WO2023092462A1

WO2023092462A1 - Use of dipyridamole in inhibiting mcl-1

Info

Publication number: WO2023092462A1
Application number: PCT/CN2021/133567
Authority: WO
Inventors: 袁曙光; 陈柳青; 崔文强; 王世玉
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2023-06-01

Abstract

The use of an inhibitor or a competitive binding agent for an Mcl-1 protein. Further provided is the use thereof as a potential drug for treating Parkinson's disease. Dipyridamole can act on anti-apoptotic protein Mcl-1, prompts the separation of the pro-apoptotic protein binding thereto by means of competitive binding to Mcl-1, and further achieves the effect of inhibiting the anti-apoptotic protein. In addition, the released pro-apoptotic protein can play a role in promoting apoptosis, wherein the IC50 of the dipyridamole for Mcl-1 is 71.27 μM.

Description

Application of dipyridamole in inhibiting Mcl-1

technical field

The invention belongs to the field of medicine, and in particular relates to the application of dipyridamole in inhibiting Mcl-1.

Background technique

Apoptosis (also known as programmed cell death) is a conserved evolutionary cellular process that plays an important role in embryonic development and tissue homeostasis. Abnormality of apoptosis function can cause various diseases, such as neurodegenerative diseases, autoimmune diseases and cancers. So far, there are two mature apoptotic pathways studied by people: death receptor-mediated pathway (extrinsic apoptotic pathway) and mitochondria-mediated pathway (intrinsic apoptotic pathway).

In the mitochondria-mediated pathway, Bcl-2 family proteins can target the mitochondrial outer membrane, change its permeability, release cytochrome c, activate apoptosis protease, and finally cause cell death. The regulation of apoptosis is mainly realized by Bcl-2 family proteins. Bcl-2 family proteins are mainly divided into two categories: one is anti-apoptotic proteins, including Bcl-2, Mcl-1, and Bcl-XL; the other is pro-apoptotic proteins, including Bax, Bak, Bim, Noxa and Puma et al. Pro-apoptotic proteins form oligomerization in the mitochondrial outer membrane, prompting the opening of the mitochondrial outer membrane pores, causing the release of cytochrome c from the mitochondria to the cytoplasm, and finally leading to apoptosis. Anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-XL inhibit apoptosis by interacting with pro-apoptotic proteins and inhibiting the formation of Bax and Bak oligomerization. Studies have shown that anti-apoptotic proteins are highly expressed in a variety of cancer cells, which is one of the important reasons for cancer cells to escape apoptosis and acquire drug resistance. Therefore, antagonizing the activity of anti-apoptotic proteins is a strategy for cancer therapy.

Dipyridamole is a non-nitrate coronary artery dilator, which can dilate coronary blood vessels, promote the formation of collateral circulation and mildly anticoagulant, and has antiviral effects. It can be used for coronary heart disease, and as an anti-platelet aggregation drug to prevent thrombosis and disseminated intravascular coagulation.

Contents of the invention

In order to overcome the problems in the prior art, the present invention provides a novel Mcl-1 protein inhibitor or competitive binding agent.

One aspect of the present invention provides the use of dipyridamole in the preparation of inhibitors of anti-apoptotic proteins.

The dipyridamole structure is shown in formula I:

Further, the anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.

Further, the anti-apoptotic protein is selected from Mcl-1.

One aspect of the present invention provides the use of dipyridamole in the preparation of a competitive binding agent for anti-apoptotic proteins.

Further, the anti-apoptotic protein is selected from Mcl-1.

Further, the competitive binding agent is a reagent that competes with the pro-apoptotic protein of the Bcl-2 protein family for binding to the anti-apoptotic protein Mcl-1.

Further, the pro-apoptotic protein of Bcl-2 protein family is selected from Bax, Bak, Bim, Noxa, Puma and derivatives thereof.

Another aspect of the present invention provides the use of dipyridamole in the preparation of a reagent for promoting apoptosis in mitochondria-mediated pathway.

Another aspect of the present invention provides the use of dipyridamole in the preparation of medicaments for treating Parkinson's disease.

Another aspect of the present invention provides a reagent for inhibiting the activity of anti-apoptotic protein, and the active ingredient in the reagent is dipyridamole.

Further, the anti-apoptotic protein is selected from Mcl-1.

Further, the preparation further includes a dispersion medium.

Further, the dispersion medium is a solution.

Another aspect of the present invention provides a reagent capable of competitively binding anti-apoptotic proteins, and the active ingredient in the reagent is dipyridamole.

Further, the anti-apoptotic protein is selected from Mcl-1.

Further, the preparation further includes a dispersion medium.

Further, the dispersion medium is a solution.

Another aspect of the present invention provides a drug for treating Parkinson's disease, the active ingredient in the drug is dipyridamole.

Further, the medicine contains pharmaceutically acceptable excipients.

Another aspect of the present invention provides a method for inhibiting the activity of an anti-apoptotic protein, the method comprising the step of contacting dipyridamole with the anti-apoptotic protein.

Further, the anti-apoptotic protein Mcl-1 of the anti-apoptotic protein Bcl-2 protein family.

Yet another aspect of the present invention provides a method for binding anti-apoptotic proteins competitively with pro-apoptotic proteins, said method comprising the step of contacting dipyridamole with said anti-apoptotic proteins and pro-apoptotic proteins.

Further, the anti-apoptotic protein Mcl-1 of the anti-apoptotic protein Bcl-2 protein family, and the pro-apoptotic protein are selected from the Bcl-2 protein family pro-apoptotic proteins Bax, Bak, Bim, Noxa, Puma and derivatives thereof.

Beneficial effect

The present invention discovers for the first time that dipyridamole can act on the anti-apoptotic protein Mcl-1, promote the separation of the pro-apoptotic protein bound to it through competitive binding to Mcl-1, and then achieve the effect of inhibiting the anti-apoptotic protein. The pro-apoptotic protein released at the same time can play a pro-apoptotic role, and then test the anti-tumor and anti-Parkinson effects, wherein the IC50 of dipyridamole against Mcl-1 is 71.27 μM.

The purpose of the present invention is to provide research on the inhibitory effect of dipyridamole on Mcl-1 protein. The present invention finds that the IC50 of dipyridamole against Mcl-1 is 71.27 μM through time-resolved fluorescence resonance energy transfer (TRF).

Description of drawings

Figure 1 is a graph showing the inhibition of Mcl-1 activity by dipyridamole compounds at different concentrations, the abscissa is the compound concentration (μM), and the ordinate is the ratio of fluorescence intensity at 520nM and 620nM. As the concentration of the compound increases, the fluorescence intensity ratio decreases, that is, the inhibitory effect increases.

Detailed ways

In order to make the above objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention will be described in detail below, but they should not be construed as limiting the scope of implementation of the present invention.

Example 1 Expression and purification of Mcl-1 protein

The Mcl-1 protein coding gene was synthesized by Shanghai Sunny Biotechnology Co., Ltd. and connected to the pET28a expression plasmid. The expression plasmid was formulated into a solution with a concentration of 100 ng/μL, and 1 μL of the plasmid solution was transformed into the expression strain Escherichia coli BL21 (DE3) by the chemically competent transformation method to express and purify the recombinant protein Mcl-1.

The specific expression steps are as follows:

1. Pick a single colony transformed on an LB plate expressing strain E.coli BL21(DE3), inoculate it into a 5mL liquid LB medium test tube containing 50μg/μL kanamycin, and culture overnight at 37°C and 220rpm .

2. According to the inoculum volume of 1%, add 1 mL of overnight cultured bacterial solution into a conical flask containing 200 mL of liquid LB medium, add kanamycin at a final concentration of 50 μg/μL, and incubate at 37°C and 220 rpm for ~3 hours , until the OD value of the bacterial solution reaches 0.4-0.6.

3. Lower the temperature of the shaker to 20° C., add IPTG with a final concentration of 0.1 mM to the culture medium to induce protein expression, and continue to culture for 16 h.

4. After the cultivation, pour the bacterial solution into a 500mL centrifuge bucket, and centrifuge at 5000rpm for 10min to collect the bacterial cells.

The specific purification steps of Mcl-1 are:

1. Cell disruption: Suspend the cells collected in the centrifuge bucket with lysis buffer (20mM Tris, 0.5M NaCl, 20mM imidazole, pH 8.0). Pre-cool the high-pressure homogenizer to 4°C in advance, remove the 20% ethanol used for storage, wash the machine 1-2 times with deionized water, and then wash the machine 1-2 times with lysis buffer. Increase the machine pressure to about 800bar to crush the bacteria.

2. Bacterial cell precipitation: put the collected broken cell liquid into a centrifuge tube, centrifuge at 12000rpm for 20min, and collect the supernatant.

3. Column loading and purification: equilibrate the column with lysis buffer. Pour the supernatant directly into the nickel column, and use gravity to make the supernatant flow through the nickel column filler, and then combine the protein with the nickel column filler. Then use lysis buffer to wash off the impurity proteins that do not specifically bind to the nickel column filler. Finally, the Mcl-1 protein was eluted with an elution buffer (lysis buffer containing 200 mM imidazole).

4. Protein concentration: put the collected Mcl-1 eluate into an ultrafiltration concentration tube, and use preservation buffer (20mM Tris, 100mM NaCl, 2mM DTT) as a diluent to remove imidazole from the target protein and concentrate Mcl-1. 1Concentration higher than 1mg/mL.

Example 2 Mcl-1 activity inhibition experiment

The dipyridamole was prepared into 10 mM, 3.33 mM, 1.11 mM, 370 μM, 123 μM, 41.2 μM, 13.7 μM, 4.57 μM, 1.52 μM and 0.50 μM stock solutions at a dilution factor of 3. Dilute Mcl-1 to 10nM with assay buffer (50mM HEPES, pH 7.2, 50mM NaCl, 1mM DTT, 0.05% Tween-20); dilute FITC-BAK-BH3 peptide to 1μM; dilute Anti-His Tb-labeled donor to 10nM. Add 12.5 μL Anti-His Tb-labeled donor, 17 μL test buffer, 12.5 μL FITC-BAK-BH3 peptide, 7.5 μL Mcl-1, and 0.5 μL dipyridamole of different concentrations to the test wells of the white 96-well plate mother solution, and then incubated at room temperature for 3 h. The test was performed with a Flex Station3 multifunctional microplate reader, the test mode was TRF, and the parameters were EX 340/30nm Em 620/10nm and Ex 340/30nm Em 520nm. The fluorescence values at 620nm and 520nm were read respectively, and the ratio of the two fluorescence intensities was used as an indicator of the inhibitory effect of the compound.

Among them, FITC-BAK-BH3 is the natural ligand of Mcl-1, Anti-His Tb-labeled donor can bind to the His tag on Mcl-1, after binding, Tb metal can bind to FITC-BAK on Mcl-1 -BH3 undergoes fluorescence resonance energy transfer (FRET); when dipyridamole competitively binds to Mcl-1, the FITC-BAK-BH3 polypeptide dissociates from Mcl-1; thus, dipyridamole can be reflected by FRET intensity Do not combine the strength of Mcl-1 ability. Therefore, the fluorescence values at 620nm and 520nm were read respectively, and the ratio of the two fluorescence intensities was used as an indicator of the inhibitory effect of the compound.

The experimental results are shown in Figure 1, and the results in Figure 1 show that dipyridamole exhibits an inhibitory effect on Mcl-1 protein, and as the concentration increases, the inhibitory effect is dose-dependent. The IC50 value of dipyridamole against Mcl-1 was further calculated based on the above experimental results. The IC50 value of dipyridamole against Mcl-1 was 71.27 μM.

Claims

Use of dipyridamole in preparing an inhibitor of anti-apoptotic protein; the anti-apoptotic protein is selected from the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family.
Use of dipyridamole in preparing a competitive binding agent for an anti-apoptotic protein; the anti-apoptotic protein is selected from the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family.
The use according to claim 2, wherein the competitive binding agent is a reagent that competitively binds to the anti-apoptotic protein Mcl-1 with the pro-apoptotic protein of the Bcl-2 protein family;

Preferably, the pro-apoptotic protein of the Bcl-2 protein family is selected from Bax, Bak, Bim, Noxa, Puma, BAK-BH3 and derivatives thereof.
Use of dipyridamole for the preparation of a reagent for promoting apoptosis in a mitochondria-mediated pathway.
Use of dipyridamole in the preparation of medicaments for treating Parkinson's disease.
A reagent for inhibiting the activity of anti-apoptotic protein, the active ingredient in the reagent is dipyridamole;

Preferably, the anti-apoptotic protein is selected from the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family;

Preferably, the formulation further includes a dispersion medium.
A reagent capable of competitively binding anti-apoptotic proteins, the active ingredient in the reagent being dipyridamole;

Preferably, the anti-apoptotic protein is selected from the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family;

Preferably, the formulation further includes a dispersion medium.
A drug for treating Parkinson's disease, the active ingredient in the drug is dipyridamole;

Preferably, the medicine contains pharmaceutically acceptable excipients.
A method of inhibiting the activity of an anti-apoptotic protein, said method comprising the step of contacting dipyridamole with said anti-apoptotic protein;

Preferably, the anti-apoptotic protein Mcl-1 of the anti-apoptotic protein Bcl-2 protein family.
A method for competitively binding an anti-apoptotic protein with a pro-apoptotic protein, the method comprising the step of contacting dipyridamole with the anti-apoptotic protein and the pro-apoptotic protein;

Preferably, the anti-apoptotic protein Mcl-1 of the anti-apoptotic protein Bcl-2 protein family, and the pro-apoptotic protein are selected from the Bcl-2 protein family pro-apoptotic proteins Bax, Bak, Bim, Noxa, Puma, BAK-BH3 and its derivatives.