observed, and by counting the number of steps, the number of fluorescence molecules concentrated at the corresponding position can be seen.

In FIG. 1K, when the fluorescence intensity decreases in 1-step corresponds to a monomer, and when the fluorescence signal decreases in 2-step corresponds to a dimer. In FIG. 1K, since the ratio of 2-step of HER2-eGFP is higher than that of eGFP and HER3-eGFP, which are relatively likely to exist as monomers, HER2-eGFP homodimer production can be demonstrated. Based on the Hidden Markov Model, the most probable step from the raw data (the step where the recursive calculation converges) was determined and analyzed.

The attached HER2 homodimer was washed with detergent. Referring to the test procedure of Example 1, the phosphorylation level (degree of activation) of HER2 Y1196 obtained as a result of washing the attached HER2 homodimer using various detergents in various concentrations is shown in FIG. 2i. As shown in Figure 2i, using DGTN (digitonin) 0.05% (w / v) or more or 0.10% (w / v) or more as detergent, or GDN 0.01% (w / v) or more to attach attached HER2 homodimer When washed, it was found that the degree of HER2 activation was high.

Example  3. HER2 - HER3 heterodimer  tyrosine kinase  Activity measurement

The immunoprecipitation add HER2-HER3 heterodimer ATP and Mg ² to the reaction chamber of (Example 1 HER2-HER3 heterodimer bound to the antibody treatment to the substrate in) ^+, which was induced, Tyr phosphorylated by incubation for 5 minutes. Subsequently, single-molecule immunolabeling with pTyr-specific antibodies was performed to observe changes in HER3 pTyr levels. The test procedure is schematically shown in FIG. 2A (1. Cell lysis, 2. Dimer attachment to the substrate, 3. Washing the attached dimer, 4. Phosphorylation by treating ATP and Mg2 +, 5. Tyr binding of HER3 or HER2 phosphorylation Antibody treatment, 6. Fluorescence signal detection), the specific test method is as follows:

In the assembly and use of (2) of Example 1, referring to the method described with biotin conjugated HER3 antibody, pull-down HER2-HER3 heterodimer from the cell lysate (1-4 mg / ml) to the surface, 0.1 Wash the remaining cell lysate with% DGTN or 0.01% GDN, 50mM HEPES [pH 7.4], 150mM NaCl, 1% glycerol, and kinase assay solution (0.1% DGTN or 0.01% GDN, 200uM ATP, 10mM magnesium chloride, 50mM HEPES [pH 7.4], 150 mM NaCl, 1% glycerol) was reacted for 5 minutes.

b. The remaining kinase assay solution is washed with PBS (or 0.1% Triton-X-100, 50mM HEPES [pH 7.4], 150mM NaCl), and a HER3 pTyr antibody solution (HER3 pTyr antibody (monoclonal rabbit host) (Cell Signaling Technology, Cat. No. # 4791S, clone: 21D3) ~ 2ug / ml, PBS or 0.1% Triton-X-100, 50mM HEPES [pH 7.4], 150mM NaCl) was reacted for 5 minutes.

c. The remaining HER3 pTyr antibody solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl), cy3 conjugated anti-rabbit FC antibody solution (polyclonal goat host anti-rabbit FC antibody (Jackson ImmunoResearch Labs, Cat.No. 111-165-046) at 5nM, PBS or 0.1% Triton-X-100, 50mM HEPES [pH 7.4], 150mM NaCl) and reacted for 5 min.

d. The remaining cy3 conjugated anti-rabbit FC antibody solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) and imaged and quantified using Total Internal Reflection Microscope.

The HER3 pTyr levels thus obtained are shown in Figure 2b. There are a total of 9 Tyr residues in the HER3 tail, and in this example, a total of 5 Tyr residues of Y1197, Y1222, Y1276, Y1289, and Y1328 were tested. As shown in Fig. 2b, the degree of phosphorylation was increased by ATP and Mg2 ⁺ culture for both pTyr residues.

From the cell lysate (lysed and immunoprecipitated using 1% digitonin or 1% GDN), the HER2-HER3 heterodimer attached to the substrate was washed with various concentrations of digitonin or GDN, respectively, and then ATP and Mg2 + were added. , Phosphorylated HER3 Y1289 was measured, and the results are shown in FIG. 2C. In addition, after washing the HER2-HER3 heterodimer attached to the substrate from the cell lysate (lysed and immunoprecipitated using 1% digitonin) using surfactants of the type and concentration shown in FIG. 2D, ATP and Mg2 + were added. After addition, phosphorylated HER3 Y1289 was measured, and the results are shown in FIG. 2D. As shown in Figure 2c and 2d, when using digitonin or GDN as a surfactant used for the HER2-HER3 heterodimer attached to the substrate, it can be confirmed that the degree of phosphorylation of HER2-HER3 heterodimer is markedly high.

2E is a schematic diagram showing the process of introducing EGFR and a corresponding single-position mutation into HER2 and HER3, respectively, to show that the HER2-HER3 heterodimer follows the same activation mechanism previously known by the EGFR homodimer. When the 714th Isoleucine (I) of HER2 is transformed into Glutamine (Q) (I714Q), the phosphatase of HER2 cannot be activated by other EGFR family proteins. When the 956th Valine (V) of HER2 is transformed to Arginine (R) (V956R), HER2 cannot activate the phosphatase of other EGFR family proteins. When the 926th Valine (V) of HER3 is changed to Arginine (R) (V926R), HER3 cannot activate the phosphatase of other EGFR family proteins.

As described above, when the single position mutations described in FIG. 2E were introduced into HER2 and HER3, respectively, the degree of activation of HER2-HER3 heterodimer was measured for the degree of phosphorylation of Y1289 of HER3, and the results are shown in FIG. 2F. In HER2-HER3 heterodimer, HER2-HER3 heterodimer phosphorylation is not inhibited even if V956R mutation exists, whereas in HER3, V926R mutation, phosphorylation is inhibited because HER3 cannot activate HER2 phosphatase. , Likewise, when there is an I714Q mutation in HER2, it can be confirmed that the phosphorylation function is inhibited because HER2 phosphatase cannot be activated by HER3. These results confirm that the immunoprecipitated HER2-HER3 heterodimer follows the activation mechanism of the previously known EGFR family.

Example  4. HER2 - HER3 heterodimer  And HER2 - HER2 homodimer  Measurement of interaction with lower signaling proteins

3A is a schematic diagram showing that the HER2-HER3 heterodimer attached to the substrate induces interaction with the eGFP-labeled sub-signaling protein when undergoing phosphorylation.

According to the test procedure described in FIG. 3A, the fluorescence signal of the lower signaling protein induced by one HER2-HER3 heterodimer was observed. More specifically, after phosphorylation of the immunoprecipitated HER2-HER3 heterodimer (trace indicated by ② in FIG. 3B) or after phosphorylation (trace indicated by ① in FIG. 3B), eGFP is labeled as described in the above example. HER2-HER3 by treatment with PLCGSH2 (PLC-gamma-SH2) (attaching eGFP (MH087225) by cloning 545-765 amino acids from PLC-gamma (NM_013187.1) or 540-765 amino acids from NP_002651.2) The interaction between heterodimer and PLCgSH2 was measured by fluorescence intensity, and the results are shown in FIG. 3B. The graph of FIG. 3b shows the fluorescence signal of PLCgSH2 bound to a single HER2-HER3 heterodimer over time, and when PLCgSH2 is treated after phosphorylation, multiple PLCgSH2 is bound to a heterodimer and a single fluorescent molecule is turned off (Example 2, 1K).

3C is a graph showing how much a single HER2-HER3 heterodimer interacts with a low-level signaling protein (PLC gamma 1) in terms of the size of the fluorescent signal and the number of single molecule off events obtained from the fluorescent signal.

Figure 3d shows how much a single HER2-HER3 heterodimer interacts with the low signaling protein (p85 a; Human p85a (NP_852664.1)) as the size of the fluorescence signal and the number of single molecule off events obtained from the fluorescence signal. It is a graph showing.

FIG. 3e is a graph showing how much a single HER2-HER3 heterodimer interacts with a sub-signaling protein (PI3K: p85 a-p110 a (NM_006218.3) complex) in the size of a fluorescence signal.

A single fluorescent molecule is denatured without fluorescence by a certain probability. If several fluorescent molecules are clustered in one place over time, multiple fluorescent molecules are turned off with a certain probability, so it can be observed that they are turned off at random rather than all at once. By measuring this signal using an electronically amplified charge-coupled device camera, it is possible to obtain several staircase-shaped signals. Counting the number of steps (the number of single fluorescent molecules turned off) reveals how many fluorescent molecules are concentrated in one place.

3C, 3D, and 3E, the top two graphs measure the intensity of fluorescence when eGFP-labeled PLCgSH2, p85a, or PI3Ks bind to a heterodimer (left before phosphorylation and right after phosphorylation). The graph below shows the count of the number of single fluorescent molecule off after measuring the fluorescent signal emitted when eGFP-labeled PI3Ks are bound to heterodimer over time.

Figure 3f is a schematic diagram showing that the HER2 homodimer attached to the substrate induces an interaction with the eGFP labeled sub-signaling protein when undergoing phosphorylation.

FIG. 3G is a graph showing how much a single HER2 homodimer interacts with a low-level signaling protein (PLC gamma 1) as the size of a fluorescence signal and the number of single molecule off events obtained from the fluorescence signal.

3H is a graph showing how much a single HER2 homodimer interacts with a low-level signaling protein (p85 a) as a fluorescent signal and the number of single molecule off events obtained from the fluorescent signal.

Figure 3i is a graph showing how much a single HER2 homodimer interacts with the amount of sub-signaling proteins (PI3K: p85a-p110a complex) as the size of the fluorescence signal and the number of single molecule off events obtained from the fluorescence signal, respectively.

In Figures 3f, 3g, and 3h, the top two graphs measure the intensity of fluorescence when eGFP-labeled PLCgSH2, p85a, or PI3Ks bind to the homodimer (left before phosphorylation, right after phosphorylation). The graph at the bottom shows the result of counting the number of single fluorescence molecules off after measuring the fluorescence signal generated when eGFP-labeled PLCgSH2, p85a, or PI3Ks are bound to the homodimer.

Figure 3j is a schematic diagram showing the process of dephosphorylation by treating PTPN1 with phosphorylated tyrosine of a HER2-HER3 heterodimer attached to a surface. It is known that phosphorylation of HER2-HER3 heterodimer occurs in cells, and at the same time, dephosphorylation by dephosphorylation enzymes is also performed. Phosphorylation is performed by treating ATP and Mg2 + with immunoprecipitated HER2-HER3 heterodimer, and processing dephosphorylation enzyme PTPN1 (Protein Tyrosine Phosphatase, Non-receptor type 1; ProSpecbio, Cat.No.PKA-219)) And the mechanism of dephosphorylation.

The graph on the left in FIG. 3K shows the result of measuring the degree of phosphorylation according to the time of ATP and Mg2 + treatment on the immunoprecipitated HER2-HER3 heterodimer (measured on the amount of pY1289 of HER3), and the graph on the right is immunoprecipitated ATP and Mg2 + are treated with HER2-HER3 heterodimer to phosphorylate tyrosine of HER3, and the results of measuring the phosphorylation level over time with PTPN1 treatment are shown (measure the amount of pY1289 in HER3).

FIG. 3L is a schematic diagram showing that ATP, Mg2 +, PTPN1, and eGFP-labeled p85a are attached to a HER2-HER3 heterodimer attached to a substrate to simulate the tyrosine signaling system in cells.

Figure 3m is a graph showing the results of measuring the fluorescent signal obtained from the experiment simulated in Figure 3 over time. After immunoprecipitation of HER2-HER3 heterodimer, ATP, Mg2 +, PTPN1, and eGFP-labeled p85a were treated at once, and then eGFP fluorescence signal generated from p85a bound to heterodimer was measured over time. In FIG. 3M, the number indicated in the trace represents the number of signal steps that increase when p85a is bound to a heterodimer, and represents the number of p85a bound to one heterodimer. The time taken for p85a to bind after the first p85a was expressed as delta (Δ) _t.

FIG. 3N is a graph showing the time taken to bind new p85a to the same HER2-HER3 heterodimer after the first p85a binding obtained in FIG. 3m. Compared to the phosphorylation and dephosphorylation over time measured in FIG. 3K and the phosphorylation and dephosphorylation rates obtained from the graph, it appears that new p85a binds after a relatively long time after the first p85a bond.

From the above results, it can be seen that the combination of phosphorylation, dephosphorylation, and p85a in the experiment devised in FIG. 3L is not sequential (ie, heterodimer phosphorylates several given tyrosine positions, and PTPN1 dephosphorylates phosphorylated tyrosine). Phosphorylation, and p85a binds to the remaining phosphorylated tyrosine).

Example  5. HER2 - HER2 homodimer  tyrosine kinase  Activity measurement

The immunoprecipitation add-HER2 HER2 heterodimer ATP and Mg ² to the reaction chamber of (Example 2-HER2 HER2 homodimer binding to the antibody treatment to the substrate in) ^+, which was induced, Tyr phosphorylated by incubation for 5 minutes. Subsequently, single-molecule immunolabeling with pTyr-specific antibodies was performed to observe changes in HER2 pTyr levels. The test procedure is schematically shown in FIG. 2G, and the specific test method is as follows:

a. In (2) assembly and use of Example 1, with reference to the method described using biotin conjugated HER2 antibody, after pulling down the HER2 homodimer from the cell lysate to the surface, 0.1% DGTN or 0.01% GDN 50mM HEPES [pH 7.4], 150mM NaCl, 1% glycerol to wash off the remaining cell lysate, kinase assay solution (0.1% DGTN or 0.01% GDN, 200uM ATP, 10mM magnesium chloride, 50mM HEPES [pH 7.4], 150mM NaCl, 1% glycerol) and reacted for 5 minutes.

b. The remaining kinase assay solution is washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl), and a HER2 pTyr antibody solution (HER2 pTyr antibody (monoclonal rabbit host) (Cell Signaling Technology, Cat. No. # 6942S, clone: D66B7) ~ 2ug / ml, PBS or 0.1% Triton-X-100, 50mM HEPES [pH 7.4], 150mM NaCl) was reacted for 5 minutes.

c. The remaining HER2 pTyr antibody solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl), cy3 conjugated anti-rabbit FC antibody solution (polyclonal goat host anti-rabbit FC antibody (Jackson ImmunoResearch Labs, Cat.No. 111-165-046) at 5nM, PBS or 0.1% Triton-X-100, 50mM HEPES [pH 7.4], 150mM NaCl) and reacted for 5 min.

d. The remaining cy3 conjugated anti-rabbit FC antibody solution was washed with PBS (or 0.1% Triton-X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl) and imaged and quantified using Total Internal Reflection Microscope.

The HER2 pTyr levels thus obtained are shown in Figure 2h. As shown in Fig. 2h, phosphorylation increased by ATP and Mg2 ⁺ culture in almost all of the Tyr residues Y1139, Y1196, Y1121 / 1222, and Y1248 of the HER2 tail (increased phosphorylation: in the case of Y1139, the control group (ATP + EDTA ) About 23 times; for Y1196, about 13 times for the control, for Y1121 / 1222, about 10 times for the control, and for Y1248, about 13 times for the control).

Example  6. HER2 - HER3 with heterodimer HER2 - HER2 homodimer  Phosphorylation rate comparison

Referring to the method of Example 3 using HER2-HER3 heterodimer, the phosphorylation degree of HER3 Y1289 with time was measured, and it is shown in FIG. 4A. It is known that HER2-HER3 heterodimer strongly induces proliferation of cells, but the enzymatic properties are unknown. 4A can be said to measure the enzymatic performance of the HER2-HER3 heterodimer (shows how quickly the HER2-HER3 heterodimer proceeds to phosphorylation at a specific concentration of ATP, a fuel used for phosphorylation). In FIG. 4A, the x-axis represents the time taken for phosphorylation, and the y-axis represents the amount of Tyr phosphorylated during the corresponding time.

In addition, referring to the method of Example 3 using a HER2-HER3 heterodimer, the phosphorylation rate of the HER3 Tyr residue was measured while increasing the ATP concentration. This is to find out how the heterodimer phosphorylation rate changes depending on the concentration of ATP. By measuring the phosphorylation rate at various ATP concentrations, K _M (Mycalis constant) and k _{cat; max} (maximum phosphorylation rate) can be measured as measures of Kinase performance. K _M is the ATP concentration at which the phosphorylation rate is half the maximum phosphorylation rate, and when the phosphorylation rate is measured at the ATP concentration corresponding to KM, half of the maximum phosphorylation rate is obtained, and k _{cat; max} is the kinase at the maximum phosphorylation rate. This is the maximum phosphorylation rate possible.

The obtained result is shown in Fig. 4B (dotted line indicates the value where the red line converges). The data in FIG. 4B mainly measured Tyr1289 of HER3, and similar levels of phosphorylation rates were measured when other Tyrs (1197, 1222, 1276, 1328) were measured, so the measured values are not specific to a specific Tyr. Can be seen. As shown in Figure 4b, as the ATP concentration increases, it can be seen that the phosphorylation rate of the HER3 Tyr residue also increases.

For comparison, referring to the procedure of Example 4 using a HER2-HER2 homodimer, the phosphorylation rate of HER2 Tyr residue (Y1196), K _M , and k _{cat; max} was measured while increasing the ATP concentration, and shown in FIG. 4C. Did.

As can be seen from the comparison of FIGS. 4B and 4C, the kcat; max of HER2-homodimer was found to be about 150 times lower than that of HER2-HER3 heterodimer, and the phosphorylation rate of HER2-HER3 heterodimer was 100 times higher than that of HER2-homodimer. It means fast. On the other hand, K _M of phosphorylation in HER2-HER3 heterodimer shows a value of 2 times or less compared to HER2-homodimer, and it can be seen that there is no significant difference in the effect of ATP between the two.

Example  7. HER2 - HER3 with heterodimer HER2 - HER2 homodimer on lapatinib  Resistance comparison

After treating the HER2 inhibitor Lapatinib with the HER2-HER3 heterodimer attached to the substrate in Example 1 and the HER2 homodimer attached to the substrate in Example 2, respectively, referring to the procedures of Examples 3 and 4, ATP (100 uM) and Magnesium chloride (10 mM) was treated (at this time, DGTN should be maintained at 0.1% and GDN should be maintained at 0.01%), and the degree of activation of each HER2 dimer was measured. The obtained results are shown in Fig. 4D. In FIG. 4D, the x-axis represents the concentration of Lapatinib used, and the y-axis represents the standardized degree of phosphorylation. Activation of HER2-HER3 heterodimer was measured using HER3 pY1289 antibody and normalized with 100% when Lapatinib was absent, and activation of HER2 homodimer was measured with HER2 pY1196 antibody with 100% when Lapatinib was absent. This is a standardized value. As shown in Fig. 4d, it was confirmed that Lapatinib showed a similar inhibition curve for HER2-homodimer and HER2-HER3 heterodimer that are not resistant to Lapatinib.

In Example 1, the HER2-HER3 heterodimer attached to the substrate and the HER2 homodimer attached to the substrate in Example 2 were treated with Lapatinib, ATP (100 uM), and magnesium chloride (10 mM) (DGTN was also 0.1%). , GDN should be maintained at 0.01%), measuring the degree of activation of HER2, the results are shown in Figure 4e. As shown in Figure 4e, compared to HER2 homodimer, in the case of HER2-HER3 heterodimer, compared to HER2 homodimer, the inhibitory effect by Lapatinib was shown to be low. These results show that HER2-HER3 heterodimer expressing cells are resistant to Lapatinib in the presence of ATP and magnesium (if Tyr phosphorylation is possible). In Example 5, since it was confirmed that the phosphorylation rate of HER2-HER3 heterodimer was significantly faster than that of HER2-homodimer, it can be said that the phosphorylation rate is related to the resistance to the inhibitor (Lapatinib) (i.e., the phosphorylation rate is The faster it is, the better the resistance of the inhibitor can be seen).

To more clearly confirm the correlation between phosphorylation rate and inhibitor resistance, in HER2-HER3 heterodimer expressing cell lysate, with Lapatinib, ATP (100uM), and magnesium chloride (10mM) (at this time DGTN is also 0.1%, GDN should be maintained at 0.01%), PTPN1 (Protein Tyrosine Phosphatase, Non-receptor type 1; ProSpecbio, Cat.No.PKA-219) was treated, and phosphorylation level of HER3 Y1289 was measured, and is shown in FIG. 4F. . PTPN1 is a dephosphorylation enzyme, which forms an equilibrium between Tyr phosphorylation and the dephosphorylation process, and serves to effectively slow down the actual phosphorylation rate without structurally changing the HER2-HER3 heterodimer. As shown in Fig. 4f, as the treatment concentration of PTPN1 increases (from the red graph (rightmost) to the purple graph (leftmost) in Fig. 4f), the actual phosphorylation rate slows, and the effect of inhibiting phosphorylation by Lapatinib It can be seen that it increases (ie, phosphorylation is inhibited even at a lower concentration of Lapatinib).

Example  8. HER2 - HER3 heterodimer  Examples of screening for drugs that inhibit phosphorylation

The screening process (HER2-HER3 heterodimer (in vitro) assay) of a candidate drug that inhibits phosphorylation of HER2-HER3 heterodimer is schematically shown in FIG. 5A.

If the HER2-HER3 heterodimer (in vitro) assay is described in more detail, referring to the process of Example 3, the cell lysate containing HER2-HER3 heterodimer prepared in Example 1 in the well (NRG1-b1 treated SKBR3 extract; concentration) Is 1mg / ml, 10 ul per well is injected) to attach HER2-HER3 heterodimer, and 0.1% DGTN or 0.01% GDN 50mM HEPES [pH 7.4], 150mM NaCl, 1% glycerol is used to dissolve the remaining cell lysate. Wash off. Phosphorylation by adding ATP (100uM) and magnesium chloride (10mM) to the HER2-HER3 heterodimer attached to the well (DGTN should be maintained at 0.1% and GDN at 0.01%), HER3 pTyr antibody (pTyr detection) Phosphorylation level was measured using. At this time, the candidate substance of the drug to be screened (used in an amount of 10 uM) was added together with ATP and magnesium chloride during phosphorylation.

As the candidate substance, 280 compounds of Tyrosine kinase inhibitor library (https://www.medchemexpress.com/screening/Protein_Tyrosine_Kinase_Compound_Library.html) provided by MCE (MedChemExpress) were used.

The phosphorylation level of HER3 Tyr1289 when measuring the 280 candidate substances in an amount of 10uM is measured and shown in FIG. 5B, and HER3 Tyr1289 phosphorylation is suppressed by 75% or more based on the drug-free treatment (ie, the degree of phosphorylation is a drug When not treated, 33 compounds were selected (25% or less). The phosphorylation inhibition value of 75% or more is a value determined in consideration that the maximum value of the measurement error is 23%, and may be determined differently for each test.

Except for using the 33 selected compounds at a concentration of 1 uM, the same test as the HER2-HER3 heterodimer (in vitro) assay described above was performed to measure the phosphorylation level of HER3 Tyr1289, and the results are shown in FIG. 5C. .

In FIG. 5C, 8 compounds showing reproducibility among 12 compounds that inhibited HER3 Tyr1289 phosphorylation by 75% or more when not treated with drugs were shown in red (Sapitinib, AEE788, Dacomitinib, PD153035, CUDC-101, PD158780, Pelitinib, and Saracatinib).

All eight compounds selected as the final candidates are tyrosine kinase inhibitors for EGFR.

To confirm the inhibitory (killing) effect of the selected compound on cells expressing HER2-HER3 heterodimer, NRG1-b1 (100 ng / ml) and a candidate compound (1 uM) were treated with each cell line and cultured for 3 days. After that, the survival rate of the cell line was measured by MTS assay. As the cell line, HCC-1419, SK-BR-3, and BT-474, which are cell lines expressing HER2-HER3 heterodimer well, and MCF-7, MDA-MB-231, which are cell lines that do not express HER2-HER3 heterodimer well , And HCC-1954, respectively. All of these cell lines were obtained from ATCC. The HCC-1419, SK-BR-3, and BT-474 cell lines show responsiveness to HER2 target treatment with the HER2 positive cell line, but when HER2-HER3 heterodimer is formed, the HER2 target treatment does not exhibit an effect. In this example, the cell line was treated with NRG1-b1 to induce HER2-HER3 heterodimer production and was used in the test. On the other hand, MCF-7 and MDA-MB-231 are HER2 negative and HER2 target treatment is not effective, HCC-1954 is a HER2 positive cell line, but there is a mutation in the lower signaling protein, so HER2 target treatment does not show effect. Does not. Cell lines of these three species were used as negative controls because HER2-HER3 heterodimer was not expected to play an important role.

The results of the MTS assay obtained by processing the candidate compounds in the cell lines (normalized by measuring the cell proliferation signal; the case where the candidate compounds are not treated are set to 100) are shown in FIG. 5D. As shown in Figure 5d, all of the eight compounds selected in Figure 5c selectively kills HER2-HER3 heterodimer positive cancer cells (HCC-1419, SK-BR-3, and BT-474), while other compounds All showed no selective killing effect on HER2-HER3 heterodimer positive cancer cells. These results show that HER2-HER3 heterodimer can be used to effectively screen therapeutic drugs that have specific therapeutic effects on specific cancers.

After treating the three drugs (Sapitinib, AEE788, Dacomitinib) with the ligand (NRG1-b1) for 1 hour or 18 hours with the ligand (NRG1-b1), the most effective effect was confirmed in the results of FIG. It was measured by immunoblot using antibodies (pHER3, pAkt, pErk antibodies), and the results are shown in Fig. 5E. Immune blots using HER3, Akt, Erk, and GAPDH antibodies were used as controls. The antibodies used were as follows:

HER3 Ab: ab32121 (abcam)

pHER3pY1289 Ab: 4791L (Cell Signaling Technology)

Akt Ab: 2920S (Cell Signaling Technology)

pAkt Ab: 4060T (Cell Signaling Technology)

Erk Ab: 4695S (Cell Signaling Technology)

pErk Ab: 9101S (Cell Signaling Technology)

GAPDH Ab: 2118S (Cell Signaling Technology).

Under the conditions of time and 18 hours, it was confirmed that all three drugs had excellent inhibitory effects on Akt phosphorylation and pErk phosphorylation, and among them, Dacomitinib showed the most excellent inhibitory effect on Akt phosphorylation and pErk phosphorylation.

In the results of FIG. 5c, the inhibition of HER3 phosphorylation (HER3 pY1289) of the three drugs (Sapitinib, AEE788, Dacomitinib) with the best effect was measured, and the results are shown in FIG. 5f. The phosphorylation change of HER3 Tyr1289 by HER2-HER3 heterodimer attached to the substrate was measured according to the concentration of drug treatment. Figure 5f shows the result of measuring the amount of increased HER3 pY1289 after immunoprecipitating the HER2-HER3 heterodimer, and then treating ATP, Mg2 + and the specified drug together at a concentration corresponding to each point.

5g to 5j show a case where lapatinib (Comparative Example; FIG. 5G), dacomitinib (FIG. 5H), CUDC-101 (FIG. 5i), and Sapitinib (FIG. 5j) were treated with SKBR3 with and without ligand (NRG1-b1). It is a graph showing the cell viability of. As shown in Fig. 5g, when NRG1-b1 is treated, the signal of HER2-HER3 heterodimer is enhanced, anti-cancer resistance occurs, and thus, the anti-cancer (cancer cell killing) effect may be reduced in the case of the existing HER2 targeted therapy, lapatinib. have. However, as shown in FIGS. 5H to 5J, NRG1-b1 is treated to induce HER2-HER3 heterodimer formation, and dacomitinib, CUDC-101, and Sapitinib (abnormally unknown drugs as targeted therapeutics for HER2 and HER3) Im), compared with the case where NRG1-b1 is not treated (HER2-HER3 heterodimer is not generated), it can be confirmed that it shows excellent or at least equal anticancer effect.

Figure 5k is a schematic diagram of an experiment for confirming the inhibitory performance (anti-tumor effect) of one of the superior drugs whose effects have been previously confirmed in SKBR3 xenograft mice. Transplanting SKBR3 to rats (SKBR3 cells inside the flank dermal layer of Balb / c nude mouse) in a 1: 1 ratio with Matrigel (Corning; Cat # 356237) at a rate of 1X10 ^ 7 per individual, dissolved in DPBS (Gibco; LS14190144) After injecting) to induce cancer, HER3 ligand NRG1-b1 was injected (intraperitoneally dissolved in DPBS at a concentration of 50 ug / kg) and dacomitinib was administered orally, and changes in cancer size were observed for 20 days. NRG1-b1 and dacomitinib were administered every day indicated by the filled triangle, and the size (volume) of the tumor tissue was measured on the date indicated by the empty triangle.

The tumor tissue size was measured when the tumor tissue size was measured in FIG. 5K, and the results are shown in FIG. 5L. The size of the tumor tissue was measured by measuring the size of the lump protruding by the tumor tissue with a vernier caliper.

Figure 5m is a graph showing the final weight of cancer tissue extracted from mice 20 days after the start of the test when lapatinib and dacomitinib were treated alone (with vehicle) or when lapatinib and dacomitinib were treated with NRG1-b1 to be. Dacomitinib was found to have a better anticancer effect when treated with NRG1-b1, as compared to lapatinib. From this, it is confirmed that dacomitinib has an excellent effect on diseases related to NRG1-b1. As an example, NRG1-b1 is expressed and the signal of the HER2-HER3 heterodimer is enhanced, so even if lapatinib (HER2 targeted therapy) is ineffective due to anticancer resistance, dacomitinib can be prescribed to overcome resistance or maximize the anticancer effect. have.

Claims (13)

HER2-HER3 hetero, comprising at least one member selected from the group consisting of safitinib, AEE788, dacomitinib, PD153035, CUDC-101, PD158780, pellitinib, saracatinib, and pharmaceutically acceptable salts thereof. Pharmaceutical composition for the prevention or treatment of dimer-related diseases. The pharmaceutical composition of claim 1, comprising at least one selected from the group consisting of safitinib, AEE788, dacomitinib, and pharmaceutically acceptable salts thereof. The pharmaceutical composition according to claim 1 or 2, wherein the disease associated with the HER2-HER3 heterodimer is a disease in which the HER2-HER3 heterodimer is present or detected. The pharmaceutical composition of claim 3, wherein the disease associated with the HER2-HER3 heterodimer is cancer. The pharmaceutical composition of claim 4, wherein the cancer is a cancer resistant to HER2 target treatment or HER3 target treatment. Safitinib, AEE788, Dacomitinib, PD153035, CUDC-101, PD158780, Pellitinib, Saracatinib, and their pharmaceutically acceptable salts require the prevention or treatment of diseases associated with HER2-HER3 heterodimers A method of preventing or treating a disease associated with HER2-HER3 heterodimer, comprising administering to a subject. The pharmaceutical composition of claim 6, comprising at least one selected from the group consisting of safitinib, AEE788, dacomitinib, and pharmaceutically acceptable salts thereof. The pharmaceutical composition according to claim 6 or 7, wherein the disease associated with the HER2-HER3 heterodimer is a disease in which HER2-HER3 heterodimer is present or is detected. The pharmaceutical composition of claim 8, wherein the disease associated with the HER2-HER3 heterodimer is cancer. The pharmaceutical composition of claim 9, wherein the cancer is a cancer resistant to HER2 target treatment or HER3 target treatment. Measuring a protein-protein interaction between a first protein and a second protein in isolated cells or tissues treated with a candidate drug targeting the first protein, wherein the first protein is HER2 and HER3 dimerized HER2-HER3 heterodimer, the second protein is a sub-protein of HER2, HER3, or both of them in a signaling pathway in a cell or tissue. The method of claim 11, wherein the anticancer agent has an anticancer effect against cancer in which the HER2-HER3 heterodimer is detected. The method of claim 12, wherein the anti-cancer agent has an anti-cancer effect against cancer resistant to a HER2 or HER3 targeted therapeutic agent.

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