WO2022089144A1 - 新穿心莲内酯在制备rab5a蛋白抑制剂中的应用 - Google Patents

新穿心莲内酯在制备rab5a蛋白抑制剂中的应用 Download PDF

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WO2022089144A1
WO2022089144A1 PCT/CN2021/121946 CN2021121946W WO2022089144A1 WO 2022089144 A1 WO2022089144 A1 WO 2022089144A1 CN 2021121946 W CN2021121946 W CN 2021121946W WO 2022089144 A1 WO2022089144 A1 WO 2022089144A1
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rab5a
protein
neoandrographolide
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protein inhibitor
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何庆瑜
汪洋
钟利叶
张静
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暨南大学
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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  • the invention belongs to the technical field of medicine, and particularly relates to the application of neoandrographolide in the preparation of RAB5A protein inhibitor.
  • Lung cancer is one of the most difficult diseases to treat in the world.
  • the progression of cancer is often accompanied by chemotherapy drugs, and once the cancer has metastasized, it will be difficult to treat.
  • the current mainstream treatment method is a combination of surgery and radiotherapy and chemotherapy.
  • the angiogenesis factor (VEGF) inhibitor bevacizumab and the epidermal growth factor receptor blocker panitumumab are widely used.
  • cetuximab, and erlotinib are widely used.
  • the clinical efficacy is still unsatisfactory, and the occurrence of chemotherapy resistance and toxic side effects affects the prognosis of cancer patients.
  • Anticancer preparations based on natural products generally have the advantages of less toxic and side effects and low prices.
  • Neoandrographolide is one of the main active ingredients in Andrographis paniculata extract [1] , and its structural formula is shown in formula I:
  • neoandrographolide exhibits different degrees of anti-inflammatory [1] , antiviral, immune stimulation, heat-clearing and detoxifying, swelling and pain-relieving and anti-cancer effects in vitro and in vivo.
  • anti-inflammatory [1] antiviral, immune stimulation, heat-clearing and detoxifying, swelling and pain-relieving and anti-cancer effects in vitro and in vivo.
  • neoandrographolide has a certain inhibitory effect on colorectal cancer [2] , oral squamous cell carcinoma [3] , cholangiocarcinoma [4] , and breast cancer. But so far, there have been no reports on new andrographolide target proteins.
  • RAB5A protein is associated with tumors, Alzheimer's disease, polycystic ovary syndrome, etc.
  • the RAB5A [5] protein is a member of the Rab GTPase family and is an important small GTPase.
  • the RAB5A protein circulates in the cytoplasm mainly in two structural forms, the activated form bound to GTP, and the one bound to GDP. inactive form. Among them, GDP interacts with Rab5-GTP protein to hydrolyze the GTP carried by it into GDP, thereby inactivating RAB5A protein. Conversely, GTP can interact with Rab5-GDP protein, thereby activating RAB5A protein to generate Rab5-GTP to promote tumor growth.
  • RAB5A has not yet reported a specific targeted small molecule drug. Therefore, it is necessary to develop inhibitors of RAB5A protein that can exert both in vitro and in vivo effects.
  • the purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide the application of neoandrographolide in the preparation of RAB5A protein inhibitors.
  • the amino acid sequence of the RAB5A protein is shown in SEQ ID NO.1.
  • the gene sequence encoding the RAB5A protein is shown in SEQ ID NO.2.
  • the neoandrographolide can directly combine with RAB5A protein to inhibit the activation of RAB5A protein.
  • the effective concentration of the neoandrographolide is 0-400 ⁇ mol/L (excluding 0); preferably, it is 12.5-400 ⁇ mol/L.
  • neoandrographolide as a RAB5A protein inhibitor (the application is for non-disease treatment purposes, such as application in an in vitro environment).
  • the RAB5A protein inhibitor includes a therapeutically effective amount of neoandrographolide and a pharmaceutically acceptable carrier.
  • the RAB5A protein inhibitor can be made into various forms of oral or injectable preparations, including capsules, tablets, granules, powders, pills, drop pills, sustained and controlled release preparations, oral liquids, mixtures, syrups, liquid injections , powder for injection and tablet for injection, etc.
  • the present invention has the following advantages and effects:
  • RAB5A protein is associated with some diseases, such as tumor, Alzheimer's disease, polycystic ovary syndrome, etc.
  • RAB5A protein is highly expressed in lung adenocarcinoma.
  • neoandrographolide can directly bind to RAB5A protein, inhibit the activation of RAB5A protein, thereby inhibiting tumor growth and proliferation, and can be used for tumor drug resistance caused by high RAB5A expression. treat.
  • neoandrographolide can bind to RAB5A protein through hydrogen bonds, C-H bonds, and Pi-Alkyl hydrophobic interactions. Therefore, neoandrographolide can be used as a RAB5A protein inhibitor, which can be used to solve the problem caused by the activation of RAB5A protein. the problem of promoting tumor growth.
  • the present invention also finds that with the increase of the concentration of neoandrographolide, the proliferation ability of tumor cells is weakened, which provides an effective way for subsequent research on RAB5A protein inhibitors or anti-tumor drugs.
  • Neoandrographolide a natural traditional Chinese medicine extract, as an inhibitor of RAB5A protein to inhibit the growth of tumor cells.
  • the point has been clearly studied; 2 Neoandrographolide exists in traditional Chinese herbal medicine, and is easy to extract and prepare, with high safety and low price, and has a good prospect of development and utilization.
  • Figure 1 is the interaction diagram between neoandrographolide and RAB5A protein; wherein, A is the titration fluorescence quenching curve; B is the fluorescence quenching degree accumulation curve.
  • Fig. 2 is a graph showing the direct interaction between neoandrographolide and RAB5A protein (Pull-down experiment result graph).
  • Figure 3 is a graph showing the experimental results of neoandrographolide's growth inhibitory effect on A549 cells.
  • Figure 4 is a graph showing the results of the clone formation experiment of neoandrographolide on the growth and proliferation of lung adenocarcinoma cells.
  • Figure 5 is a schematic diagram of the binding mode of neoandrographolide and RAB5A protein simulated by molecular docking; wherein, A and B are schematic diagrams of molecular docking; C is a predicted map of the binding site of neoandrographolide and RAB5A protein.
  • the present invention will be described in further detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the test methods that do not specify specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions suggested by the manufacturer.
  • the reagents and raw materials used in the present invention can be obtained commercially.
  • RAB5A protein sequence involved in the present invention is shown in SEQ ID NO.1, and the gene sequence encoding the RAB5A protein is shown in SEQ ID NO.2:
  • RAB5A protein sequence (SEQ ID NO.1):
  • RAB5A gene sequence (SEQ ID NO.2):
  • RAB5A protein was obtained by corresponding immunoblotting analysis and staining analysis.
  • GST-Rab5A binding domain(R5BD) pull down assay can be used to detect the direct interaction between neoandrographolide and RAB5A protein, specifically:
  • A549 cells were plated into 96-well plates, 3000 cells per well; andrographolide was dissolved in DMSO (dimethyl sulfoxide) to prepare a stock solution with a concentration of 10 mM, and then cultured with DMEM cells Base (purchased from Life Technologies, Gaithersburg, MD, USA) diluted stock solutions to prepare working solutions of different concentrations; after step A549 cells adhered, add different concentrations (12.5, 25, 50, 100, 12.5, 25, 50, 100, 200, 400 ⁇ M) working solution was added to the corresponding well, and an equal amount of DMSO was added as a blank control. After the working solution was treated for 48 hours, the WST-1 cell proliferation and cytotoxicity detection kit (purchased from Biyuntian Biotechnology Co., Ltd.) was used. Cell viability was detected. The above three experimental steps were repeated three times.
  • DMSO dimethyl sulfoxide
  • neoandrographolide as an inhibitor of RAB5A protein. It indicates that neoandrographolide is an effective lung cancer treatment by inhibiting RAB5A enzyme activity, and has broad application value in the clinical treatment of lung cancer.

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Abstract

一种新穿心莲内酯在制备RAB5A蛋白抑制剂中的应用。通过实验发现，RAB5A蛋白在肺腺癌中高表达，而新穿心莲内酯通过氢键、C-H键、Pi-Alkyl疏水作用直接与RAB5A蛋白结合，抑制RAB5A蛋白激活，进而抑制肿瘤生长和增殖，因此，新穿心莲内酯可用于RAB5A高表达所引起的肿瘤药物耐受方面的治疗，且还发现随着新穿心莲内酯浓度升高，肿瘤细胞增值能力减弱，为后续RAB5A蛋白抑制剂方面的研究提供有效途径。

Description

新穿心莲内酯在制备RAB5A蛋白抑制剂中的应用 技术领域
本发明属于医药技术领域，特别涉及新穿心莲内酯在制备RAB5A蛋白抑制剂中的应用。
背景技术
肺癌是世界上最难治疗的疾病之一。癌症的恶化常常伴随着化疗药物，癌症一旦发生转移，将难以治疗。现在主流的治疗方法是手术加放化疗结合的方法，在靶向治疗领域，应用比较多的有血管生成因子(VEGF)抑制剂贝伐单抗和表皮生长因子受体阻滞剂帕尼单抗、西妥昔单抗和艾洛替尼。尽管如此，临床疗效仍不理想，化疗耐药和毒副作用的发生影响了癌症病人的预后。从天然中草药中筛选抗癌药物已被证实是一种切实可行的思路，紫杉醇、长春新碱等研究已取得显著成绩。自然产物类的抗癌制剂，一般具有毒副作用小、价格低廉的优点。
新穿心莲内酯(Neoandrographolide)是穿心莲提取物中主要的活性成分之一 [1]，其结构式如式I所示：
Figure PCTCN2021121946-appb-000001
早先有相关报道新穿心莲内酯在体外和体内表现出不同程度的抗炎 [1]、抗病毒、免疫刺激、清热解毒、消肿止痛以及抗癌等功效。在肿瘤相关的研究领域中，新穿心莲内酯对结直肠癌 [2]、口腔鳞状细胞癌 [3]、胆内胆管癌 [4]、乳腺癌等都有一定的抑制效果。但到目前为止，尚未有新穿心莲内酯靶标蛋白的相关报道。
RAB5A蛋白与肿瘤、阿尔兹海默症、多囊性卵巢綜合症等有关系。RAB5A [5]蛋白是Rab GTP酶家族成员，是一种重要的小GTP酶，RAB5A蛋白在胞质内 的循环主要有两种结构形式，分别是与GTP结合的活化形式，以及与GDP结合的失活形式。其中，GDP通过与Rab5-GTP蛋白相互作用，将其携带的GTP水解成为GDP，从而使RAB5A蛋白失活。相反，GTP则能够与Rab5-GDP蛋白相互作用，从而使RAB5A蛋白激活，生成Rab5-GTP促进肿瘤生长。近几年，人们发现很多参与调控RAB5蛋白GTP/GDP循环的因子。该蛋白的活性的激活，能够直接影响下游的Ras-MAPK通路的激活，从而促进肿瘤发生，因此相比药物开发难度较大的Ras-MAPK通路而言，靶向RAB5蛋白能够为肿瘤治疗提供一个新的契机。目前，RAB5A仍未报导有特异性的靶向小分子药物。因此，开发在体内外均能发挥效应的RAB5A蛋白的抑制剂，是很有必要的。
发明内容
本发明的目的在于克服现有技术的缺点与不足，提供新穿心莲内酯在制备RAB5A蛋白抑制剂中的应用。
本发明的目的通过下述技术方案实现：
新穿心莲内酯在制备RAB5A蛋白抑制剂中的应用。
所述的RAB5A蛋白的氨基酸序列如SEQ ID NO.1所示。
编码所述RAB5A蛋白的基因序列如SEQ ID NO.2所示。
所述的新穿心莲内酯可直接与RAB5A蛋白结合，抑制RAB5A蛋白激活。
所述的新穿心莲内酯的有效浓度为0～400μmol/L(不包括0)；优选为12.5～400μmol/L。
新穿心莲内酯作为RAB5A蛋白抑制剂的应用(所述的应用为非疾病治疗目的，如体外环境下进行应用)。
所述的RAB5A蛋白抑制剂包括治疗有效量的新穿心莲内酯和药学上可接受的载体。
所述RAB5A蛋白抑制剂可制成各种形式的口服或注射制剂，包括胶囊剂、片剂、颗粒剂、散剂、丸剂、滴丸剂、缓控释制剂、口服液、合剂、糖浆剂、液体注射剂、注射用粉剂和注射用片剂等。
本发明相对于现有技术具有如下的优点及效果：
(1)RAB5A蛋白与一些疾病相关，如肿瘤、阿尔兹海默症、多囊性卵巢綜合症等。RAB5A蛋白在肺腺癌中高表达，本发明中发现新穿心莲内酯可直接与RAB5A蛋白结合，抑制RAB5A蛋白激活，进而抑制肿瘤生长和增殖，可用于RAB5A高表达所引起的肿瘤药物耐受方面的治疗。
(2)本发明中发现新穿心莲内酯可以通过氢键、C-H键、Pi-Alkyl疏水作用与RAB5A蛋白结合，因此，新穿心莲内酯可作为RAB5A蛋白抑制剂，可用于解决RAB5A蛋白激活所引起的促进肿瘤生长的问题。另外，本发明还发现随着新穿心莲内酯浓度升高，肿瘤细胞增值能力减弱，为后续RAB5A蛋白抑制剂或抗肿瘤药物方面的研究提供有效途径。
(3)本发明利用天然中药提取物新穿心莲内酯作为RAB5A蛋白的抑制剂，抑制肿瘤细胞生长，主要具备如下优点：①新穿心莲内酯直接作用于RAB5A蛋白抑制该蛋白活性，且相互作用位点已经研究清楚；②新穿心莲内酯存在于传统中草药中，提取制备方便，安全性高，价格低廉，开发利用的前景好。
附图说明
图1是新穿心莲内酯与RAB5A蛋白之间的相互作用图；其中，A为滴定荧光淬灭曲线；B为荧光淬灭程度累积曲线。
图2是新穿心莲内酯与RAB5A蛋白存在直接相互作用图(Pull-down实验结果图)。
图3是新穿心莲内酯对A549细胞的生长抑制作用实验结果图。
图4是新穿心莲内酯对肺腺癌细胞生长增殖的克隆形成实验结果图。
图5是分子对接模拟新穿心莲内酯与RAB5A蛋白的结合模式图；其中，A、B为分子对接示意图；C为新穿心莲内酯与RAB5A蛋白结合位点预测图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明，本发明所用试剂和原材料均可通过市售获得。
本发明中涉及到RAB5A蛋白序列如SEQ ID NO.1所示，编码RAB5A蛋白的基因序列，如SEQ ID NO.2所示：
RAB5A蛋白序列(SEQ ID NO.1)：
Figure PCTCN2021121946-appb-000002
Figure PCTCN2021121946-appb-000003
RAB5A基因序列(SEQ ID NO.2)：
Figure PCTCN2021121946-appb-000004
实施例1新穿心莲内酯与RAB5A蛋白结合抑制肿瘤细胞生长
1、首先确定新穿心莲内酯与RAB5A蛋白是否结合，我们采用荧光滴定实验，Pull-down实验来分析：
(1)荧光滴定实验
我们纯化出RAB5A蛋白，具体过程为：以A549细胞(购自购于美国标准生物品收藏中心细胞库)的RNA逆转录而成的cDNA为模板，进行PCR扩增(扩增引物见表1)，获得RAB5A基因，然后将其构建至pGEX-4T-1表达载体(Addgene)中，并将构建好的载体(RAB5A-WT载体)转化于BL-21表达菌中，用IPTG(异丙基-β-D-硫代半乳糖苷)以终浓度为0.5mM的条件诱导表达4～6h后收集菌体。采用GE公司的GST亲和层析纯化柱对重组蛋白进行纯化，采用GE公司的Thrombin酶切除GST标签。通过相应的免疫印迹分析及考染分析，获得RAB5A蛋白。
获得重组的RAB5A蛋白后，采用荧光滴定实验 [6]实验手段[参考文献：L.Zhang et al.,Crucial residue Trp158 of lipoprotein PiaA stabilizes the ferrichrome-PiaA complex in Streptococcus pneumoniae.Journal of inorganic  biochemistry 167,150(Feb,2017)]进行分析，结果如图1所示，表明新穿心莲内酯与RAB5A蛋白存在直接相互作用，即新穿心莲内酯可与RAB5A蛋白结合。
表1.RAB5A质粒的引物序列
Figure PCTCN2021121946-appb-000005
(2)Pull-down实验
GST-Rab5A binding domain(R5BD)pull down实验可以用来检测新穿心莲内酯与RAB5A蛋白直接相互作用，具体为：
1)将A549细胞株用终浓度为0、50、100μM的新穿心莲内酯处理24h后，用Western及IP细胞裂解液(碧云天，Cat#P0013)按照1ml/皿的量加入到培养皿中，冰上裂解15min后，12000g离心30min，取上清进行蛋白浓度测定。
2)采用GE公司的GST亲和层析纯化柱纯化GST-R5BD珠子，具体步骤如下：将GST亲和层析树脂(GE；Glutathione Sepharose 4B；17-0756-01)混匀后，取1mL上柱，室温静置30min；用10倍柱体积dd H2O清洗柱子；用10倍柱体积的Binding buffer(1×PBS缓冲液，pH 7.4)平衡柱子；将收集的上清样液缓缓装入层析柱中(重复挂柱一次)；用10倍柱体积的Binding buffer(1×PBS缓冲液，pH 7.4)洗下杂蛋白得到GST-R5BD珠子。
3)取1mg不同处理组的蛋白裂解液与纯化好的GST-R5BD珠子进行共孵育4℃过夜，用PBS缓冲液进行洗涤3次后，取等体积的产物进行WB检测(Western Blot蛋白质印迹法)，用anti-Rab5抗体(proteintech,Cat#11947-1-AP)检测。
采取Pull-down实验手段分析新穿心莲内酯与RAB5A蛋白的直接相互作用，结果如图2所示。
2、细胞增殖及细胞毒性实验
(1)分别将A549细胞铺入96孔板内，每孔3000个细胞；将穿心莲内酯溶于DMSO(二甲基亚砜)中，配制成浓度为10mM的储备液，再用DMEM细胞培养基(购自于Life Technologies,Gaithersburg,MD,USA)稀释储备液配制成不同浓度的工作液；待步骤A549细胞贴壁后，按每孔100μL体积加入不同浓度(12.5、25、50、100、200、400μM)的工作液到对应孔中，以加入等量DMSO作为空白对照，工作液处理48小时后，用WST-1细胞增殖及细胞毒性检测试剂 盒(购自碧云天生物技术有限公司)检测细胞活性。以上3个实验步骤进行3次生物学重复。
结果如图3所示：结果表明随着新穿心莲内酯的浓度升高，细胞的生存能力降低，新穿心莲内酯浓度越大，细胞生存能力越弱，即新穿心莲内酯可以抑制肺腺癌细胞生长。
(2)另外，我们还采用了克隆形成实验评估不同浓度新穿心莲内酯对细胞的增值能力。向6孔板中加2ml DMEM培养基，取2000个A549细胞于6孔板中，加入不同浓度(25、50、100μM)的新穿心莲内酯工作液(配制方法同上)，培养箱培养2周，期间换液两次。克隆形成检测加入一定量甲醇(覆盖细胞表面)，固定10min，吸出甲醇，加入结晶紫染色5min，用自来水冲洗干净后，倒扣6孔板，晾干，扫描。
结果如图4所示，表明随着新穿心莲内酯浓度升高，细胞的增值能力减弱。
3、为了进一步研究新穿心莲内酯与RAB5A蛋白的结合模式，使用Discovery Studio软件对新穿心莲内酯和RAB5A的相互作用情况进行计算模拟，并得出潜在作用位点。结果如图5所示，预测新穿心莲内酯可以通过氢键、C-H键、Pi-Alkyl疏水作用与RAB5A蛋白结合，预测结合位点如图5所示。
以上结果确定了新穿心莲内酯可作为RAB5A蛋白的抑制剂。预示着新穿心莲内酯通过抑制RAB5A酶活是一个有效的肺癌治疗手段，在肺癌临床治疗方面具有广阔的应用价值。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
参考文献
[1]Xiao Xia Cong,Xiu Kui Gao,Xi Sheng Rao,Jie Wen,Xiao Ceng Liu,Yin Pu Shi,Min Yi He,Wei Liang Shen,Yue Shen,Hongwei Ouyang,Ping Hu,Boon Chuan Low,Zhuo Xian Meng,Yue Hai Ke,Ming Zhu Zheng,Lin Rong Lu,Yong Heng Liang,Li Ling Zheng,Yi Ting Zhou.Rab5a activates IRS1to coordinate IGF-AKT-mTOR signaling and myoblast differentiation during muscle regeneration[J].Cell Death&amp；Differentiation:Official journal of the ADMC Associazione Differenziamento e Morte Cellulare,2020,27(8).
[2]Sharma Venu,Qayum Arem,Kaul Sanjana,Singh Ajeet,Kapoor Kamal K,Mukherjee Debaraj,Singh Shashank  K,Dhar Manoj K. Carbohydrate Modifications of Neoandrographolide for Improved Reactive Oxygen Species-Mediated Apoptosis through Mitochondrial Pathway in Colon Cancer.[J]. ACS omega,2019,4(24).
[3]Liang Zhang,Nan Li,Kun Cao,Xiao-Yan Yang,Guandi Zeng,Xuesong Sun,Qing-Yu He. Crucial residue Trp158 of lipoprotein PiaA stabilizes the ferrichrome-PiaA complex in Streptococcus pneumoniae[J]. Journal of Inorganic Biochemistry,2017,167.
[4]Suzuki Ryuichiro,Matsushima Yasuaki,Okudaira Noriyuki,Sakagami Hiroshi,Shirataki Yoshiaki. Cytotoxic Components Against Human Oral Squamous Cell Carcinoma Isolated from Andrographis paniculata.[J].Anticancer research,2016,36(11).
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Claims (7)

  1. 新穿心莲内酯在制备RAB5A蛋白抑制剂中的应用。
  2. 根据权利要求1所述的应用，其特征在于：
    所述的新穿心莲内酯直接与RAB5A蛋白结合，抑制RAB5A蛋白激活。
  3. 根据权利要求1或2所述的应用，其特征在于：
    所述的RAB5A蛋白的氨基酸序列如SEQ ID NO.1所示。
  4. 根据权利要求3所述的应用，其特征在于：
    编码所述RAB5A蛋白的基因序列如SEQ ID NO.2所示。
  5. 根据权利要求1或2所述的应用，其特征在于：
    所述的新穿心莲内酯的有效浓度为0～400μmol/L，不包括0。
  6. 根据权利要求5所述的应用，其特征在于：
    所述的新穿心莲内酯的有效浓度为12.5～400μmol/L。
  7. 新穿心莲内酯作为RAB5A蛋白抑制剂的应用，其特征在于：所述的应用的环境为体外环境。
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