Application of benzenesulfonamide derivative in preparation of preparation for inhibiting eIF3a protein expression

Abstract

The invention belongs to the field of medicines, and particularly relates to application of a benzenesulfonamide derivative in inhibition of eIF3a protein, the structure of the benzenesulfonamide derivative is shown in the following formula: # imgabs0 #, the research finds that the benzenesulfonamide derivative shown in the formula 1 can be specifically combined with the eIF3a protein, and Western Bolt proves that the benzenesulfonamide derivative shown in the formula 1 can significantly down-regulate expression of eIF3a; a plate cloning experiment and an EDU experiment prove that the benzenesulfonamide derivative shown in the formula 1 inhibits cancer cell proliferation. The invention provides a brand new thought for cancer treatment, especially oral cancer treatment, and has potential medical value and important medical prospect.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of benzenesulfonamide derivatives in inhibiting eIF3a protein. Background Art

[0002] Eukaryotic protein synthesis includes three stages: initiation, elongation, and termination. This complex physiological process is carried out on the ribosomes in the cytoplasm, and a series of protein factors called eukaryotic translation initiation factors (eIFs) are involved. So far, 13 initiation factors have been discovered, among which the largest molecular weight eIF3 contains 8-11 subunits and plays an important role in the protein synthesis initiation pathway. Eukaryotic Translation Initiation Factors 3a (eIF3a) is the largest subunit in the eIF3 family and plays a key role in controlling gene expression. eIF3a participates in the occurrence and development of tumors and various diseases by regulating cell cycle, apoptosis, differentiation, fibrosis, etc. Many studies have confirmed that eIF3a plays an important role in the occurrence and development of tumors. For example, the high expression of eIF3a in breast cancer, cervical cancer, esophageal cancer, gastric cancer, bladder cancer, non-small cell lung cancer and other tumors is conducive to maintaining the malignant phenotype of the tumor. The effective inhibition of eIF3a factor is of great significance for the treatment of these tumors. Summary of the invention

[0003] The purpose of the present invention is to provide the use of benzenesulfonamide derivatives in the preparation of preparations for inhibiting eIF3a protein expression.

[0004] In order to achieve the above object, the technical solution of the present invention is as follows: The use of benzenesulfonamide derivatives in inhibiting the expression of eIF3a protein, the structure of the benzenesulfonamide derivatives is shown in the following formula: Formula 1.

[0005] The compound of formula 1 is named N1,N6-bis{4-[(mesitylamino)sulfonyl]phenyl}hexanediamide.

[0006] The invention discloses an application of a benzenesulfonamide derivative in preparing a reagent binding to eIF3a protein, wherein the structure of the benzenesulfonamide derivative is shown in Formula 1.

[0007] The invention discloses an application of a benzenesulfonamide derivative in preparing a reagent for degrading eIF3a protein, wherein the structure of the benzenesulfonamide derivative is shown in Formula 1.

[0008] The present invention studies show that the benzenesulfonamide derivatives shown in Formula 1 can bind to the eIF3a protein and promote the degradation of the eIF3a protein, and have the function of inhibiting the activity of eIF3a. Therefore, the compound of Formula 1 has anti-tumor activity.

[0009] The invention discloses an application of a benzenesulfonamide derivative in the preparation of an anti-tumor drug. The structure of the benzenesulfonamide derivative is shown in Formula 1.

[0010] In a preferred embodiment, the tumor includes one or more of breast cancer, cervical cancer, esophageal cancer, gastric cancer, bladder cancer, non-small cell lung cancer, and oral cancer.

[0011] In a preferred embodiment, the tumor cells are HN-30 or Cal-33.

[0012] An anti-tumor drug comprises a benzenesulfonamide derivative as shown in Formula 1, a pharmaceutically acceptable salt thereof or an acyl derivative thereof.

[0013] The present invention has found that the benzenesulfonamide derivatives shown in Formula 1 can inhibit tumor cell proliferation by promoting the degradation of eIF3a protein, thereby achieving an anti-cancer effect.

[0014] In a preferred embodiment, the anti-tumor drug further comprises other anti-cancer active ingredients with non-conflicting medicinal properties.

[0015] In a preferred embodiment, the anti-tumor drug further comprises a pharmaceutically acceptable excipient.

[0016] In a preferred embodiment, the anti-tumor drug has any pharmaceutically acceptable dosage form.

[0017] The present invention has found that the benzenesulfonamide derivatives shown in Formula 1 can specifically bind to the eIF3a protein, and Western Bolt proves that the benzenesulfonamide derivatives shown in Formula 1 can significantly downregulate the expression of eIF3a; plate cloning experiments and EDU experiments prove that the benzenesulfonamide derivatives shown in Formula 1 inhibit cancer cell proliferation. The present invention provides a new idea for cancer treatment drugs, especially oral cancer treatment drugs, with potential medical value and important medical prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A is a diagram showing the binding of the compound of formula 1 to eIF3a; A is a diagram showing the direct binding of the compound of formula 1 to the purified eIF3a protein as demonstrated by an SPR experiment; B is a diagram showing the absence of a binding signal between the compound of formula 1 and PBS as demonstrated by an SPR experiment; Figure 2A is a result diagram showing that the compound of formula 1 down-regulates the protein expression of eIF3a; A is a result diagram showing that the compound of formula 1 can down-regulate the protein expression of eIF3a in a dose-dependent manner; B is a result diagram showing that the compound of formula 1 can down-regulate the protein expression of eIF3a in a time-dependent manner; Figure 3 A is a result diagram showing that the compound of formula 1 inhibits the proliferation of oral cancer cells; A is a result diagram showing that the compound of formula 1 inhibits the proliferation of oral cancer cells as detected by a plate cloning experiment; B is a result diagram showing that the compound of formula 1 inhibits the proliferation of oral cancer cells as demonstrated by an EDU experiment; Figure 4 The figure is a Western Blot experimental result showing that the compound of formula 1 can induce changes in the expression of apoptosis-related proteins; Figure 5 The result diagram shows that the compound of formula 1 inhibits the proliferation of oral cancer cells depending on the expression of eIF3a. DETAILED DESCRIPTION

[0019] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, they will be described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0020] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0021] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0022] It should be noted that the benzenesulfonamide derivatives of formula 1 of the present invention were purchased from MedChemExpress: Formula 1.

[0023] Experimental Example 1 SPR experiments demonstrated that the compound of formula 1 directly binds to the eIF3a protein.

[0024] eIF3a protein was purchased from Mybiosource, catalog number: MBS2903047 Compound of formula 1: N1, N6-bis{4-[(mesitylamino)sulfonyl]phenyl}hexanediamide 1. Experimental Methods 1.1 Experimental treatment The sample of compound 1 and eIF3a protein were taken out of the -80°C refrigerator and returned to room temperature. The compound of formula 1 was diluted to a concentration of 100 μM using dimethyl sulfoxide (DMSO) as the stationary phase printing working solution. The working solution was then printed onto the surface of the 3D photo-crosslinked chip using a BioDot-1520 array printer (Biodot, USA) and the coupling operation was completed. The eIF3a protein sample stock solution was then added to phosphate buffered saline-Tween (PBST) (pH=7.4, 0.1% Tween 20) and diluted to 5 concentration gradients: 10 nM, 40 nM, 160 nM, 640 nM, 2560 nM.

[0025] 1.2 Experimental Procedure All mobile phase samples flowed in sequence, and the binding and dissociation process were detected by Berthold bScreen LB 991 SPR analyzer (Berthold, Germany), and the signals were collected to draw signal curves. PBST was used as a mobile carrier throughout the experiment. In the interaction test phase, the analyte flowed through the chip surface at 0.5 μL / s; after each interaction pair test was completed, the chip surface was regenerated, that is, Glycine-HCl (pH=2.0) solution was used as the regeneration solution, and the flow rate was 2 μL / s. The data analysis software LightCompass (Version.20240711, Berthold Technologies GmbH) and the Langmuir model were used to fit the curve and export the affinity data.

[0026] 2. Experimental results The experimental results are as follows Figure 1 And as shown in Table 1.

[0027] like Figure 1 -A, the compound of formula 1 has a binding signal with the eIF3a protein, and the binding signal increases with the increase of concentration ( Figure 1 A).

[0028] like Figure 1 -B, the compound of formula 1 has no binding signal with PBS ( Figure 1 B).

[0029] As shown in Table 1, the affinity constant of the compound of Formula 1 to the eIF3a protein is KD = 4.31E-08 M (4.31 nM); the affinity constant of the compound of Formula 1 to PBS is KD = 3.87E-01 M (387.00 mM).

[0030]

[0031] 3. Conclusion The compound of formula 1 can directly bind to the eIF3a protein.

[0032] Example 2 The compound of formula 1 down-regulates the expression of eIF3a.

[0033] The cells used in this experiment were human oral cancer cells HN-30 and Cal-33.

[0034] Compound of formula 1: N1, N6-bis{4-[(mesitylamino)sulfonyl]phenyl}hexanediamide 1 Experimental methods 1.1 Experimental treatment: Oral cancer cells were treated with solvent (DMSO) and compound 1 (1, 2, 5, 10 μM) and cultured in a cell culture incubator (37°C, 5% CO 2 ).

[0035] 1.2 Detection: After 48 hours of experimental treatment, extract total cell protein, add pre-cooled PBS buffer to each well and rinse gently 1-2 times, add appropriate amount of strong RIPA lysis buffer (Biyuntian), lyse for 30 minutes, centrifuge at 15000rpm and 4℃ for 15 minutes. Use Biyuntian BCA protein concentration determination reagent to quantify protein and denature. Perform Western Blot protein immunoblotting by gel electrophoresis-transfer-blocking-primary antibody incubation-secondary antibody incubation-washing membrane-scanning and development, etc. to detect the expression of eIF3a and Tubulin (as internal reference).

[0036] 1.3 Experimental Results like Figure 2 As shown, protein immunoblotting showed that the compound of formula 1 can significantly inhibit the expression of eIF3a. Figure 2 A shows that Western Blot experiments prove that the compound of formula 1 can down-regulate the protein expression of eIF3a in a dose-dependent manner. The concentration of the compound of formula 1 is 10µM, and the expression level is the lowest. Figure 2 B shows that Western Blot experiments prove that 5 μM of the compound of formula 1 can down-regulate the protein expression of eIF3a in a time-dependent manner, and the expression level decreases significantly after 24 hours. This shows that the compound of formula 1 can down-regulate the expression of eIF3a.

[0037] 3. Conclusion The compound of formula 1 can down-regulate the expression of eIF3a in oral cancer cells.

[0038] Example 3 The compound of formula 1 inhibits the proliferation of oral cancer cells in vitro.

[0039] Cells used in this experiment: Human oral cancer cells were HN-30 and Cal-33; Compound of formula 1: N1, N6-bis{4-[(mesitylamino)sulfonyl]phenyl}hexanediamide 1. Experimental Methods 1.1 Experimental treatment: Oral cancer cells were treated with solvent (DMSO) and compound 1 (2, 5, 10 μM) respectively and cultured in a cell culture incubator (37°C, 5% CO 2 ).

[0040] 1.2 Detection: (1) Observation of cell clone formation After 10 days of experimental treatment, the cells were fixed with paraformaldehyde and stained with crystal violet to observe the number of clones.

[0041] (2) EdU detection of cell proliferation After 48 hours of cell treatment, the experiment was completed according to the instructions of Ruibo EDU cell proliferation assay (Cat#: C0310-1), as follows: After cell treatment, the EDU solution was diluted at a ratio of complete culture medium: EDU solution (reagent A) = 1000: 1 (v / v), replaced with the original culture medium and placed in a cell culture incubator for 2 hours, fixed with fixative for 15 minutes, treated with 2 mg / mL glycine working solution for 5 minutes, washed with PBS buffer for 5 minutes, 100 μL of permeabilization agent (TritonX-100) was added to each well, ApDUollo staining solution was prepared according to the instructions, added to the cells and incubated for 30 minutes, and the cells were treated with TritonX-100 (0.5%, 100 μL / well) permeabilization solution for 3 times, each time for 10 minutes, and Hoechst33342 working solution was added. Finally, the cells were washed with PBS buffer for 5 minutes and placed under an inverted fluorescence microscope for observation.

[0042] 2. Experimental results The experimental results are as follows Figure 3 As shown, 3A is a plate cloning experiment to detect that the compound of formula 1 inhibits the proliferation of oral cancer cells; 3B is an EDU experiment to prove that the compound of formula 1 inhibits the proliferation of oral cancer cells. Figure 3 As shown in A, the concentration of the compound of formula 1 is 10 μM, which significantly inhibits the proliferation of Cal-33 cells; the concentration of the compound of formula 1 is 5 μM, which significantly inhibits the proliferation of HN-30 cells; this indicates that the compound of formula 1 significantly inhibits the formation of cell clones. Figure 3 The results of B and Figure 3 Similar to A, it indicates that the compound of formula 1 significantly inhibits the proliferation of tumor cells.

[0043] 3. Conclusion The compound of formula 1 can inhibit the proliferation of oral cancer cells.

[0044] Example 4 The compound of formula 1 induces apoptosis in oral cancer cells.

[0045] Cells used in this experiment: Human oral cancer cells were HN-30 and Cal-33; Compound of formula 1: N1, N6-bis{4-[(mesitylamino)sulfonyl]phenyl}hexanediamide 1. Experimental Methods 1.1 Experimental treatment: Oral cancer cells were treated with solvent (DMSO) and compound 1 (1, 2, 5, 10 μM) and cultured in a cell culture incubator (37°C, 5% CO 2 ).

[0046] 1.2 Detection: After 48 hours of experimental treatment, total cell protein was extracted, and each well was gently rinsed 1-2 times with pre-cooled PBS buffer, and an appropriate amount of strong RIPA lysis buffer (Biyuntian) was added for lysis for 30 minutes, and centrifuged at 15000rpm and 4℃ for 15 minutes. Protein quantification and denaturation were performed using Biyuntian BCA protein concentration determination reagent. Equal amounts of protein were subjected to gel electrophoresis-transfer-blocking-primary antibody incubation-secondary antibody incubation-washing membrane-scanning and development steps for Western Blot protein immunoblotting to detect the expression of PARP, Bcl2, Bax and Tubulin (as an internal reference).

[0047] 2. Experimental results like Figure 4 As shown, after treatment with the compound of formula 1, the expression of the cleavage form of the apoptosis-related protein PARP and Bax in oral cancer increased, while the expression of the anti-apoptotic protein Bcl2 was downregulated.

[0048] 3. Conclusion This indicates that the compound of formula 1 can induce apoptosis of oral cancer cells.

[0049] Example 5 The inhibition of oral cancer cell proliferation by the compound of formula 1 depends on the expression of eIF3a.

[0050] Cells used in this experiment: Human oral cancer cells were HN-30 and Cal-33; Compound of formula 1: N1, N6-bis{4-[(mesitylamino)sulfonyl]phenyl}hexanediamide 1. Experimental Methods 1.1 Experimental treatment: Oral cancer cells were transfected with siRNA (eIF3a siRNA sequence is 5′-CGTGCTGATGATGATCGGTTT-3′). The RNA-specific transfection reagent LipRNAiMax was used as the transfection reagent. First, the siRNA-LipRNAiMax premix was prepared. The siRNA stock solution was taken out of the -20℃ refrigerator, thawed on ice, and gently pipetted to mix. 4μL of siRNA stock solution was added to 350μL Opti-MEM culture medium (Gibco, Cat#: 31985-070), and allowed to stand at room temperature for 5 minutes. At the same time, LipRNAiMax was gently pipetted to mix, 4μL was added to another 350μL Opti-MEM culture medium, mixed, and allowed to stand at room temperature for 5 minutes. After 5 minutes, the LipRNAiMax mixture was added to the diluted siRNA mixture, mixed, and the timer was set for 15 minutes. After the incubation, the six-well cell culture plate was taken out and the original culture medium was discarded, and the mixed solution was added and gently shaken to mix. After culturing for 6 hours, the complete culture medium was replaced and continued to be cultured. After 48 hours of transfection, the above cells were digested and re-plated. After the cells adhered to the wall, the above oral cancer cells were treated with a solvent (DMSO) and the compound of formula 1, respectively.

[0051] 1.2 Detection: After 10 days of experimental treatment, the cells were fixed with paraformaldehyde and stained with crystal violet to observe the number of clones.

[0052] 2. Experimental results like Figure 5 As shown, the compound of formula 1 can inhibit the proliferation of oral cancer. However, after silencing eIF3a, the inhibitory effect of the compound of formula 1 on oral cancer cells is weakened.

[0053] 3. Conclusion The compound of formula 1 inhibits the proliferation of oral cancer cells in a manner dependent on the expression of eIF3a, and exerts an anti-tumor effect by targeting eIF3a.

[0054] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. The use of benzenesulfonamide derivatives in the preparation of a preparation for inhibiting eIF3a protein expression, characterized in that: The structure of the benzenesulfonamide derivatives is shown below: Formula 1.

2. Use of benzenesulfonamide derivatives in the preparation of reagents binding to eIF3a protein, characterized in that: The structure of the benzenesulfonamide derivative is shown in Formula 1.

3. The use of benzenesulfonamide derivatives in the preparation of reagents for degrading eIF3a protein, characterized in that: The structure of the benzenesulfonamide derivative is shown in Formula 1.

4. The use of benzenesulfonamide derivatives in the preparation of anti-tumor drugs, characterized in that: The structure of the benzenesulfonamide derivative is shown in Formula 1.

5. The use according to claim 4, characterized in that: The tumor includes one or more of breast cancer, cervical cancer, esophageal cancer, gastric cancer, bladder cancer, non-small cell lung cancer, and oral cancer.

6. The use according to claim 4, characterized in that: The tumor cells were HN-30 or Cal-33.

7. An anti-tumor drug, characterized in that: It includes benzenesulfonamide derivatives as shown in Formula 1, pharmaceutically acceptable salts thereof or acyl derivatives thereof.

8. The anti-tumor drug according to claim 7, characterized in that: The anti-tumor drug also contains other anti-cancer active ingredients with non-conflicting medicinal properties.

9. The anti-tumor drug according to claim 7, characterized in that: The anti-tumor drug further comprises pharmaceutically acceptable excipients.

10. The anti-tumor drug according to claim 7, characterized in that: The anti-tumor drug has any pharmaceutically acceptable dosage form.

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