WO2023092464A1 - Use of ellipticine and pharmaceutically acceptable salt thereof in inhibiting mcl-1 - Google Patents
Use of ellipticine and pharmaceutically acceptable salt thereof in inhibiting mcl-1 Download PDFInfo
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- WO2023092464A1 WO2023092464A1 PCT/CN2021/133570 CN2021133570W WO2023092464A1 WO 2023092464 A1 WO2023092464 A1 WO 2023092464A1 CN 2021133570 W CN2021133570 W CN 2021133570W WO 2023092464 A1 WO2023092464 A1 WO 2023092464A1
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- protein
- apoptotic protein
- mcl
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- apoptotic
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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
Definitions
- the invention belongs to the field of medicines, and in particular relates to the application of ellipticine and its pharmaceutically acceptable salts in inhibiting Mcl-1.
- Apoptosis also known as programmed cell death
- 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).
- 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.
- the anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-XL inhibit apoptosis by interacting with pro-apoptotic proteins and inhibiting the oligomerization of Bax and Bak.
- 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.
- Mcl-1 has a role in the survival of dopamine neurons, and these neurons are selectively killed in the progression of Parkinson's disease, suggesting that Mcl-1 is a potential target for the treatment of Parkinson's disease.
- the selective Bcl-2 inhibitor ABT-199 has been approved by the US Food and Drug Administration for the treatment of patients with chronic lymphocytic leukemia who lack chromosome 17p.
- ABT-199 was not effective in cancer patients with Mcl-1 overexpression. Therefore, the development of new Mcl-1 protein inhibitors is a very valuable research topic in the research of anti-tumor drugs or anti-Parkinson's disease.
- Ellipticine is a cytotoxic alkaloid fused with indole and isoquinoline. According to related reports, it can inhibit the activity of RNA polymerase. It can also inhibit the activity of acetylcholinesterase and butyrylcholinesterase in humans and experimental animals; it has antioxidant activity.
- the present invention provides a novel Mcl-1 protein inhibitor or competitive binding agent.
- One aspect of the present invention provides the use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of inhibitors of anti-apoptotic proteins.
- anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.
- anti-apoptotic protein is selected from Mcl-1.
- One aspect of the present invention provides the use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of a competitive binding agent for anti-apoptotic proteins.
- anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.
- anti-apoptotic protein is selected from Mcl-1.
- 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.
- 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 ellipticine or a pharmaceutically acceptable salt thereof in the preparation of an agent for promoting apoptosis in a mitochondria-mediated pathway.
- Another aspect of the present invention provides the use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating Parkinson's disease.
- Another aspect of the present invention provides a reagent for inhibiting the activity of an anti-apoptotic protein, and the active ingredient in the reagent is ellipticine or a pharmaceutically acceptable salt thereof.
- anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.
- anti-apoptotic protein is selected from Mcl-1.
- the preparation further includes a dispersion medium.
- the dispersion medium is a solution.
- Another aspect of the present invention provides a reagent capable of competitively binding anti-apoptotic proteins, the active ingredient in the reagent being ellipticine or a pharmaceutically acceptable salt thereof.
- anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.
- anti-apoptotic protein is selected from Mcl-1.
- the preparation further includes a dispersion medium.
- 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 ellipticine or a pharmaceutically acceptable salt thereof.
- 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 ellipticine or a pharmaceutically acceptable salt thereof with the anti-apoptotic protein.
- the anti-apoptotic protein Mcl-1 of the anti-apoptotic protein Bcl-2 protein family is the anti-apoptotic protein Mcl-1 of the anti-apoptotic protein Bcl-2 protein family.
- Another aspect of the present invention provides a method for competitively binding an anti-apoptotic protein with a pro-apoptotic protein, the method comprising combining ellipticine or a pharmaceutically acceptable salt thereof with the anti-apoptotic protein and the pro-apoptotic protein contact steps.
- 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.
- the present invention discovers for the first time that ellipticine or its pharmaceutically acceptable salt can act on the anti-apoptotic protein Mcl-1, and promote the separation of the pro-apoptotic protein bound to it by competitively binding to Mcl-1, thereby achieving inhibition of anti-apoptosis The role of 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 ellipticine or its pharmaceutically acceptable salt against Mcl-1 is 34.52 ⁇ M.
- Figure 1 is a diagram showing the inhibition of Mcl-1 activity by different concentrations of ellipticine compounds, the abscissa is the compound concentration ( ⁇ M), 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.
- 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/ ⁇ L, and 1 ⁇ L 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.
- Mcl-1 The specific purification steps of Mcl-1 are:
- 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.
- lysis buffer 20mM Tris, 0.5M NaCl, 20mM imidazole, pH 8.0.
- Bacterial cell precipitation put the collected broken cell liquid into a centrifuge tube, centrifuge at 12000rpm for 20min, and collect the supernatant.
- 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.
- Ellipticine was diluted by 3 times to prepare stock solutions of 10 mM, 3.33 mM, 1.11 mM, 370 ⁇ M, 123 ⁇ M, 41.2 ⁇ M, 13.7 ⁇ M, 4.57 ⁇ M, 1.52 ⁇ M and 0.50 ⁇ M respectively.
- FITC-BAK-BH3 is the natural ligand of Mcl-1
- Anti-His Tb-labeled donor can bind to the His tag on Mcl-1
- Tb metal can bind to FITC-BAK on Mcl-1 -BH3 undergoes fluorescence resonance energy transfer (FRET);
- FRET fluorescence resonance energy transfer
- FITC-BAK-BH3 polypeptide dissociates from Mcl-1; thus, ellipticine binding can be reflected by FRET intensity
- FRET fluorescence resonance energy transfer
- the experimental results are shown in Figure 1.
- the results in Figure 1 show that ellipticine exhibits an inhibitory effect on the Mcl-1 protein, and as the concentration increases, the inhibitory effect is dose-dependent.
- the IC50 value of ellipticine against Mcl-1 was further calculated based on the above experimental results.
- the IC50 value of ellipticine against Mcl-1 was 34.52 ⁇ M.
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Abstract
The use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of an anti-apoptotic protein Mcl-1 inhibitor, specifically the use as an inhibitor of or a competitive binding agent to the Mcl-1 protein. Further provided is the use thereof as a potential drug for treating Parkinson's disease. Research finds that ellipticine or the pharmaceutically acceptable salt thereof can act on the anti-apoptotic protein Mcl-1, and competitively binds to Mcl-1 to prompt the separation of a pro-apoptotic protein binding thereto, so that the effect of inhibiting the anti-apoptotic protein is achieved. In addition, the released pro-apoptotic protein can play a role in promoting apoptosis. IC50 of ellipticine or the pharmaceutically acceptable salt thereof for Mcl-1 is 34.52 μM.
Description
本发明属于药物领域,具体涉及玫瑰树碱及其可药用盐在抑制Mcl-1中的应用。The invention belongs to the field of medicines, and in particular relates to the application of ellipticine and its pharmaceutically acceptable salts in inhibiting Mcl-1.
细胞凋亡(又称程序性细胞死亡)是一种保守进化的细胞过程,对胚胎的发育和组织稳态起着十分重要的作用。细胞凋亡功能的异常会引起多种疾病,比如神经退行性疾病、自身免疫性疾病和癌症等。目前为止,人们研究的比较成熟的凋亡途径主要有两种:死亡受体介导途径(外源性凋亡途径)和线粒体介导途径(内源性凋亡途径)。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).
在线粒体介导的途径中,Bcl-2家族蛋白可通过靶向线粒体外膜,改变其通透性,进而释放细胞色素c,激活细胞凋亡蛋白酶,最终引起细胞死亡。细胞凋亡的调控主要通过Bcl-2家族蛋白实现。Bcl-2家族蛋白主要分为两类:一类是抗凋亡蛋白,包括Bcl-2、Mcl-1、和Bcl-XL;另一类是促凋亡蛋白,包含Bax、Bak、Bim、Noxa和Puma等。促凋亡蛋白在线粒体外膜形成寡聚化,促使线粒体外膜孔道打开,引起细胞色素c从线粒体释放到细胞质,最终导致凋亡发生。而抗凋亡蛋白Bcl-2、Mcl-1、和Bcl-XL则通过与促凋亡蛋白相互作用,抑制Bax和Bak寡聚化的形成而抑制凋亡。研究表明,抗凋亡蛋白在多种癌细胞中高度表达,是癌细胞逃避凋亡和获得耐药性的重要原因之一。因此,拮抗抗凋亡蛋白的活性是一种癌症治疗的策略。近年来,有研究表明Mcl-1对多巴胺神经元的存活有作用,而这些神经元在帕金森疾病进展中选择性死亡,这表明Mcl-1是治疗帕金森疾病的潜在靶点。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. The anti-apoptotic proteins Bcl-2, Mcl-1, and Bcl-XL inhibit apoptosis by interacting with pro-apoptotic proteins and inhibiting the oligomerization of Bax and Bak. 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. In recent years, studies have shown that Mcl-1 has a role in the survival of dopamine neurons, and these neurons are selectively killed in the progression of Parkinson's disease, suggesting that Mcl-1 is a potential target for the treatment of Parkinson's disease.
目前,已有多种针对抗凋亡蛋白的小分子抑制剂被报道,并显示出良好的抗癌活性。比如,选择性Bcl-2抑制剂ABT-199被美国食品药品监督管理局批准上市,用于治疗17p染色体缺少的慢性淋巴细胞白血病患者。然而,ABT-199对Mcl-1过表达的癌症患者效果不理想。因此,发展新型Mcl-1蛋白抑制剂是抗肿瘤药物或抗帕金森疾病研究中极有价值的研究课题。At present, a variety of small molecule inhibitors targeting anti-apoptotic proteins have been reported, and have shown good anticancer activity. For example, the selective Bcl-2 inhibitor ABT-199 has been approved by the US Food and Drug Administration for the treatment of patients with chronic lymphocytic leukemia who lack chromosome 17p. However, ABT-199 was not effective in cancer patients with Mcl-1 overexpression. Therefore, the development of new Mcl-1 protein inhibitors is a very valuable research topic in the research of anti-tumor drugs or anti-Parkinson's disease.
玫瑰树碱(ellipticine)是一种细胞毒生物碱,由吲哚与异喹啉稠合。据相关报道,其能抑制RNA聚合酶的活性。还能抑制人和实验动物的乙酰胆碱酯酶和丁酰胆碱酯酶活性;具有抗氧化剂活性。Ellipticine is a cytotoxic alkaloid fused with indole and isoquinoline. According to related reports, it can inhibit the activity of RNA polymerase. It can also inhibit the activity of acetylcholinesterase and butyrylcholinesterase in humans and experimental animals; it has antioxidant activity.
发明内容Contents of the invention
本发明为克服现有技术中的问题,提供了一种新的Mcl-1蛋白的抑制剂或竞争性结合剂。In order to overcome the problems in the prior art, the present invention provides a novel Mcl-1 protein inhibitor or competitive binding agent.
本发明一个方面提供了玫瑰树碱或其可药用盐在制备抗凋亡蛋白的抑制剂中的用途。One aspect of the present invention provides the use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of inhibitors of anti-apoptotic proteins.
所述玫瑰树碱或其可药用盐结构如式I所示:The structure of the ellipticine or its pharmaceutically acceptable salt is shown in formula I:
进一步地,抗凋亡蛋白选自Bcl-2蛋白家族的抗凋亡蛋白。Further, the anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.
进一步地,抗凋亡蛋白选自Mcl-1。Further, the anti-apoptotic protein is selected from Mcl-1.
本发明一个方面提供了玫瑰树碱或其可药用盐在制备抗凋亡蛋白的竞争性结合剂中的用途。One aspect of the present invention provides the use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of a competitive binding agent for anti-apoptotic proteins.
进一步地,抗凋亡蛋白选自Bcl-2蛋白家族的抗凋亡蛋白。Further, the anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.
进一步地,抗凋亡蛋白选自Mcl-1。Further, the anti-apoptotic protein is selected from Mcl-1.
进一步地,所述竞争性结合剂为与Bcl-2蛋白家族促凋亡蛋白竞争性结合抗凋亡蛋白Mcl-1的试剂。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.
进一步地,Bcl-2蛋白家族促凋亡蛋白选自Bax、Bak、Bim、Noxa、Puma及其衍生物。Further, the pro-apoptotic protein of Bcl-2 protein family is selected from Bax, Bak, Bim, Noxa, Puma and derivatives thereof.
本发明另一个方面提供了玫瑰树碱或其可药用盐在制备促进线粒体介导途径的细胞凋亡的试剂中的用途。Another aspect of the present invention provides the use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of an agent for promoting apoptosis in a mitochondria-mediated pathway.
本发明另一个方面提供了玫瑰树碱或其可药用盐在制备治疗帕金森症的药物中的用途。Another aspect of the present invention provides the use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating Parkinson's disease.
本发明再一个方面提供了一种抑制抗凋亡蛋白活性的试剂,所述试剂中的活性成分为玫瑰树碱或其可药用盐。Another aspect of the present invention provides a reagent for inhibiting the activity of an anti-apoptotic protein, and the active ingredient in the reagent is ellipticine or a pharmaceutically acceptable salt thereof.
进一步地,抗凋亡蛋白选自Bcl-2蛋白家族的抗凋亡蛋白。Further, the anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.
进一步地,抗凋亡蛋白选自Mcl-1。Further, the anti-apoptotic protein is selected from Mcl-1.
进一步地,所述制剂中还进一步包含分散介质。Further, the preparation further includes a dispersion medium.
进一步地,所述的分散介质为溶液。Further, the dispersion medium is a solution.
本发明再一个方面提供了一种能够竞争性结合抗凋亡蛋白的试剂,所述试剂中的活性成分为玫瑰树碱或其可药用盐。Another aspect of the present invention provides a reagent capable of competitively binding anti-apoptotic proteins, the active ingredient in the reagent being ellipticine or a pharmaceutically acceptable salt thereof.
进一步地,抗凋亡蛋白选自Bcl-2蛋白家族的抗凋亡蛋白。Further, the anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family.
进一步地,抗凋亡蛋白选自Mcl-1。Further, the anti-apoptotic protein is selected from Mcl-1.
进一步地,所述制剂中还进一步包含分散介质。Further, the preparation further includes a dispersion medium.
进一步地,所述的分散介质为溶液。Further, the dispersion medium is a solution.
本发明再一个方面提供了一种治疗帕金森症的药物,所述药物中的活性成分为玫瑰树碱或其可药用盐。Another aspect of the present invention provides a drug for treating Parkinson's disease, the active ingredient in the drug is ellipticine or a pharmaceutically acceptable salt thereof.
进一步地,所述药物中包含可药用辅料。Further, the medicine contains pharmaceutically acceptable excipients.
本发明再一个方面提供了一种抑制抗凋亡蛋白活性的方法,所述方法包括将玫瑰树碱或其可药用盐与所述抗凋亡蛋白接触的步骤。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 ellipticine or a pharmaceutically acceptable salt thereof with the anti-apoptotic protein.
进一步地,抗凋亡蛋白Bcl-2蛋白家族的抗凋亡蛋白Mcl-1。Further, the anti-apoptotic protein Mcl-1 of the anti-apoptotic protein Bcl-2 protein family.
本发明再一个方面提供了一种与促凋亡蛋白竞争性结合抗凋亡蛋白的方法,所述方法包括将玫瑰树碱或其可药用盐与所述抗凋亡蛋白以及促凋亡蛋白接触的步骤。Another aspect of the present invention provides a method for competitively binding an anti-apoptotic protein with a pro-apoptotic protein, the method comprising combining ellipticine or a pharmaceutically acceptable salt thereof with the anti-apoptotic protein and the pro-apoptotic protein contact steps.
进一步地,抗凋亡蛋白Bcl-2蛋白家族的抗凋亡蛋白Mcl-1,促凋亡蛋白选自Bcl-2蛋白家族促凋亡蛋白Bax、Bak、Bim、Noxa、Puma及其衍生物。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.
本发明首次发现了玫瑰树碱或其可药用盐能够作用于抗凋亡蛋白Mcl-1,通过竞争性结合Mcl-1,促使与之结合的促凋亡蛋白分离,进而达到抑制抗凋亡蛋白的作用。同时释放的促凋亡蛋白能够发挥促凋亡的作用,进而试验抗肿瘤以及抗帕金森的作用,其中玫瑰树碱或其可药用盐针对Mcl-1的IC50为34.52μM。The present invention discovers for the first time that ellipticine or its pharmaceutically acceptable salt can act on the anti-apoptotic protein Mcl-1, and promote the separation of the pro-apoptotic protein bound to it by competitively binding to Mcl-1, thereby achieving inhibition of anti-apoptosis The role of 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 ellipticine or its pharmaceutically acceptable salt against Mcl-1 is 34.52 μM.
图1为不同浓度的玫瑰树碱化合物抑制Mcl-1活性图,横坐标为化合物浓度(μM),纵坐标为520nM和620nM处荧光强度比值。随着化合物浓度增高,荧光强度比值降低,即抑制作用增强。Figure 1 is a diagram showing the inhibition of Mcl-1 activity by different concentrations of ellipticine compounds, 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.
为了使本发明的上述目的、特征和优点能够更加明显易懂,下面对本发明的具体实施方式做详细的说明,但不能理解为对本发明的可实施范围的限定。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.
实施例1Mcl-1蛋白的表达及纯化Expression and purification of embodiment 1Mcl-1 protein
Mcl-1蛋白编码基因由上海桑尼生物科技有限公司合成并连接到pET28a表达质粒。将该 表达质粒配制成浓度100ng/μL溶液,取1μL质粒溶液通过化学感受态转化法,转化到表达菌株Escherichia coli BL21(DE3)中,进行重组蛋白Mcl-1的表达及纯化。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.
具体表达步骤如下:The specific expression steps are as follows:
1.挑取转化于表达菌株E.coli BL21(DE3)的LB平板上的单菌落,接种到含50μg/μL卡那霉素的5mL液体LB培养基试管中,37℃,220rpm条件下过夜培养。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.按照1%接种量,将1mL过夜培养的菌液加入到含200mL液体LB培养基的三角锥形瓶中,加入终浓度50μg/μL卡那霉素,37℃,220rpm条件下培养~3h,至菌液OD值达到0.4-0.6。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.将摇床温度降低至20℃,向培养液中添加终浓度为0.1mM的IPTG以诱导蛋白表达,并继续培养16h。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 hours.
4.培养结束后,将菌液倒入500mL离心桶中,5000rpm离心10min收集菌体。4. After the cultivation, pour the bacterial solution into a 500mL centrifuge bucket, and centrifuge at 5000rpm for 10min to collect the bacterial cells.
Mcl-1具体纯化步骤为:The specific purification steps of Mcl-1 are:
1.菌体破碎:利用裂解缓冲液(20mM Tris,0.5M NaCl,20mM imidazole,pH 8.0)将收集于离心桶的菌体悬浮。提前将高压均质机预冷至4℃,去除保存用的20%乙醇,用去离子水清洗机器1-2遍,再用裂解缓冲液清洗机器1-2遍。将机器压力调高至800bar左右,破碎菌体。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.菌体沉淀:将收集的菌体破碎液放入离心管中,12000rpm离心20min,收集上清液。2. Bacterial cell precipitation: put the collected broken cell liquid into a centrifuge tube, centrifuge at 12000rpm for 20min, and collect the supernatant.
3.上柱和纯化:用裂解缓冲液将柱子平衡。将上清液直接倒入镍柱中,利用重力的作用,使上清液流过镍柱填料,进而使蛋白和镍柱填料结合。再用裂解缓冲液将不与镍柱填料特异性结合的杂蛋白洗涤下来。最后,用洗脱缓冲液(包含200mM imidazole的裂解缓冲液)洗脱Mcl-1蛋白。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.蛋白浓缩:收集的Mcl-1洗脱液放入超滤浓缩管中,用保存缓冲液(20mM Tris,100mM NaCl,2mM DTT)作为稀释液,使目的蛋白最终去除咪唑,并浓缩Mcl-1浓度高于1mg/mL。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.
实施例2 Mcl-1活性抑制性实验Example 2 Mcl-1 activity inhibition experiment
将玫瑰树碱以3倍的稀释倍数,分别配制成10mM,3.33mM,1.11mM,370μM,123μM,41.2μM,13.7μM,4.57μM,1.52μM,0.50μM的母液。利用测试缓冲液(50mM HEPES,pH 7.2,50mM NaCl,1mM DTT,0.05%Tween-20)将Mcl-1稀释至10nM;将FITC-BAK-BH3肽稀释至1μM;Anti-His Tb-labeled donor稀释至10nM。向白色96孔板测试孔中加入12.5μL Anti-His Tb-labeled donor、17μL测试缓冲液、12.5μL FITC-BAK-BH3肽、7.5μL Mcl-1及0.5μL 不同浓度的上述玫瑰树碱母液,然后在室温下孵育3h。用Flex Station3多功能酶标仪进行测试,测试模式为TRF,参数为EX 340/30nm Em 620/10nm和Ex 340/30nm Em 520nm。分别读取620nm和520nm处荧光值,以二者荧光强度比作为化合物抑制效果的指标。Ellipticine was diluted by 3 times to prepare stock solutions of 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 respectively. 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 of the above-mentioned ellipticine mother solution of different concentrations to the test wells of the white 96-well plate, Then incubated at room temperature for 3h. 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.
其中,FITC-BAK-BH3是Mcl-1的天然配体,Anti-His Tb-labeled donor能够与Mcl-1上的His标签结合,结合后,Tb金属可以和Mcl-1上结合的FITC-BAK-BH3发生荧光共振能量转移(FRET);当玫瑰树碱竞争性结合到Mcl-1后,FITC-BAK-BH3多肽会从Mcl-1解离;因而,通过FRET强度可以反应出玫瑰树碱结合Mcl-1能力的强弱。因此,分别读取620nm和520nm处荧光值,以二者荧光强度比作为化合物抑制效果的指标。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 ellipticine competitively binds to Mcl-1, FITC-BAK-BH3 polypeptide dissociates from Mcl-1; thus, ellipticine binding can be reflected by FRET intensity 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.
实验结果见图1,图1结果显示了玫瑰树碱显示了对Mcl-1蛋白的抑制作用,且随着浓度的上升,显示出抑制效果具有剂量依赖性。通过上述实验结果进一步计算玫瑰树碱针对Mcl-1的IC50值玫瑰树碱针对Mcl-1的IC50值为34.52μM。The experimental results are shown in Figure 1. The results in Figure 1 show that ellipticine exhibits an inhibitory effect on the Mcl-1 protein, and as the concentration increases, the inhibitory effect is dose-dependent. The IC50 value of ellipticine against Mcl-1 was further calculated based on the above experimental results. The IC50 value of ellipticine against Mcl-1 was 34.52 μM.
Claims (10)
- 玫瑰树碱或其可药用盐在制备抗凋亡蛋白的抑制剂中的用途;所述抗凋亡蛋白选自Bcl-2蛋白家族的抗凋亡蛋白Mcl-1。Use of ellipticine or a pharmaceutically acceptable salt thereof in preparing an inhibitor of an anti-apoptotic protein; the anti-apoptotic protein is selected from the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family.
- 玫瑰树碱或其可药用盐在制备抗凋亡蛋白的竞争性结合剂中的用途;所述抗凋亡蛋白选自Bcl-2蛋白家族的抗凋亡蛋白Mcl-1。Use of ellipticine or a pharmaceutically acceptable salt thereof 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.
- 根据权利要求2所述的用途,所述竞争性结合剂为与Bcl-2蛋白家族促凋亡蛋白竞争性结合抗凋亡蛋白Mcl-1的试剂;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;优选地,Bcl-2蛋白家族促凋亡蛋白选自Bax、Bak、Bim、Noxa、Puma、BAK-BH3及其衍生物。Preferably, the pro-apoptotic protein of the Bcl-2 protein family is selected from Bax, Bak, Bim, Noxa, Puma, BAK-BH3 and derivatives thereof.
- 玫瑰树碱或其可药用盐在制备促进线粒体介导途径的细胞凋亡的试剂中的用途。Use of ellipticine or a pharmaceutically acceptable salt thereof in the preparation of a reagent for promoting apoptosis in a mitochondria-mediated pathway.
- 玫瑰树碱或其可药用盐在制备治疗帕金森症的药物中的用途。Use of ellipticine or a pharmaceutically acceptable salt thereof in preparing a medicine for treating Parkinson's disease.
- 一种抑制抗凋亡蛋白活性的试剂,所述试剂中的活性成分为玫瑰树碱或其可药用盐;A reagent for inhibiting the activity of anti-apoptotic protein, the active ingredient in the reagent is ellipticine or a pharmaceutically acceptable salt thereof;优选地,抗凋亡蛋白选自Bcl-2蛋白家族的抗凋亡蛋白Mcl-1;Preferably, the anti-apoptotic protein is selected from the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family;优选地,所述制剂中还进一步包含分散介质。Preferably, the formulation further includes a dispersion medium.
- 一种能够竞争性结合抗凋亡蛋白的试剂,所述试剂中的活性成分为玫瑰树碱或其可药用盐;A reagent capable of competitively binding to an anti-apoptotic protein, the active ingredient in the reagent being ellipticine or a pharmaceutically acceptable salt thereof;优选地,抗凋亡蛋白选自Bcl-2蛋白家族的抗凋亡蛋白Mcl-1;Preferably, the anti-apoptotic protein is selected from the anti-apoptotic protein Mcl-1 of the Bcl-2 protein family;优选地,所述制剂中还进一步包含分散介质。Preferably, the formulation further includes a dispersion medium.
- 一种治疗帕金森症的药物,所述药物中的活性成分为玫瑰树碱或其可药用盐;A medicine for treating Parkinson's disease, the active ingredient in the medicine is ellipticine or a pharmaceutically acceptable salt thereof;优选地,所述药物中包含可药用辅料。Preferably, the medicine contains pharmaceutically acceptable excipients.
- 一种抑制抗凋亡蛋白活性的方法,所述方法包括将玫瑰树碱或其可药用盐与所述抗凋亡蛋白接触的步骤;A method for inhibiting the activity of an anti-apoptotic protein, the method comprising the step of contacting ellipticine or a pharmaceutically acceptable salt thereof with the anti-apoptotic protein;优选地,抗凋亡蛋白Bcl-2蛋白家族的抗凋亡蛋白Mcl-1。Preferably, the anti-apoptotic protein Mcl-1 of the anti-apoptotic protein Bcl-2 protein family.
- 一种与促凋亡蛋白竞争性结合抗凋亡蛋白的方法,所述方法包括将玫瑰树碱或其可药用盐与所述抗凋亡蛋白以及促凋亡蛋白接触的步骤;A method for competitively binding an anti-apoptotic protein with a pro-apoptotic protein, the method comprising the step of contacting ellipticine or a pharmaceutically acceptable salt thereof with the anti-apoptotic protein and the pro-apoptotic protein;优选地,抗凋亡蛋白Bcl-2蛋白家族的抗凋亡蛋白Mcl-1,促凋亡蛋白选自Bcl-2蛋白家族促凋亡蛋白Bax、Bak、Bim、Noxa、Puma、BAK-BH3及其衍生物。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.
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