A method for screening inhibitors of glutaminase based on nuclear magnetic resonance targeting

Abstract

This invention discloses a method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance (NMR). This invention utilizes NMR technology to achieve in-situ, non-destructive, and real-time detection of specific glutamine metabolic pathways within cells, establishing a cell-based screening technology for anticancer active substances targeting specific glutamine metabolic pathways. This technology is based on […]. 13 C5, 15 [N2]-L-glutamine was used as a tracer to characterize the metabolic process of glutamine in liver cancer cells. Its downstream metabolite, glutamate, was detected using NMR. The inhibitory effect of the target active substance (such as herbal extracts) on glutamine metabolism was evaluated using glutamine consumption level and glutamate production level as indicators. The feasibility of this screening method was confirmed through validation of known inhibitors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method and approach for detecting glutamate, a metabolite of glutamine in cells, using nuclear magnetic resonance spectroscopy, and then screening inhibitors targeting glutaminase, providing new methods and ideas for the development of anticancer drugs. Background Technology

[0002] Metabolic reprogramming is a hallmark feature of malignant tumors, specifically referring to the adaptive changes in metabolic pathways that occur in cells during rapid proliferation and invasion to meet the increased demands for energy, biosynthetic precursors, and redox homeostasis. Unlike normal cells, which primarily rely on oxidative phosphorylation for energy under aerobic conditions, tumor cells often switch to glycolysis (the Warburg effect) even in the presence of oxygen, exhibiting abnormal activation of multiple anabolic pathways, including glutamine metabolism, hexosamine biosynthesis, and nucleotide metabolism. Among these, glutamine, as a key regulator, provides important carbon and nitrogen sources for tumor cell survival and proliferation, while also helping tumor cells resist oxidative stress damage by generating glutathione. Due to the multiple supporting roles of glutamine metabolism in tumorigenesis and development, targeting this metabolic pathway has become a highly anticipated cutting-edge direction in the field of anticancer drug development.

[0003] Glutaminase is the rate-limiting enzyme in the first step of the conversion of glutamine to glutamate. The glutamate produced by glutamate is a metabolic hub crucial for the malignant survival of cancer cells. It not only replenishes the tricarboxylic acid cycle by converting glutamine to α-ketoglutarate, providing energy and biosynthetic raw materials for tumors, but also serves as a precursor for glutathione synthesis, building an antioxidant defense to inhibit ferroptosis. More importantly, glutamate participates in maintaining the integrity of the mitochondrial electron transport chain and regulating protein translation, while simultaneously reshaping the immune microenvironment to promote drug resistance. Therefore, targeting glutaminase can selectively disrupt the critical metabolic lifeline of tumor cells, inducing metabolic stress, oxidative damage, and growth inhibition.

[0004] Glutaminase inhibitors are among the most closely watched categories. For example, CB-839 has entered clinical trials in various solid tumors and hematologic malignancies, demonstrating its ability to effectively inhibit tumor cell proliferation by blocking glutaminase catalysis. This indicates that glutaminase inhibitors hold significant potential for anti-tumor applications. However, the clinical translation of glutaminase inhibitors still faces numerous challenges, including drug resistance due to tumor metabolic plasticity and issues related to drug safety. Therefore, finding inhibitors with both high safety and efficacy is of significant clinical importance.

[0005] Traditional drug screening strategies for targeting glutamine metabolism in cancer therapy are mainly divided into two categories: phenotypic screening and targeted affinity screening. Phenotypic screening typically assesses the activity of test molecules by detecting endpoints such as cell proliferation, apoptosis, or metabolite accumulation. This method can directly reflect the overall efficacy of a compound, but it has limitations such as a high false positive rate and the inability to dynamically track metabolic changes in real time. While targeted affinity screening has higher throughput, it is divorced from the real metabolic network environment within cells and cannot reflect the metabolic stability of drugs in living cells, membrane permeability, and the influence of intracellular metabolic compensation mechanisms. This may lead to poor efficacy of the screened active molecules at the cellular level. Therefore, developing a new technology that can monitor specific metabolic pathways of glutamine in living cells in real time and in situ is crucial for overcoming existing screening bottlenecks and accelerating the development of anti-tumor drugs.

[0006] Nuclear magnetic resonance (NMR) allows for in-situ, non-destructive, and long-term real-time monitoring of cell samples. These unique advantages make NMR a robust and reliable tool for screening and validating the function of highly effective anticancer drugs targeting specific glutamine metabolic pathways. In recent years, traditional Chinese medicine (TCM) and its active ingredients have demonstrated unique advantages in the field of antitumor metabolism research due to their multi-target and multi-pathway characteristics. However, current research on TCM intervention in tumors by regulating glutamine metabolic pathways is still in its early stages, and systematic research on the mechanisms by which TCM active ingredients regulate glutamine metabolic pathways is insufficient. Therefore, screening more safe, effective, and phenotypically defined glutamine metabolism regulators from TCM resources holds promise for bringing new breakthroughs in tumor intervention and treatment. Summary of the Invention

[0007] Based on the aforementioned existing technologies, this invention provides a method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance (NMR). This invention utilizes NMR technology to achieve in-situ, non-destructive, and real-time detection of specific glutamine metabolic pathways within cells, establishing a cell-based screening technology for anticancer active substances targeting specific glutamine metabolic pathways. This technology is based on […]. 13 C5, 15 [N2]-L-glutamine was used as a tracer to characterize the metabolic process of glutamine in liver cancer cells. Its downstream metabolite glutamate was detected by NMR. The inhibitory effect of the test active substances (such as herbal extracts) on glutamine metabolism was evaluated by using the glutamine consumption level and glutamate production level as indicators. The feasibility of this screening method was confirmed by the validation of known inhibitors.

[0008] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows: A method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance imaging includes the following steps: S1. Culture liver cancer cells and embed them as live cell samples; S2. Prepare a mixture of isotopically labeled glutamine metabolic precursors; S3. Add the metabolic precursor mixture to the live cell sample and then incubate it to allow the liver cancer cells to take up and metabolize the isotope-labeled glutamine. S4. Add the test substance or test substance solution to the live cell sample treated in step S3, and then continue incubation to obtain the experimental sample. S5. The experimental samples were detected using nuclear magnetic resonance (NMR) technology to obtain and quantify the characteristic NMR signals of isotopically labeled glutamine, glutamic acid, and UDP-N-acetylglucosamine. S6. Add an equal amount of DMSO to the test substance or test substance solution to the live cell sample treated in step S3, and then continue incubation to obtain the control sample. S7. The control sample was detected using nuclear magnetic resonance (NMR) technology to obtain and quantify the characteristic NMR signals of isotopically labeled glutamine, glutamic acid, and UDP-N-acetylglucosamine. S8. Compare the characteristic NMR signal intensity of isotopically labeled glutamic acid in the experimental sample with that in the control sample to evaluate the inhibitory effect of the analyte on glutamic acid production. S9. If the characteristic NMR signal intensity of isotopically labeled glutamate in the experimental sample is significantly lower than that of isotopically labeled glutamate in the control sample, then the analyte exhibits an inhibitory effect on glutamate synthesis and is identified as an inhibitor targeting glutaminase.

[0009] Furthermore, the isotope-labeled glutamine is [ 13 C5, 15 [N2]-L-glutamine.

[0010] Furthermore, the liver cancer cells are HepG2 cells.

[0011] Furthermore, the substance to be tested is an extract of traditional Chinese medicine.

[0012] Furthermore, the Chinese herbal medicines mentioned are selected from Scutellaria baicalensis extract, Codonopsis pilosula extract, or Astragalus membranaceus extract.

[0013] Furthermore, the nuclear magnetic resonance techniques mentioned include HMBC and HCNCH.

[0014] Furthermore, during the HCNCH detection, 13 C- 15 N-transfer delay optimized to 25 ms Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention utilizes [ 13C5, 15 [N2]-L-glutamine, as a tracer, can highly specifically monitor the specific metabolic pathway of isotopically labeled glutamine to glutamate conversion.

[0015] 2. This invention uses NMR detection technology to observe the production process of glutamate, a metabolite of glutamine, in living cells, which can improve screening efficiency and accuracy. It is also suitable for the precise screening of complex active substances such as extracts of traditional Chinese medicine.

[0016] 3. This invention, through verification of known inhibitors (CB-839), confirms the feasibility of this screening method and successfully screens out traditional Chinese medicines that inhibit glutaminase activity. Attached Figure Description

[0017] Figure 1 for[ 13 C5, 15 Dynamic changes over time of HCNCH spectrum signals of glutamine, UDPHexNAc (downstream metabolite of glutamine), and glutamate in HepG2 live cells as traced by N2-L-glutamine metabolism.

[0018] Figure 2 The graph shows the changes in glutamine and glutamate levels in HepG2 live cells over time after treatment with inhibitors and different herbal extracts. Figure 2 a is a graph showing the change in glutamine content. Figure 2 b is a graph showing the changes in glutamic acid content. Detailed Implementation

[0019] To facilitate understanding and implementation of the present invention by those skilled in the art, the present invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0020] The invention adopts [ 13 C5, 15 [N2]-L-glutamine was used as a tracer to track the metabolic conversion of glutamine to glutamate in HepG2 cells. The inhibitor CB-839 acts on glutaminase 1 (GLS1), a key enzyme in glutamine breakdown, inhibiting the conversion of glutamine to glutamate. The results showed that the inhibitor CB-839 was significantly effective. Through verification of known inhibitors like CB-839, the traditional Chinese medicine Astragalus membranaceus was screened and found to have a certain inhibitory effect on the conversion of glutamine to glutamate. Example 1

[0021] 1. Contains [ 13 C5, 15 Preparation of the [N2]-L-glutamine metabolic precursor mixture: Weigh out 61.25mg [ 13 C5, 15 [N2]-L-glutamine, dissolved in 10 mL of high-glucose DMEM medium (complete medium) supplemented with 10% (v:v) fetal bovine serum and 1% (v:v) penicillin-streptomycin, so that [ 13 C5, 15 The final concentration of [N2]-L-glutamine was 40 mM. The resulting mixture was filtered through a 0.22 μm microporous membrane, and the filtrate was diluted to 100 mL with complete culture medium to obtain [ 13 C5, 15 A mixture of metabolic precursors with a concentration of 4 mM of N2-L-glutamine.

[0022] 2. Preparation of extracts from traditional Chinese medicine herbs: 2.1 Weigh an appropriate amount of Astragalus powder and immerse it completely in 80% (v / v) ethanol solution. Let it stand at room temperature for 5 days. After immersion, perform ultrasonic-assisted extraction for 15 min using an ultrasonic cleaner. Filter the crude extract through a 0.22 μm microporous membrane under vacuum to remove residues, insoluble particles, and other impurities, obtaining a clear filtrate. Place the clear filtrate in a rotary evaporator and evaporate under reduced pressure at 76℃, 20 rpm to remove most of the ethanol solvent, obtaining a viscous concentrate. Transfer the concentrate to a freeze dryer and freeze-dry until completely powdered to thoroughly remove residual solvent. Seal the obtained Astragalus freeze-dried powder and store it in a -20℃ freezer protected from light for later use. Before the experiment, add an appropriate amount of Astragalus freeze-dried powder to dimethyl sulfoxide (DMSO) and vortex thoroughly to dissolve it. Then, centrifuge at 12000 rpm for 5 min in a high-speed centrifuge, discard the insoluble precipitate at the bottom, and collect the supernatant, which is the Astragalus extract.

[0023] 2.2. Scutellaria baicalensis and Codonopsis pilosula were extracted and prepared according to the above method to obtain Scutellaria baicalensis extract and Codonopsis pilosula extract, respectively.

[0024] 3. Preparation of live cell samples: 3.1 Immediately after removing HepG2 cells from the liquid nitrogen tank, thaw them in a 37°C water bath. Then, disperse the thawed HepG2 cell solution in a 1:9 volume ratio in high-glucose DMEM medium supplemented with 10% fetal bovine serum (v:v) and 1% (v:v) penicillin-streptomycin. Centrifuge at 200 rcf to remove the supernatant and obtain cell pellet. Redisperse the obtained cell pellet in complete medium and incubate at 37°C with 5% CO2 for continuous amplification.

[0025] 3.2 After culturing in an incubator for 48 hours, the cells were harvested to obtain a cell pellet. The obtained cell pellet was preheated at 37°C for 20 seconds, resuspended in agarose solution, and squeezed into a PEEK tube. After standing at room temperature for 2 minutes and 20 seconds, the agarose gel line formed in the PEEK tube was transferred to a specially designed NMR tube using a 5 mL syringe. This NMR tube is used for sample loading in the bioreactor.

[0026] 4. Loading live cell samples: 4.1 Following the method reported by Enrico Luchinat et al. in the literature, an NMR bioreactor was constructed. The NMR bioreactor consisted of a thermostat and a circulating flow device to maintain the nutrients required by the cells. The bioreactor was thoroughly rinsed before sample loading to ensure sterility. The rinsing solutions used in the following order were: 0.2 M sodium hydroxide solution, 3 M citric acid solution, 0.2 M sodium hydroxide solution, 75% (v / v) ethanol, and sterile water. Each rinsing solution was used at the same flow rate for 10 minutes.

[0027] 4.2 Connect the NMR tube containing the live cell sample obtained in step 3 into the bioreactor, and then add 30 mL of 4 mM metabolic precursor mixture to the NMR tube to incubate the live cell sample in the NMR bioreactor.

[0028] 5. Real-time detection of metabolism in live cell samples: The NMR tubes of the live cell samples processed in step 4 were placed in a 5 mm TCI (Temperature Compressor). 13 C 1 H, 15 HCNCH spectra were continuously acquired at 310 K while incubating on a 600 MHz Bruker Avance III spectrometer with a low-temperature probe. An experiment was set up to detect HCNCH in live cells, with spectral widths of 12 ppm (1H) and 185 ppm (N). 13 C), the spectral center was also set at 4.7 ppm and 90 ppm. Data acquisition used 64 cumulative summations. 1 H and 13 The time-domain data points in dimension C are 2048 and 128, respectively. The polarization transfer delay is optimized based on the HC coupling constant of 140 Hz, while... 13 C- 15 The N-transfer delay is specifically set to 25 ms (corresponding to 1 / (4J) of the CN coupling constant of 10 Hz) to ensure optimal signal transfer efficiency.

[0029] In order to observe in real time [ 13 C5, 15A control experiment was conducted to determine whether [N2]-L-glutamine could enter and be metabolized in HepG2 cells. HepG2 cells were compared with cells containing [N2]-L-glutamine. 13 C5, 15 The metabolic precursor mixture of [N2]-L-glutamine was incubated in a bioreactor and monitored in real time. The results are as follows: Figure 1 As shown, Figure 1 Prove that as time changes, [ 13 C5, 15 [N2]-L-glutamine can enter HepG2 cells and be metabolized into glutamate (Glu), while driving the synthesis and accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc).

[0030] Real-time detection of experimental samples: The NMR tubes of the live cell samples processed in step 4 were placed in a 5 mm TCI (Temperature Compressor). 13 C 1 H, 15 HCNCH spectra were continuously acquired at 310 K while incubating on a 600 MHz Bruker Avance III spectrometer with a low-temperature probe. After 4 hours of incubation, Astragalus extract was added to an NMR tube containing the live cell sample treated in step 4, and incubation continued while HCNCH spectra were continuously acquired.

[0031] The experiment was conducted using equal volumes of Scutellaria baicalensis extract, Codonopsis pilosula extract, inhibitor CB-839 solution (CB-839 dissolved in DMSO to prepare a 10 mM CB-839 solution), and dimethyl sulfoxide instead of Astragalus membranaceus extract, following steps 4-6.

[0032] 8. Treatment with Astragalus membranaceus extract, Scutellaria baicalensis extract, Codonopsis pilosula extract, inhibitor CB-839 solution, and dimethyl sulfoxide [ 13 C5, 15 HepG2 cells were used for N2]-L-glutamine metabolism tracking. The signal dynamics of glutamate and glutamine in the HCNCH spectrum were measured. Signal integration of glutamate and glutamine in the HCNCH spectrum yielded the changes in glutamine and glutamate levels over time, as shown in the graph. Figure 2 As shown, by Figure 2 (a) It is known that treatment with Astragalus membranaceus extract, Scutellaria baicalensis extract, Codonopsis pilosula extract, inhibitor CB-839 solution, and dimethyl sulfoxide [ 13 C5, 15 Following N2]-L-glutamine metabolism tracing in HepG2 cells, the glutamine content in HepG2 cells gradually decreased over time. Figure 2 (b) It can be seen that dimethyl sulfoxide treatment [ 13 C5,15 Following N2]-L-glutamine metabolism tracing in HepG2 cells, the glutamate content in HepG2 cells initially increased slowly over time, then decreased slowly, and finally increased sharply. Treatment with Scutellaria baicalensis extract and Codonopsis pilosula extract... 13 C5, 15 After N2]-L-glutamine metabolism tracing in HepG2 cells, the glutamine content in HepG2 cells first gradually decreased and then gradually increased over time. At 10 hours, the glutamine content in both treatment groups was significantly higher than that in the dimethyl sulfoxide treatment group; Astragalus extract and inhibitor CB-839 solution treatment [ 13 C5, 15 After tracing HepG2 cells with N2]-L-glutamine metabolism, the glutamate content in HepG2 cells gradually decreased over time. The Astragalus extract group showed almost no significant change in the last two hours. The glutamine content in these two treatment groups was much lower than that in the other three treatment groups.

[0033] It can be seen that treatment with Astragalus extract [ 13 C5, 15 Following N2]-L-glutamine metabolism tracing in HepG2 cells, the upward trend of glutamate was suppressed, and its level was even lower than that in the CB-839 treatment group (e.g., Figure 2 (b) This indicates that the traditional Chinese medicine Astragalus membranaceus contains active ingredients that inhibit the production of glutaminase, thereby inhibiting the conversion of glutamine to glutamate.

Claims

1. A method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance, characterized in that... Includes the following steps: S1. Culture liver cancer cells and embed them as live cell samples; S2. Prepare a mixture of isotopically labeled glutamine metabolic precursors; S3. Add the metabolic precursor mixture to the live cell sample and then incubate it to allow the liver cancer cells to take up and metabolize the isotope-labeled glutamine. S4. Add the test substance or test substance solution to the live cell sample treated in step S3, and then continue incubation to obtain the experimental sample. S5. The experimental samples were detected using nuclear magnetic resonance (NMR) technology to obtain and quantify the characteristic NMR signals of isotopically labeled glutamine, glutamic acid, and UDP-N-acetylglucosamine. S6. Add an equal amount of DMSO to the test substance or test substance solution to the live cell sample treated in step S3, and then continue incubation to obtain the control sample. S7. The control sample was detected using nuclear magnetic resonance (NMR) technology to obtain and quantify the characteristic NMR signals of isotopically labeled glutamine, glutamic acid, and UDP-N-acetylglucosamine. S8. Compare the characteristic NMR signal intensity of isotopically labeled glutamic acid in the experimental sample with that in the control sample to evaluate the inhibitory effect of the analyte on glutamic acid production. S9. If the characteristic NMR signal intensity of isotopically labeled glutamate in the experimental sample is significantly lower than that of isotopically labeled glutamate in the control sample, then the analyte exhibits an inhibitory effect on glutamate synthesis and is identified as an inhibitor targeting glutaminase.

2. The method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance according to claim 1, characterized in that: The isotope-labeled glutamine is [ 13 C5, 15 [N2]-L-glutamine.

3. The method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance according to claim 1, characterized in that: The liver cancer cells were HepG2 cells.

4. The method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance according to claim 1, characterized in that: The substance to be tested is an extract of traditional Chinese medicine.

5. The method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance according to claim 4, characterized in that: The Chinese herbal medicines mentioned are selected from Scutellaria baicalensis extract, Codonopsis pilosula extract, or Astragalus membranaceus extract.

6. The method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance according to claim 1, characterized in that: The nuclear magnetic resonance techniques mentioned include HMBC and HCNCH.

7. The method for screening inhibitors targeting glutaminase based on nuclear magnetic resonance according to claim 6, characterized in that: During the HCNCH detection 13 C- 15 The N-transfer delay has been optimized to 25 ms.

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