Selective inhibitor of balanced nucleoside transporter 2, pharmaceutical composition, kit and application of selective inhibitor

Abstract

The invention belongs to the technical field of biology, and particularly relates to a selective inhibitor of balanced nucleoside transporter 2, a pharmaceutical composition, a kit and application of the selective inhibitor. The inhibitor is a compound as shown in a formula (I), and a stereoisomer, a tautomer, a solvate or a pharmaceutically acceptable salt thereof. The inhibitor disclosed by the invention can be specifically combined with the equilibrium nucleoside transporter 2, shows obvious inhibitory activity on the equilibrium nucleoside transporter 2, and can be used for improving the thermal stability of the equilibrium nucleoside transporter 2 by combining the equilibrium nucleoside transporter 2.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a selective inhibitor of equilibrative nucleoside transporter 2, a pharmaceutical composition, a kit and applications thereof. Background Art

[0002] Equilibrative nucleoside transporter 2 (ENT2), encoded by the SLC29A2 gene, is a bidirectional transmembrane transporter and a member of the SLCs superfamily of solute proteins. It is widely distributed on the cell membranes of various tissues and cell types. Although the specific biological functions of ENT2 remain to be fully elucidated, existing studies indicate that it plays a key role in the transport of endogenous and pharmaceutical nucleoside substrates, particularly in the transmembrane delivery of nucleoside analogs used in antiviral and anticancer drugs. Furthermore, ENT2 plays an essential role in the endogenous adenosine signaling pathway. By regulating extracellular adenosine concentrations, ENT2 indirectly influences the activation state of adenosine receptors (ARs), including Adora1, Adora2A, Adora2B, and AdoraA3, thereby modulating the adenosine signaling pathway. This pathway plays a central role in managing various physiological and pathological conditions, such as infection and inflammation, and is particularly crucial in combating infection and inflammatory responses. In summary, ENT2 is not only a key mediator of nucleoside transport, but also plays an important role in multiple physiological and pathological processes such as drug delivery and adenosine signaling, making it an important target for drug action and potential therapeutic intervention.

[0003] Currently, no potent and selective ENT2 small molecule inhibitors have been reported. The development of ENT2 small molecule drugs is extremely difficult for the following reasons: (1) ENT2 characterization is difficult, and many physiological and pathological functions need further study; (2) ENT2 protein is a membrane protein, and its isolation, purification, and structural elucidation are extremely difficult. The lack of ENTs membrane protein structure hinders the rational design of small molecule drugs; (3) ENT2 transporters are widely expressed and have poor substrate specificity, making specific drug screening strategies more challenging. Summary of the Invention

[0004] In view of this, the present invention provides a selective inhibitor of equilibrative nucleoside transporter 2, a pharmaceutical composition, a kit and uses thereof.

[0005] To achieve the above solution, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present application provides a compound, which is a compound represented by formula (I), a stereoisomer, a tautomer, a solvate or a pharmaceutically acceptable salt thereof.

[0007]

[0008] Optionally, the compound is capable of specifically binding to equilibrative nucleoside transporter 2.

[0009] Optionally, the compound enhances the thermal stability of equilibrative nucleoside transporter 2 by binding to equilibrative nucleoside transporter 2.

[0010] In a second aspect, the present application provides a pharmaceutical composition comprising the compound as described above and pharmaceutically acceptable excipients.

[0011] Optionally, the pharmaceutically acceptable excipients include at least one of a dispersant, an emulsifier, a solvent, a cosolvent, a solubilizer, a latent solvent, a carrier, a filler, a stabilizer, a wetting agent, a penetrant, an adhesive and a defoaming agent.

[0012] In the present application, examples of dispersants include stearic acid dispersants, aliphatic amide dispersants, ester dispersants, paraffin dispersants, metal soap dispersants, and low molecular weight wax dispersants. Examples of stearic acid dispersants include stearic acid, linolenic acid, and oleic acid. Examples of aliphatic amide dispersants include vinyl bisstearamide, stearamide, and hexenyl bisstearamide. Examples of ester dispersants include stearic acid monoglyceride and tristearin. Examples of paraffin dispersants include liquid paraffin and microcrystalline paraffin. Examples of metal soap dispersants include stearates. Examples of low molecular weight wax dispersants include polyethylene homopolymers, ethylene-acrylic acid copolymers, ethylene-vinyl acetate copolymers, and low molecular weight ionomers.

[0013] In the present application, emulsifiers include, but are not limited to, anionic emulsifiers, cationic emulsifiers, and nonionic emulsifiers. Examples of anionic emulsifiers include fatty acid soaps, alkyl sulfates, alkylbenzene sulfonates, and phosphates. Examples of cationic emulsifiers include N-dodecyldimethylamine and its amine derivatives, quaternary ammonium salts, and the like. Examples of nonionic emulsifiers include polyoxyethylene ethers, polyoxypropylene ethers, ethylene oxide and propylene oxide block copolymers, and polyol fatty acid esters.

[0014] In the present application, solvents include, but are not limited to, water, alcohols, and fatty oils. Examples of fatty oils include castor oil, soybean oil, peanut oil, and olive oil.

[0015] In the present application, the cosolvent includes, but is not limited to, organic acids and their salts, amide compounds (such as urea, acetamide, etc.), etc. Examples of organic acid salts include sodium benzoate, sodium salicylate, and the like.

[0016] In the present application, examples of the solubilizer include polysorbate compounds, polyoxyethylene fatty acid ester compounds, and the like.

[0017] In the present application, the cosolvent may include, for example, ethanol, propylene glycol, glycerol and the like.

[0018] In the present application, carriers include but are not limited to micelles, microemulsions, gels, liquid crystals, vesicles, and the like.

[0019] In the present application, fillers include, for example, starch, powdered sugar, dextrin, lactose, microcrystalline cellulose, inorganic salts, mannitol and the like.

[0020] In the present application, examples of stabilizers include glycerol, syrup, gum arabic, sodium alginate, methylcellulose, sodium carboxymethylcellulose, polysorbates (i.e., Tweens), polyoxyethylene castor oil, acetates, phosphates, and the like.

[0021] In the present application, the wetting agent may include, for example, water, ethanol, starch paste, syrup, gum arabic, gelatin, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, magnesium aluminum silicate, bletilla striata gum, polyethylene glycol (PEG) 4000 and the like.

[0022] In the present application, examples of the penetrant include azone, propylene glycol, dimethyl sulfoxide (DMSO), dimethyl isosorbide (DMIS), and the like.

[0023] In the present application, the adhesive can include, for example, starch slurry, methyl cellulose (MC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (CMC-Na), ethyl cellulose (EC), polyvidone (PVP), gelatin, sodium alginate, magnesium aluminum silicate, bletilla striata gum, polyethylene glycol 4000, polyethylene glycol 6000, dextrin and the like.

[0024] In the present application, the defoaming agent may include, for example, vegetable oil, turpentine, polymethyl silicone, polyether fatty alcohol defoaming agent, polyether modified defoaming agent, organosilicon defoaming agent and the like.

[0025] In a third aspect, the present application provides a kit comprising the compound as described above and instructions.

[0026] In a fourth aspect, the present application provides use of the compound or pharmaceutical composition as described above in the preparation of a drug for a disease associated with equilibrative nucleoside transporter 2.

[0027] Optionally, the disease is an inflammatory disease or a tumor.

[0028] Optionally, the inflammatory disease is inflammatory bowel disease.

[0029] Optionally, the disease is a neurodegenerative disease.

[0030] As described above, the selective inhibitor of equilibrative nucleoside transporter 2, pharmaceutical composition, kit and use thereof of the present invention have the following beneficial effects:

[0031] The compounds of the present application can specifically bind to equilibrative nucleoside transporter 2, exhibit significant inhibitory activity on equilibrative nucleoside transporter 2, and can improve the thermal stability of equilibrative nucleoside transporter 2 by binding to equilibrative nucleoside transporter 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the specificity test result of compound 1, Drugs represents drugs, and the vertical axis represents ba 3 H-adenosine substrate uptake;

[0033] Figure 2 The figure shows the expression results of Flag-ENT1 and Flag-ENT2 proteins in HEK293T cells;

[0034] Figure 3 The figure shows the expression results of Flag-ENT2 protein in HEK293T cells after corresponding treatment;

[0035] Figure 4 This is a graph showing the results of a concentration-dependent test;

[0036] Figure 5 This is a graph of thermal stability test results, where Temp represents temperature. DETAILED DESCRIPTION

[0037] The present invention is further illustrated below through specific examples. However, it should be noted that the specific material ratios, process conditions and results described in the embodiments of the present invention are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

[0038] The present invention is described in detail below by specific examples. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific numerical values ​​exemplified below.

[0039] (1) Compound screening

[0040] use 3 A high-throughput screening of the rapafucin compound library was performed using a H-labeled adenosine substrate uptake experiment to obtain compound 1 (hereinafter referred to as ZG11). The structure of the compound is shown in formula (I):

[0041]

[0042] (2) Specificity test

[0043] To explore the specificity of ZG11, this application conducted a 3H-Adenosine substrate uptake experiment. The specific steps are as follows:

[0044] PK15-ENT1 (specifically expressing human ENT1), PK15-ENT2, and PK15-ENT2 cells (specifically expressing human ENT2) were seeded into 96-well plates at a seeding density of 5000 cells / well, and 180 μL of culture medium was added for overnight culture.

[0045] ZG11 was added to each well according to the concentration gradient and incubated for 1 h;

[0046] join in 3 H-Adenosine treatment for 6 h;

[0047] The signal value was measured using a MicroBeta2 scintillation counter; the data were processed and the IC was calculated. 50 , determine the selectivity, the results are as follows Figure 1 shown.

[0048] Depend on Figure 1 It can be seen that ZG11 has the highest inhibitory activity against ENT2 (IC 50 =77.9 nM), and had no significant inhibitory effect on ENT1. This result shows that the compound of the present application has good selectivity (ENT2 / ENT1>80 times).

[0049] (3) Biotin-ZG11 pulldown experiment

[0050] To verify the binding of ZG11 to ENT2 protein, the present application transiently transfected pCDNA-Flag-ENT1 and pCDNA-Flag-ENT2 plasmids into HEK293T cells to overexpress Flag-ENT1 and Flag-ENT2 proteins. After 48 hours, the cells were collected by centrifugation. The results are as follows: Figure 2 As shown;

[0051] Wash the cells with Buffer A, centrifuge, resuspend the cells with 400 μL of Buffer B, and gently grind the cells on ice using a glass homogenizer until there are no cell clumps;

[0052] The cell lysate was placed on a shaker at 4°C for 2 h, then centrifuged at 13,000 rpm and 4°C for 10 min. The protein supernatant was collected and the protein concentration was quantified using BCA.

[0053] The protein sample was diluted 5-fold with Buffer C to reduce the DDM concentration;

[0054] Add 25 μL of streptavidin magnetic beads to the protein supernatant and shake on a shaker at 4°C for 30 min to remove endogenous biotin in the protein;

[0055] The streptavidin magnetic beads were removed by centrifugation, and the supernatant was collected for subsequent experiments;

[0056] The above protein samples were dispensed into new EP tubes at a volume of 300 μL / tube. DMSO, ZG11 (20 μM, final concentration), rapamycin (20 μM, final concentration), and FK506 (20 μM, final concentration) were added to each tube, respectively, and pretreated at 4°C for 30 min.

[0057] DMSO or 1 μM Biotin-ZG11 was added to each tube and incubated at 4°C for 1 h;

[0058] Add 25 μL of streptavidin magnetic beads and incubate at 4°C for 2 h;

[0059] Centrifuge at high speed at 4°C, remove the supernatant, and wash the beads three times with Buffer A, each wash for 5 minutes;

[0060] Add 50 μL of 1× Loading buffer to each tube of sample and resuspend;

[0061] The cells were heated in a metal bath at 100°C for 10 min, and the Flag-ENT2 level was detected by Western blot using Input as a control. The results were as follows: Figure 2 、 Figure 3 As shown, different concentrations of ZG11 were used to test whether Biotin-ZG11 was concentration-dependent. The results are shown in Figure 4 shown.

[0062] like Figure 2 As shown, Flag-ENT1 and Flag-ENT2 proteins were overexpressed in HEK293T cells.

[0063] like Figure 3As shown, Biotin-ZG11 can specifically pull down Flag-ENT2, and pretreatment with ZG11 (20 μM) can block the binding of Biotin-ZG11 to Flag-ENT2. This result shows that the compound of the present application can directly bind to the ENT2 protein. FK506 and rapamycin can compete with ZG11 for binding to the FKBP12 protein. If the binding of ZG11 and ENT2 depends on the FKBP12 protein, then the pre-addition of high concentrations of FK506 or rapamycin will prevent ZG11 from binding to ENT2. High concentrations of FK506 (20 μM) and rapamycin (20 μM) have no effect on the binding of Biotin-ZG11 to Flag-ENT2. This result shows that the binding of ZG11 to ENT2 is not only specific, but may also not depend on the FKBP12 protein.

[0064] like Figure 4 As shown, Biotin-ZG11 could pull down Flag-ENT2 protein in a concentration-dependent manner.

[0065] (4) Thermal stability test

[0066] To further explore the thermal stability of ZG11, this application conducted CETSA (Cellular Thermal Shift Assay), the specific steps are as follows:

[0067] Two ENT2 overexpressing cells of the same density were treated with 10 μM ZG11 (final concentration) (0.1% DMSO) and 0.1% DMSO, respectively, and incubated at 37°C and 5% CO2 in a cell culture incubator for 3 hours. The two cells were collected at the same time and the operation steps were the same. The specific steps were: after aspirating the culture medium, rinse with PBS, and then resuspended by pipetting with PBS buffer containing 1% protease inhibitors. After mixing, the cell suspension was divided into equal volumes and placed in PCR tubes. The cells were heated at different temperatures for 3 minutes using a PCR instrument. After the samples returned to room temperature, they were repeatedly frozen and thawed three times with liquid nitrogen to fully lyse the cells. The cells were centrifuged at a speed of 12000 rpm and a temperature of 4°C for 10 minutes using a low-temperature centrifuge. An equal amount of supernatant was aspirated and stored in another new centrifuge tube. The differences between the two were detected by western blot. The results are shown in the figure. Figure 5 shown.

[0068] like Figure 5As shown, in the absence of ZG11 (i.e., the group without ZG11), ENT2 protein began to precipitate at 60°C as the temperature increased, and the protein was almost completely precipitated at 65°C. However, after adding ZG11 (10 μM), the protein hardly precipitated at 60°C, and even at 65°C, the protein was still not completely precipitated. This result indicates that the compounds of the present application can enhance the thermal stability of ENT2 protein by binding to the ENT2 protein.

[0069] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A compound, characterized in that The compound is a compound represented by formula (I), a stereoisomer, a tautomer, a solvate or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein The compound can specifically bind to equilibrative nucleoside transporter 2.

3. The compound according to claim 1, wherein The compound enhances the thermal stability of equilibrative nucleoside transporter 2 by binding to equilibrative nucleoside transporter 2.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition according to claim 4, wherein The pharmaceutically acceptable excipients include at least one of a dispersant, an emulsifier, a solvent, a cosolvent, a solubilizer, a latent solvent, a carrier, a filler, a stabilizer, a wetting agent, a penetrant, a sticker and a defoaming agent.

6. A kit, characterized in that The kit comprises the compound according to any one of claims 1 to 3 and instructions.

7. Use of the compound according to any one of claims 1 to 3 in the preparation of a medicament for treating a disease associated with equilibrative nucleoside transporter 2.

8. The use according to claim 7, characterized in that The disease is an inflammatory disease or a tumor.

9. The use according to claim 8, characterized in that The inflammatory disease is inflammatory bowel disease.

10. The use according to claim 7, characterized in that The disease is a neurodegenerative disease.

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