KR101119995B1 - Screening method of protein-protein interaction inhibitor - Google Patents

Abstract

The present invention relates to a method for screening protein interaction inhibitors.

According to the present invention, even a small amount of protein can be selected a number of inhibitor candidates that affect the interaction between proteins can significantly reduce the cost and time. In addition, when using FLOW NMR rather than the conventional 2D NMR, it is more effective to select a plurality of inhibitor candidates without removing the column from a separate NMR tube.

Description

Screening method of protein-protein interaction inhibitor

The present invention relates to a method for screening protein interaction inhibitors, and more particularly, to a method for screening protein interaction inhibitors capable of selecting a plurality of inhibitor candidates even with a small amount of protein.

Cells maintain life by performing various biological functions such as gene expression, cell growth, cell cycle, metabolism, and signaling through diverse and complex protein-protein interactions. Thus, understanding protein-protein interactions and their functions within cells is the cornerstone of understanding life phenomena and an important foundation for drug development and disease treatment. Methods for investigating protein-protein interactions in vitro or in vivo include affinity chromoatography, coimmunoprecipitation, phage display, and two-hybrid assays. While conventional methods exist, such as assays, GST-fusion protein pulldown, and immunohistochemistry, these methods have several advantages, but they provide rapid intracellular protein-protein interactions. There are several drawbacks to the identification.

Specifically, in the case of protein affinity chromatography, there is a difficulty in preparing purified protein. In addition, since protein-to-protein interactions occur in vitro, there is a risk that false-positive results may be produced that may appear to interact by electrostatic interactions while non-interacting proteins in the cell pass through the column. . Coimmunoprecipitation requires highly sensitive, purified antibodies that can recognize the type of protein in the cell. Therefore, it is difficult to prove the interaction between proteins when the sensitivity and specificity of the antibody is low. In the case of phage display, since the protein is expressed in a fused form with the capsid or outer membrane protein of the phage, the size of the protein that can be expressed is limited. Many proteins in mammalian cells undergo many modifications after translation, but phage are difficult to study because they do not undergo the same folding and post-translational modifications as eukaryotic proteins. Since a single protein undergoes various modifications to interact with various proteins to perform intracellular functions, what kind of modification does it take and after various modifications?

Studying interacting pairs is the cornerstone of understanding the regulation of complex cell functions. However, when studying interaction using phage display, various modifications that occur in mammalian cells do not occur, so it is difficult to study the interaction between proteins that interact after various modifications.

Two-hybrid stems are commonly used in yeast and mammalian cells, where yeast proteins must be made identical to those in eukaryotic cells and fold and modify. In the two-hybrid system using mammalian cells, folding or modification occurs properly after protein synthesis, but the interaction of proteins in the cytoplasm is confirmed through transcriptional activation in the nucleus using the DNA binding domain. Protein-to-protein interactions are difficult to identify in the cytoplasm. In addition, when the interaction between proteins does not sufficiently activate the reporter gene, in the case of interacting rather inhibit the transcription, such as the difference between the activation and the control group does not have a large difference is difficult to prove the interaction. Immunohistochemistry involves the immobilization of paraffin and formalin during the preparation of the sample, during which the cells may be affected, the cells in which the sensitive antibody is required, and only the target proteins present are stained with dyes. Later, based on these results, it is difficult to study the exact protein-protein interactions because the interactions between these proteins are inferred by their location. The GST pull-down assay involves the process of expressing and purifying target proteins in bacteria, which makes it difficult to express the proteins soluble in water and may have a different structure from that expressed in mammalian cells. In addition, since protein degradation may occur during and after purification, continuous protein status should be monitored. In addition, the binding between proteins is greatly influenced by the composition of the buffer used. Therefore, proper buffer composition studies should be accompanied and in vitro experiments could yield different results from in vivo interactions.

Therefore, the time spent in the cloning process of the conventional methods for analyzing protein-protein interactions, the need for purified antigen-specific antibodies, contamination during the process such as cell destruction in the course of the experiment and changes in the cell environment, Overcoming limitations such as difficulty in protein purification and high cost, a new analysis system is required to analyze protein interactions easily and accurately at high speed.

In order to meet these demands, methods of identifying protein interactions and screening inhibitors through NMR introduced are effective in minimizing the aforementioned side effects. However, the method requires a new protein to test another inhibitor candidate after testing one inhibitor candidate that inhibits the interaction of the protein, thus increasing the amount of protein required and much more cost and effort. There was a necessary problem.

The present invention has been made to solve the above problems, the first problem to be solved by the present invention is to provide a protein interaction inhibitor screening method that can select a plurality of inhibitor candidates with a small amount of protein.

The second problem to be solved of the present invention is to provide a protein interaction inhibitor selection method that can select a plurality of inhibitor candidates without removing the column from the NMR tube.

The present invention to achieve the first object,

1) preparing two interacting proteins with an affinity tag attached to at least one protein and linked with a linker, 2) labeling the protein with an isotope, 3) the affinity tag is Adsorbing the two proteins attached to the column, 4) inserting the two protein-adsorbed columns into an NMR tube, and then measuring 2D nuclear magnetic resonance spectra, and 5) inserting the two proteins into the NMR tube. Adding at least one inhibitor candidate to inhibit the interaction of the protein, 6) measuring the 2D nuclear magnetic resonance spectrum for the NMR tube to which the inhibitor candidate is added, and measuring the 2D nuclear magnetic in step 4) It provides a method for screening protein interaction inhibitors, including selecting candidates through active site signal changes compared to resonance spectra.

According to another preferred embodiment of the invention, the two interacting proteins may be enzymes or receptors, more preferably mdm2 and p53.

According to another preferred embodiment of the present invention, the isotope may be ¹³ N, ¹⁵ N.

According to another preferred embodiment of the present invention, the linker ~ ~ amino acid sequence of the linker may be composed of 16 amino acids "GGGSGGGSGGGSGGGS".

According to another preferred embodiment of the present invention, the affinity tag may be a GST resin.

According to another preferred embodiment of the present invention, the length of the column is 4-5cm, the diameter may be 0.2 ~ 0.6cm.

According to another preferred embodiment of the present invention, the 2D nuclear magnetic resonance spectrum may be HSQC-NMR.

According to another preferred embodiment of the present invention, after step 6), 7) removing the inhibitor candidate from the column of the NMR tube, 8) two proteins in the NMR tube in which the inhibitor candidate is removed Adding at least one kind of new inhibitor candidate which inhibits the interaction of the same, 9) measuring the 2D nuclear magnetic resonance spectrum for the NMR tube to which the new inhibitor candidate is added, and measuring the 2D nucleus in step 4) Comparing the magnetic resonance spectrum with the step of screening the candidate substance through the active site signal change, the steps 7) to 9) may be repeated.

According to another preferred embodiment of the present invention, the steps 7) to 9) may be repeated 1 to 20 times.

In order to achieve the second object of the present invention, 1) preparing two proteins interacting with an affinity tag attached to at least one protein and linked by a linker, 2) any one or more of the proteins Labeling the protein with an isotope, 3) adsorbing the two proteins with the affinity tag attached to the column, 4) inserting the two protein-adsorbed column into the NMR tube and then performing 2D nuclear magnetic resonance spectra. Measuring, 5) adding at least one inhibitor candidate to inhibit the interaction of two proteins in a column inserted into the NMR tube, 6) 2D nuclei for the NMR tube to which the inhibitor candidate is added Measuring a magnetic resonance spectrum and comparing candidate 2D nuclear magnetic resonance spectra measured in step 4) to select candidate substances through change of active site signal; Provided that the protein interaction inhibitor screening method.

According to another preferred embodiment of the present invention, after step 6), 7) washing the FLOW NMR tube to remove the inhibitor candidate, 8) binding two proteins to the column inserted into the FLOW NMR tube. Adding at least one new inhibitor candidate to inhibit and 9) measuring the 2D nuclear magnetic resonance spectrum for the FLOW NMR tube to which the other inhibitor candidate is added and measuring the 2D nuclear magnetic resonance in step 5) Including the step of selecting a candidate material through the active site signal change compared to the spectrum, steps 7) to 9) may be repeated 1 to 20 times.

Hereinafter, the terms used in the present specification will be briefly described.

Unless stated otherwise, the interaction of proteins means that the proteins do not act alone in vivo but exhibit their function through binding to other proteins.

'FLOW NMR' refers to an NMR technique that can be measured by changing the solute while flowing the solution without taking the sample out of the NMR tube.

'Peptide' means a peptide having a sequence similar to the substrate peptide sequence of the protein of interest and capable of binding to the active site of the protein, and a 'peptide analog' is a compound substantially similar to amino acids in such peptide sequence (amino acid analog) Peptides and fragments thereof, including those that retain the property of binding to the active site of the protein.

 According to the present invention, even a small amount of protein can be selected a number of inhibitor candidates that affect the interaction between proteins can significantly reduce the cost and time. In addition, when using FLOW NMR rather than the conventional 2D NMR, it is more effective to select a plurality of inhibitor candidates without removing the column from a separate NMR tube.

Furthermore, analyzing the three-dimensional structure of the protein bound to the inhibitor not only reveals how the inhibitor binds to the protein, but also uses this information to design new inhibitors that affect the interactions between proteins. .

Hereinafter, the present invention will be described in more detail.

As described above, by analyzing the three-dimensional structure of the active site of the interacting protein, it is possible to design a new drug at a very high speed. For example, analysis of the three-dimensional structure of a protein bound to an inhibitor not only reveals how the inhibitor binds to the protein, but also uses this information to design new inhibitors that affect the interactions between proteins. This can be analyzed using NMR. However, screening a large number of inhibitor candidates requires proteins for each inhibitor candidate, resulting in a large amount of isotopically substituted proteins when each of them is analyzed using NMR. It will lead to a dramatic rise.

Accordingly, in one embodiment of the present invention, 1) preparing two proteins interacting with an affinity tag attached to at least one protein and linked with a linker, and 2) labeling the protein with an isotope. 3) adsorbing the two proteins with the affinity tag attached to the column, 4) measuring the 2D nuclear magnetic resonance spectrum after placing the two protein-adsorbed columns into the NMR tube, 5) the NMR Adding at least one inhibitor candidate that inhibits the interaction of two proteins to a column inserted in the tube, 6) measuring 2D nuclear magnetic resonance spectra for the NMR tube to which the inhibitor candidate is added Providing a protein interaction inhibitor screening room comprising screening candidates through active site signal change compared to 2D nuclear magnetic resonance spectrum measured in step 4) W was seeking to overcome the above problems.

Referring to the screening method of the present invention in more detail, first, as step 1), an affinity tag is attached to at least one protein and two interacting proteins connected by a linker are prepared. The two proteins that can be used in the present invention may be applied as long as the interaction between the proteins occurs, but it is advantageous to design a three-dimensional structure of the inhibitor candidate by using a known protein with a known three-dimensional structure. . On the other hand, the two interacting proteins may be enzymes and / or receptors or peptides or peptide analogs capable of binding to the active site of one protein. Examples thereof may be mdm2 and p53, but are not limited thereto and may be applied as long as the technical principles of the present invention are applicable.

Meanwhile, two proteins used in the present invention have interactions, and separately link the two proteins with a linker. Through this, even if the inhibitor candidates described below are combined with one protein and the interaction of the two proteins is inhibited, the binding of the two proteins can be maintained by the linker. Even when washed with water, the two proteins are bound to the column and bound by a linker, so that they can be used for screening another inhibitor candidate.

The linker usable in the present invention can be used as long as it can connect two proteins, without any limitation, and may be appropriately selected. Preferably, a linker having a number of amino acids less than 50 may be used, but is not limited thereto. . The linker used in the present invention may be commercially available, or may be manufactured by itself. Specifically, it may be a peptide composed of amino acids such as “GGGSGGGSGGGSGGGS (SEQ ID NO: 1)”.

In the present invention, an affinity tag may be used to adhere the protein to the column. The affinity tag is attached to at least one of the two proteins and the affinity tag usable in the present invention is not limited in kind as long as it can be attached to a column, but preferably from the group consisting of GST and His-tag. It may be any one or more selected. Meanwhile, the affinity tag may be expressed with a protein or attached by a method such as cross linking, but is not limited thereto.

On the other hand, the affinity tag can be attached to the protein by a conventional method, preferably can be attached by cloning to a commercial vector containing the affinity tag.

Next, in step 2), at least one protein of the two proteins is labeled with an isotope. In this case, ¹³ C or ¹⁵ N may be preferably used as the protein isotope, and in this case, both proteins may be labeled or only one protein may be labeled. Furthermore, although all amino acids constituting the protein can be labeled, but if there is information on the amino acid sequence of the active site of the protein of interest, it is preferable to label only the corresponding amino acids, and some amino acids among the amino acids constituting the protein (for example, For example, lysine alone is used to simplify the detection of the NMR spectrum as compared to the case of labeling all amino acids, making it easier to determine the active site.

The type and concentration of the solvent that can be used in the present invention is not limited as long as it can be used in conventional NMR, but preferably a buffer solution of pH 6.8 ~ 8 can be used, 0.1mM protein can be added but not limited thereto. Do not.

Next, in step 3) the two affinity-tagged proteins are adsorbed onto the column. The column is not limited as long as it is a material capable of binding to an affinity tag and can be used for NMR. If the GST resin is used as the affinity tag, it is advantageous to use a GST column. In addition, the size and shape of the column usable in the present invention is sufficient that it can be inserted into the NMR tube, preferably the length of the column is 4-5 cm, the diameter may be 0.2 ~ 0.6 cm.

On the other hand, the method of adsorbing two proteins on the column is not limited. Preferably, the protein is purified and then equilibrated with a buffer and then adsorbed on the column.

Next, in step 4), the two protein-adsorbed columns are placed in an NMR tube, and then 2D nuclear magnetic resonance spectra are measured. Specifically, the study of interaction between proteins using NMR has various advantages compared with the above-described method using X-ray. X-rays must first obtain a co-complex single crystal of the protein, which is not easy. In particular, the interactions between proteins are strong enough that the basic conditions that must be maintained during the protein crystallization process must be met. However, protein interaction studies using NMR are all performed in solution, so that the interaction between weak proteins can be measured relatively easily by increasing the concentration of binding protein. In order to identify the structure using NMR, the chemical shift of each amino acid must be determined first. The chemical shift information thus obtained can reveal the tertiary interaction site between proteins in great detail. NMR techniques, which are widely used to measure interactions between proteins, may include chemical shift perturbation and cross-saturation. On the other hand, 2D nuclear magnetic resonance spectrum for each protein can be measured to facilitate the contrast of the activation signal, and when the interaction between proteins occurs, the peak of the pattern different from that of the individual protein appears.

Next, step 5) adds at least one inhibitor candidate to inhibit the binding of two proteins to the column inserted into the NMR tube. In this case, any inhibitor candidates that can be added may be added regardless of the kind of the substance that is expected to inhibit the interaction of two proteins, and may be added regardless of the types of proteins, peptides, compounds, and natural products. can do. At this time, the kind of inhibitor candidate added may be one or several kinds, and when only one kind is added, it is possible to determine whether the inhibitor candidate is a target inhibitor. . When there are many kinds of inhibitor candidates, various kinds of inhibitor candidates may be mixed and added to determine whether there is a target inhibitor in the added mixture. If it is found that there is a target inhibitor in the mixture, it can be analyzed one by one to find the target inhibitor. On the other hand, it is advantageous to detect the target inhibitor, preferably, the inhibitor candidates are added alone or in combination within 10 kinds.

Next, in step 6), the 2D nuclear magnetic resonance spectrum of the NMR tube to which the inhibitor candidate is added is measured, and the candidate substance is selected through the active site signal change by comparing with the 2D nuclear magnetic resonance spectrum measured in step 5). do. Specifically, when an inhibitor candidate is added to two interacting proteins, if the added candidate is an inhibitor that inhibits interaction, the active site signal of the 2D nuclear magnetic resonance spectrum is changed by interfering with the interaction. In other words, the interaction between the proteins is stopped, so that the NMR peak of each protein is reduced, not the NMR peak of the binding protein. However, if the added candidates are not actually inhibitors, they do not interfere with protein interactions, so the activation site signal is hardly changed. This can determine whether the inhibitor candidate can inhibit the interaction between the actual protein.

Meanwhile, in order to test another inhibitor candidate after step 6), 7) removing the inhibitor candidate from the column of the NMR tube, 8) removing the inhibitor candidate from the column of the two proteins in the NMR tube. Adding at least one new inhibitor candidate which inhibits the interaction; 9) measuring the 2D nuclear magnetic resonance spectrum for the NMR tube to which the new inhibitor candidate is added and measuring the 2D nucleus in step 4) Comparing the magnetic resonance spectrum with the step of screening the candidate material through the active site signal change, the steps 7) to 9) may be repeated.

Specifically, after step 6) and step 7), the column may be removed from the NMR tube and washed with water to remove the inhibitor candidate. In this case, the inhibitor candidate is removed, but the protein is still attached to the column because it has an affinity tag attached to it. It is not removed together with the material. On the other hand, if the inhibitor candidate material is removed, the interaction between the proteins occurs again. If the linker is not connected between the proteins, the binding between the proteins is broken by the inhibitor. It is removed together with the substance.

Then, at step 8), at least one kind of new inhibitor which inserts the column from which the inhibitor candidate has been removed into the NMR tube and inhibits binding of two proteins to the column inserted into the NMR tube as in step 6). After the addition of the candidate, step 9) measures the 2D nuclear magnetic resonance spectrum of the NMR tube to which another inhibitor candidate is added and compares the active site signal with the 2D nuclear magnetic resonance spectrum measured in step 4). Changes can be used to screen candidates. This allows for the reuse of proteins that have already been used for inhibitor screening without preparing the proteins freshly. Steps 7) to 9) may be repeated, and preferably steps 7) to 9) may be repeated 1 to 20 times, but the present invention is not limited thereto.

The principle of the present invention described above will be described with reference to FIG. When the GST_Mdm2 hybrid protein having GST resin attached as an affinity tag is reacted with the p53 protein, the two proteins bind by the interaction between the proteins. Then, two proteins are linked by adding a linker having an amino acid sequence (GGGSGGGSGGGSGGGS). Then, adding it to the column causes the GST resin to adsorb to the column, resulting in both proteins adsorbing to the column. Subsequent addition of Nutlin-3 as an inhibitor maintains adsorption on the column but inhibits the interaction between the two proteins, separating both proteins and linking only to the linker. Subsequently, washing the column to remove Nutlin-3 leaves the protein adsorbed on the column and other proteins linked by the linker to remain on the column. Then, new inhibitor candidates can be added to the column to repeat the same experiment.

Protein interaction inhibitor screening method according to a second embodiment of the present invention comprises the steps of 1) preparing two proteins with an affinity tag attached to at least one protein and linked by a linker, 2) the protein Labeling any one or more proteins with isotopes, 3) adsorbing the two proteins with the affinity tag attached to the column, 4) inserting the two protein adsorbed columns into an NMR tube and then placing the 2D nucleus in a 2D nucleus. Measuring magnetic resonance spectra, 5) adding at least one inhibitor candidate to inhibit the interaction of two proteins in a column inserted into the NMR tube, 6) an NMR tube to which the inhibitor candidate is added 2D nuclear magnetic resonance spectra for the target are selected and candidates are selected through active site signal changes compared to the 2D nuclear magnetic resonance spectra measured in step 4). A differentiation step is included.

Hereinafter, only the characteristic contents of the second embodiment will be described except for the contents overlapping with the first embodiment. Specifically, the two embodiments are different in that they use FLOW NMR rather than conventional NMR as 2D NMR. FLOW NMR will not remove the inhibitor used after removing the column from the NMR tube to add new inhibitor candidates, but will remove the inhibitor used inside the NMR tube without removing the column. This allows for the continuous screening of another inhibitor candidate without the inconvenience of taking out the separately used column.

As a result, according to the present invention, even a small amount of protein can select a plurality of inhibitor candidates that affect the interaction between proteins, thereby significantly reducing the cost and time. In addition, in the case of using FLOW NMR rather than the conventional 2D NMR, a plurality of inhibitor candidates can be selected without removing the column from a separate R tube, which is more effective.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are not intended to limit the scope of the present invention, which should be construed as to help the understanding of the present invention.

Example 1 Preparation and Purification of Proteins

 The portions corresponding to 3 to 109 of the residues of the Mdm2 protein (NCBI ABT17086) and the portions corresponding to 1 to 73 of the residues of the p53 protein (NCBI BAC16799) were amplified by PCR. Thereafter, Mdm2 (residue 3-109) was cloned into the pGEX4T-1 expression vector, and then a linker (GGGSGGGSGGGSGGGS) was connected to Mdm2 (residue 3-109), and p53 (residue 1-73) was cloned later. In this manner, the mdm2-linker-p53 protein with an affinity tag was expressed.

The prepared plasmid was transformed into E. coli BL21 (DE3) and inoculated in M9 minimal media (1 L) labeled with ¹⁵ N. Then, when the absorbance reached OD 0.6, the temperature was lowered to 25 ° C., 1 mM IPTG was added thereto, and then cultured at 25 ° C. for 6 hours. The culture was then centrifuged at 5000 rpm for 20 minutes at 4 ° C. to recover the transformed strain, followed by PBS buffer (pH = 7.3, 140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO _4). The strain was transformed with an ultrasonic mill while maintaining a cold (4 ° C.) state. The sample was centrifuged at 15000 rpm for 30 minutes at 4 ° C. to separate cell debris containing a supernatant containing cellular endogenous protein and solid components such as cell wall-membrane components, and then the supernatant was taken to obtain protein. (Mdm2_linker_p53) was purified.

Example 2 Measurement of 2D NMR

The equilibrated GST resin and the purified protein were mixed and placed in an NMR tube to measure 2D NMR. The column was washed with water in the NMR tube to remove inhibitor candidates. After adding at least one new inhibitor candidate that inhibits binding of two proteins to the NMR tube from which the inhibitor candidate has been removed, 2D nuclear magnetic resonance spectra of the NMR tube to which another inhibitor candidate is added are measured. Candidates were selected through the change of active site signal compared with the 2D nuclear magnetic resonance spectrum measured above.

<Experimental Results>

2 is an HSQC NMR spectrum of GST Resin_p53. In the above, GST resin means a material of a column. 3 is an HSQC NMR spectrum for GST Resin_GST_mdm2_linker_p53. Specifically, in the case of mdm2, GST, which is a macromolecule, is attached so that the volume becomes so large that almost no NMR peak occurs except for a binding portion with p53. In addition, in the case of p53 it can be confirmed that due to the interaction with mdm2, the NMR activation signal is weakened compared with the p53 alone.

 4 is a spectrum of HSQC NMR measured after adding 0.5 mM Nutlin-3 as an inhibitor. The circled part in FIG. 4 means a new peak that did not exist in FIG. 3, which is interpreted as the interaction of mdm2 and p53 was interrupted by the addition of Nutlin-3. It can be seen that it corresponds to a compound that inhibits.

Meanwhile, the HSQC NMR spectrum of the GST resin _GST_mdm2_linker_p53 may be substantially identical to the active signal region of FIG. This means that when the inhibitor is removed, p53 is not removed like the inhibitor and is adsorbed on the column, causing interaction with mdm2. It is interpreted that the minimum is minimized.

As a result, only the inhibitors already used are removed from the column, and thus the GST resin_GST_mdm2_linker_p53 still remains, so that the screening process can be performed again by adding a new inhibitor candidate.

The present invention is useful for finding new drug substances with low cost and high efficiency, and for new drug research and development such as structure-based drug design.

1 is a schematic diagram showing a method for screening an inhibitor candidate according to a preferred embodiment of the present invention.

2 is an HSQC NMR spectrum for GST Resin_p53.

3 is an HSQC NMR spectrum for GST Resin_GST_mdm2_linker_p53.

4 is an HSQC NMR spectrum after adding Nutlin3 to GST resin_GST_mdm2_linker_p53.

5 is an HSQC NMR spectrum for GST resin _GST_mdm2_linker_p53 after washing the column.

<110> KOREA BASIC SCIENCE INSTITUTE <120> Screening method of protein-protein interaction inhibitor <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> artificial linker <400> 1 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser   1 5 10 15  

Claims (14)

1) preparing two interacting proteins with an affinity tag attached to at least one protein and linked with a linker; 2) labeling the protein with an isotope, 3) adsorbing the two proteins with the affinity tag attached to the column; 4) measuring 2D nuclear magnetic resonance spectra after placing the two protein-adsorbed columns in an NMR tube; 5) adding at least one kind of inhibitor candidate to inhibit the interaction of two proteins in the column inserted into the NMR tube; 6) measuring the 2D nuclear magnetic resonance spectrum of the NMR tube to which the inhibitor candidate is added, and selecting the candidate through active site signal change by comparing with the 2D nuclear magnetic resonance spectrum measured in step 4) Protein interaction inhibitor screening method. The method of claim 1, Method for screening protein interaction inhibitors, characterized in that the two interacting proteins are enzymes or receptors. 3. The method of claim 2, The two interacting proteins are mdm2 and p53, characterized in that the protein interaction inhibitor selection method. The method of claim 1, The isotope is ¹³ C or ¹⁵ N, characterized in that the protein interaction inhibitor selection method. The method of claim 1, Wherein said linker has an amino acid sequence represented by SEQ ID NO: 1. The method of claim 1, The affinity tag is GST, His-tag protein interaction inhibitor selection method, characterized in that any one or more selected from the group consisting of. The method of claim 1, The length of the column is 4 ~ 5 cm, the diameter is 0.2 ~ 0.6 cm protein interaction inhibitor screening method, characterized in that. The method of claim 1, Wherein said 2D nuclear magnetic resonance spectrum is HSQC-NMR. The method of claim 1, In step 6), the inhibitor candidate material is a protein interaction inhibitor selection method, characterized in that 1 to 10 kinds are added. The method of claim 1, After step 6), 7) removing the inhibitor candidate from the column of the NMR tube; 8) adding at least one new inhibitor candidate to the NMR tube where the inhibitor candidate has been removed to inhibit interaction of two proteins; 9) measuring the 2D nuclear magnetic resonance spectrum for the NMR tube to which the new inhibitor candidate is added and selecting the candidate substance through active site signal change by comparing with the 2D nuclear magnetic resonance spectrum measured in step 4). Including; Step 7) to 9) is a protein interaction inhibitor selection method, characterized in that it is repeated. The method of claim 10, The 7) to 9) step is a protein interaction inhibitor selection method, characterized in that it is performed 1 to 20 times. 1) preparing two interacting proteins with an affinity tag attached to at least one protein and linked with a linker; 2) labeling at least one protein of the protein with an isotope, 3) adsorbing the two proteins with the affinity tag attached to the column; 4) measuring 2D nuclear magnetic resonance spectra after placing the two protein-adsorbed columns in an NMR tube; 5) adding at least one kind of inhibitor candidate to inhibit the interaction of two proteins in the column inserted into the NMR tube; 6) measuring the 2D nuclear magnetic resonance spectrum of the NMR tube to which the inhibitor candidate is added, and selecting the candidate through active site signal change by comparing with the 2D nuclear magnetic resonance spectrum measured in step 4) Protein interaction inhibitor screening method. The method of claim 12, After step 6), 7) washing the FLOW NMR tube to remove the inhibitor candidate; 8) adding at least one new inhibitor candidate to inhibit the interaction of two proteins in a column inserted into the FLOW NMR tube; And 9) The 2D nuclear magnetic resonance spectrum of the FLOW NMR tube to which another inhibitor candidate is added is measured, and the candidate substance is selected by changing the active site signal compared with the 2D nuclear magnetic resonance spectrum measured in step 5). Including steps; The 7) to 9) step is a protein interaction inhibitor selection method, characterized in that it is performed 1 to 20 times. delete

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