KR101538517B1 - A METHOD FOR EVALUATION OF WILD TYPE AND MUTANT TYPE ISOCITRATE DEHYDROGENASE ACTIVITY USING 13C LABELED α-KETOGLUTARATE AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY - Google Patents

Abstract

The present invention relates to a method for evaluating the activity of a circular or mutant IDH enzyme by selectively monitoring only α-KG and 2-HG in cell lysates using α-KG labeled with 13 C and a nuclear magnetic resonance spectroscopy technique. The present invention can selectively monitor only α-KG and 2-HG among many compounds in HDF cell lysates using α-KG labeled with 13C, thereby confirming the presence of IDH prototype and mutant type and observing enzyme activity And can be useful for the development of inhibitors of enzymes. In addition, the inhibitor thus developed can be used as a treatment for brain glioma.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for evaluating the activity of circular and mutant forms of isocitrate dehydrogenase enzyme using alpha-ketoglutarate labeled with < 13 > C and a nuclear magnetic resonance spectrometer LABELED α-KETOGLUTARATE AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY}

The present invention is based on the finding that by selectively monitoring only alpha-ketoglutarate and 2-hydroxyglutarate in a complex mixture such as cell lysate using isotope-labeled alpha-ketoglutarate and nuclear magnetic resonance spectroscopy, The present invention relates to a method for evaluating the activity of a citrate dehydrogenase of a prototype and a mutant form. The present invention also relates to a method for screening a therapeutic agent for brain glioma using the same.

Brain cancer is a type of glioma that occurs within the brain parenchyma. Glioblastoma is a typical refractory disease with a median survival of one year, even if a combination of surgery, radiotherapy, and chemotherapy is applied. Isocitrate dehydrogenase (abbreviated as IDH) gene mutation was detected in more than 80% of secondary gliomas and WHO grade II-III brain gliomas in brain glioma, and IDH gene mutation was clinically diagnosed . The IDH prototype enzyme is known to oxidatively decarboxylate isocitric acid to produce α-ketoglutarate (hereinafter abbreviated as α-KG) and NADH or NADPH have. The enzyme is also an intermediate metabolite that synthesizes amino acid biosynthesis of glutamic acid, glutamine, arginine and proline, as well as energy metabolism through the electron transport system. Therefore, the TCA circuit for energy metabolism, protein biosynthesis and nitrogen metabolism It acts as an important regulatory enzyme. IDH enzymes of higher animals are classified into three types of isoforms: cytoplasmic NADP + -specific IDH1, mitochondrial NADP + -specific IDH2, and mitochondrial NAD + -specific IDH3, depending on the type of cofactor and the location of the cofactor It is divided.

Although the circular IDH enzyme mediates α-KG conversion from isocitrate, the IDH mutant enzyme does not mediate the mechanism of the circular IDH enzyme, and 2-hydroxyglutarate (hereinafter referred to as' 2-HG Quot;). ≪ / RTI > Therefore, 2-HG is expressed as a specific "onco-metabolite" in IDH gene mutation brain tumors as the expression of 2-HG is increased in brain glioma patients, This is being raised. Therefore, the development of a technique for determining the IDH gene mutation in patients with brain glioma by selectively quantifying 2-HG and measuring the activity of the IDH gene is clinically significant in terms of effective treatment design and monitoring of patient's prognosis and treatment response I have.

Currently, MRS (magnetic resonance spectroscopy) is used in vivo to quantify 2-HG, but lack of information about the baseline of the spectrum and the intrinsic methyl groups and fatty acids in lysates of brain glioma cells and tissues The overlapping of a signal of a small molecule and a target signal becomes a problem. In addition, the 2-HG assays using LC-MS have a disadvantage in that retention time is changed, and therefore it is necessary to perform the operation using the standard product every time to judge whether 2-HG is present or not. Although an enzyme assay kit is commercially available, there is a limit to measure only the circular IDH activity. Above all, it is not a direct measurement of 2-HG by measuring the change of NADH. Therefore, a method for measuring 2-HG more easily is demanded.

The present invention provides a method comprising: obtaining a sample in an individual; Contacting the obtained sample with a sample containing isotopically labeled? -KG; Obtaining a HCACO spectrum using a nuclear magnetic resonance spectrometer (NMR) of a sample containing? -KG in contact with the sample; And confirming the presence of 2-HG through the obtained spectrum. The present invention also provides a method for providing information for brain glioma diagnosis.

In addition, the present invention relates to a method for detecting a mutant IDH, which comprises mixing and reacting a sample containing an isotope labeled? -KG, a mutant IDH and a sample; Obtaining a HCACO spectrum using the sample and a sample containing? -KG reacted with the mutant IDH using nuclear magnetic resonance spectroscopy (NMR); Confirming the presence and content of 2-HG through the obtained spectrum; And selecting a sample that is less than the peak value of 2-HG obtained by contacting only mutant IDH with isotopically labeled? -GK, or in which 2-HG is not detected, to the mutant IDH inhibitor screening method .

In addition, the present invention relates to a method for preparing a sample, which comprises mixing a sample containing isotope labeled? -KG, a mutant IDH and a sample; Obtaining the mixture with a change of time using a nuclear magnetic resonance spectrometer (NMR) and HCACO spectra; Measuring the presence and content of? -GG, the presence and content of 2-HG through the obtained spectrum; And ascertaining an increase in the content of 2-HG according to time. The present invention also provides a method for measuring the activity of a mutant IDH enzyme.

An aspect includes obtaining a sample from an individual; Contacting the obtained sample with a sample containing isotopically labeled? -KG; Obtaining a spectrum using a nuclear magnetic resonance spectroscopy (NMR) of a sample containing? -KG in contact with the sample; And confirming the presence of 2-HG through the obtained spectrum. The present invention also provides a method for providing information for brain glioma diagnosis.

The above method will be described in detail as follows.

First, a step of obtaining a sample from an object is included.

An individual can be an animal and can be a human. It may also be a human having a disease. At this time, the disease may be a brain disease, and it may be a brain glioma. Samples can be obtained in human blood. Blood samples can be plasma or serum. In addition, samples can be obtained in tissue. At this time, tissues can be obtained in various parts of the human body. Skin tissue, brain tissue, lung tissue, and the like. At this time, the tissue can be peeled as soon as possible to minimize degradation and minimize contamination from blood cells. In addition, enzymes may be included if they are present, such as in urine, tears and saliva.

Next, the method can include the step of contacting the obtained sample with a sample containing isotopically labeled? -GK. The obtained sample can be contacted with isotopically labeled? -KG without separate purification or separation. As used herein, the term "isotope labeled a-KG" means that at least one atom is replaced by an isotope. Also, carbon can be replaced by a non-stable isotope. At this time, it is preferable that four carbon atoms of the five carbon atoms of? -KG are substituted (Fig. 1A). The contact may be mixed in a separate container, but may be done in a tube containing the obtained sample. At this time, 0.0001 mg to 50 mg of? -KG may be used per 1 mL of the sample. Also, 0.001 mg to 10 mg per 1 mL of the sample may be used. Also, 0.01 mg to 5 mg per 1 mL of the sample may be used. Also, 0.1 mg to 1 mg per 1 mL of sample may be used.

At this time, it is possible to additionally contact NADPH together. As used herein, the term "NADPH" is a reduced form of the nicotinamide adenine dinucleotide phosphate and may also be referred to as "nicotinamide adenine dinucleotide phosphate (reduced form). At this time, NADPH may be contained in an amount of 0.01 mg to 100 mg per 1 mg of? -KG administered. It may also contain from 0.1 mg to 10 mg per mg of α-KG administered.

Next, the sample containing alpha-KG contacted with the sample may be subjected to nuclear magnetic resonance spectroscopy (NMR) to obtain the HCACO spectrum. As used herein, the term "nuclear magnetic resonance" is referred to as "Nuclear Magnetic Resonance" or "NMR ", and nuclear magnetic resonance spectroscopy measures chemical shifts Means an analysis device. Dimensional HCACO (correlation between 1HA, 13CA and 13CO chemical shifts) technique while obtaining the spectrum. The principle of the 1D-HCACO technique is to add a selective pulse to the intrinsic chemical shift value of the carbonyl carbon and the alpha carbon in a compound in which the carbonyl carbon and the alpha carbon are directly linked by a covalent bond, Obtaining the spectroscopic spectrum selectively detects only the hydrogen peak of the alpha carbon. Each of α-KG and 2-HG has its own specific H-CA-CO chemical shift, and is characterized by obtaining a single hydrogen peak (signal) by applying a selective pulse according to the H-CA-CO chemical shift .

Finally, the presence of 2-HG may be confirmed through the obtained spectrum.

2-HG combines intrinsic H-CA-CO specific chemical shifts to selectively present a 1H peak at 3.5 ppm to 4.5 ppm when selectively pulsed and a 1H peak at 4.04 ppm. When peaks are formed in this section, it can be confirmed that 2-HG exists.

In another aspect, there is provided a method for detecting a protein comprising the steps of: reacting a sample containing an isotope-labeled? -KG, a mutant IDH and a sample; Obtaining a HCACO spectrum using the sample and a sample containing? -KG reacted with the mutant IDH using nuclear magnetic resonance spectroscopy (NMR); Confirming the presence and content of 2-HG through the obtained spectrum; And selecting a sample that is less than the peak value of 2-HG obtained by contacting only mutant IDH with isotopically labeled? -GK, or in which 2-HG is not detected, to the mutant IDH inhibitor screening method .

First, a sample containing isotope labeled α-KG, a mutant IDH, and a sample may be mixed and reacted.

Isotopically labeled? -Ge is replaced by one or more atomic isotopes. At this time, 0.0001 mg to 50 mg of? -KG may be used per 1 mL of the sample. Also, 0.001 mg to 10 mg per 1 mL of the sample may be used. Also, 0.01 mg to 5 mg per 1 mL of the sample may be used. Also, 0.1 mg to 1 mg per 1 mL of sample may be used. In addition, the NADPH can be contacted together. At this time, the amount of NADPH may be 0.01 mg to 100 mg per 1 mg of α-KG to be administered. It may also contain from 0.1 mg to 10 mg per mg of α-KG administered.

The sample may be a compound, a mixture, an extract or a fraction, or a mixture thereof, which is expected to inhibit the activity of IDH. At this time, AGI-5198, which is known as an inhibitor of mutant IDH1, can be used as a positive control. Also, it may be Rutaecarpine, Wuweizisu B, Rutaevin, Oroxylin A, Umbelliferone, or Paeonol, But any material can be used as a sample. When the sample is treated, it can be treated with 0.001 μM to 1000 μM, treated with 0.01 μM to 100 μM, and treated with 0.5 μM to 50 μM.

Next, the sample containing α-KG reacted with the sample and the mutant IDH may be subjected to a nuclear magnetic resonance spectroscopy (NMR) to obtain a HCACO spectrum. A method of obtaining the HCACO spectrum using a nuclear magnetic resonance spectroscope can be performed as described above.

Next, the step of confirming the presence and content of 2-HG through the obtained spectrum may be included. 2-HG combines intrinsic H-CA-CO specific chemical shifts to selectively present a 1H peak at 3.5 ppm to 4.5 ppm when selectively pulsed and a 1H peak at 4.04 ppm. When peaks are formed in this section, it can be confirmed that 2-HG exists.

Next, selection may be made of a sample that is less than the peak value of 2-HG obtained by contacting only mutant IDH with isotopically labeled? -GG, or 2-HG was not detected. At this time, as a positive control group, a substance known to inhibit the activity of mutant IDH can be used. In particular, AGI-5198 can be used. When AGI-5198 was treated, the activity of the mutant IDH was inhibited, so that? -KG was not converted to 2-HG.

In addition, the negative control group was not treated with a mutant IDH activity inhibitor. At this time, all? -GG is converted to 2-HG. Therefore, it can be judged that the activity of the mutant IDH is suppressed when a peak of low intensity is detected as compared with the negative control group.

Yet another aspect provides a mutant IDH inhibitor screened by the screening method. At this time, the mutant IDH inhibitor may be used as a composition for treating brain glioma.

The pharmaceutical composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredient for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Can further be suitably formulated according to the respective disease or ingredient, using appropriate methods in the art or as disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

The pharmaceutical composition used in the present invention can be manufactured for oral administration, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intramuscular, transdermal, and the like. May be mixed with any pharmaceutically acceptable vehicle for injectable compositions for direct injection. The pharmaceutical compositions of the present invention may comprise a freeze-dried composition which allows for the composition of an injectable solution, in particular by addition of an isotonic sterile solution or dry, in particular sterile water, or an appropriate physiological saline. Direct injection of the nucleic acid into the tumor of the patient can be advantageous because it allows the therapeutic efficiency to be concentrated in the infected tissue. The dosage can be adjusted by various parameters, in particular by the gene, the vector, the mode of administration employed, the disease in question, or alternatively, the required duration of treatment. Also, the range varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. The daily dose is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and is preferably administered once to several times a day.

Yet another aspect is a method of identifying a sample, comprising the steps of mixing a sample containing an isotope labeled? -KG, a mutant IDH and a sample; Obtaining the mixture with a change of time using a nuclear magnetic resonance spectrometer (NMR) and HCACO spectra; Measuring the presence and content of a-KG and the presence and content of 2-HG through the obtained spectrum; Determining the activity of the mutant IDH enzyme comprising identifying a change in the content of 2-HG according to time.

First, it may include mixing a sample containing the isotope labeled? -KG, a mutant IDH, and a sample.

Isotopically labeled? -Ge is replaced by one or more atomic isotopes. At this time, 0.0001 mg to 50 mg of? -KG may be used per 1 mL of the sample. Also, 0.001 mg to 10 mg per 1 mL of the sample may be used. Also, 0.01 mg to 5 mg per 1 mL of the sample may be used. Also, 0.1 mg to 1 mg per 1 mL of sample may be used. In addition, the NADPH can be contacted together. At this time, the amount of NADPH may be 0.01 mg to 100 mg per 1 mg of α-KG to be administered. It may also contain from 0.1 mg to 10 mg per mg of α-KG administered.

Next, the mixture may be subjected to a nuclear magnetic resonance spectroscopy (NMR) to obtain the HCACO spectrum according to the change of time.

Experiments may include proceeding at 37 [deg.] C, at which the activity of the enzyme can operate normally. A method of obtaining the HCACO spectrum using a nuclear magnetic resonance spectroscope can be performed as described above. Each of α-KG and 2-HG has its own specific H-CA-CO chemical shift, and it is characterized by obtaining one hydrogen peak (signal) by applying selective pulse according to H-CA-CO chemical shift do. And mixing the mixture to obtain the HCACO spectrum from the time when? -KG is converted to 2-HG by the mutant IDH enzyme. The spectrum is obtained by crossing the 2-HG HCACO spectrum with? -GK.

Next, the presence and content of α-KG and the presence and content of 2-HG can be measured through the obtained spectrum. Specifically, the presence and content of α-KG and the presence and content of 2-HG are increased through the spectrum.

2-HG combines intrinsic H-CA-CO specific chemical shifts to selectively present a 1H peak at 3.5 ppm to 4.5 ppm when selectively pulsed and a 1H peak at 4.04 ppm. When peaks are formed in this section, it can be confirmed that 2-HG exists. It can be seen that as the size of the formed peak increases, the content increases.

α-KG combines unique H-CA-CO specific chemical shifts, and when selectively pulsed, a 1H peak may exist at 2.5 ppm to 3.5 ppm, and a 1H peak may exist at 2.96 ppm. When peaks are formed in this region, it can be confirmed that a-KG exists. It can be confirmed that when the size of the formed peak decreases, the content decreases.

Finally, we provide a method for measuring the activity of mutant IDH enzymes through the change of 2-HG content over time.

The activity of an enzyme can be measured by varying the concentration of the enzyme product over time. By comparing with the standard product, the change of the 2-HG concentration with time can be confirmed, and if the changed concentration is divided by the correspondingly changed time, the enzyme activity unit comes out. In addition, the activity of the mutant IDH enzyme can be judged to be high as the content of 2-HG increases.

The present invention allows the selective measurement of 2-HG produced by the mutant IDH enzyme from 13C-labeled α-KG by a nuclear magnetic resonance spectroscope HCACO method among many compounds in the cell lysate, thereby observing the prognosis of brain glioma Alternatively, mutant IDH enzymes can be useful for the development of inhibitors and the activity of mutant IDH enzymes can be measured.

Figure 1 shows the reaction of circular IDH (abbreviated as "WT-IDH") and mutant IDH (abbreviated as "MT-IDH" hereinafter) and optimization of the HCACO pulse sequence It is showing. Figure 1 (a) shows the reaction result by IDH. 13C4 The labeled carbon is marked with an asterisk, and the detectable HC-CO spin system is marked with a dotted line. The chemical shifts of the spin system are also indicated. FIG. 1 (b) shows an HCACO pulse sequence optimized for an off-resonance type pulse in an open shape. This optional pulse has a Q3 form with a 12.5 kHz B1 field. Hard pulses are indicated by vertical lines.
Figure 2 shows the spectrum of the reaction product of WT-IDH and MT-IDH. The left graph of FIG. 2 (a) shows the conventional one-dimensional noesy-presaturation spectrum of the reaction product by MT-IDH in the cell lysate. The middle plot shows a one-dimensional HCACO spectrum optimized for a-KG, a substrate for MT-IDH in cell lysates. The right graph shows the 1-dimensional HCACO spectrum optimized for 2-HG, the substrate of MT-IDH in cell lysates. All spectra were obtained from a single sample. The middle spectrum was obtained before the reaction, and after the reaction, the spectrum was changed to the 2-HG spectrum. Figure 2 (b) shows the characteristics of the HCACO-based essay. The left is the HCACO spectrum for the MT-IDH reaction, the middle is the HCACO spectrum for the WT-IDH reaction, and the right is the HCACO spectrum for the reaction without the enzyme.
Figure 3 shows the results of experiments on enzyme inhibition experiments. The figure at the top left shows the one-dimensional HCACO spectrum of 2-HG, which is the MT-IDH-reacted product. Figure 3 (a) shows the spectral results obtained after adding 0.7 μM of AGI-5198, which is known as an inhibitor of MT-IDH. 3 (b) to 3 (g) show spectral results obtained after treatment of 50 μM of the indicated compound (b: Rutaecarpine, c: Wuweizisu B, d: Rutaevin, e: Oroxylin A, f: Umbelliferone, g: Paeonol). FIG. 3 (h) shows the spectral results obtained after the treatment of the material in which all of the above materials are mixed.
Fig. 4 shows an NMR spectrum for observing changes of? -KG and 2-HG with time as a one-dimensional HCACO technique. Cycle number is indicated and it takes about 22 minutes per cycle.
Figure 5 shows a heterogeneous nuclear HCACO spectrum that is modified when MT-IDH is reacted and α-KG is converted to 2-HG.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1. Isotopes Labeled  2- KG Wow NMR  The 2- HG Detection  Confirm

1.1. Preparation of sample

Α-KG (1,2,3,4-13C4, 99%) labeled with isotope 13C4 was purchased from Cambridge Isotope Labratories (Andover, MA, U.S.A.). Recombinant WT-IDH1 (TP710042) and recombinant MT-IDH1 (TP710043) were also purchased from Origene (Rockville, MD, U.S.A.).

1.2. Cell line preparation and culture

Human dermal fibroblast (HDF) was obtained from Professor Jeong Jin Ho of Seoul National University College of Medicine. The cell lines were cultured in DMEM medium (Hyclone, UT, USA). At this time, heat-inactivated fetal bovine serum (FBS) and penicillin (100 U / mL, Hyclone, UT, USA) were added to the medium. The cell lines were cultured in an incubator having a humidity of about 5% CO ₂ and 90% at 37 ° C.

1.3. NMR  Sample preparation for spectrometer measurement

A cell lysate was obtained in about 10 ⁶ HDF cells for enzyme assay. Specifically, roughly counted cells were centrifuged at 1,000 rpm for 5 minutes to obtain cell pellets. The obtained cell pellet was washed three times with PBS buffer. And then resuspended in 200 [mu] l of cell lysis buffer (Invitrogen, USA). After resuspending, the cells were pulverized by ultrasonication. The ultrasonic treatment was repeated 10 times on the ice for 1 second after 3 seconds of ultrasonic treatment. The pulverized cells were then centrifuged at 15,000 g for 20 minutes at 4 ° C. The supernatant obtained after centrifugation was transferred to a new tube. To the supernatant obtained above, 0.1 mg of the recombinant enzyme,? -GG, and 0.858 mg of NADPH were added. After that, finally, 5 vol% of D2O for holding the lock signal was put and transferred to the NMR tube. After that, the cells were incubated overnight at 37 ° C, and then a spectrum was obtained using an NMR spectrometer. In addition, a spectrum was obtained using NMR spectroscopy before the reaction to obtain the spectrum of? -KG. However, monitoring of changes in α-KG and 2-HG over time showed that the HCACO spectra were obtained directly at NMR spectroscopy without incubation time at 37 ° C.

1.4. NMR  Spectrum measurement using spectrometer

The NMR spectrometer was a 600-MHz Bruker Avance spectrometer with a cryogenic probe. One-dimensional NMR spectra were performed with a noesy-presaturation parameter (Fig. 2a, left). However, since there is a large amount of lysate in the HDF cell lysate, the overlap with the target signal is severe, so that it is impossible to distinguish and quantify α-KG and 2-HG in the conventional 1D 1H-NMR spectrum.

In addition, the HCACO spectrum was obtained as a gradient-selected HCACO pulse sequence at an absolute temperature of 310K. Compared to the off-resonance, the center of chemical shifts was at 178 ppm and 74 ppm for 2-HG for carbonyl carbon and alpha carbon, 208 ppm and 34 for alpha-KG, respectively ppm.

When the sample was subjected to HCACO technique with selective pulses at 178 and 74 ppm, the intrinsic chemical shifts of 2-HG carbonyl carbon and alpha carbon, the spectrum of 2-HG alpha carbon Peaks are detected and when the selective pulses at 208 and 38 ppm are applied on the same principle, the alpha carbon-hydrogen peak of α-KG can be detected at around 3 ppm, and even if α-KG and 2-HG are mixed, Respectively. In addition, by detecting the peak every 11 minutes, it is possible to observe quantitative changes of? -KG and 2-HG with time, and it is possible to monitor the activity of the gene mutant enzyme (Fig. 4).

Example  2. MT - IDH  Screening inhibitor

2.1. Preparation of sample

In order to confirm that inhibitor screening of MT-IDH was possible using NMR, AGI-5198 (see Chemical Formula 1), which is known as inhibitor of R132H MT-IDH in MT-IDH, was obtained from Xcessbio (San Diego, Respectively. In addition, in order to confirm whether other substances inhibit the activity of MT-IDH, compounds having the following formulas (2) to (7) were prepared.

2.2. NMR  Sample preparation for spectrometer measurement

A cell lysate was obtained in about 10 ⁶ HDF cells for enzyme assay. Specifically, roughly counted cells were centrifuged at 1,000 rpm for 5 minutes to obtain cell pellets. The obtained cell pellet was washed three times with PBS buffer. And then resuspended in 200 [mu] l of cell lysis buffer (Invitrogen, USA). After resuspending, the cells were pulverized by ultrasonication. The ultrasonic treatment was repeated 10 times on the ice for 1 second after 3 seconds of ultrasonic treatment. The pulverized cells were then centrifuged at 15,000 g for 20 minutes at 4 ° C. The supernatant obtained after centrifugation was transferred to a new centrifuge tube.

To the supernatant obtained above, 0.1 mg of the recombinant enzyme,? -GG, and 0.858 mg of NADPH were added. In addition, 0.5 μl of each of the above-mentioned Formulas 2 to 7 was added to the final concentration of 50 μM, and the final sample was completely mixed with 0.5 μl of each of the 2 to 7 mixture. After incubation at 37 ° C overnight, HCACO spectra were obtained using NMR spectroscopy.

2.3. NMR  Spectrum measurement using spectrometer

The HCACO spectrum was obtained using an NMR spectrometer in the same manner as the method performed in 1.4. As shown in FIG. 3, it was confirmed that 2-HG was produced by MT-IDH when the inhibitor was not treated. However, it was confirmed that 2-HG was hardly produced when AGI-5198, which is known as an inhibitor of MT-IDH, was treated. In addition, it was confirmed that the production amount of 2-HG was lower than that of the control group in which none of the compounds of the formulas (2) to (7) was different. In particular, when the compounds of formulas (2) to (7) were mixed, it was confirmed that the activity of MT-IDH was inhibited to a degree similar to that of AGI-5198.

Example  3. MT - IDH  Active measurement

3.1. NMR  Sample preparation for spectrometer measurement

A cell lysate was obtained in about 10 ⁶ HDF cells for enzyme assay. Specifically, roughly counted cells were centrifuged at 1,000 rpm for 5 minutes to obtain cell pellets. The obtained cell pellet was washed three times with PBS buffer. And then resuspended in 200 [mu] l of cell lysis buffer (Invitrogen, USA). After resuspending, the cells were pulverized by ultrasonication. The ultrasonic treatment was repeated 10 times on the ice for 1 second after 3 seconds of ultrasonic treatment. The pulverized cells were then centrifuged at 15,000 g for 20 minutes at 4 ° C. The supernatant obtained after centrifugation was transferred to a new centrifuge tube.

To the supernatant obtained above, 0.1 mg of the recombinant enzyme, α-KG and 0.858 mg of NADPH were added and the HCACO spectrum was obtained at 37 ° C. using an NMR spectrometer.

3.2. NMR  Measurement of spectrum and measurement of enzyme activity using spectroscope

The HCACO spectrum was obtained by NMR spectroscopy in the same manner as in the above-mentioned method in which the above mixture was mixed and subjected to the conversion from α-KG to 2-HG by the mutant IDH enzyme at 1.4. As shown in FIG. 4, a peak of 1H? -GH at 2.5 ppm to 3.5 ppm and a 1H-HG peak at 3.5 ppm to 4.5 ppm were confirmed. The content of α-KG decreased with time and increased with 2-HG time. The concentration of 2-HG was increased by comparison with the standard. The activity of the enzyme was calculated by subtracting the changed 2-HG concentration with the corresponding time, and the MT-IDH activity value of 0.03 milliunit / mL was obtained.

When the screening method of the present invention is used, the mutant IDH inhibitor can be screened effectively, so that an effective therapeutic agent for brain glioma can be easily found, so that it can be usefully used in the development of new drugs.

Claims (12)

Obtaining a sample in the subject;
Contacting the obtained sample with a sample containing isotopically labeled? -KG (? -Ketoglutarate);
Obtaining a spectrum using a nuclear magnetic resonance spectroscopy (NMR) HCACO technique with a sample containing? -GK contacted with said sample; And
And confirming the presence of 2-HG (2-hydroxyglutarate) through the obtained spectrum. The method according to claim 1, wherein the isotope labeled α-KG is labeled with 13C. The method of claim 1, wherein identifying the presence of 2-HG (2-hydroxyglutarate) is performed by confirming that the 1H-13C peak is present at 3.5 ppm to 4.5 ppm. Way. 4. The method of claim 3, wherein the 1H-13C peak is present at 4.04 ppm. Mixing a sample containing isotope-labeled? -KG (? -Ketoglutarate), a mutation isocitrate dehydrogenase, and a sample;
Obtaining a spectrum of the sample containing α-KG which has reacted with the sample and the mutant isocitrate dehydrogenase using nuclear magnetic resonance spectroscopy (NMR);
Confirming the presence and content of 2-HG (2-hydroxyglutarate) through the obtained spectrum; And
Selecting a sample that is less than or equal to the peak value of 2-HG obtained by contacting the mutant isocitrate dehydrogenase with an isotopically labeled? -GG, or 2-HG was not detected, Screening method. 6. The method according to claim 5, wherein the isotope labeled α-KG is labeled with 13C. 6. The method of claim 5, wherein confirming the presence of 2-HG (2-hydroxyglutarate) is performed by confirming that the 1H-13C peak is present at 3.5 ppm to 4.5 ppm. 8. The method of claim 7, wherein the 1H-13C peak is present at 4.04 ppm. A mutant IDH inhibitor obtained by the screening method of claim 5. 9. A composition for treating brain glioma comprising the mutant IDH inhibitor of claim 9. Mixing a sample containing isotope labeled? -KG, a mutant IDH and a sample to prepare a mixture thereof;
Obtaining the mixture with a change of time using a nuclear magnetic resonance spectrometer (NMR) and HCACO spectra;
Measuring the presence and content of? -GG, the presence and content of 2-HG through the obtained spectrum; And
Determining the change in the content of 2-HG according to time; and determining the activity of the mutant IDH enzyme. 12. The method according to claim 11, wherein the activity of the IDH enzyme is determined to be high as the content of 2-HG increases.

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