WO2021029565A1 - Method for screening binding inhibiting substance of api5 and fgf2 - Google Patents

Abstract

The present invention relates to a method for screening an FGF2 binding inhibiting substance and the use thereof, the method comprising the steps of: i) introducing cysteine into the outside of FGF2; ii) labeling the cysteine-introduced FGF2 of step i) with a fluorescent material; iii) mixing the FGF2 labeled with the fluorescent material of step ii), a protein which can bind to the FGF2 and a candidate substance which can inhibit the binding therebetween; and iv) measuring the fluorescence intensity of the mixed substances of step iii). By using the screening method of the present invention, cysteine is additionally introduced into FGF2 to enable more efficient fluorescent material labeling, and thus a fluorescence polarization value measured by means of a fluorescence polarization assay and the like is amplified and the binding (interaction) force between the FGF2 and API5 or the substance inhibiting same can be efficiently screened.

Description

API5 and FGF2 binding inhibitor screening method

This application claims priority to Korean Patent Application No. 10-2019-0097467, filed on August 09, 2019, and the entire specification is a reference to this application.

The present invention comprises the steps of: i) introducing cysteine to the outside of FGF2;

ii) labeling a fluorescent material on FGF2 into which the cysteine of step i) has been introduced;

iii) mixing the fluorescent substance-labeled FGF2 of step ii), a protein capable of binding to FGF2, and a candidate substance capable of inhibiting the binding thereof; And

iv) measuring the fluorescence intensity of the mixed materials of step iii);

It relates to a method for screening a FGF2 binding inhibitor comprising a and use thereof.

Protein-protein, nucleic acid (DNA, RNA)-protein interactions, or appropriate nucleases and proteolytic enzymes activity, in addition to normal physiological reactions such as cell development, growth, and homeostasis, can lead to the occurrence of various diseases including cancer. Even related abnormal physiological responses are involved in almost all cellular signal transductions. Therefore, materials such as organic or inorganic small molecules, aptamers, peptides, therapeutic proteins, and antibodies are rapidly being developed for diagnosis and treatment. Therefore, in order to verify the efficacy of these pharmacological candidates, quantitative analysis of small molecule compound-protein, nucleic acid-protein, peptide-protein, protein-protein avidity (interaction) or nuclease, protease activity is very important. I can.

The currently used methods for analyzing the binding strength (interaction) or enzyme activity between molecules can be largely divided into a heterogeneous method and a homogeneous method. Representative examples of heterogeneity methods include enzyme linked immunosorbent assay (ELISA), microarray, and surface plasmon resonance (SPR). These heterogeneous methods require a step of immobilizing a molecule on a substrate and a washing step for analysis of avidity (interaction) or enzyme activity. Furthermore, it is also essential to purely separate substances and reaction products bound to substances (low molecular weight compounds or nucleic acids or proteins) to be analyzed for final avidity (interaction) analysis or enzyme activity measurement.

However, since the homogeneous method does not require the immobilization, washing, or separation process required in the heterogeneous method, it has the advantage of being able to easily and quickly analyze the binding strength (interaction) or enzyme activity between molecules. have. Representative examples of homogeneous methods include fluorescence resonance energy transfer (FRET) and fluorescence polarization (FP) technologies. Currently, the fluorescence resonance energy transfer measurement method is widely used to measure the binding force (interaction) or enzyme activity between molecules, but due to the demands of a donor and an acceptor, a fluorescent substance is added to both molecules to be analyzed. There is a fatal drawback that must be labeled.

On the other hand, the fluorescence polarization measurement method, which enables analysis of the binding strength (interaction) or enzyme activity between molecules even when a fluorescent substance is labeled on only one molecule, is a method of measuring the fluorescence polarization that occurs when the fluorescent substance in a solution is excitation. . When a fluorescent substance is excited and is in a stable state, it emits fluorescence polarized light, and when the fluorescent molecule is excited, it rotates by Brownian motion to emit fluorescence to a plane different from the excitation plane, resulting in loss of fluorescent polarization. It is to use the principle. Based on this principle, fluorescence polarization increases when molecular weight increases due to binding between molecules, and fluorescence polarization decreases when molecular weight decreases due to dissociation or decomposition. Therefore, low molecular weight compounds-protein, nucleic acid-protein, peptide-protein, protein -It can be said to be a powerful technology in the analysis of binding (interaction) and enzyme activity of proteins, receptors-ligands, enzymes-substrates, nucleic acids-nucleic acids, etc.

Republic of Korea Patent Registration No. 10-1576363 relates to a method of measuring fluorescence polarization in which a signal is amplified. By amplifying the fluorescence polarization value using biotin and streptavidin, it is possible to analyze the binding force (interaction) between two molecules or measure enzyme activity. Is being disclosed.

However, there are no studies or descriptions of using fluorescent polarization to screen for inhibitors that inhibit the binding of FGF2, particularly FGF2 and API5.

Accordingly, the present inventors made diligent efforts to provide a method for effectively screening the binding (interaction) power of a low molecular weight compound and a protein, etc., particularly the binding power of FGF2 and API5 or inhibitors that inhibit it.As a result, by introducing cysteine into FGF2, API5 In the case of screening after reacting with, it was confirmed that the binding strength of FGF2 and API5 or inhibitors that inhibit it can be screened with high efficiency, and the present invention was completed.

Accordingly, an object of the present invention is the steps of i) introducing cysteine outside FGF2;

ii) labeling a fluorescent material on FGF2 into which the cysteine of step i) has been introduced;

iii) mixing the fluorescent substance-labeled FGF2 of step ii), a protein capable of binding to FGF2, and a candidate substance capable of inhibiting the binding thereof; And

iv) measuring the fluorescence intensity of the mixed materials of step iii);

It is to provide a method for screening an FGF2 binding inhibitor comprising a, and a use thereof.

In order to achieve the object of the present invention, the present invention comprises the steps of: i) introducing cysteine to the outside of FGF2;

ii) labeling a fluorescent material on FGF2 into which the cysteine of step i) has been introduced;

iii) mixing the fluorescent substance-labeled FGF2 of step ii), a protein capable of binding to FGF2, and a candidate substance capable of inhibiting the binding thereof; And

iv) measuring the fluorescence intensity of the mixed materials of step iii);

It provides a FGF2 binding inhibitor screening method comprising a.

According to a preferred embodiment of the present invention, the cysteine in step i) may be introduced at the C-terminus of FGF2.

According to a preferred embodiment of the present invention, the fluorescent material of step ii) is FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine), FAM (fluorescein amidite), Rhodamine, TAMRA (Carboxytetramethylrhodamine), Cy™, CF™ (Cyanine- based fluorescent dye) and Alexa Fluor may be any one or more selected from the group consisting of.

According to a preferred embodiment of the present invention, the protein capable of binding to FGF2 in step iii) may have a molecular weight greater than or equal to FGF2.

According to a preferred embodiment of the present invention, the protein capable of binding to FGF2 of iii) is any selected from the group consisting of FLAG, histidine, CBD, MBP, TRX and GST (Glutathione S-transferase). It may be a protein to which one or more tags are fused.

According to a preferred embodiment of the present invention, the protein capable of binding to FGF2 in step iii) may be API5.

According to a preferred embodiment of the present invention, measuring the fluorescence intensity in step iv) is FRET (Fluorescence Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer), FP (Fluorescence Polarization), HCS (High-content Screening). And it may be measured using one or more methods selected from the group consisting of HTS (High Throughput Screening).

According to a preferred embodiment of the present invention, when v) the fluorescence intensity measured in step iv) decreases to a level of 50% to 100% compared to before mixing the candidate material of step iii), it is determined as an FGF2 binding inhibitor. It may be to further include the step of.

In addition, the present invention provides a FGF2 binding inhibitor selected by the screening method.

According to a preferred embodiment of the present invention, the binding may be a combination of FGF2 and API5.

In addition, the present invention provides a composition for inhibiting anticancer drug resistance comprising the FGF2 binding inhibitor.

In the present invention, “FGF2 (protein) outside” refers to a portion of the FGF2 protein that is directed to the outside of the FGF2 protein rather than the inside of the protein. Correspondingly, “inside FGF2 (protein)” refers to a portion of the FGF2 protein directed toward the inside of the FGF2 protein rather than outside the protein.

"Inhibition of anticancer drug resistance" of the present invention refers to all effects of reducing or alleviating the resistance or resistance of cancer cells to anticancer agents so that the anticancer effect of the anticancer agent against cancer cells can be fully exhibited.

"API5 (Apoptosis inhibitor 5)" of the present invention was discovered as an increased gene through cDNA analysis of cells that did not undergo apoptosis without growth factor, and was named AAC-11. API5 has been reported to be involved in increasing cancer survival in several cancer cells. Through physical binding with Acinus (ACIN1), it inhibits the induction of apoptosis through Acinus-mediated DNA fragmentation and blocks the activity of Caspase-3, a apoptosis protein. In addition, it was confirmed that the cytotoxicity of the anticancer drug was increased by effectively inhibiting E2F1-induced apoptosis and lowering the expression of API5. In addition, it has been found that it is involved in the migration and invasion of cancer cells, which is deeply related to cancer metastasis, and plays an important role in immune evasion of cancer cells.

The "fibroblast growth factor 2 (FGF2)" of the present invention is one of a large family of fibroblast growth factors consisting of 23 FGF members, and is the most abundant among FGF members in the central nervous system. It plays an important role as a regulatory mediator of proliferation, differentiation, development and survival in various cell types and has mitogenic activity in the nervous system. In addition, FGF2 has a potential angiogenic effect, promotes the growth of smooth muscle cells, wound healing and tissue repair, as well as the pluripotent of human and mouse embryonic stem (ES) cells. It has been reported to remain in state. There are two types of FGF2: high molecular weight (22, 22.5, 24 or 34 kDa) and low molecular weight (18 kDa). High molecular weight FGF2 has a nuclear localization signal (NLS) at the N-terminus, allowing high molecular weight FGF2 to be located in the nucleus, whereas low-molecular weight FGF2 does not have NLS at the N-terminus, whereas atypical dichotomy at the C-terminus NLS regulates some low molecular weight FGF2 to be located in the nucleus.

The "FGF2 binding inhibitor" of the present invention refers to a substance that inhibits FGF2 from binding to other proteins, and in the present invention, it may mean a substance that inhibits the binding of FGF2 and API5.

In the FGF2 binding inhibitor screening method of the present invention, when cysteine was introduced to be located outside the FGF2 protein, a fluorescent substance such as Alexa Fluor 488 could be labeled on FGF2 with higher efficiency (Example 2). The Alexa Fluor 488-labeled FGF2, His, and GST-tagged API5 and API5 and FGF2 binding inhibitor candidates were reacted at once and analyzed by fluorescence polarization analysis. As a result, the fluorescence polarization value was significantly increased, resulting in various API5 and FGF2 binding inhibitor candidates were effectively screened (Example 3).

Accordingly, the present invention comprises the steps of: i) introducing cysteine to the outside of FGF2;

ii) labeling a fluorescent material on FGF2 into which the cysteine of step i) has been introduced;

iii) mixing the fluorescent substance-labeled FGF2 of step ii), a protein capable of binding to FGF2, and a candidate substance capable of inhibiting the binding thereof; And

iv) measuring the fluorescence intensity of the mixed materials of step iii);

It can provide a method for screening a FGF2 binding inhibitor comprising a.

The cysteine in step i) of the present invention may be introduced at the C-terminus of FGF2. FGF2 originally contained cysteine at the C167 and C234 positions, but this was directed to the inside of the FGF2 protein, so it was difficult to bind fluorescent substances such as Alexa Fluor 488. Accordingly, when cysteine is introduced (C289) at the C-terminus of FGF2, the binding efficiency with fluorescent materials such as Alexa Fluor 488 may increase.

The fluorescent material of step ii) of the present invention is FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine), FAM (fluorescein amidite), Rhodamine, TAMRA (Carboxytetramethylrhodamine), Cy™, CF™ (Cyanine-based fluorescent dye), and Alexa Fluor. It is preferably any one or more selected from the group consisting of, more preferably FITC (fluorescein isothiocyanate) or Alexa Fluor, and most preferably Alexa Fluor. The Alexa Fluor may be Alexa Fluor 488.

When screening for protein-protein (peptide) binding or inhibitors that inhibit it based on the fluorescence polarization analysis method, the fluorescence polarization value increases if protein-protein (peptide) binding occurs, and protein-protein (peptide) binding is If suppressed, it can be predicted that the fluorescence polarization value suppresses.

In the present invention, the introduction of cysteine into FGF2 means that in order to further maximize the difference in fluorescence polarization values in the fluorescence polarization analysis method, a fluorescent material must be labeled on a material having a small molecular weight. It is to amplify (Fig. 2). Accordingly, the protein capable of binding to FGF2 in step iii) may have a molecular weight greater than or equal to that of FGF2, preferably having a molecular weight greater than that of FGF2. The protein capable of binding to FGF2 may be FGFR or API5, preferably API5. The full length of FGF2 (residue 1-288) has a molecular weight of about 30.8 kDa, the molecular weight of FGF2 (residue 135-288) is 17.1 kDa, and FGF2 (residue 135-288) in which a His-tag at the N-terminus and cysteine are introduced at the C-terminus ) Has a molecular weight of about 19.8 kDa. The molecular weight of His-API5 is about 61 kDa, and the molecular weight of His-API5 labeled with GST is about 170 kDa.

To fuse any one or more tags selected from the group consisting of FLAG, histidine, CBD, MBP, TRX and GST (Glutathione S-transferase) to the protein capable of binding to FGF2 of iii) of the present invention. I can. It is preferable to fuse all of histidine, GST (Glutathione S-transferase) or histidine and GST.

Measuring the fluorescence intensity in step iv) of the present invention may preferably be measuring the intensity of fluorescence polarization, and FRET (Fluorescence Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer), FP (Fluorescence Polarization), HCS It may be measured using one or more methods selected from the group consisting of (High-content Screening) and High Throughput Screening (HTS). More preferably, it is preferable to measure using FP (Fluorescence Polarization) or HTS (High Throughput Screening) method.

The screening method of the present invention comprises the step of determining as an FGF2 binding inhibitor when v) the fluorescence intensity measured in step iv) decreases to a level of 50% to 100% compared to before mixing the candidate material of step iii). It may be additionally included. Preferably, when the fluorescence intensity is reduced to a level of 55% to 90%, it is preferable to determine it as an FGF2 binding inhibitor, and more preferably, when it is reduced to a level of 60% to 70%, it is preferable to determine it as an FGF2 binding inhibitor.

As a result, the present invention can provide a FGF2 binding inhibitor selected by the screening method.

The binding of the present invention may be a combination of FGF2 and API5.

In addition, the present invention can provide a composition for inhibiting anticancer drug resistance comprising the FGF2 binding inhibitor.

The anticancer agent may be any one or more selected from the group consisting of doxorubicin, 5-fluorouracil, carboplatin, cisplatin, carmustine, dacarbazine, etoposide, vinorelbine, topotecan, irinotecan, and estramustine. .

In the case of using the screening method of the present invention, by introducing additional cysteine to FGF2, the fluorescent substance can be more efficiently labeled, thereby amplifying the fluorescence polarization value measured by the fluorescence polarization analysis method, etc., and binding (interaction) of FGF2 and API5 It is possible to effectively screen the power or substances that inhibit it.

1 shows the principle of a method for analyzing or screening a protein-protein (peptide) binding or binding inhibitor based on an FP analysis method. When analyzing protein-protein (peptide) binding, when the FP signal increases, it means that the protein and protein (peptide) are combined.When screening for a protein-protein (peptide) binding inhibitor, when the FP signal decreases, It means that the binding of protein (peptide) is inhibited.

Figure 2 shows the principle of the method for screening substances that inhibit the binding of API5 and FGF2 of the present invention.

3 shows the FGF2 protein into which cysteine is introduced at the C-terminus. The red color represents the newly introduced cysteine outside the FGF2 protein, and the yellow color represents the existing cysteine toward the inside of the FGF2 protein.

4 shows the API5 protein (green color) bound to the FGF2 protein (light blue) into which cysteine was introduced at the C-terminus. In FGF2, cysteine at positions 167 (C167) and 234 (C234) previously existed (yellow), but this is toward the inside of the FGF2 protein, and the newly introduced cysteine at position 289 (C289, red) is located outside the FGF2 protein. do.

5 shows the results of measuring API5 and FGF2 binding based on the fluorescence polarization analysis method. When the His-API5 protein was bound to His-FGF2 labeled with Alexa Fluor 488, the FP value increased from 188 to 272. When the GST-API5 protein was combined with His-FGF2 labeled with Alexa Fluor 488, the FP value increased from 183 to 258.

6 shows the results of screening candidates for API5 and FGF2 binding inhibitors using the method of the present invention.

[Example 1]

Preparation of recombinant protein

In order to increase the screening efficiency of inhibitors that inhibit the binding of FGF2 and API5, it was attempted to amplify the affinity of FGF2 and API5 to fluorescent substances.

Specifically, it was mutated from FGF2 to C211S and C229S for mass expression in E. coli. A cysteine (Cys) residue was introduced as the 289th amino acid at the C-terminus outside the FGF2 protein (Cys289, FIG. 3). In order to increase the efficiency of protein purification, both FGF2 and API5 were tagged with His. In order to increase the efficiency of protein purification and to increase the molecular weight difference from FGF2, a recombinant API5 in which GST (Glutathion S-transferase) was fused to API5 was also prepared.

[Example 2]

Fluorescent labeling on proteins

We tried to determine whether the introduction of cysteine (Cys) affects the labeling efficiency of FGF2 and fluorescent substances (dyes).

Specifically, a TCEP solution (pH 7.5) was added to FGF2 without Cys or 100 times (molar ratio) of FGF2 prepared in Example 1, and then reacted at room temperature for 10 minutes. After 10 minutes, put the dye Alexa Fluor 488 (Dye/protein ratio = 0.5-3), mix by pipetting immediately, and spin down with a table top centrifuge. And it is allowed to react at room temperature for 2 hours, at that time, pipetting every 30 minutes. Finally, after wrapping with foil, it is reacted at 4°C for 15 hours (protein-fluorescent substance reaction). Dye that does not bind to FGF2 is removed using a HiTrap Desalting column (excess fluorescent substance removed). Check the profile peak obtained by performing the desalting column, and collect the tube that enters the peak. After measuring the absorbance of each solution in each tube at 280nm and 494nm, respectively, the labeling efficiency was confirmed through the following [Equation 1] and [Equation 2]. When the dye (M)/protein (M) value of [Equation 2] was 1 or less, it was determined that the labeling efficiency was high, and when the value was greater than 1, it was determined that the protein and the overreacted or unreacted dye were mixed.

The protein molar extinction coefficient (protein molar extinction coefficient) was 16,055 cm ^-1 M ^-1 ), and the dye molar extinction coefficient (Alexa Fluor 488) was 71,000 cm ^-1 M ^-1 . Amax is the absorbance of the dye measured at the maximum wavelength (Alexa Fluor 488: Abs 495), CF is the correction factor (Alexa Fluor 488: 0.11), and the dilution factor is diluted before measuring absorbance. Indicate the amount of sample.

[Equation 1]

Protein concentration (M) = [A ₂₈₀ -(A ₄₉₄ × 0.11)] × dilution ratio / protein molar extinction coefficient

[Equation 2]

Dye (M)/protein (M) = A ₄₉₄ × dilution factor / dye water extinction coefficient × protein concentration (M)

As a result, in the case of FGF2 without introducing Cys, the fluorescence polarization value was very low after a certain period of time. This is because the existing Cys residues present inside FGF2 are not sufficiently exposed on the protein surface, so that the fluorescent substance is shared. It was determined that labeling through binding was not properly performed. On the other hand, in the case of FGF2 in which Cys was exposed to the outside of the protein by introducing Cys at the C-terminus, it was confirmed that the fluorescent label was maintained for a long period of time. In addition, the labeling efficiency values of FGF2 with Cys introduced at the C-terminus were mostly about 0.3 to 0.8, and it was determined that Alexa Fluor 488 was appropriately covalently bonded to the introduced Cys to be labeled.

[Example 3]

Protein binding inhibitor screening

Fluorescence polarization analysis of binding of purified API5 full length (1.2 mM) stored in 1×PBS and FGF2 (residue 135-288; 10 nM) labeled with Alexa Fluor 488 prepared in Example 2 above It was confirmed by the method. Then, the conjugate was mixed with the small molecule compounds to be screened and a fluorescence polarization competition assay was performed to identify a substance that inhibits FGF2-API5 (FGF2 binding site) binding.

More specifically, a 1.2 mM His tag or GST fused API5 protein, 10 nM Alexa Fluor 488 labeled FGF2, and 6 candidate compounds of 1.5 to 2.25 mM were prepared at each concentration, and a 96-well black plate Each was put into each of the mixture and reacted at room temperature for 30 minutes, and then fluorescence polarization (mp) was measured at room temperature using Infinte F200 Pro (TECAN Group Ltd, Switzerland). The excitation wavelength was 485 nm, and the emission wavelength was 535 nm. The six candidate compounds are respectively API5 derived peptide (1), FGF2 derived peptide (2), API5 binding substance derived from thermal shift assay (3), a derivative of candidate 3 (4), and other candidate substances of 3 It is a derivative (5) and a negative control group (6).

As a result, when the compound was not added (No inhibitor), the fluorescence polarization value was measured as 279, compared to the measurement of 279, and when the inhibitory candidate material was added, various fluorescence polarization values in the range of about 152 to 250 appeared, especially candidate 3 In the case of, it was found that the fluorescence polarization value decreased to about 50% level. When the level of reduction in fluorescence polarization value is expressed as a percentage of inhibition, as shown in [Fig. 6], the degree of binding inhibition between candidate substances can be clearly distinguished, thereby inhibiting the binding of FGF2 and API5. It was confirmed that candidates for inhibition can be screened effectively.

In the case of using the screening method provided by the present invention, by introducing an additional cysteine into FGF2, the fluorescent substance can be labeled more efficiently, thereby amplifying the fluorescence polarization value measured by the fluorescence polarization analysis method, etc., and binding of FGF2 and API5 It has high industrial applicability because it can effectively screen for action) or substances that inhibit it.

Claims (11)

1. i) introducing cysteine to the outside of FGF2;

   ii) labeling a fluorescent material on FGF2 into which the cysteine of step i) has been introduced;

   iii) mixing the fluorescent substance-labeled FGF2 of step ii), a protein capable of binding to FGF2, and a candidate substance capable of inhibiting the binding thereof; And

   iv) measuring the fluorescence intensity of the mixed materials of step iii);

   FGF2 binding inhibitor screening method comprising a.
2. The screening method according to claim 1, wherein the cysteine of step i) is introduced at the C-terminus of FGF2.
3. The method of claim 1, wherein the fluorescent material in step ii) is FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine), FAM (fluorescein amidite), Rhodamine, TAMRA (Carboxytetramethylrhodamine), Cy™, CF™ (Cyanine-based fluorescent dye). And Alexa Fluor?, screening method, characterized in that at least any one selected from the group consisting of.
4. The screening method according to claim 1, wherein the protein capable of binding to FGF2 in step iii) has a molecular weight greater than or equal to FGF2.
5. The method of claim 1, wherein the protein capable of binding to FGF2 of iii) is any one or more tags selected from the group consisting of FLAG, histidine, CBD, MBP, TRX and GST (Glutathione S-transferase). A screening method, characterized in that it is a fused protein.
6. The method of claim 1, wherein the protein capable of binding to FGF2 in step iii) is API5.
7. The method of claim 1, wherein measuring the fluorescence intensity in step iv) is FRET (Fluorescence Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer), FP (Fluorescence Polarization), HCS (High-content Screening), and HTS (High Throughput Screening) screening method, characterized in that the measurement using any one or more methods selected from the group consisting of.
8. According to claim 1, v) If the fluorescence intensity measured in step iv) is reduced to a level of 50% to 100% compared to before mixing the candidate material of step iii), the step of determining as an FGF2 binding inhibitor is additionally performed. Screening method comprising a.
9. FGF2 binding inhibitors selected by the screening method of claim 1.
10. The inhibitor of claim 9, wherein the binding is a combination of FGF2 and API5.
11. A composition for inhibiting anticancer drug resistance comprising the FGF2 binding inhibitor of claim 9.

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