Cyclic peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of tumor drugs, and particularly relates to cyclic peptide as well as a preparation method and application thereof. The cyclic peptide provided by the invention, as an inhibitor of membrane protein, has a natural structure advantage and is high in bioavailability; besides, the cyclic peptide can effectively inhibit the transport activity of GLUT5 on the molecular level and inhibit fructose uptake of tumor cells so as to inhibit cell proliferation, for example, IC50 for inhibiting proliferation of K562 leukemia cells is equal to 14.57 mu M; in addition, generation of fructose metabolite pyruvic acid can be effectively reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor drugs, and in particular relates to a cyclic peptide and a preparation method and application thereof. Background Art

[0002] Fructose is widely found in various fruits and honey. With advances in sugar production technology, high-fructose corn syrup, a sweetener containing a high concentration of fructose, has become widely used worldwide, leading to a dramatic increase in fructose intake. However, numerous studies have shown that excessive fructose intake is highly correlated with obesity, gut microbial imbalance, colon cancer, ovarian cancer, prostate cancer, breast cancer, and leukemia. Unlike glucose, fructose absorption is not regulated by insulin, resulting in a faster absorption rate. Furthermore, fructose can enter cells through the GLUT5 transporter. Once inside, fructose is first phosphorylated by fructokinase to fructose-1-phosphate, consuming one ATP molecule. Fructose-1-phosphate is then converted to fructose-6-phosphate by fructose-1-phosphate aldolase. Fructose-6-phosphate is then converted to fructose-1,6-diphosphate through a series of enzymatic reactions. Fructose-1,6-diphosphate then enters the glycolysis pathway to produce energy.

[0003] Tumor cells exhibit the Warburg effect, whereby they metabolize glucose through glycolysis, rather than the more efficient tricarboxylic acid cycle, even in the presence of oxygen. Because glycolysis produces less ATP, tumor cells require increased glucose intake to meet their energy needs. Upon depletion of glucose, tumor cells switch to fructose as a carbon source. Studies have shown that increased fructose intake increases tumor malignancy, migration, and proliferation. Blocking fructose uptake or utilization by tumor cells can effectively slow tumor progression and inhibit tumor cell proliferation. GLUT5 is a key transporter for fructose entry into cells. Its expression is tissue-specific, with high expression primarily on the apical and lateral plasma membranes of intestinal epithelial cells, while expression is lower in red blood cells, kidney, adipose tissue, muscle, and brain. Studies have shown that SLC2A5 is upregulated in various tumors and positively correlated with poor prognosis, highlighting the role of GLUT5 in tumor cell growth. For example, fructose promotes GLUT5 expression in colorectal cancer cells, enhancing their ability to adapt to hypoxia. These observations suggest that GLUT5 may play a key role in maintaining tumor cell survival. Gene knockout or silencing of SLC2A5 can inhibit cancer cell proliferation and metastasis, indicating that GLUT5 is a potential target for tumor therapy.

[0004] Therefore, the development of targeted inhibitors against GLUT5 is expected to become a new strategy for tumor treatment. Summary of the Invention

[0005] Based on this, the present invention synthesized a polypeptide (also called polypeptide B38), which can effectively inhibit GLUT5 activity, thereby inhibiting the uptake of fructose by tumor cells to achieve the purpose of inhibiting cell proliferation, and showed excellent activity in inhibiting the proliferation of K562 leukemia cells and reducing pyruvate production.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a cyclic peptide, the structural formula of which is shown below:

[0008]

[0009] Another aspect of the present invention provides a method for preparing the cyclic peptide of the present invention, comprising: combining a tetrapeptide fragment and eFKBD to obtain a cyclic peptide, wherein the structural formula of the tetrapeptide fragment is as follows:

[0010]

[0011] The structural formula of eFKBD is shown below:

[0012]

[0013] In another aspect, the present invention provides a pharmaceutical composition of the present invention, comprising the cyclic peptide of the present invention.

[0014] In another aspect, the present invention provides a pharmaceutical preparation comprising the cyclic peptide or the pharmaceutical composition of the present invention.

[0015] Preferably, the dosage forms of the above-mentioned pharmaceutical preparation include injection, oral solution, tablet, powder, granule, capsule, patch or suppository.

[0016] In another aspect, the present invention provides a use of the cyclic peptide or the pharmaceutical composition of the present invention in the preparation of a drug for treating tumors.

[0017] Preferably, in the above application, the cyclic peptide or pharmaceutical composition has the effect of inhibiting tumor cell proliferation.

[0018] Preferably, in the above applications, the cyclic peptide or pharmaceutical composition has the following effects:

[0019] (i) It has the effect of inhibiting the activity of GLUT5 protein in tumor cells;

[0020] (ii) It has the effect of reducing fructose uptake by tumor cells;

[0021] (iii) It has the effect of reducing pyruvate production.

[0022] Preferably, in the above application, the tumor is a leukemia hematological malignancy.

[0023] The beneficial effects of the present invention include:

[0024] (1) The cyclic peptide B38 provided by the present invention has natural structural advantages as a membrane protein inhibitor and high bioavailability;

[0025] (2) The cyclic peptide B38 provided by the present invention can effectively inhibit the transport activity of GLUT5 at the molecular level (binding K of GLUT5) D 16.3 μM), and inhibited the uptake of fructose by tumor cells, thereby inhibiting cell proliferation. For example, the IC 50 =14.57μM; in addition, it can also effectively reduce the production of fructose metabolite pyruvate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The affinity between MSNBA and GLUT5-EGFP;

[0027] Figure 2 The affinity between the cyclic peptide B38 and GLUT5-EGFP in the present invention;

[0028] Figure 3 The cyclic peptide B38 in the present invention changes the thermal stability of GLUT5;

[0029] Figure 4 The inhibitory effect of the cyclic peptide B38 of the present invention on the proliferation of K562 cells under the culture conditions of 6 mM fructose and glucose;

[0030] Figure 5 The inhibitory effect of MSNBA on K562 cells under 6 mM fructose culture conditions is shown.

[0031] Figure 6 The inhibition of isotope fructose transport by cyclic peptide B38 in K562 cells;

[0032] Figure 7 This is the effect of cyclic peptide B38 on pyruvate production in K562 cells. DETAILED DESCRIPTION

[0033] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0034] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0035] An embodiment of the present invention provides a cyclic peptide, the structural formula of which is shown below:

[0036]

[0037] The present invention also provides a method for preparing the cyclic peptide of the present invention, which comprises combining a tetrapeptide fragment and eFKBD to obtain a cyclic peptide, wherein the structural formula of the tetrapeptide fragment is as follows:

[0038]

[0039] The structural formula of eFKBD is shown below:

[0040]

[0041] An embodiment of the present invention further provides a pharmaceutical composition of the present invention, which includes the cyclic peptide of the present invention.

[0042] It should be noted that the cyclic peptide in the present invention can be combined with other active ingredients to form a pharmaceutical composition to achieve the purpose of improving the therapeutic effect. The other active ingredients can be small molecule compounds or protein small molecules.

[0043] An embodiment of the present invention further provides a pharmaceutical preparation, which includes the cyclic peptide of the present invention or the pharmaceutical composition of the present invention.

[0044] It should be noted that the cyclic peptide or pharmaceutical composition of the present invention can be combined with a carrier to prepare different pharmaceutical dosage forms, such as injections, oral liquids, tablets, powders, granules, capsules, patches or suppositories; in addition, the preparation methods of different pharmaceutical dosage forms are well known in the art.

[0045] The embodiments of the present invention also provide a use of the cyclic peptide of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating tumors.

[0046] In some specific examples, in the above applications, the cyclic peptide or pharmaceutical composition has the effect of inhibiting tumor cell proliferation.

[0047] In some specific examples, in the above applications, the cyclic peptide or pharmaceutical composition has the following effects:

[0048] (i) It has the effect of inhibiting the activity of GLUT5 protein in tumor cells;

[0049] (ii) It has the effect of reducing fructose uptake by tumor cells;

[0050] (iii) It has the effect of reducing pyruvate production.

[0051] In some specific examples, in the above application, the tumor is a leukemia hematological malignancy.

[0052] It should be noted that the cyclic peptide of the present invention can effectively inhibit the transport activity of GLUT5 at the molecular level and inhibit the uptake of fructose by tumor cells, thereby inhibiting cell proliferation. For example, IC 50 =14.57μM; in addition, it can also effectively reduce the production of fructose metabolite pyruvate (GLUT5 is a membrane protein that mainly transports fructose, pyruvate is a fructose metabolite, and inhibition of GLUT5 will lead to a decrease in pyruvate. Multiple aspects have verified that the compound inhibits GLUT5 uptake).

[0053] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0054] 1. Synthesis of Cyclic Peptide B38

[0055] Example 1

[0056] The B38 structure can be divided into two fragments: the structural domain eFKBD fragment and the effector domain tetrapeptide fragment. The specific preparation method is as follows:

[0057] (1) Synthesis of the eFKBD domain fragment

[0058] According to the paper "Rapamycin-inspired macrocycles with new target specificity"

[0059] The synthesis was carried out according to the synthesis method described in (DOI:10.1038 / s41557-018-0187-4).

[0060] (2) Synthesis of tetrapeptide fragments

[0061] The synthetic route of the tetrapeptide fragment is as follows:

[0062]

[0063] The specific synthesis steps are as follows:

[0064] 1) The dried resin beads were shaken with CH2Cl2 (8×, 8 times the volume of the resin beads) for 20 min (orbital shaking, 500 rpm-600 rpm) to fully swell the resin, and then the solvent was drained and washed with DMF (1×);

[0065] 2) An amino acid (L-4-fluorophenylalanine) protected with Fmoc (9-fluorenylmethoxycarbonyl) (3.0 equivalents) and HATU (condensing agent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (3.0 equivalents) were added sequentially to the resin preparation container and quickly mixed, followed by the addition of DIPEA (N,N-diisopropylethylamine) (6.0 equivalents);

[0066] 3) After shaking for 3 hours, the reaction mixture was drained, briefly washed with DMF, and fresh reaction mixture was added a second time to ensure high conversion;

[0067] 4) After draining the solvent, shake with a mixed solvent of piperidine and DMF (piperdine:DMF=1:4 (v / v)) for 20 minutes to deprotect the Fmoc group. After draining the solvent, rinse the resin with DMF (1×) and CH2Cl2 (4×) five times, respectively, and then dry the resin under vacuum;

[0068] 5) Then, 4-tert-butoxy-L-proline, L-4-hydroxyphenylalanine, and N-methyl-L-glycine were sequentially coupled to the cis-C6 linker-conjugated resin beads according to the above method to obtain a tetrapeptide fragment.

[0069] (3) Synthesis of cyclic peptides

[0070] The synthetic route of cyclic peptide is as follows:

[0071]

[0072] The specific synthesis steps are as follows:

[0073] 1) eFKBD (1.5 equivalents), HATU (2.0 equivalents), and DIPEA (3.0 equivalents) synthesized in step (1) were sequentially added to a container containing resin, and after stirring for 6 hours, the reaction mixture was drained to obtain a resin to which the eFKBD fragment was attached;

[0074] 2) The tetrapeptide-linked resin prepared in step (2) and the eFKBD-linked resin were placed in a microwave reactor, and then DCE (dichloroethane) (2 mL / 100 mg, meaning 2 mL DCE was added to 100 mg of the resin mixture) and Hoveyda-Grubbs II (purchased from Bidex Pharmaceuticals, purity 98.1%) (30 mmol%, the amount of Hoveyda-Grubbs II in the resin mixture was 30 mmol%) were added;

[0075] 3) The reactor was sealed and the reaction program was set to stir at 140°C for 30 minutes under microwave irradiation;

[0076] 4) After the reaction is completed, the solvent is removed by rotary evaporation;

[0077] 5) The crude product was separated and purified by silica gel column chromatography and eluted with MeOH:DCM=1:10 to obtain cyclic peptide B38.

[0078] 2. Affinity test of cyclic peptide B38 and GLUT5

[0079] The affinity of cyclic peptide B38 to GLUT5-EGFP was verified by surface plasmon resonance (SPR), and the specific steps are as follows.

[0080] (1) Run Buffer Preparation

[0081] Prepare the running buffer according to Table 1.

[0082] Table 1 Preparation of running buffer

[0083] Reagents Final concentration NaCl 150mM Hepes (4-hydroxyethylpiperazineethanesulfonic acid) buffer, pH 8.0 10mM Tween20 0.1％ DDM (dodecyl-β-D-maltoside) 0.5mM CHS (cholesterol sulfate sodium salt) 0.5mM DMSO (dimethyl sulfoxide) 2％

[0084] After the running buffer is prepared, filter it using a 0.2μm filter membrane. In the Biacore 8K molecular interaction instrument, remove the maintenance chip and replace it with the CM5 chip that needs to be coupled with protein, and run the Change solution program.

[0085] (2) Coupling ligand

[0086] (1) A CM5 chip was selected and GLUT5-EGFP protein (GLUT5 protein linked to green fluorescent protein EGFP) was pre-enriched. The purpose was to adsorb and increase the concentration of the ligand (GLUT5-EGFP protein) near the chip surface through electrostatic interaction during the chemical coupling process, thereby improving the coupling efficiency and reducing ligand consumption. The ligand (GLUT5-EGFP protein) was diluted in 10 mM sodium acetate buffer at pH 4.0, pH 4.5, pH 5.0, and pH 5.5, respectively, and flowed over a blank chip surface to test the effect of electrostatic adsorption.

[0087] (2) Take out sodium acetate solutions of different pH values from the 4°C refrigerator and add about 20 μg of purified GLUT5-EGFP protein. Mix thoroughly and add to the well plate according to the instrument prompts. Select the pre-set program: Contact time 120 s, Flow rate 10 μL / min, start the program, and select the sodium acetate solution with the highest response value for the next step of formal protein coupling.

[0088] (3) After preliminary experiments, it was found that sodium acetate solution with pH 4.0 was more suitable for coupling GLUT5-EGFP. About 20 μg of GLUT5-EGFP protein was added to the sodium acetate solution with a final volume of 60 μL. 89 μL of EDC-NHS and 129 μL of Ethanolamine were added to the corresponding positions of a 96-well U-bottom plate. The Immobilization program was run on the instrument: Contact time 600 s, Flow rate 10 μL / min.

[0089] (3) Sample injection

[0090] (1) Due to the high refractive index of DMSO (dimethyl sulfoxide) (1200 RU for 1% DMSO), a solvent calibration solution was prepared according to the instrument instructions. The solution was run at the beginning and end of the experimental run and every 20-30 cycles. The solvent calibration solution was prepared as shown in Table 2 below.

[0091] Table 2 Solvent calibration solutions

[0092] name Run Buffer DMSO 3% DMSO 4.9mL 150μL 1.5% DMSO 4.9mL 75μL name 3% DMSO 1.5% DMSO 2% DMSO 750mL 1.5mL 2.5% DMSO 1.5mL 750μL

[0093] (2) Take 50mM MSNBA (a specific inhibitor of GLUT5 fructose transport in proteoliposomes,

[0094]

[0095] ) was dissolved in DMSO and then diluted with DMSO to 2.5 mM as the MSNBA stock solution; 10 μL of the 2.5 mM MSNBA stock solution was taken and thoroughly mixed with 10 μL of DMSO for dilution, and 10 μL was taken for the next round of dilution, and five rounds of dilution were performed continuously; 4 μL of the diluted small molecule compound MSNBA was added to 196 μL of DMSO-free running buffer, mixed thoroughly, and then added to a 96-well plate according to the prompts of the Biacore 8K instrument, and the pre-set single-cycle run program was run.

[0096] (3) After all programs are completed, remove the CM5 chip and replace it with a maintenance chip. Change the solution to dH2O and perform desalination maintenance.

[0097] The affinity test results of MSNBA and GLUT5 are as follows Figure 1 The results showed that cyclic peptide B38 has a certain affinity for GLUT5, K D It is 50.4μM.

[0098] In addition, the affinity of cyclic peptide B38 to GLUT5 was tested according to the above test method, and the results were as follows: Figure 2 The results showed that cyclic peptide B38 has a certain affinity for GLUT5, K D The affinity was 16.3 μM, which was better than that of MSNBA.

[0099] 3. Thermal stability test of cyclic peptide B38 and GLUT5

[0100] In the following tests, the electrophoresis and staining steps are as follows:

[0101] (1) Preparation of polyacrylamide gel and electrophoresis solution

[0102] 1) Prepare 50×TAE Buffer according to Table 3 below.

[0103] Table 3 50×TAEBuffer Preparation

[0104] Reagent name Mass / Volume Tris 242g glacial acetic acid 57.1mL <![CDATA[Na2EDTA·H2O]]> 37.2g HCl Adjust to pH 8.0 <![CDATA[dH2O]]> Adjust volume to 1L

[0105] 2) Prepare 10× electrophoresis buffer according to Table 4 below.

[0106] Table 410× electrophoresis fluid preparation

[0107]

[0108]

[0109] 3) Dilute 50×TAE Buffer to 1×TAE with dH2O. Weigh 0.25 g of agarose and place it in a 200 mL conical flask. Add 25 mL of 1×TAE dilution buffer and heat in a microwave oven until the agarose is completely melted. Remove and shake well. This is a 1% agarose gel solution.

[0110] 4) When the agarose gel has cooled slightly, add 2.5 μL of Gel-red dye and mix thoroughly. Pour the warm agarose solution into the mold; allow the gel solution to completely solidify at room temperature for 20 minutes. Carefully remove the comb and add 1× TAE buffer to the electrophoresis tank.

[0111] 5) Mix 3 μL DNA sample and 0.5 μL Loading. Add all the prepared samples to the wells. Cover the electrophoresis tank and connect the electrode plugs. The DNA should migrate from the negative electrode to the positive electrode (red plug). Apply a voltage of 120 V and a current of 100 mA-200 mA for 45 minutes until the sample reaches the bottom.

[0112] (2) Dilute the above 10× electrophoresis buffer 10 times with dH2O, slowly pour an appropriate amount of electrophoresis buffer into the electrophoresis tank, add the target protein to the sample well, and perform electrophoresis at a constant voltage of 210V for 50 minutes.

[0113] (3) Prepare Coomassie Brilliant Blue staining solution according to Table 5 below.

[0114] Table 5 Coomassie Brilliant Blue Staining Solution Preparation

[0115] Reagent name Mass / Volume Coomassie Brilliant Blue R250 2.5g Anhydrous ethanol 600mL glacial acetic acid 700mL dH2O Adjust volume to 1L

[0116] (4) After electrophoresis, remove the gel from the electrophoresis tank and place it in a gel box. Add an appropriate amount of Coomassie Brilliant Blue staining solution, heat in a microwave for 30 seconds, and stain on a shaker at room temperature for 10 minutes.

[0117] (5) Pour off the staining solution, wash with dH2O, add 20% anhydrous ethanol, heat in a microwave oven for 2.5 minutes, and decolorize on a shaker at room temperature overnight.

[0118] Thermal stability test of cyclic peptide B38 and GLUT5

[0119] (1) Take the purified GLUT5-EGFP and adjust the protein concentration to 0.08 mg / mL using MB buffer (25 mM Tris, pH 8.0, 150 mM NaCl, 10% Glycerol) containing 0.5 mM DDM + 0.5 mM CHS. Add PPase (PPase purified by our laboratory) at a mass ratio of GLUT5-EGFP:PPase = 10:1, and digest on ice for 2 h.

[0120] (2) Add 1% (mass fraction) OG (octylglucoside) to the protein solution after enzyme digestion, mix thoroughly, and dispense into 1.5 mL EP tubes;

[0121] (3) Add DMSO (final concentration of 1% (volume fraction)) and 100 μM cyclic peptide B38 to the aliquoted protein and incubate on ice for 1 h;

[0122] (4) After incubation, the tubes were aliquoted into 1.5 mL EP tubes and then heated in a metal bath at 35°C, 38°C, 41°C, 44°C, 47°C, 50°C, 53°C, and 56°C for 10 min, and then immediately placed on ice;

[0123] (5) After centrifugation at 13000 rpm and 4°C for 20 min, carefully remove the supernatant with a pipette and discard the precipitate. Take 15 μL of the supernatant and add 5 μL of 4×Loading to mix thoroughly. After SDS-PAGE verification, stain with Coomassie Brilliant Blue.

[0124] The results showed that the molecular level experiment Protein thermal shift also showed that cyclic peptide B38 changed the thermal stability of GLUT5 (see Figure 3 ).

[0125] 4. Test of the inhibitory effect of cyclic peptide B38 on cell proliferation

[0126] (1) K562 cell culture

[0127] Cell recovery:

[0128] (1) Quickly remove K562 cells (human chronic myeloid leukemia cells) from the liquid nitrogen tank and place on ice. Thaw the cells by gently shaking in a 37°C water bath until the solution in the cryovial is only partially thawed.

[0129] (2) Centrifuge at 900 rpm for 4 min, discard the supernatant, add the corresponding culture medium (RPMI 1640 medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin) to resuspend the cells, transfer the cells to a cell culture flask with a pipette, shake gently, and place in a 37°C, 5% CO2 cell culture incubator. Change the medium after 24 h.

[0130] Cell passaging and cell cryopreservation:

[0131] (1) K562 cells were cultured in T75 flasks at 37°C, 5% CO2 (RPMI 1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin). Cells were passaged after approximately 3 days of culture.

[0132] (2) Use a Pasteur pipette to transfer one-third of the K562 cells (one-third of the culture medium) to a new T75 culture flask and add fresh culture medium to a volume of 50 mL. Mix gently and place in a cell culture incubator (37°C, 5% CO2, the same below);

[0133] (3) K562 cells were centrifuged once every three passages. K562 cells were centrifuged at 900 rpm for 4 min in a 50 mL centrifuge tube and the original culture medium was discarded. Approximately 5 mL of RPMI1640 culture medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin was added. After gentle pipetting to mix, the cells were added to a T75 culture flask containing 45 mL of 1640 complete culture medium and cultured in a cell culture incubator.

[0134] (4) Before freezing, the K562 cells were centrifuged at 900 rpm for 4 min to collect the cell pellet. The cells were resuspended in a freezing solution containing 90% FBS and 10% DMSO, and then aliquoted into 1 mL cell cryovials. The cells were transferred to a programmed cooling box and placed in a -80°C refrigerator for complete cooling. After that, the cells were transferred to liquid nitrogen for long-term storage.

[0135] (II) Cell plating

[0136] (1) Remove K562 cells in the logarithmic growth phase from the incubator and wash twice with PBS solution preheated to 37°C;

[0137] (2) Adjust the K562 cell density to 2.6×10 cells using sugar-free RPMI 1640 complete medium (sugar-free RPMI 1640 with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin). 4 cells / mL, add 180 μL of K562 cell suspension adjusted to the target density of 5000 cells / well (5000 cells / well) to each well of a 96-well cell culture plate;

[0138] (3) Take 10 μL of 1200 mM fructose solution, add it to 90 μL of sugar-free RPMI 1640 complete medium, mix thoroughly, and then take 10 μL of the mixture and add it to the plated K562 cells. In the fructose-free control group, add 10 μL of sugar-free RPMI 1640 complete medium to the plated K562 cells.

[0139] (4)IC 50 During the assay, 8 μL of DMSO solution containing cyclic peptide B38 (the concentration of cyclic peptide B38 is 10 mM) was taken, and the dilution was obtained after 2-fold dilution with DMSO. 4 μL of DMSO solution containing cyclic peptide B38 (the concentration of cyclic peptide B38 is 10 mM) and the 2-fold dilution were respectively added to 36 μL of sugar-free 1640 complete culture medium, and the mixture was thoroughly blown and mixed with a spray gun. 10 μL was taken and added to the plated cells.

[0140] (5) After all 96-well plates are loaded, 200 μL of PBS solution should be added to the side wells of the 96-well plate to prevent the side well effect caused by evaporation of the culture medium. Then the 96-well plate is placed in a cell culture incubator and cultured for 3 days to obtain a cell suspension (the final fructose concentration in the cell suspension is 6 mM). Three replica wells were set up for each group of experiments in this experiment.

[0141] (3) Detection of viable cell number

[0142] The number of live cells was detected using the Cell prof luminescence viability detection reagent to quantify ATP to detect live cells in the culture medium. During the experiment, care was taken to avoid light, including: taking the Cell prof solution out of the -80°C refrigerator to thaw, using a dispenser to add samples, adding 80 μL of Cell prof solution to each well of the well plate where the cells were cultured in the previous step, using a dispenser to thoroughly pipette and mix (be careful not to produce bubbles), then taking 200 μL into a white 96-well plate, leaving it at room temperature for 8 minutes until the luminescence signal stabilizes, and detecting the chemiluminescence signal on a microplate reader.

[0143] The fructose solution was replaced with glucose solution, and the experiment was carried out according to the above method to test the number of living cells. The effects of fructose and glucose on cell viability in combination with cyclic peptide B38 were as follows: Figure 4 shown.

[0144] In addition, the cyclic peptide B38 was replaced with MSNBA, and the experiment was carried out according to the above method to test the number of living cells. The effect of fructose and MSNBA on cell viability was as follows: Figure 5 shown.

[0145] pass Figure 4 and Figure 5 It can be seen that the results show that the cyclic peptide B38 in the present invention has a good proliferation inhibitory function compared with MSNBA. The IC 50 It is 14.57μM.

[0146] 5. Uptake and transport experiments

[0147] (1) Cell plating and 13 C isotope fructose incubation

[0148] (1) Remove K562 cells in the logarithmic growth phase from the incubator and wash three times with PBS solution preheated to 37°C;

[0149] (2) Count the cells using a trypan blue cell counter and adjust the cell density to 6.67×10 6 cells / mL, plated into 24-well plates, 300 μL per well, i.e. 2×10 6 cells / well;

[0150] (3) Place the 24-well plate with cells in a cell culture incubator and starve for 30 minutes; add 1.5 μL of 10 mM MA10 DMSO stock solution to the cells; add 1.5 μL of DMSO to the control group, mix quickly and gently with a 200 μL pipette, and then place in a cell culture incubator and incubate for 10 minutes;

[0151] (4) Take the 24-well plate out of the cell culture incubator and quickly add 5 μL of 600 mM 13 C6-fructose, and use a 200 μL pipette to quickly and gently pipette to mix, and place in a cell culture incubator and incubate for 30 minutes.

[0152] (2) Mass spectrometry detection

[0153] (1) Take the cells incubated with fructose in the previous step out of the incubator, aspirate as much of the K562 cells as possible, transfer them to a 1.5 mL centrifuge tube, and centrifuge at 12000 rpm for 30 seconds;

[0154] (2) Discard the supernatant and add pre-chilled PBS solution containing 6 mM fructose to mix the cell pellet quickly and gently. Centrifuge again at 12,000 rpm and 4°C for 30 s. Repeat this washing step three times. Be careful not to aspirate the cells during the supernatant aspiration process and aspirate the supernatant as cleanly as possible to reduce errors.

[0155] (3) Since fructose is absorbed, transported and metabolized quickly, the operation time of this step should be reduced as much as possible;

[0156] (4) The washed K562 cell pellet was resuspended in a methanol:acetonitrile:ultrapure water = 1:1:1 (v / v / v) resuspension solution. 50 μL of the above resuspension solution was added to each tube of cell pellet. The cells were vortexed for 2 min to fully lyse the cells and then placed in a −80°C refrigerator for overnight extraction.

[0157] (5) Thaw the cell lysate extracted overnight at -80°C, centrifuge at 4°C, 13,000 rpm for 1 hour, carefully aspirate the supernatant (be careful not to aspirate the precipitate, otherwise it will clog the mass spectrometer pipeline) and place it in a sample vial for testing; the sample can be stored in a -80°C refrigerator before LC-MS / MS analysis;

[0158] (6) Before injection, equilibrate the column with the initial mobile phase of 95% acetonitrile and 5% 5 mM ammonium formate for 30 min until the column pressure stabilizes. The column pressure of the chromatographic column used in the present invention is generally stable at around 230 bar. Note that ultrapure water must be used when preparing the buffer used for mass spectrometry.

[0159] (7) Chromatographic analysis parameters are as follows:

[0160] Mobile phase A: 5 mM ammonium formate, mobile phase B: acetonitrile, flow rate 0.4 mL / min, column temperature 40°C, elution program as shown in Table 3 below.

[0161] Table 6 Chromatographic analysis elution program

[0162]

[0163]

[0164] Run for 2 minutes according to the above parameters. 13 The C6-fructose ion pairs are shown in Table 7 below.

[0165] Table 7 13 C6-fructose ion pair

[0166] Q1Mass Q3Mass DwellTime(ms) CE (volts) 185.100 61.00 30.0 -35.00

[0167] in addition, 13 The C6-fructose parameters are as follows:

[0168] Curtain Gas (CUR): 40; Collision Gas (CAD): Medium; Ion Spray Voltage (IS); Temperature (TEM): 450.0; Ion Source Gas1 (GS1): 50.0; Ion Source Gas2 (GS2): 50.0; Declustering Potential (DP): -10; Entrance Potential (EP): -10; Collision Cell Exit Potential (CXP): -10.

[0169] During the sample injection process, methanol was injected every 9 samples to clean the pipeline and prevent contamination between samples.

[0170] Test results such as Figure 6 and Figure 7 The results showed that cyclic peptide B38 inhibited 13 C isotope-labeled fructose enters K562 cells ( Figure 6 ), and reduce intracellular 13 Production of C isotope-labeled pyruvate ( Figure 7 ).

[0171] VI. Verification of the anti-tumor function of cyclic peptide B38 in vivo

[0172] To systematically evaluate the in vivo antitumor activity of the novel compound B38 against K562 cells, this study established a human K562 cell xenograft model for pharmacodynamic studies. Three immunodeficient NOD / SCID mice were subcutaneously inoculated with K562 cells to induce tumor formation. B38 was then administered intraperitoneally as a single agent, at doses of 20 mg / kg and 5 mg / kg, every other day. Results showed that after three weeks of continuous treatment, tumor volume in the B38-treated group was significantly reduced compared to the blank control group, while tumors in the control group continued to grow. The in vivo antitumor effect of B38 was comparable to that of the positive control drug, with the high-dose group exhibiting significantly superior tumor inhibition compared to the untreated control group. Safety assessments revealed that body weight changes in all dose groups remained within normal physiological fluctuations throughout the dosing period. No abnormalities in hematological, hepatic, or renal function indicators, nor significant toxicity, were observed, suggesting that B38 possesses a favorable therapeutic window and safety profile in vivo.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. Cyclic peptide, the structural formula of which is shown below:

2. The method for preparing the cyclic peptide according to claim 1, characterized in that include: The tetrapeptide fragment and eFKBD are combined to obtain a cyclic peptide, wherein the structural formula of the tetrapeptide fragment is as follows: The structural formula of eFKBD is shown below:

3. A pharmaceutical composition, characterized in that Including the cyclic peptide according to claim 1.

4. A pharmaceutical preparation, characterized in that The invention comprises the cyclic peptide according to claim 1 or the pharmaceutical composition according to claim 3.

5. The pharmaceutical preparation according to claim 4, characterized in that The dosage forms of pharmaceutical preparations include injections, oral solutions, tablets, powders, granules, capsules, patches or suppositories.

6. Use of the cyclic peptide according to claim 1 or the pharmaceutical composition according to claim 3 in the preparation of a drug for treating tumors.

7. The use according to claim 6, characterized in that The cyclic peptide or the pharmaceutical composition has the effect of inhibiting the proliferation of tumor cells.

8. The use according to claim 7, characterized in that Applications include: Cyclic peptides or pharmaceutical compositions having the following effects: (i) It has the effect of inhibiting the activity of GLUT5 protein in tumor cells; (ii) It has the effect of reducing fructose uptake by tumor cells; (iii) It has the effect of reducing pyruvate production.

9. The use according to any one of claims 6 to 8, characterized in that The tumor is a malignant tumor of the blood system called leukemia.

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