阅读说明：本技术 原儿茶醛在抑制CtBP1中的应用 (Application of protocatechualdehyde in inhibiting CtBP1 ) 是由 李福伦 邓禹 徐志建 郭婉军 华亮 朱圣杰 郭冬婕 冯心怡 李斌 于 2019-11-02 设计创作，主要内容包括：本发明涉及原儿茶醛在制备CtBP1抑制剂中的应用,功能研究表明,PA可抑制乳腺癌细胞的增殖和迁移。此外,我们发现在乳腺癌细胞中,PA可提高CtBP1的靶基因P21和E-cadherin的表达水平,同时能够降低P21和E-cadherin启动子区域的CtBP1结合强度。然而,在我们采用CRISPR/Cas9构建的CtBP1基因敲除的乳腺癌细胞中,PA不能对P21和E-cadherin的表达水平产生影响。另外,在CtBP1基因敲除的乳腺癌细胞中可观察到对PA诱导的增殖和迁移的抑制作用的抵抗。我们的研究结果表明,PA可直接与CtBP1结合,其抗癌活性的分子机制之一是靶向CtBP1,表明PA是一种潜在的CtBP1抑制剂。(The invention relates to application of protocatechualdehyde in preparation of a CtBP1 inhibitor, and functional research shows that PA can inhibit proliferation and migration of breast cancer cells. In addition, we found that in breast cancer cells, PA can increase the expression level of target genes P21 and E-cadherin of CtBP1, and can reduce the CtBP1 binding strength of P21 and E-cadherin promoter regions. However, in the breast cancer cells with CtBP1 gene knockout constructed by using CRISPR/Cas9, PA can not influence the expression level of P21 and E-cadherin. In addition, resistance to inhibition of PA-induced proliferation and migration was observed in CtBP1 knock-out breast cancer cells. Our research results show that PA can be directly combined with CtBP1, and one of molecular mechanisms of the anticancer activity of PA is targeting CtBP1, which shows that PA is a potential CtBP1 inhibitor.)

1. Application of protocatechualdehyde in preparation of CtBP1 inhibitor is provided.

2. The use according to claim 1, wherein the protocatechualdehyde further comprises pharmaceutically acceptable salts and esters thereof, optionally substituted analogs thereof, or combinations of one or more compounds comprising protocatechualdehyde, further comprising derivatives of protocatechualdehyde.

3. Use according to claim 1 or 2, characterized in that the protocatechualdehyde is used as single active ingredient of a medicament or as active ingredient together with other CtBP1 inhibitors.

4. Application of protocatechualdehyde in preparation of medicines for inhibiting cell proliferation and migration related to CtBP1 overexpression and/or treating diseases of CtBP1 overexpression.

5. Use according to claim 4, wherein the protocatechuic aldehyde is used as the sole active ingredient of the medicament or together with other medicaments as the active ingredient.

6. The use of claim 4, wherein the disease in which CtBP1 is overexpressed is a tumor.

7. An inhibitor of CtBP1, wherein the active ingredient of the inhibitor comprises a therapeutically effective amount of protocatechualdehyde.

8. The inhibitor according to claim 7, wherein the medicament further comprises a component which has a positive effect on the inhibition of cell proliferation and migration associated with the overexpression of CtBP1 and/or a medicament for treating diseases in which CtBP1 is overexpressed, and/or a pharmaceutically acceptable carrier for improving the stability of protocatechualdehyde, when the medicament is administered simultaneously with protocatechualdehyde.

9. The inhibitor according to claim 7, wherein the pharmaceutically acceptable carrier comprises an emulsifier, excipient, filler, binder, humectant, disintegrant, absorption enhancer, flavoring agent, coloring agent, or solubilizing agent.

Technical Field

The invention relates to the technical field of new indications of medicines, in particular to application of protocatechualdehyde in inhibiting CtBP 1.

Background

Carboxy-terminal binding proteins (CtBP) are evolutionarily conserved transcription co-repressors. The study shows that CtBP is related to tumorigenesis and tumor progression, and the CtBP can promote epithelial-mesenchymal transition (EMT) and is used as an apoptosis antagonist to participate in the inhibition of a plurality of tumor suppressor genes. Deletion of epithelial cadherin (E-cadherin) expression is a characteristic molecular event of various epithelial tumor EMTs, CtBP-mediated E-cadherin inhibition shows its important role in promoting tumor EMT, which is a step leading to malignant characteristics of tumor cells: loss of cell adhesion molecules to the tumor, acquisition of migration and erosion phenotypes, resistance to apoptosis, and the like.

CtBP of vertebrate is coded by CtBP1 gene and CtBP2 gene, and expressed to produce two proteins CtBP1 and CtBP 2. C-terminal binding protein 1 (CtBP 1), a transcriptional co-repressor, is overexpressed in a variety of cancers. CtBP1 can promote cancer cell proliferation, migration, invasion and apoptosis by inhibiting a plurality of tumor inhibiting factors.

Protocatechualdehyde (PA), with chemical name of 3, 4-dihydroxybenzaldehyde, is derived from water-soluble extract of blood circulation promoting and blood stasis removing medicine (such as Saviae Miltiorrhizae radix, folium Ilicis Purpureae, and herba Salvia officinalis). Because the antioxidant activity of protocatechualdehyde is derived from the mother nucleus of its catechol, it has various physiological activities, such as the actions of dilating artery, reducing myocardial oxygen consumption, inhibiting platelet aggregation, resisting lipid peroxidation and scavenging free radicals.

In recent years, as more and more protocatechualdehyde is researched and reported, new pharmacological effects of protocatechualdehyde are discovered successively. XuY et al found that bolus tail vein injection of PA reduced the mortality of caecal ligation and perforation induced sepsis rats by blocking the inflammatory site through HMGBl and NF-jB signaling pathways. ZouZ and the like firstly prove that PA has the effect of resisting HBV, can effectively inhibit the replication of the HBV of the cell strain in vitro and can also effectively inhibit the replication of DHBV in vivo. Gao J et al found that PA protected human neuroblastoma cells by modulating the DJ-1 gene. Chinese patent document CN201910615961.6 discloses the application of protocatechualdehyde in preparing inhibitory drugs for bacterial quorum sensing systems. Chinese patent document CN 201610711489.2 discloses the application of protocatechualdehyde in the preparation of medicine for treating acute kidney injury. However, the action mechanism of PA on CtBP1 has not been reported yet.

Disclosure of Invention

The invention aims to overcome the defects and technical shortcomings of the prior art and provide an application of protocatechualdehyde in preparation of a CtBP1 inhibitor.

The invention also aims to provide a new application of protocatechuic aldehyde in inhibiting cell proliferation and migration related to CtBP1 overexpression and/or treating diseases of CtBP1 overexpression.

It is still another object of the present invention to provide a CtBP1 inhibitor.

In order to achieve the first purpose, the invention adopts the technical scheme that:

application of protocatechualdehyde in preparation of CtBP1 inhibitor is provided.

After learning the inhibitory effect of the protocatechualdehyde of the present invention on CtBP1, one skilled in the art can use various conventional methods to process other protocatechualdehyde-containing materials into pharmaceutically acceptable salts and esters, optionally substituted analogs of synthetic or semi-synthetic protocatechualdehyde, or combinations of one or more compounds comprising protocatechualdehyde, including derivatives of protocatechualdehyde. Such processing includes, but is not limited to: pulverizing, extracting with water, extracting with organic solvent, etc. More specifically, the process comprises, for example, the steps of: weighing, pulverizing, decocting, etc.

Preferably, the protocatechualdehyde is used as a single active ingredient of a medicament or is used as an active ingredient together with other CtBP1 inhibitors.

In order to achieve the second object, the invention adopts the technical scheme that:

application of protocatechualdehyde in preparation of medicines for inhibiting cell proliferation and migration related to CtBP1 overexpression and/or treating diseases of CtBP1 overexpression.

Preferably, the protocatechualdehyde is used as a single active ingredient of a medicament or as an active ingredient in combination with other medicaments.

Preferably, the disease in which CtBP1 is overexpressed is a tumor.

The invention also discloses the application of protocatechuic aldehyde in designing, developing and preparing related inhibitors aiming at CtBP1 overexpression or using the same as biochemical reagents, analytical reagents or detection reagents, or the application of protocatechuic aldehyde in a novel method and a technical aspect for researching cell signaling pathway regulated by CtBP1 overexpression and related target protein biological functions.

In order to achieve the third object, the invention adopts the technical scheme that:

an inhibitor of CtBP1, the active ingredient of which comprises a therapeutically effective amount of protocatechualdehyde.

Preferably, the medicament also comprises a component which has positive effects on inhibiting cell proliferation and migration related to the over-expression of CtBP1 and/or treating a medicament for a disease of the over-expression of CtBP1 after being simultaneously administered with protocatechualdehyde and/or a pharmaceutically acceptable carrier for improving the stability of protocatechualdehyde.

Preferably, the pharmaceutically acceptable carrier includes an emulsifier, excipient, filler, binder, humectant, disintegrant, absorption enhancer, flavoring agent, coloring agent or solubilizing agent

By "pharmaceutically acceptable" is meant a substance that is not substantially biologically or otherwise undesirable, i.e., the substance can be administered to an individual without causing any substantially undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The "carrier", also referred to as "excipient", includes any commonly used excipient in pharmacy and should be selected based on compatibility and the desired release profile properties of the dosage form. Exemplary carrier materials include, for example, emulsifiers, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, flavoring agents, coloring agents, or solubilizing agents, and the like. "pharmaceutically acceptable carriers" may include, for example, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, dextrin-maltose complexing agent, glycerin, magnesium silicate, sodium caseinate, soybean lecithin, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like. The pharmaceutical composition can be prepared into oral preparations, such as tablets, hard capsules, soft capsules, dripping pills, granules, micro-capsule tablets, suspensions, dripping pills or oral liquids, by adopting methods known in the art.

Functional studies have shown that PA inhibits proliferation and migration of breast cancer cells. In addition, we found that in breast cancer cells, PA can increase the expression level of target genes P21 and E-cadherin of CtBP1, and can reduce the CtBP1 binding strength of P21 and E-cadherin promoter regions. However, in the breast cancer cells with CtBP1 gene knockout constructed by using CRISPR/Cas9, PA can not influence the expression level of P21 and E-cadherin. In addition, resistance to inhibition of PA-induced proliferation and migration was observed in CtBP1 knock-out breast cancer cells. Our research results show that PA can be directly combined with CtBP1, and one of molecular mechanisms of the anticancer activity of PA is targeting CtBP1, which shows that PA is a potential CtBP1 inhibitor.

Drawings

FIG. 1 is a graphical representation of PA in combination with CtBP 1. Wherein A is a butt joint mode of PA and CtBP1, CtBP1 is in a mode diagram, a is PA, b is phenylpyruvate, and a dashed line is a distance; b is a binding affinity map for detecting PA to CtBP1 using the MST assay.

Figure 2A is a growth curve of MDA-MB-231 and MCF-7 cells treated with 0.1% DMSO or 100 μ MPA, respectively, indicating P < 0.05; b is an image of the ability of breast cancer cells to migrate as measured by the scratch test, showing scratches with or without PA treatment. The percentage of scratch closure area shown in the bar graph is the mean ± standard deviation from triplicate experimental data, representing P < 0.05; c is the ability of breast cancer cells to migrate as measured by transwell experiments, the number of migrated cells was calculated from 3 randomly selected microscopic fields, the results shown in the bar graph are the mean ± standard deviation of data from triplicates, indicating P < 0.05.

FIG. 3A is the expression of P21 and E-cadherin mRNA from each group of cells after 48 hours of drug treatment; b is the expression level of P21 and E-cadherin protein after 48 hours of PA treatment; c is CtBP1 chromatin co-immunoprecipitation plot, P < 0.05.

FIG. 4A shows Crispr-Cas9 construction of MDA-MB-231 and MCF-7CtBP1 knock-out cell Sanger sequencing, confirming 2 knock-out cell lines with nucleotide 'A' insertion between the 57 th and 58 th nucleotides of the 2 nd CDS exon of CtBP 1; b is the mRNA and protein expression level of P21 and E-cadherin after the CtBP1 is knocked out by breast cancer cells, and represents that P is less than 0.05; c is a complete knock-out of CtBP1 in MDA-MB-231 and MCF-7 cells increased the IC50 value of PA-treated breast cancer cells; d is an image for detecting the migration capacity of the MDA-MB-231 and MCF-7 cells knocked out by CtBP1 through a scratch test, and E is an image for detecting the migration capacity of the MDA-MB-231 and MCF-7 cells knocked out by CtBP1 through a transwell test; f is the coverage area and the percentage of migrated cells for the DMSO group/100 μ MPA group, calculated from the mean of the groups; g is the expression level of P21 and E-cadherin proteins in CtBP1 knockout cells.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention; furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.