CN114377003A - Application of otilonium bromide in resisting tumor - Google Patents

Abstract

The invention relates to the technical field of medicines, and relates to a new application of Otilonium Bromide (Otilonium Bromide) in tumor treatment. Specifically, the invention discovers for the first time that the compound (otilonium) shown in the formula (I) has an enzyme activity inhibition effect on deubiquitinating hydrolase USP28, and therefore, the compound can reduce the level of USP28 substrate protein c-Myc in tumor cells and inhibit the proliferation and growth of the tumor cells, and is expected to be developed into an anti-tumor lead compound or a medicine targeting deubiquitinating hydrolase USP 28.

Description

Application of otilonium bromide in resisting tumor

Technical Field

The invention relates to the technical field of medicines, in particular to application of otilonium bromide in tumor resistance.

Background

Since the twentieth century, the living environment of human beings is continuously worsened, the pollution to the living environment is gradually increased, the contact between people and carcinogenic factors is more and more tight, the incidence rate of malignant tumors is gradually increased year by year, and the malignant tumors exceed cardiovascular and cerebrovascular diseases and become the biggest enemies of human health. According to the live reports of the world health organization, tumors, i.e., cancers, are the second leading cause of death in the world, and 1400 million people were diagnosed as cancers in 2012. It is estimated that by 2023, new cancer cases will increase to 2200 ten thousand per year and 2400 ten thousand per year by 2035, i.e., the number of cancer cases will increase by five in the next 20 years. The reported data show that new cases and death cases of cancer in China are located in the global position. Therefore, there is an urgent need in the art for the development of effective antitumor drugs.

The deubiquitinating hydrolase USP28 has important biological functions and is involved in the regulation of various tumor-related signal pathways. The targeted inhibition of USP28 can be an effective idea for inhibiting the proliferation of tumor cells.

Otilonium bromide is generally used as an antispasmodic in clinic and can relieve abdominal pain, flatulence and relapse of irritable bowel syndrome. The medicine is an acetylcholine receptor inhibitor, and can inhibit intracellular acetylcholine-induced Ca²⁺A signal. However, no literature reports on the inhibition of the enzyme activity of the deubiquitinase USP28 and the inhibition of the proliferation of tumor cells by otilonium bromide exist at present.

Disclosure of Invention

The invention aims to provide a new application of otilonium bromide in the aspect of tumor resistance. Specifically, the invention discloses that the thionium bromide can reduce the level of USP28 substrate protein c-Myc in tumor cells by inhibiting the activity of USP28, so that the proliferation and growth of the tumor cells are inhibited, and the thionium bromide has an effective anti-tumor effect.

In a first aspect of the invention there is provided the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a formulation containing said compound, in the manufacture of a medicament for use in the treatment of a disease or condition associated with cancer

a) Agents that inhibit the enzymatic activity of USP 28;

b) drugs that inhibit tumor cell proliferation;

c) a medicine for treating, relieving and preventing related diseases caused by tumors.

In another preferred embodiment, the tumor is a tumor associated with high expression of USP 28.

In another preferred embodiment, the tumor is selected from the group consisting of: rectal cancer, breast cancer, non-small cell lung cancer, and glioma, or a combination thereof.

In another preferred embodiment, the formulation is oral or non-oral.

In another preferred embodiment, the formulation comprises: powder, granule, capsule, injection, tincture, oral liquid, tablet, buccal tablet, or dripping pill.

In another preferred embodiment, the formulation further comprises:

an anti-tumor drug selected from the group consisting of: capecitabine, irinotecan, oxaliplatin, trifluridine compound tablet, canofir, regorafenib, pimari cilari or temozolomide.

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising:

i) a compound of formula I or a pharmaceutically acceptable salt thereof, or a prodrug thereof;

ii) other antineoplastic agents, which may include, but are not limited to, capecitabine, irinotecan, oxaliplatin, trifluridine, bevacizumab, cetuximab, panitumumab, kanafenib

Nini, regorafenib, pipabrizide, temozolomide, and astuzumab;

iii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is an injection, tablet, capsule, pill, suspension or emulsion.

In another preferred embodiment, the pharmaceutical composition is in an oral dosage form, a transdermal dosage form, an intravenous or intramuscular injection dosage form.

In a third aspect of the invention, there is provided a use of the pharmaceutical composition of the second aspect for the preparation of:

a) agents that inhibit the enzymatic activity of USP 28;

b) drugs that inhibit tumor cell proliferation;

c) a medicine for treating, relieving and preventing related diseases caused by tumors.

In a fourth aspect of the present invention, there is provided a method of inhibiting USP28 enzyme activity comprising the step of contacting a sufficient amount of a compound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the second aspect with a cell to inhibit USP28 enzyme activity.

In another preferred example, the cell is a cell with high expression of USP 28.

In a fifth aspect of the invention, there is provided a method of inhibiting tumor cell proliferation comprising the steps of:

1) administering to a subject in need thereof a medically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 5;

2) inhibiting activity of USP28 enzyme of tumor cells, thereby inhibiting proliferation of tumor cells.

In another preferred embodiment, the method is non-diagnostic, non-therapeutic.

In another preferred embodiment, the method is in vitro.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 otilonium inhibits the deubiquitinating hydrolytic activity of USP 28.

Figure 2 otilonium inhibits the proliferation and growth of a549, MCF7, U87, Ls174T and HCT116 cells.

FIG. 3 Otifolium bromide decreased the stability of the c-Myc protein in HCT116 and Ls174T cells.

Detailed Description

The present inventors have made extensive and intensive studies and have surprisingly found that otilonium bromide has an effect of inhibiting tumor proliferation for the first time, and have completed the present invention on this basis.

Specifically, the research of the invention shows that the otilonium bromide can reduce the level of USP28 substrate protein c-Myc by inhibiting the activity of deubiquitinating hydrolase USP28, thereby achieving the effect of inhibiting tumor proliferation.

Term(s) for

Active ingredient

As used herein, the term "compounds of the present invention" refers to compounds of formula (I). The term also includes compounds of formula (I) and various pharmaceutically acceptable salts thereof.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid. One preferred class of salts is that formed by reacting a compound of the present invention with a base. Suitable bases for salt formation include, but are not limited to: inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate and sodium phosphate, and organic bases such as ammonia, triethylamine and diethylamine. These salts can be prepared from the compounds of formula (I) by known salt-forming methods.

Pharmaceutical compositions and methods of administration

As the compound has obvious USP28 enzyme activity inhibition effect and inhibits the proliferation and growth of tumor cells, the compound, the pharmaceutically acceptable inorganic or organic salt thereof and the pharmaceutical composition containing the compound as the main active ingredient can be used for treating tumors related to the high expression of USP 28. The pharmaceutical composition of the present invention comprises a compound of the present invention or a pharmaceutically acceptable salt thereof in a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 5-100mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 5 to 100 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Deubiquitinated hydrolase USP28

The deubiquitinating hydrolase USP28 has important biological functions and is involved in the regulation of various tumor-related signal pathways. USP28 is the first deubiquitinase found to antagonize the function of ubiquitin ligase FBW7^[4]. Overexpression of USP28 in the cell antagonizes ubiquitination modification of c-Myc and Cyclin E1 by FBW7, and USP28 in the knockout tumor cell reduces the level of c-Myc protein, so USP28 promotes c-Myc-mediated tumor cell proliferation. In addition, the stability of some other non-FBW 7-related proteins was also regulated by USP 28. USP28 can improve the intracellular stability of LSD1, LSD1 regulates the pluripotency and differentiation of cells by participating in the demethylation process of H3K4me1/2, and LSD1 overexpression is related to the formation of various tumors. USP28 escapes the fate degraded by proteasome by removing the ubiquitination modification of LSD1, and thus improves the intracellular stability of LSD1 and makes the breast cancer cell pluripotent as a stem cell^[5]. In addition to regulating the stability of the above protooncoproteins, USP28 is also involved in the regulation of other tumor formation-associated signaling pathway proteins. A new study result shows that USP28 allows HIF-1 α to escape from degraded fate by antagonizing ubiquitination modification of HIF-1 α by GSK-3 β and FBW 7. Regulation of HIF-1 alpha by GSK-3 beta/FBW 7/USP28 affects cell proliferation, differentiation and apoptosis^[6]. USP28 is closely related to the occurrence, development of various tumors, especially colorectal cancer: the research finds that USP28 has the effects of maintaining intestinal homeostasis and promoting colorectal cancer formation. After analyzing the cancer cell samples of the patients with colorectal cancer, the levels of USP28, c-Myc, NICD1 and c-Jun in the cells are remarkably increased compared with the colon cells of normal individuals^[7]. In addition, studies on bladder cancer, non-small cell lung cancer, glioma also showed that USP28 expression was observed in these cancer cellsPhenomenon of up-regulation^[8-10]. Thus, USP28 has become a potential new target for cancer therapy.

The main advantages of the invention include:

(1) the invention discloses that the otilonium bromide has the inhibition effect of the deubiquitinating hydrolase USP28 for the first time, and can obviously inhibit the proliferation and growth of cells A549, MCF7, U87, Ls174T and HCT 116. The otilonium bromide provides a structural skeleton reference for the research and development of the medicine targeting the deubiquitinase USP 28.

(2) The otilonium bromide is a drug on the market, and has better prospect for preparing antineoplastic drugs.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1 test method and results for the inhibition of enzymatic Activity of USP28 by Othionium Bromide

In this example, the enzyme activity inhibition effect of otilonium bromide on USP28 was tested by Ub-AMC hydrolysis inhibition assay, and the specific assay method and steps are as follows:

the experiment is mainly divided into 2 systems, namely a control group and an experimental group. Control group: the total reaction volume was 200. mu.l, USP28 final concentration of 20nM, Ub-AMC final concentration of 250nM, and DMSO content of 2%. Experimental groups: the total reaction was 200. mu.l, USP28 at 20nM, Ub-AMC at 250nM, otilonium bromide at 1,2,5,10,20,50,100 and 200. mu.M, and DMSO at 2%. And sequentially adding each group of experimental samples into a black micropore plate (96 micropore plate), setting excitation light of a SpectraMax M5 microplate reader to be 380nm, setting emission light to be 460nm, setting the reaction temperature to be 37 ℃, and setting the reaction time to be 10 min. Calculating the thionium bromide pairs with different concentrations according to the value of the catalytic reaction ratePercentage inhibition of enzymatic activity of deubiquitinase USP 28. The enzyme activity inhibition IC of otilonium bromide to USP28 was calculated by non-linear fitting the otilonium bromide concentration to the percentage of enzyme activity inhibition using GraphPad Prism 5 software₅₀The value is obtained.

The test results are shown in fig. 1: otilonium bromide significantly inhibits the enzymatic activity of USP28 catalyzed hydrolysis of Ub-AMC, and IC thereof₅₀The value was 6.90 ± 0.90 μ M, with n being 3.

Example 2 method and results for testing the inhibitory effect of otilonium bromide on the proliferation and growth of tumor cells

In this embodiment, the SRB method is used to determine the proliferation and growth inhibitory effect of otilonium bromide on tumor cells, and the specific experimental method and steps are as follows:

(1) a549, MCF7, U87, Ls174T and HCT116 cells in good condition and in the logarithmic phase of growth were digested with pancreatin and counted, respectively. Diluting to a certain concentration with culture medium, mixing, inoculating the cells into 60 wells of the center of a 96-well plate with a row gun, adding 180. mu.L of cell suspension into each well, and counting 5000-6000 cells in each well. After cell plating, 200. mu.L of sterilized PBS solution was added to a circle of wells around the periphery of a 96-well plate, and the cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator overnight;

(2) after overnight adherent growth of cells in 96-well plates, respective concentrations of otilonium bromide were added per well, with 0.5% DMSO as a control. In the experiments of A549 and HCT116 cells, the final concentration of otilonium bromide is set to be 1,5,10,15,20,25,30,50 and 80 mu M, and the DMSO content is 0.5%. In the MCF7 cell experiments, the final concentration of otilonium bromide was set to 1,3,6,10,15,20,25,30 and 60 μ M, with a DMSO content of 0.5%. In the U87 cell experiments, final concentrations of otilonium bromide were set at 0.5,1,3,6,10,15,30,60 and 100 μ M with a DMSO content of 0.5%. In Ls174T cell experiments, the final concentration of otilonium bromide was set to 2,5,10,15,20,25,30,60 and 100 μ M with a DMSO content of 0.5%. 3 multiple wells were set for each concentration condition. The cells were incubated at 37 ℃ with 5% CO₂Culturing for 72h in an incubator;

(3) after 72h of cell culture, 50. mu.L of 50% trichloroacetic acid (wt/vol) was added to each well. Cells were fixed at 4 ℃ for about 1 h. After cell fixation is finished, pouring out liquid in the 96-well plate, slowly washing the holes by tap water, and placing the 96-well plate in an oven for drying;

(4) adding 100 μ L of SRB dye (4mg/mL) into each well, dyeing for 15min, washing unbound dye with 1% glacial acetic acid, and drying the 96-well plate in an oven;

(5) add 150. mu.L 10mM Tris solution per well, place 96-well plate on shaker and shake slowly to dissolve SRB dye well and mix well. The 96-well plate was then placed in a SpectraMax M5 microplate reader to read the OD₅₁₀And (4) light absorption value. And respectively calculating the proliferation inhibition percentages of the otilonium bromide with different concentrations on the 5 tumor cells according to the light absorption values. Performing nonlinear fitting on the concentration and the proliferation inhibition percentage of the otilonium bromide by utilizing GraphPad Prism 5 software, and calculating the proliferation inhibition IC of the otilonium bromide on the 5 tumor cells₅₀The value is obtained.

The test results are shown in fig. 2: the otilonium bromide can obviously inhibit the proliferation and growth of A549 cells, MCF7 cells, U87 cells, Ls174T cells and HCT116 cells, and the otilonium bromide can inhibit the proliferation of the above 5 tumor cells by IC₅₀The values were 17.60 ± 0.26 μ M, 9.19 ± 0.84 μ M, 26.30 ± 1.33 μ M, 18.60 ± 0.42 μ M and 13.20 ± 0.49 μ M, respectively, with n being 3.

Example 3 detection method and results of the reduction of the stability of the USP28 substrate protein c-Myc in HCT116 and LS174T cells by otilonium bromide

This example uses immunoblotting to examine the effect of otilonium bromide on the stability of c-Myc protein in HCT116 and Ls174T cells.

The antibody information used in this example is as follows:

Anti-USP28(AbCam,Ab126604,1:1000),Anti-c-Myc(AbCam,Ab32072,1:1000),Anti-GAPDH(Cell Signaling Technology,#2118,1:1000)。

the three primary antibodies are Rabbit-derived antibodies, and the secondary antibody Anti-Rabbit used in the present example was purchased from Absin and prepared at a dilution ratio of 1: 4000.

The specific experimental method and steps are as follows:

3.1 Effect of Ortibromamine on c-Myc protein levels in tumor cells:

(1) will be in good condition and in raw stateLong log phase Ls174T and HCT116 cells were trypsinized and counted. The cell suspension is mixed evenly and inoculated into a 6-well plate, and the number of cells in each well is 80-90 ten thousand. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator;

(2) after the cell density is grown to about 90%, the culture medium is aspirated, the cells are washed by PBS buffer solution, then the culture medium containing the otilonium bromide is added, the final concentration of the otilonium bromide is set to be 10,30,50,80 and 100 mu M, and 0.5% DMSO is set as a control group at the same time, and the medicine is added for 2 hours;

(3) after drug treatment, the cells were removed from the incubator, washed twice with PBS buffer, placed on ice and added with an appropriate amount of RIPA (radio immunopropraction assay), the cell lysates were collected into 1.5mL EP tubes, centrifuged at 12000rpm at 4 ℃ for 10min, cell lysate supernatant was aspirated, total protein concentration of the cell lysates was determined with Bradford kit, total protein concentration of all samples was adjusted to unity with RIPA lysate, the corresponding volume of 5 × loading buffer was added, and the samples were boiled at 100 ℃ for 10 min. The prepared samples were used to perform subsequent western blot experiments.

3.2 Effect of otilonium on ubiquitin proteasome degradation pathway of c-Myc protein in tumor cells:

to demonstrate that the down-regulation of c-Myc protein levels in tumor cells by otitidium bromide is achieved by promoting the ubiquitin proteasome degradation process of c-Myc, we treated tumor cells with proteasome inhibitor MG 132.

(1) The Ls174T and HCT116 cells in good condition and in the logarithmic phase of growth were separately trypsinized and counted. The cell suspension is mixed evenly and inoculated into a 6-well plate, and the number of cells in each well is 80-90 ten thousand. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator.

(2) After the cell density had grown to about 90%, the medium was aspirated, the cells were washed with PBS buffer, and then the media containing the different experimental system components were added separately. In the experiment system without MG132, the control group contained only 0.5% DMSO, the experiment group contained only otilonium bromide, and in the HCT116 cell experiment, the final concentration of otilonium bromide was set to 25 μ M, and in the Ls174T cell experiment, the final concentration of otilonium bromide was set to 40 μ M; in the experiment system containing MG132, the control group contained only 10. mu.M of MG132 (0.5% DMSO), the experiment group contained oltipraz and 10. mu.M of MG132 (0.5% DMSO), and in the HCT116 cell experiment, the final concentration of oltipraz was set to 25. mu.M, and in the Ls174T cell experiment, the final concentration of oltipraz was set to 40. mu.M.

(3) After 3h of drug treatment, the cells were removed from the incubator, washed twice with PBS buffer, placed on ice and added with an appropriate amount of RIPA lysate, the cell lysate was collected into a 1.5mL EP tube, centrifuged at 12000rpm at 4 ℃ for 10min, the cell lysate supernatant was aspirated, the total protein concentration of the cell lysate was determined with Bradford kit, the total protein concentration of all samples was adjusted to be uniform with RIPA lysate, the corresponding volume of 5 × loading buffer was added, and the samples were boiled at 100 ℃ for 10 min. The prepared samples were used to perform subsequent western blot experiments.

3.3 Western blot experiment:

(1)SDS-PAGE

centrifuging the prepared sample, adding the sample into each pore channel by using a pipette gun, setting 2 Marker pore channels in each block of glue, adding 2 mu L of protein Marker into each pore channel, and filling the residual volume with a loading buffer solution. After the sample adding is finished, covering an electrode cover, firstly concentrating the sample into lines in the laminated gel at a lamination low voltage of 60V, then separating the sample at a separation voltage of 120V quickly, and ending the electrophoresis when the bromophenol blue moves to the bottom of the separation gel;

(2) rotary film

During SDS-PAGE, the PVDF membrane can be immersed in methanol for activation, and after the activation is finished, the PVDF membrane is placed in a membrane transferring liquid for stabilization for 10min so as to be used in subsequent experiments. Assembling a transfer film sandwich: the black surface of the black-and-white clip is placed at the bottom, and the sandwich clip is prepared from the negative electrode to the positive electrode according to the sequence of sponge sheet-filter paper-glue-PVDF membrane-filter paper-sponge sheet. In order to avoid influencing the experimental result, the whole sandwich clip preparation process is carried out in the membrane transferring liquid, and air bubbles need to be avoided. And (3) placing the assembly in a film rotating groove, adding a film rotating liquid and an ice box which are stored at low temperature, and preventing the film rotating process from being overhigh in temperature and constant current of 0.25A for film rotating for 3 hours. After the membrane is switched, the power supply is turned off, the PVDF membrane is taken out, the Marker strip position is taken as the reference of the protein molecular weight, the target protein strip is cut, the mark is written, and the PVDF membrane is placed in an incubation box;

(3) antibody incubation and development

The PVDF membrane containing the target protein band is washed by TBST buffer solution and then put into TBST buffer solution containing 5% skimmed milk for blocking for 1 h. Pouring out milk, adding appropriate amount of TBST buffer solution, washing for 3 times, and shaking and washing for 10min on a side shaking table each time. The primary antibody is diluted by a primary antibody dilution liquid according to the dilution ratio in the specification. Clean PVDF membrane added corresponding primary antibody, 4 degrees C shaking incubation overnight. And (4) absorbing primary antibodies, adding a proper amount of TBST buffer solution to wash for 3 times, washing for 10min each time, and removing non-specifically bound antibodies. Secondary antibodies were prepared with TBST buffer containing 5% skim milk. The corresponding secondary antibody was selected according to the species source of the primary antibody and incubated with PVDF membrane for 1.5h at room temperature. The secondary antibody is absorbed, and the mixture is washed for 3 times by adding a proper amount of TBST buffer solution, each time for 10min, and the non-specifically bound antibody is removed. And mixing the developing solution A and the developing solution B in equal proportion, dripping the mixture on a PVDF membrane at a constant speed, slowly shaking the carrying plate at a constant speed to ensure that the developing solution on the strips is uniformly distributed, and taking a picture by using a gel imaging photographing system to obtain an immunoblotting result picture.

3.4 results of the experiment

The experimental results are shown in fig. 3: otilonium bromide decreases the stability of the c-Myc protein in HCT116 and Ls174T cells. Figure 3A shows that otilonium down-regulates the intracellular level of USP28 substrate protein c-Myc: as the concentration of otilonium bromide is gradually increased, the level of c-Myc protein in the cells is gradually reduced, and the degree of reduction shows dependence on the otilonium bromide concentration. Figure 3B shows that the down-regulation of c-Myc protein levels in HCT116 and Ls174T cells by otilonium bromide is dependent on the ubiquitin proteasome degradation pathway, thus indicating that: downregulation of c-Myc protein levels was caused by inhibition of USP28 activity by otilonium bromide. c-Myc protein levels in cells not treated and treated with otilonium were comparable after MG132 addition during cell culture.

Experimental results show that otilonium significantly down-regulates the c-Myc protein level in HCT116 and Ls174T cells, and that the down-regulation of c-Myc protein content by otilonium is achieved by facilitating its degradation process via the ubiquitin proteasome pathway. Therefore, the compound is expected to be developed into an anti-tumor lead compound or a medicine targeting deubiquitinase USP 28.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a formulation containing said compound, for the preparation of a medicament for the treatment of a disease or condition associated with cancer

a) Agents that inhibit the enzymatic activity of USP 28;

b) drugs that inhibit tumor cell proliferation;

c) a medicine for treating, relieving and preventing related diseases caused by tumors.

2. The use of claim 1, wherein said tumor is a tumor associated with high expression of USP 28.

3. The use of claim 1, wherein the formulation is oral or non-oral.

4. The use of claim 1, wherein the formulation further comprises:

an anti-tumor drug selected from the group consisting of: capecitabine, irinotecan, oxaliplatin, trifluridine compound tablet, canofir, regorafenib, pimari cilari or temozolomide.

5. A pharmaceutical composition, comprising:

i) a compound of formula I or a pharmaceutically acceptable salt thereof, or a prodrug thereof;

ii) other antineoplastic agents that may include, but are not limited to, capecitabine, irinotecan, oxaliplatin, trifluridine, bevacizumab, cetuximab, panitumumab, cannelfenib, regorafenib, pipabrizide, temozolomide, and astemizumab;

iii) a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is an injection, tablet, capsule, pill, suspension, or emulsion.

7. Use of a pharmaceutical composition according to claim 5 for the preparation of:

a) agents that inhibit the enzymatic activity of USP 28;

b) drugs that inhibit tumor cell proliferation;

c) a medicine for treating, relieving and preventing related diseases caused by tumors.

8. A method of inhibiting the enzymatic activity of USP28 comprising the step of contacting a sufficient amount of a compound of formula I or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 5 with a cell such that the activity of USP28 enzyme is inhibited.

9. The method of claim 8, wherein the cell is a cell with high expression of USP 28.

10. A method of inhibiting tumor cell proliferation comprising the steps of:

1) administering to a subject in need thereof a medically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 5;

2) inhibiting activity of USP28 enzyme of tumor cells, thereby inhibiting proliferation of tumor cells.

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