CN116173035A - Use of dipyridamole in inhibiting Mcl-1 - Google Patents
Use of dipyridamole in inhibiting Mcl-1 Download PDFInfo
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- CN116173035A CN116173035A CN202111422793.2A CN202111422793A CN116173035A CN 116173035 A CN116173035 A CN 116173035A CN 202111422793 A CN202111422793 A CN 202111422793A CN 116173035 A CN116173035 A CN 116173035A
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention relates to application of dipyridamole in preparation of an anti-apoptotic protein Mcl-1 inhibitor, and particularly discloses application of dipyridamole as an inhibitor of Mcl-1 protein or a competitive binding agent. Further provides application of the compound as a medicament for potential treatment of parkinsonism. The dipyridamole can act on the anti-apoptosis protein Mcl-1, and the separation of the combined pro-apoptosis protein is promoted by competitively combining with the anti-apoptosis protein Mcl-1, so that the effect of inhibiting the anti-apoptosis protein is achieved. The simultaneously released pro-apoptotic proteins were able to exert a pro-apoptotic effect, with an IC50 of dipyridamole against Mcl-1 of 71.27 μm.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of dipyridamole in Mcl-1 inhibition.
Background
Apoptosis (also known as programmed cell death) is a conserved evolutionary cellular process that plays a very important role in embryo development and tissue homeostasis. Abnormalities in apoptotic function can cause a variety of diseases such as neurodegenerative diseases, autoimmune diseases, and cancers. So far, there are two main relatively mature apoptosis pathways studied: death receptor-mediated pathways (exogenous apoptotic pathways) and mitochondrial-mediated pathways (endogenous apoptotic pathways).
In a mitochondrial mediated pathway, bcl-2 family proteins can alter their permeability by targeting the mitochondrial outer membrane, thereby releasing cytochrome c, activating apoptotic proteases, ultimately leading to cell death. The regulation of apoptosis is mainly achieved by Bcl-2 family proteins. Bcl-2 family proteins are largely divided into two classes: one class is anti-apoptotic proteins, including Bcl-2, mcl-1, and Bcl-XL; the other class is pro-apoptotic proteins, including Bax, bak, bim, noxa and Puma, among others. The pro-apoptotic proteins form an oligomerization in the mitochondrial outer membrane, causing the opening of the mitochondrial outer membrane pore, causing cytochrome c release from the mitochondria to the cytoplasm, ultimately leading to apoptosis. The anti-apoptotic proteins Bcl-2, mcl-1, and Bcl-XL inhibit apoptosis by interacting with pro-apoptotic proteins to inhibit the formation of Bax and Bak oligomerization. Studies have shown that anti-apoptotic proteins are highly expressed in a variety of cancer cells, one of the important reasons for cancer cells to evade apoptosis and acquire resistance. Thus, antagonizing the activity of apoptotic proteins is a strategy for cancer treatment.
Dipyridamole is a non-nitrate coronary artery dilator, has effects of dilating coronary vessel, promoting formation of collateral circulation and slightly anticoagulating, and has antiviral effect. Can be used for treating coronary heart disease, and can be used as anti-platelet aggregation medicine for preventing and treating thrombosis and disseminated intravascular coagulation.
Disclosure of Invention
The present invention provides a novel inhibitor or competitive binding agent of Mcl-1 protein to overcome the problems of the prior art.
In one aspect the invention provides the use of dipyridamole in the manufacture of an inhibitor of an anti-apoptotic protein.
The structure of the dipyridamole is shown as a formula I:
further, the anti-apoptotic protein is selected from the Bcl-2 family of proteins.
Further, the anti-apoptotic protein is selected from Mcl-1.
In one aspect the invention provides the use of dipyridamole in the preparation of a competitive binding agent for an anti-apoptotic protein.
Further, the anti-apoptotic protein is selected from the Bcl-2 family of proteins.
Further, the anti-apoptotic protein is selected from Mcl-1.
Further, the competitive binding agent is an agent that competes with the Bcl-2 protein family pro-apoptotic protein for binding to the anti-apoptotic protein Mcl-1.
Further, the Bcl-2 protein family pro-apoptotic proteins are selected from Bax, bak, bim, noxa, puma and derivatives thereof.
In another aspect the invention provides the use of dipyridamole in the manufacture of an agent for promoting apoptosis of a mitochondrial-mediated pathway.
In another aspect, the invention provides the use of dipyridamole in the manufacture of a medicament for the treatment of parkinson's disease.
In a further aspect, the invention provides an agent for inhibiting the activity of an anti-apoptotic protein, wherein the agent comprises dipyridamole as the active ingredient.
Further, the anti-apoptotic protein is selected from the Bcl-2 family of proteins.
Further, the anti-apoptotic protein is selected from Mcl-1.
Further, the preparation further comprises a dispersion medium.
Further, the dispersion medium is a solution.
In a further aspect the invention provides an agent capable of competitively binding to anti-apoptotic proteins, wherein the active ingredient in said agent is dipyridamole.
Further, the anti-apoptotic protein is selected from the Bcl-2 family of proteins.
Further, the anti-apoptotic protein is selected from Mcl-1.
Further, the preparation further comprises a dispersion medium.
Further, the dispersion medium is a solution.
In a further aspect the invention provides a medicament for the treatment of parkinson's disease, wherein the active ingredient of the medicament is dipyridamole.
Further, the medicine contains pharmaceutically acceptable auxiliary materials.
In a further aspect the invention provides a method of inhibiting the activity of an anti-apoptotic protein, the method comprising the step of contacting dipyridamole with said anti-apoptotic protein.
Further, the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family.
In a further aspect the invention provides a method of competitively binding an anti-apoptotic protein with a pro-apoptotic protein, said method comprising the step of contacting dipyridamole with said anti-apoptotic protein and with a pro-apoptotic protein.
Further, the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family is an anti-apoptotic protein selected from the group consisting of Bcl-2 protein family pro-apoptotic protein Bax, bak, bim, noxa, puma and derivatives thereof.
Advantageous effects
The dipyridamole can act on the anti-apoptosis protein Mcl-1, and the separation of the combined pro-apoptosis protein is promoted by competitively combining with the anti-apoptosis protein Mcl-1, so that the effect of inhibiting the anti-apoptosis protein is achieved. The simultaneously released pro-apoptotic proteins were able to exert pro-apoptotic effects, thus testing for anti-tumor and anti-parkinsonism effects, with dipyridamole having an IC50 of 71.27 μm against Mcl-1.
The invention aims to provide a study on the inhibitory effect of dipyridamole on Mcl-1 protein. The present invention found that dipyridamole had an IC50 of 71.27. Mu.M for Mcl-1 by time resolved fluorescence resonance energy Transfer (TRF).
Drawings
FIG. 1 is a graph of the Mcl-1 inhibition activity of dipyridamole compounds at various concentrations, with compound concentration (. Mu.M) on the abscissa and fluorescence intensity ratio at 520nM and 620nM on the ordinate. As the concentration of the compound increases, the ratio of fluorescence intensities decreases, i.e., the inhibition increases.
Detailed Description
The following detailed description of the present invention will be made in detail to make the above objects, features and advantages of the present invention more apparent, but should not be construed to limit the scope of the present invention.
EXAMPLE 1 expression and purification of Mcl-1 protein
The Mcl-1 protein encoding gene was synthesized by Shanghai Sanny Biotechnology Co.Ltd and ligated into the pET28a expression plasmid. The expression plasmid is prepared into a solution with the concentration of 100 ng/. Mu.L, 1 mu.L of the plasmid solution is transformed into an expression strain Escherichia coli BL (DE 3) by a chemocompetent transformation method, and the expression and purification of the recombinant protein Mcl-1 are carried out.
The specific expression steps are as follows:
1. single colonies transformed on LB plates of expression strain E.coli BL21 (DE 3) were picked up and inoculated into 5mL liquid LB medium tubes containing 50. Mu.g/. Mu.L kanamycin and incubated overnight at 37℃and 220 rpm.
2. According to the inoculation amount of 1%, 1mL of the overnight cultured bacterial liquid is added into a triangular conical flask containing 200mL of liquid LB culture medium, 50 mug/mug kanamycin with the final concentration is added, and the bacterial liquid is cultured for 3 hours at 37 ℃ and 220rpm until the OD value of the bacterial liquid reaches 0.4-0.6.
3. The shaker temperature was lowered to 20℃and IPTG was added to the culture broth at a final concentration of 0.1mM to induce protein expression, and the culture was continued for 16 hours.
4. After the completion of the culture, the bacterial liquid was poured into a 500mL centrifuge bowl, and centrifuged at 5000rpm for 10min to collect bacterial cells.
The specific purification steps of Mcl-1 are as follows:
1. and (3) thallus crushing: the cells collected in the centrifuge bowl were suspended in lysis buffer (20mM Tris,0.5M NaCl,20mM imidazole,pH 8.0). Pre-cooling the high-pressure homogenizer to 4 ℃ in advance, removing 20% ethanol for preservation, washing the machine with deionized water for 1-2 times, and washing the machine with lysis buffer for 1-2 times. The pressure of the machine is regulated to about 800bar, and the thalli are crushed.
2. Bacterial precipitation: the collected cell disruption solution was placed in a centrifuge tube, centrifuged at 12000rpm for 20min, and the supernatant was collected.
3. Loading on a column and purifying: the column was equilibrated with lysis buffer. Pouring the supernatant directly into a nickel column, and allowing the supernatant to flow through the nickel column packing by utilizing the action of gravity so as to combine the protein and the nickel column packing. The impure proteins which do not specifically bind to the nickel column packing are washed off with lysis buffer. Finally, the Mcl-1 protein was eluted with an elution buffer (lysis buffer containing 200mM imidozole).
4. Protein concentration: the collected Mcl-1 eluent is placed into an ultrafiltration concentration tube, and a preservation buffer (20mM Tris,100mM NaCl,2mM DTT) is used as a diluent to finally remove imidazole from the target protein, and the concentration of the Mcl-1 is higher than 1mg/mL.
Example 2Mcl-1 Activity inhibition assay
Dipyridamole was formulated into 10mM,3.33mM,1.11mM, 370. Mu.M, 123. Mu.M, 41.2. Mu.M, 13.7. Mu.M, 4.57. Mu.M, 1.52. Mu.M, 0.50. Mu.M mother liquors at 3-fold dilutions, respectively. Mcl-1 was diluted to 10nM with test buffer (50mM HEPES,pH 7.2, 50mM NaCl,1mM DTT,0.05%Tween-20); FITC-BAK-BH3 peptide was diluted to 1. Mu.M; anti-His Tb-labeled donor was diluted to 10nM. To a white 96-well plate test well, 12.5. Mu.L of Anti-His Tb-labeled donor, 17. Mu.L of test buffer, 12.5. Mu.L of FITC-BAK-BH3 peptide, 7.5. Mu.L of Mcl-1, and 0.5. Mu.L of the above dipyridamole mother liquor at various concentrations were added, followed by incubation at room temperature for 3 hours. The test was performed with a Flex Station3 multifunctional microplate reader in TRF with parameters EX 340/30nm Em 620/10nm and Ex 340/30nm Em 520nm. And respectively reading fluorescence values at 620nm and 520nm, and taking the fluorescence intensity ratio of the two as an index of the inhibition effect of the compound.
Wherein FITC-BAK-BH3 is a natural ligand of Mcl-1, anti-His Tb-delayed donor can be combined with a His tag on Mcl-1, and after combination, tb metal can generate Fluorescence Resonance Energy Transfer (FRET) with FITC-BAK-BH3 combined on Mcl-1; upon competitive binding of dipyridamole to Mcl-1, the FITC-BAK-BH3 polypeptide dissociates from Mcl-1; thus, the strength of dipyridamole's ability to bind Mcl-1 can be reflected by FRET strength. Therefore, fluorescence values at 620nm and 520nm were read, respectively, and the ratio of the fluorescence intensities was used as an index of the inhibitory effect of the compound.
The results of the experiment are shown in FIG. 1, and the results of FIG. 1 show that dipyridamole shows inhibition of Mcl-1 protein and that the inhibition effect is dose dependent with increasing concentration. Further calculation of IC50 value of dipyridamole against Mcl-1 by the above experimental results dipyridamole against Mcl-1 the IC50 value of dipyridamole against Mcl-1 was 71.27. Mu.M.
Claims (10)
1. Use of dipyridamole in the preparation of an inhibitor of an anti-apoptotic protein; the anti-apoptotic proteins are selected from the Bcl-2 family of proteins, the anti-apoptotic protein Mcl-1.
2. Use of dipyridamole in the preparation of a competitive binding agent to an anti-apoptotic protein; the anti-apoptotic proteins are selected from the Bcl-2 family of proteins, the anti-apoptotic protein Mcl-1.
3. The use of claim 2, the competitive binding agent being an agent that competes with Bcl-2 protein family pro-apoptotic proteins for binding to anti-apoptotic protein Mcl-1;
preferably, the Bcl-2 family of protein pro-apoptotic proteins are selected from Bax, bak, bim, noxa, puma, BAK-BH3 and derivatives thereof.
4. Use of dipyridamole in the manufacture of an agent for promoting apoptosis of a mitochondrial-mediated pathway.
5. Use of dipyridamole in the manufacture of a medicament for the treatment of parkinson's disease.
6. An agent for inhibiting the activity of an anti-apoptotic protein, wherein the active ingredient in the agent is dipyridamole;
preferably, the anti-apoptotic protein is selected from the group consisting of Mcl-1, an anti-apoptotic protein of the Bcl-2 protein family;
preferably, the formulation further comprises a dispersion medium.
7. An agent capable of competitively binding anti-apoptotic proteins, wherein the active ingredient in said agent is dipyridamole;
preferably, the anti-apoptotic protein is selected from the group consisting of Mcl-1, an anti-apoptotic protein of the Bcl-2 protein family;
preferably, the formulation further comprises a dispersion medium.
8. A medicament for treating parkinson's disease, wherein the active ingredient in the medicament is dipyridamole;
preferably, the medicament comprises pharmaceutically acceptable auxiliary materials.
9. A method of inhibiting the activity of an anti-apoptotic protein, the method comprising the step of contacting dipyridamole with the anti-apoptotic protein;
preferably, the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family is an anti-apoptotic protein.
10. A method of 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 a pro-apoptotic protein;
preferably, the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family is an anti-apoptotic protein selected from the group consisting of Bcl-2 protein family pro-apoptotic protein Bax, bak, bim, noxa, puma, BAK-BH3 and derivatives thereof.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111422793.2A CN116173035A (en) | 2021-11-26 | 2021-11-26 | Use of dipyridamole in inhibiting Mcl-1 |
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| CN202111422793.2A CN116173035A (en) | 2021-11-26 | 2021-11-26 | Use of dipyridamole in inhibiting Mcl-1 |
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| CN116173035A true CN116173035A (en) | 2023-05-30 |
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| CN202111422793.2A Pending CN116173035A (en) | 2021-11-26 | 2021-11-26 | Use of dipyridamole in inhibiting Mcl-1 |
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