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WO2023168771A1 - Mtor inhibitor and use thereof - Google Patents

Mtor inhibitor and use thereof Download PDF

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WO2023168771A1
WO2023168771A1 PCT/CN2022/084001 CN2022084001W WO2023168771A1 WO 2023168771 A1 WO2023168771 A1 WO 2023168771A1 CN 2022084001 W CN2022084001 W CN 2022084001W WO 2023168771 A1 WO2023168771 A1 WO 2023168771A1
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protein
rheb
mtor
atp6ap1
mtor inhibitor
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松阳洲
冯然
刘峰
吴苏
周志芬
李若菲
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Sun Yat Sen University
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to an mTOR inhibitor and its application.
  • mTOR (mechanistic mammalian target of rapamycin) is a type of protein kinase involved in intracellular signal transmission in mammals and other animals. It plays an important role in protein translation, autophagy, immune regulation, cytoskeleton regulation, gene transcription, etc. Closely related to basic life phenomena such as cell growth, division and death. mTOR is a protein discovered as an intracellular target of rapamycin that exhibits potent immunosuppressive and anti-cancer effects, and is a giant serine-threonine kinase with a molecular weight of 290,000.
  • mTOR inhibitors have been extensively studied, but as chemotherapy drugs, their application has different shortcomings.
  • the development of mTOR inhibitors can be roughly divided into three stages.
  • the first is rapamycin, which was first discovered, but because they only partially block the 4E-BP-dependent translation pathway and cannot inhibit the mTORC2–Akt-regulated pro-survival pathway, At the same time, it is only effective against renal cancer cells, so the clinical treatment effect is not ideal.
  • the second generation drugs (Torin1, PP242, Ku-0063794) mainly rely on competing with ATP to occupy the kinase active site to inhibit mTORC1 and mTORC2 substrates.
  • early clinical data indicate the emergence of resistance to catalytic mTOR inhibitors.
  • the third generation of drugs mainly relies on combined drugs and uses dual inhibition of PI3K and mTOR to work.
  • drug strategies that use proteins to inhibit mTOR to treat tumors have not yet been developed.
  • the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide an mTOR inhibitor and its application.
  • the present invention uses a new proximity labeling technology screening method to find the C-terminal fragment (C tail) of ATP6AP1 protein as Rheb. Guanylate exchange factor (GEF) can activate Rheb. On this basis, amino acids at positions 14, 17, and 18 of the 30 amino acids at the C-terminus are mutated. The mutants can bind to and block the effect of endogenous ATP6AP1 on Rheb. inhibition, thereby inhibiting the mTORC1 signaling pathway.
  • C tail C-terminal fragment
  • GEF Guanylate exchange factor
  • the present invention provides an mTOR inhibitor, and the mTOR inhibitor is:
  • the mTOR inhibitor is a protein with at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared to an ATP6AP1 protein mutant, and the C-terminus of the ATP6AP1 protein mutant has an amino acid sequence identity that is greater than that of ATP6AP1
  • the aspartic acids at positions 14, 17, and 18 of the protein were all mutated to alanine.
  • the mTOR inhibitor is the ATP6AP1 protein mutant or the polypeptide.
  • the ATP6AP1 protein mutant was obtained by mutating aspartic acid at positions 14, 17, and 18 in the C-terminal 30 amino acids of the ATP6AP1 protein to alanine.
  • the sequence of the 30 amino acids is as SEQ ID NO. :1 shown.
  • the present invention found the ATP6AP1 protein through a new proximity labeling technology screening method, mutated the 14th, 17th, and 18th aspartic acid in the 30 C-terminal amino acids to alanine, and obtained a short peptide that inhibits Rheb-mTORC1 (mTOR inhibitor, SEQ ID NO:2), inhibits the mTORC1 signaling pathway.
  • mTOR inhibitor SEQ ID NO:2
  • Rheb is unique to mTORC1.
  • Traditional inhibitors that directly target mTOR can simultaneously inhibit mTORC1 and mTORC2 downstream signals, but the mTOR inhibitor obtained in the present invention does not affect mTORC2 downstream signals.
  • the mTOR inhibitor is an mTORCl inhibitor.
  • the second purpose of the present invention is to provide the application of the above-mentioned ATP6AP1 protein mutant in inhibiting the activity of the mTOR signaling pathway.
  • the mTOR signaling pathway includes the mTORC1 signaling pathway and the mTORC2 signaling pathway.
  • the present invention provides a method for screening mTOR inhibitors, including the following steps:
  • the guanine nucleotide exchange factor screening conditions include the following conditions:
  • GEF renal nucleotide exchange factor
  • the present invention obtains Rheb protein activator-ATP6AP1 protein through novel proximity labeling technology screening.
  • the C-terminal 30 amino acids of ATP6AP1 protein can directly target and activate Rheb.
  • the concentration of insulin is 0.9 ⁇ M, and the concentration of biotin is 50 ⁇ M.
  • the growing cell lines can function better.
  • the stimulation time is 15 minutes.
  • GEF protein can be captured more accurately.
  • the biotin ligase includes biotin ligase xxID, and the fusion expression vector of the biotin ligase and Rheb is HAFlag-xxID-Rheb.
  • the biotin ligase mentioned in the present invention is not limited to the present invention, and can also be a biotin ligase commonly used in this field.
  • the cell line stably expressing Rheb protein is a HeLa-xxID-Rheb stably expressing cell line
  • the control cell line is a HeLa-xxID stably expressing cell line.
  • the host cells include HeLa cells, and may also be other cells.
  • the fourth object of the present invention is to provide a tumor suppressor drug, which includes the above-mentioned mTOR inhibitor and a medically acceptable carrier.
  • the fifth object of the present invention is to provide an anti-aging drug, including the above-mentioned mTOR inhibitor, and a medically acceptable carrier.
  • the sixth object of the present invention is to provide the application of the above-mentioned mTOR inhibitor in preparing drugs/reagents that increase the phosphorylation level of substances in the mTOR signaling pathway, wherein the substances in the mTOR signaling pathway are substrate S6K and its downstream S6.
  • the phosphorylation level of substances in the mTOR signaling pathway is reduced.
  • the seventh object of the present invention is to provide the use of the above-mentioned mTOR inhibitor in the preparation of drugs for preventing and/or treating cancer and anti-aging by inhibiting the activity of the mTORC1 signaling pathway.
  • the cancer is breast cancer or pancreatic cancer.
  • the present invention has the following beneficial effects:
  • the present invention provides an mTOR inhibitor and its application.
  • the C-terminal fragment (C tail) of the ATP6AP1 protein is found as a guanylate exchange factor (GEF) of Rheb and can activate Rheb.
  • GEF guanylate exchange factor
  • aspartic acid at positions 14, 17, and 18 of the 30 amino acids at the C-terminus were all mutated to alanine to obtain a new mTOR inhibitor that can bind to and block the effect of endogenous ATP6AP1 on Rheb Inhibition, thereby inhibiting the mTORC1 signaling pathway.
  • the mTOR inhibitor obtained in the present invention has higher specificity and can exert its inhibitory effect in a more targeted manner.
  • the mTOR inhibitor obtained by the present invention has broad medicinal prospects in treating diabetes and tumors.
  • Figure 1 shows the construction process diagram and mass spectrometry detection chart of HeLa-xxID-Rheb stable expression cell line and HeLa-xxID stable expression cell line;
  • Figure 2 is a fold analysis chart of the Come/GO/Stay group in Example 1;
  • Figure 3 is a schematic diagram of overexpression of mTOR inhibitor inhibiting the activity of mTOR signaling pathway
  • Figure 4 is a schematic diagram showing how the overexpressed mTOR inhibitor inhibits the activity of the mTOR signaling pathway.
  • Example 1 A method for screening mTOR inhibitors
  • a method for screening mTOR inhibitors includes the following steps:
  • the enriched proteins in the HeLa-xxID-Rheb stable expression cell line were subtracted from the enriched proteins in the HeLa-xxID stable expression cell line to obtain the conditions with/without insulin stimulation.
  • Rheb neighboring proteins further analysis of this part of the enriched proteins (Rheb neighboring proteins) can be divided into Come/GO/Stay groups, that is, the protein group that appears after stimulation/the stable existence group before and after stimulation/the protein group that leaves after stimulation (such as As shown in Figure 2), the Come group includes ATP6AP1 protein, REEPS protein, and SC22B protein.
  • the GO group includes VAMP3 protein and SCD protein.
  • the Stay group includes RAB7A protein and TSC1 protein. Since GEF protein should appear after insulin stimulation, the Come group is The protein was validated as a GEF candidate protein.
  • GEF renal nucleotide exchange factor
  • the ATP6AP1 protein (C-terminal 30 amino acids, TYGLHMILSLKTMDRFDDHKGPTISLTQIV) can well meet the above conditions, proving that the ATP6AP1 protein is a potential GEF for Rheb.
  • overexpressing ATP6AP1 (2 ⁇ g) in the HeLa cell line; C-terminal (2 ⁇ g) can significantly increase the activity of the mTOR signaling pathway; complementing the mTOR inhibitor formed by the C-terminal 3D/A (8 ⁇ g) mutation (in the 30 amino acids of the C-terminal Aspartic acids at positions 14, 17, and 18 were all mutated to alanine), which could not rescue the decrease in phosphorylation caused by knockdown of ATP6AP1, significantly reducing the ability of the C terminus to perform GEF functions (refer to Figure 4 ).
  • the present invention uses a new proximity labeling technology screening method to find that the C-terminal fragment (C tail) of ATP6AP1 can activate Rheb as a guanylate exchange factor (GEF) of Rheb.
  • C tail C-terminal fragment
  • GEF guanylate exchange factor
  • aspartic acid at positions 14, 17, and 18 of the 30 amino acids at the C-terminus were mutated to alanine to obtain a new mTOR inhibitor that can bind to and block endogenous ATP6AP1 inhibits Rheb, thereby inhibiting the mTORC1 signaling pathway. This is the first short peptide discovered to inhibit Rheb-mTORC1.
  • mTOR inhibitors Since both mTORC1 and mTORC2 complexes contain mTOR proteins, and Rheb is unique to mTORC1, traditional inhibitors that directly target mTOR can simultaneously inhibit mTORC1 and mTORC2 downstream signals, while short peptides that inhibit Rheb-mTORC1 do not affect mTORC2 downstream.
  • the mTOR inhibitor obtained in the present invention has higher specificity and can exert inhibitory effects in a more targeted manner.
  • mTOR signaling pathway inhibitors have broad medicinal prospects in the treatment of diabetes and tumors.

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Abstract

The present invention relates to the field of biotechnology. Provided are an mTOR inhibitor and the use thereof. The mTOR inhibitor is obtained by means of mutating aspartic acids at positions 14, 17 and 18 in 30 amino acids at the C terminal of an ATP6AP1 protein into alanines, and the sequence of the 30 amino acids is as shown in SEQ ID NO: 1. By means of a new proximity labeling and screening method, a C-terminal fragment (C tail) of an ATP6AP1 protein is found as a guanylate exchange factor (GEF) of Rheb which can activate Rheb. On this basis, aspartic acids at positions 14, 17 and 18 in 30 amino acids at the C-terminal are all mutated into alanines, so as to obtain a new mTOR inhibitor. The mTOR inhibitor can bind to and block the inhibition of Rheb by means of endogenous ATP6AP1, thereby inhibiting the mTORC1 signaling pathway.

Description

一种mTOR抑制剂及其应用An mTOR inhibitor and its application 技术领域Technical field

本发明涉及生物技术领域，尤其是涉及一种mTOR抑制剂及其应用。The present invention relates to the field of biotechnology, and in particular to an mTOR inhibitor and its application.

背景技术Background technique

mTOR(mechanistic mammalian target of rapamycin)，是哺乳类等动物的参与细胞内信号传递的蛋白激酶的一种，其在蛋白质转译、细胞自噬、免疫调节、细胞骨格调节、基因转录等发挥重要作用，与细胞成长、分裂及死亡等基本生命现象密切相关。mTOR，已知是作为表现出强效免疫抑制作用及抗癌作用的雷帕霉素的细胞内标靶而被发现的蛋白质，且是分子量29万的巨大丝氨酸-苏氨酸激酶。mTOR (mechanistic mammalian target of rapamycin) is a type of protein kinase involved in intracellular signal transmission in mammals and other animals. It plays an important role in protein translation, autophagy, immune regulation, cytoskeleton regulation, gene transcription, etc. Closely related to basic life phenomena such as cell growth, division and death. mTOR is a protein discovered as an intracellular target of rapamycin that exhibits potent immunosuppressive and anti-cancer effects, and is a giant serine-threonine kinase with a molecular weight of 290,000.

目前mTOR抑制剂已有广泛研究，但作为化疗药物，其适用均存在不同的缺陷。mTOR抑制剂的发展大致可分为三种阶段，首先是最早发现的雷帕霉素，但因为它们仅部分阻断4E-BP依赖性翻译途径，并且不能抑制mTORC2–Akt调节的促生存途径，同时仅对于肾癌细胞有疗效，因此临床上治疗效果并不理想。第二代药物(Torin1，PP242,Ku-0063794)主要依靠与ATP竞争占据激酶活性位点抑制mTORC1和mTORC2底物发挥作用。但早期临床数据表明催化mTOR抑制剂会出现耐药性。第三代药物主要是依靠联合用药，使用PI3K和mTOR双重抑制发挥作用。而利用蛋白抑制mTOR，进而治疗肿瘤的用药策略至今尚未被开发。At present, mTOR inhibitors have been extensively studied, but as chemotherapy drugs, their application has different shortcomings. The development of mTOR inhibitors can be roughly divided into three stages. The first is rapamycin, which was first discovered, but because they only partially block the 4E-BP-dependent translation pathway and cannot inhibit the mTORC2–Akt-regulated pro-survival pathway, At the same time, it is only effective against renal cancer cells, so the clinical treatment effect is not ideal. The second generation drugs (Torin1, PP242, Ku-0063794) mainly rely on competing with ATP to occupy the kinase active site to inhibit mTORC1 and mTORC2 substrates. However, early clinical data indicate the emergence of resistance to catalytic mTOR inhibitors. The third generation of drugs mainly relies on combined drugs and uses dual inhibition of PI3K and mTOR to work. However, drug strategies that use proteins to inhibit mTOR to treat tumors have not yet been developed.

发明内容Contents of the invention

本发明的目的在于克服上述现有技术的不足之处而提供一种mTOR抑制剂及其应用，本发明通过新型邻近标记技术筛选方法，找到了ATP6AP1蛋白的C端片段(C tail)作为Rheb的鸟苷酸交换因子(GEF)可激活Rheb，在此基础上，将C末端30个氨基酸中第14、17、18位的氨基酸进行突变，其突变体可结合并阻断内源性ATP6AP1对Rheb的抑制，进而抑制mTORC1信号通路。The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide an mTOR inhibitor and its application. The present invention uses a new proximity labeling technology screening method to find the C-terminal fragment (C tail) of ATP6AP1 protein as Rheb. Guanylate exchange factor (GEF) can activate Rheb. On this basis, amino acids at positions 14, 17, and 18 of the 30 amino acids at the C-terminus are mutated. The mutants can bind to and block the effect of endogenous ATP6AP1 on Rheb. inhibition, thereby inhibiting the mTORC1 signaling pathway.

为实现上述目的，本发明采取的技术方案为：In order to achieve the above objects, the technical solutions adopted by the present invention are:

第一目的，本发明提供了一种mTOR抑制剂，所述mTOR抑制剂为：First object, the present invention provides an mTOR inhibitor, and the mTOR inhibitor is:

1)多肽，所述多肽具有与SEQ ID NO:2所示的氨基酸序列至少9/10、14/15或29/30的序列一致性；或1) A polypeptide having a sequence identity of at least 9/10, 14/15 or 29/30 with the amino acid sequence shown in SEQ ID NO: 2; or

2)C端为所述多肽的蛋白质。2) A protein whose C-terminus is the polypeptide.

优选地，所述mTOR抑制剂为相比ATP6AP1蛋白突变体具有至少95％、96％、97％、98％或99％氨基酸序列一致性的蛋白质，所述ATP6AP1蛋白突变体的C末端相比ATP6AP1蛋白在第14、17、18位的天冬氨酸均突变为丙氨酸。Preferably, the mTOR inhibitor is a protein with at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared to an ATP6AP1 protein mutant, and the C-terminus of the ATP6AP1 protein mutant has an amino acid sequence identity that is greater than that of ATP6AP1 The aspartic acids at positions 14, 17, and 18 of the protein were all mutated to alanine.

更优选地，所述mTOR抑制剂为所述ATP6AP1蛋白突变体或所述多肽。More preferably, the mTOR inhibitor is the ATP6AP1 protein mutant or the polypeptide.

需要说明的是，ATP6AP1蛋白的C末端30个氨基酸中的第14、17、18位的天冬氨酸均突变为丙氨酸获得所述ATP6AP1蛋白突变体，30个氨基酸的序列如SEQ ID NO:1所示。It should be noted that the ATP6AP1 protein mutant was obtained by mutating aspartic acid at positions 14, 17, and 18 in the C-terminal 30 amino acids of the ATP6AP1 protein to alanine. The sequence of the 30 amino acids is as SEQ ID NO. :1 shown.

本发明通过新型邻近标记技术筛选方法，找到了ATP6AP1蛋白，将C末端30个氨基酸中的第14、17、18位的天冬氨酸均突变为丙氨酸，获得抑制Rheb-mTORC1的短肽(mTOR抑制剂，SEQ ID NO:2)，抑制mTORC1信号通路。The present invention found the ATP6AP1 protein through a new proximity labeling technology screening method, mutated the 14th, 17th, and 18th aspartic acid in the 30 C-terminal amino acids to alanine, and obtained a short peptide that inhibits Rheb-mTORC1 (mTOR inhibitor, SEQ ID NO:2), inhibits the mTORC1 signaling pathway.

由于mTORC1、mTORC2复合物均含有mTOR蛋白，而Rheb为mTORC1特有。传统直接靶向mTOR的抑制剂，能同时抑制mTORC1、mTORC2下游信号，而本发明获得的mTOR抑制剂并不影响mTORC2下游。Since both mTORC1 and mTORC2 complexes contain mTOR protein, Rheb is unique to mTORC1. Traditional inhibitors that directly target mTOR can simultaneously inhibit mTORC1 and mTORC2 downstream signals, but the mTOR inhibitor obtained in the present invention does not affect mTORC2 downstream signals.

更优选地，mTOR抑制剂为mTORC1抑制剂。More preferably, the mTOR inhibitor is an mTORCl inhibitor.

第二目的，本发明提供了上述ATP6AP1蛋白突变体在抑制mTOR信号通路活性中的应用。The second purpose of the present invention is to provide the application of the above-mentioned ATP6AP1 protein mutant in inhibiting the activity of the mTOR signaling pathway.

作为本发明所述ATP6AP1蛋白突变体在抑制mTOR信号通路活性中的应用的优选实施方式，所述mTOR信号通路包括mTORC1信号通路和mTORC2信号通路。As a preferred embodiment of the application of the ATP6AP1 protein mutant of the present invention in inhibiting the activity of the mTOR signaling pathway, the mTOR signaling pathway includes the mTORC1 signaling pathway and the mTORC2 signaling pathway.

第三目的，本发明提供了一种筛选mTOR抑制剂的方法，包括以下步骤：As a third object, the present invention provides a method for screening mTOR inhibitors, including the following steps:

1)构建生物素连接酶与Rheb融合表达载体，将载体转入宿主细胞内，药物筛选稳定表达Rheb蛋白的细胞株，同时构建对照细胞株；1) Construct a biotin ligase and Rheb fusion expression vector, transfer the vector into host cells, screen cell lines that stably express Rheb protein, and construct control cell lines;

2)向稳定表达Rheb蛋白的细胞株中加入胰岛素和生物素进行刺激，向对照细胞株中加入生物素刺激，然后裂解样品、提取蛋白、富集蛋白，再进行质 谱检测，分析蛋白；2) Add insulin and biotin to the cell line stably expressing Rheb protein for stimulation, add biotin to the control cell line for stimulation, then lyse the sample, extract the protein, enrich the protein, and then perform mass spectrometry detection to analyze the protein;

3)分别将稳定表达Rheb蛋白的细胞株中的富集蛋白和对照细胞株中的富集蛋白之间的重叠蛋白扣除(扣除表示为将两组数据中重叠的蛋白去掉，以达到去除对照组干扰的目的)，获得有/无进行胰岛素刺激条件下的Rheb邻近蛋白，分析Rheb邻近蛋白，经鸟嘌呤核苷酸交换因子筛选条件，得ATP6AP1蛋白；以及3) Subtract the overlapping proteins between the enriched proteins in the cell lines stably expressing Rheb protein and the enriched proteins in the control cell lines respectively (subtraction means removing the overlapping proteins in the two sets of data to achieve the purpose of removing the control group). (Purpose of interference), obtain Rheb neighboring proteins with/without insulin stimulation, analyze Rheb neighboring proteins, and obtain ATP6AP1 protein through guanine nucleotide exchange factor screening conditions; and

4)将ATP6AP1蛋白C末端30个氨基酸中的第14、17、18位的天冬氨酸均突变为丙氨酸，获得mTOR抑制剂。4) Mutation of aspartic acid at positions 14, 17, and 18 in the 30 amino acids at the C-terminus of the ATP6AP1 protein to alanine to obtain mTOR inhibitors.

更优选地，所述鸟嘌呤核苷酸交换因子筛选条件包括以下条件：More preferably, the guanine nucleotide exchange factor screening conditions include the following conditions:

(1)GEF(鸟嘌呤核苷酸交换因子)与Rheb存在体内外相互作用；(1) GEF (guanine nucleotide exchange factor) interacts with Rheb in vivo and in vitro;

(2)过表达GEF后可以激活mTORC1下游信号通路；(2) Overexpression of GEF can activate the mTORC1 downstream signaling pathway;

(3)敲低或敲除GEF后，mTORC1下游信号通路活性受到大幅度抑制；(3) After knocking down or knocking out GEF, the activity of the mTORC1 downstream signaling pathway is greatly inhibited;

(4)体外实验证明GEF可以直接促进Rheb完成GDP-GTP转换过程。(4) In vitro experiments prove that GEF can directly promote Rheb to complete the GDP-GTP conversion process.

本发明通过新型邻近标记技术筛选得到Rheb蛋白激活剂-ATP6AP1蛋白，ATP6AP1蛋白的C末端30个氨基酸可以直接靶向激活Rheb。The present invention obtains Rheb protein activator-ATP6AP1 protein through novel proximity labeling technology screening. The C-terminal 30 amino acids of ATP6AP1 protein can directly target and activate Rheb.

作为本发明所述筛选mTOR抑制剂的方法的优选实施方式，所述胰岛素的浓度为0.9μM，所述生物素的浓度为50μM。As a preferred embodiment of the method for screening mTOR inhibitors of the present invention, the concentration of insulin is 0.9 μM, and the concentration of biotin is 50 μM.

胰岛素、生物素选用上述浓度时，使得生长的细胞株可以较好的发挥功能。When the above concentrations of insulin and biotin are selected, the growing cell lines can function better.

作为本发明所述筛选mTOR抑制剂的方法的优选实施方式，所述刺激的时间为15min。采用上述刺激时间时，可以更准确的捕捉到GEF蛋白。As a preferred embodiment of the method for screening mTOR inhibitors of the present invention, the stimulation time is 15 minutes. When using the above stimulation time, GEF protein can be captured more accurately.

作为本发明所述筛选mTOR抑制剂的方法的优选实施方式，所述生物素连接酶包括生物素连接酶xxID，所述生物素连接酶与Rheb融合表达载体为HAFlag-xxID-Rheb。本发明中提到的生物素连接酶不仅限于本发明，还可以为本领域中常规使用的生物素连接酶。As a preferred embodiment of the method for screening mTOR inhibitors of the present invention, the biotin ligase includes biotin ligase xxID, and the fusion expression vector of the biotin ligase and Rheb is HAFlag-xxID-Rheb. The biotin ligase mentioned in the present invention is not limited to the present invention, and can also be a biotin ligase commonly used in this field.

优选地，所述稳定表达Rheb蛋白的细胞株为HeLa-xxID-Rheb稳定表达细胞株，所述对照细胞株为HeLa-xxID稳定表达细胞株。Preferably, the cell line stably expressing Rheb protein is a HeLa-xxID-Rheb stably expressing cell line, and the control cell line is a HeLa-xxID stably expressing cell line.

更优选地，所述宿主细胞包括HeLa细胞，还可以为其他细胞。More preferably, the host cells include HeLa cells, and may also be other cells.

第四目的，本发明提供了一种抑癌药物，包含上述mTOR抑制剂，以及医学上可接受的载体。The fourth object of the present invention is to provide a tumor suppressor drug, which includes the above-mentioned mTOR inhibitor and a medically acceptable carrier.

第五目的，本发明提供了一种抗衰老药物，包含上述mTOR抑制剂，以及 医学上可接受的载体。The fifth object of the present invention is to provide an anti-aging drug, including the above-mentioned mTOR inhibitor, and a medically acceptable carrier.

第六目的，本发明提供了上述mTOR抑制剂在制备提高mTOR信号通路中物质磷酸化水平的药物/试剂中的应用，其中，所述mTOR信号通路中物质为底物S6K及其下游S6。The sixth object of the present invention is to provide the application of the above-mentioned mTOR inhibitor in preparing drugs/reagents that increase the phosphorylation level of substances in the mTOR signaling pathway, wherein the substances in the mTOR signaling pathway are substrate S6K and its downstream S6.

优选地，由于ATP6AP1蛋白敲低或降低，导致了所述mTOR信号通路中物质磷酸化水平的下降。Preferably, due to knockdown or reduction of ATP6AP1 protein, the phosphorylation level of substances in the mTOR signaling pathway is reduced.

第七目的，本发明提供了上述mTOR抑制剂在制备通过抑制mTORC1信号通路的活性预防和/或治疗癌症、以及抗衰老药物中的应用，所述癌症为乳腺癌或胰腺癌。The seventh object of the present invention is to provide the use of the above-mentioned mTOR inhibitor in the preparation of drugs for preventing and/or treating cancer and anti-aging by inhibiting the activity of the mTORC1 signaling pathway. The cancer is breast cancer or pancreatic cancer.

与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

本发明提供了一种mTOR抑制剂及其应用，利用新型邻近标记技术筛选方法找到了ATP6AP1蛋白的C端片段(C tail)作为Rheb的鸟苷酸交换因子(GEF)可激活Rheb，在此基础上，将C末端30个氨基酸中第14、17、18位的天冬氨酸均突变为丙氨酸，获得新的mTOR抑制剂，该mTOR抑制剂可结合并阻断内源性ATP6AP1对Rheb的抑制，进而抑制mTORC1信号通路，相比于现存的mTOR抑制剂，本发明获得的mTOR抑制剂特异性较高，更能有针对性的发挥抑制剂作用。本发明获得的mTOR抑制剂在治疗糖尿病、肿瘤中具有广泛的药用前景。The present invention provides an mTOR inhibitor and its application. Using a novel proximity labeling technology screening method, the C-terminal fragment (C tail) of the ATP6AP1 protein is found as a guanylate exchange factor (GEF) of Rheb and can activate Rheb. On this basis On the other hand, aspartic acid at positions 14, 17, and 18 of the 30 amino acids at the C-terminus were all mutated to alanine to obtain a new mTOR inhibitor that can bind to and block the effect of endogenous ATP6AP1 on Rheb Inhibition, thereby inhibiting the mTORC1 signaling pathway. Compared with existing mTOR inhibitors, the mTOR inhibitor obtained in the present invention has higher specificity and can exert its inhibitory effect in a more targeted manner. The mTOR inhibitor obtained by the present invention has broad medicinal prospects in treating diabetes and tumors.

附图说明Description of the drawings

图1为HeLa-xxID-Rheb稳定表达细胞株和HeLa-xxID稳定表达细胞株构建过程图及质谱检测图；Figure 1 shows the construction process diagram and mass spectrometry detection chart of HeLa-xxID-Rheb stable expression cell line and HeLa-xxID stable expression cell line;

图2为实施例1中Come/GO/Stay组的差异倍数分析图；Figure 2 is a fold analysis chart of the Come/GO/Stay group in Example 1;

图3为过表达mTOR抑制剂抑制mTOR信号通路活性的示意图；Figure 3 is a schematic diagram of overexpression of mTOR inhibitor inhibiting the activity of mTOR signaling pathway;

图4为回补过表达mTOR抑制剂抑制mTOR信号通路活性的示意图。Figure 4 is a schematic diagram showing how the overexpressed mTOR inhibitor inhibits the activity of the mTOR signaling pathway.

具体实施方式Detailed ways

为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。In order to better explain the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the drawings and specific embodiments.

在以下实施例中，所使用的实验方法如无特殊说明，均为常规方法，所用 的材料、试剂等，如无特殊说明，均可从商业途径得到。In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used can be obtained from commercial sources unless otherwise specified.

实施例1、一种筛选mTOR抑制剂的方法Example 1. A method for screening mTOR inhibitors

一种筛选mTOR抑制剂的方法，包括以下步骤：A method for screening mTOR inhibitors includes the following steps:

1)构建生物素连接酶(xxID)与Rheb融合表达载体(HAFlag-xxID-Rheb)，将载体转入HeLa细胞内，药物筛选HeLa-xxID-Rheb稳定表达细胞株，同时构建对照细胞系HeLa-xxID稳定表达细胞株，利用含10％透析血清培养基培养；实验前16小时，使用无血清培养基培养，规避胰岛素的干扰。1) Construct biotin ligase (xxID) and Rheb fusion expression vector (HAFlag-xxID-Rheb), transfer the vector into HeLa cells, drug screen HeLa-xxID-Rheb stable expression cell line, and construct a control cell line HeLa- The xxID stable expression cell line was cultured using a medium containing 10% dialyzed serum; 16 hours before the experiment, a serum-free medium was used to avoid interference from insulin.

2)然后向HeLa-xxID-Rheb稳定表达细胞株中加入胰岛素(0.9μM)和生物素(50μM)进行刺激，刺激15min，向HeLa-xxID稳定表达细胞株中加入生物素(50μM)刺激，不加入胰岛素，刺激15min，对样品进行裂解，提取蛋白后，利用链霉亲和素下拉，富集生物素化的蛋白，对链霉亲和磁珠进行还原烷基化，胰酶酶解，脱盐，最后将样品真空干燥后干粉保存，进行质谱检测(如图1所示)，分析蛋白。2) Then add insulin (0.9 μM) and biotin (50 μM) to the HeLa-xxID-Rheb stable expression cell line for stimulation, and stimulate for 15 minutes. Add biotin (50 μM) to the HeLa-xxID stable expression cell line for stimulation. Add insulin, stimulate for 15 minutes, lyse the sample, extract the protein, use streptavidin pull-down to enrich the biotinylated protein, perform reductive alkylation on streptavidin magnetic beads, trypsin digestion, and desalting , finally, the sample was vacuum dried and stored as dry powder, and mass spectrometry was performed (as shown in Figure 1) to analyze the protein.

3)在有/无进行胰岛素刺激条件下，分别将HeLa-xxID-Rheb稳定表达细胞株中的富集蛋白扣除HeLa-xxID稳定表达细胞株中的富集蛋白，获得有/无进行胰岛素刺激条件下的Rheb邻近蛋白，将这部分富集蛋白(Rheb邻近蛋白)进一步分析，可分为Come/GO/Stay组，即刺激后出现蛋白组/刺激前后稳定存在组/刺激后离开蛋白组(如图2所示)，Come组包括ATP6AP1蛋白、REEPS蛋白、SC22B蛋白，GO组包括VAMP3蛋白、SCD蛋白，Stay组包括RAB7A蛋白、TSC1蛋白，由于GEF蛋白应该在胰岛素刺激后出现，因此将Come组蛋白作为GEF候选蛋白进行验证。3) With/without insulin stimulation, the enriched proteins in the HeLa-xxID-Rheb stable expression cell line were subtracted from the enriched proteins in the HeLa-xxID stable expression cell line to obtain the conditions with/without insulin stimulation. Under the Rheb neighboring proteins, further analysis of this part of the enriched proteins (Rheb neighboring proteins) can be divided into Come/GO/Stay groups, that is, the protein group that appears after stimulation/the stable existence group before and after stimulation/the protein group that leaves after stimulation (such as As shown in Figure 2), the Come group includes ATP6AP1 protein, REEPS protein, and SC22B protein. The GO group includes VAMP3 protein and SCD protein. The Stay group includes RAB7A protein and TSC1 protein. Since GEF protein should appear after insulin stimulation, the Come group is The protein was validated as a GEF candidate protein.

4)对质谱表单的候选蛋白进行验证，Rheb的GEF候选蛋白需满足以下条件：4) Verify the candidate proteins on the mass spectrometry form. Rheb’s GEF candidate proteins must meet the following conditions:

(1)GEF(鸟嘌呤核苷酸交换因子)与Rheb存在体内外相互作用；(1) GEF (guanine nucleotide exchange factor) interacts with Rheb in vivo and in vitro;

(2)过表达GEF后可以激活mTOR下游信号通路；(2) Overexpression of GEF can activate the mTOR downstream signaling pathway;

(3)敲低或敲除GEF后，mTOR下游信号通路活性受到大幅度抑制；(3) After knocking down or knocking out GEF, the activity of the mTOR downstream signaling pathway is greatly inhibited;

(4)体外实验证明GEF可以直接促进Rheb完成GDP-GTP转换过程。(4) In vitro experiments prove that GEF can directly promote Rheb to complete the GDP-GTP conversion process.

通过筛选，发现ATP6AP1蛋白(C末端30个氨基酸，TYGLHMILSLKTMDRFDDHKGPTISLTQIV)可以很好的满足上述条件，证明ATP6AP1蛋白是Rheb的潜在GEF。Through screening, it was found that the ATP6AP1 protein (C-terminal 30 amino acids, TYGLHMILSLKTMDRFDDHKGPTISLTQIV) can well meet the above conditions, proving that the ATP6AP1 protein is a potential GEF for Rheb.

5)将ATP6AP1蛋白C末端30个氨基酸中的第14、17、18位的天冬氨酸均突变为丙氨酸，获得mTOR抑制剂。5) Mutation of aspartic acid at positions 14, 17, and 18 in the 30 amino acids at the C-terminus of the ATP6AP1 protein to alanine to obtain mTOR inhibitors.

通过NCBI对C末端进行序列保守性分析，发现C末端(30aa)十分保守，在HeLa细胞系中敲低ATP6AP1并过表达ATP6AP1全长蛋白(4μg)，在胰岛素刺激15min后，发现可以挽救由于ATP6AP1敲低而导致的mTOR信号通路被抑制的现象(mTOR底物S6K及其下游S6的磷酸化水平明显降低)；而过表达C末端3D/A(4μg)突变形成的mTOR抑制剂(C末端30个氨基酸中的第14、17、18位的天冬氨酸均突变为丙氨酸)则会抑制mTOR信号通路的活性(参考图3)。同时，在HeLa细胞系中过表达ATP6AP1(2μg)；C末端(2μg)可以明显提升mTOR信号通路活性；回补C末端3D/A(8μg)突变形成的mTOR抑制剂(C末端30个氨基酸中的第14、17、18位的天冬氨酸均突变为丙氨酸)，并不能挽救ATP6AP1敲低导致的磷酸化降低的现象，使C末端行使GEF功能的能力大幅度降低(参考图4)。Sequence conservation analysis of the C terminus through NCBI found that the C terminus (30aa) is very conserved. Knocking down ATP6AP1 and overexpressing ATP6AP1 full-length protein (4 μg) in the HeLa cell line, after insulin stimulation for 15 minutes, it was found that ATP6AP1 can be rescued. The mTOR signaling pathway is inhibited due to knockdown (the phosphorylation level of mTOR substrate S6K and its downstream S6 is significantly reduced); while the mTOR inhibitor formed by overexpression of C-terminal 3D/A (4μg) mutation (C-terminal 30 Mutations of aspartic acid at positions 14, 17, and 18 of each amino acid to alanine will inhibit the activity of the mTOR signaling pathway (see Figure 3). At the same time, overexpressing ATP6AP1 (2μg) in the HeLa cell line; C-terminal (2μg) can significantly increase the activity of the mTOR signaling pathway; complementing the mTOR inhibitor formed by the C-terminal 3D/A (8μg) mutation (in the 30 amino acids of the C-terminal Aspartic acids at positions 14, 17, and 18 were all mutated to alanine), which could not rescue the decrease in phosphorylation caused by knockdown of ATP6AP1, significantly reducing the ability of the C terminus to perform GEF functions (refer to Figure 4 ).

本发明利用新型邻近标记技术筛选方法找到了ATP6AP1的C端片段(C tail)作为Rheb的鸟苷酸交换因子(GEF)可激活Rheb。在此基础上，将C末端30个氨基酸中第14、17、18位的天冬氨酸均突变为丙氨酸，获得新的mTOR抑制剂，该mTOR抑制剂可结合并阻断内源性ATP6AP1对Rheb的抑制，进而抑制mTORC1信号通路，这是首个发现可抑制Rheb-mTORC1的短肽。由于mTORC1、mTORC2复合物均含有mTOR蛋白，而Rheb为mTORC1特有，传统直接靶向mTOR的抑制剂，能同时抑制mTORC1、mTORC2下游信号，而抑制Rheb-mTORC1的短肽并不影响mTORC2下游。相比于现存的mTOR抑制剂，本发明获得的mTOR抑制剂特异性较高，更能有针对性的发挥抑制剂作用。此外，mTOR信号通路抑制剂在治疗糖尿病、肿瘤中具有广泛的药用前景。The present invention uses a new proximity labeling technology screening method to find that the C-terminal fragment (C tail) of ATP6AP1 can activate Rheb as a guanylate exchange factor (GEF) of Rheb. On this basis, aspartic acid at positions 14, 17, and 18 of the 30 amino acids at the C-terminus were mutated to alanine to obtain a new mTOR inhibitor that can bind to and block endogenous ATP6AP1 inhibits Rheb, thereby inhibiting the mTORC1 signaling pathway. This is the first short peptide discovered to inhibit Rheb-mTORC1. Since both mTORC1 and mTORC2 complexes contain mTOR proteins, and Rheb is unique to mTORC1, traditional inhibitors that directly target mTOR can simultaneously inhibit mTORC1 and mTORC2 downstream signals, while short peptides that inhibit Rheb-mTORC1 do not affect mTORC2 downstream. Compared with existing mTOR inhibitors, the mTOR inhibitor obtained in the present invention has higher specificity and can exert inhibitory effects in a more targeted manner. In addition, mTOR signaling pathway inhibitors have broad medicinal prospects in the treatment of diabetes and tumors.

最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention may be modified or equivalently substituted without departing from the essence and scope of the technical solution of the present invention.

Claims (12)

一种mTOR抑制剂，其特征在于，所述mTOR抑制剂为:An mTOR inhibitor, characterized in that the mTOR inhibitor is: 1)多肽，所述多肽具有与SEQ ID NO:2所示的氨基酸序列至少9/10、14/15或29/30的序列一致性；或1) A polypeptide having a sequence identity of at least 9/10, 14/15 or 29/30 with the amino acid sequence shown in SEQ ID NO: 2; or 2)C端为所述多肽的蛋白质。2) A protein whose C-terminus is the polypeptide. 如权利要求1所述的mTOR抑制剂，其特征在于，所述mTOR抑制剂为相比ATP6AP1蛋白突变体具有至少95％、96％、97％、98％或99％氨基酸序列一致性的蛋白质，所述ATP6AP1蛋白突变体的C末端相比ATP6AP1蛋白在第14、17、18位的天冬氨酸均突变为丙氨酸。The mTOR inhibitor of claim 1, wherein the mTOR inhibitor is a protein with at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared to the ATP6AP1 protein mutant, The C-terminus of the ATP6AP1 protein mutant has aspartic acid at positions 14, 17, and 18 compared to the ATP6AP1 protein, all mutated to alanine. 如权利要求2所述的mTOR抑制剂，其特征在于，所述mTOR抑制剂为所述ATP6AP1蛋白突变体或所述多肽。The mTOR inhibitor of claim 2, wherein the mTOR inhibitor is the ATP6AP1 protein mutant or the polypeptide. 权利要求2所述的ATP6AP1蛋白突变体在抑制mTOR信号通路活性中的应用。Application of the ATP6AP1 protein mutant described in claim 2 in inhibiting the activity of the mTOR signaling pathway. 如权利要求4所述的应用，其特征在于，所述mTOR信号通路包括mTORC1信号通路和mTORC2信号通路。The application of claim 4, wherein the mTOR signaling pathway includes the mTORC1 signaling pathway and the mTORC2 signaling pathway. 一种筛选mTOR抑制剂的方法，其特征在于，包括以下步骤：A method for screening mTOR inhibitors, characterized by comprising the following steps: 1)构建生物素连接酶与Rheb融合表达载体，将载体转入宿主细胞内，药物筛选稳定表达Rheb蛋白的细胞株，同时构建对照细胞株；1) Construct a biotin ligase and Rheb fusion expression vector, transfer the vector into host cells, screen cell lines that stably express Rheb protein, and construct control cell lines; 2)向稳定表达Rheb蛋白的细胞株中加入胰岛素和生物素进行刺激，向对照细胞株中加入生物素刺激，然后裂解样品、提取蛋白、富集蛋白，再进行质谱检测，分析蛋白；2) Add insulin and biotin to the cell line stably expressing Rheb protein for stimulation, add biotin to the control cell line for stimulation, then lyse the sample, extract the protein, enrich the protein, and then perform mass spectrometry detection to analyze the protein; 3)分别将稳定表达Rheb蛋白的细胞株中的富集蛋白和对照细胞株中的富集蛋白之间重叠的蛋白扣除，获得有/无进行胰岛素刺激条件下的Rheb邻近蛋白，分析Rheb邻近蛋白，经鸟嘌呤核苷酸交换因子筛选条件，得ATP6AP1蛋白；以及3) Subtract overlapping proteins between enriched proteins in cell lines stably expressing Rheb protein and enriched proteins in control cell lines to obtain Rheb neighboring proteins with or without insulin stimulation and analyze Rheb neighboring proteins. , through guanine nucleotide exchange factor screening conditions, ATP6AP1 protein was obtained; and 4)将ATP6AP1蛋白C末端30个氨基酸中的第14、17、18位的天冬氨酸均突变为丙氨酸，获得mTOR抑制剂。4) Mutation of aspartic acid at positions 14, 17, and 18 in the 30 amino acids at the C-terminus of the ATP6AP1 protein to alanine to obtain mTOR inhibitors. 如权利要求6所述的方法，其特征在于，所述胰岛素的浓度为0.9μM，所述生物素的浓度为50μM，所述刺激的时间为15min。The method of claim 6, wherein the concentration of insulin is 0.9 μM, the concentration of biotin is 50 μM, and the stimulation time is 15 min. 如权利要求6所述的方法，其特征在于，所述生物素连接酶包括生物素连接酶xxID，所述生物素连接酶与Rheb融合表达载体为HAFlag-xxID-Rheb。The method of claim 6, wherein the biotin ligase includes biotin ligase xxID, and the biotin ligase and Rheb fusion expression vector is HAFlag-xxID-Rheb. 一种抑癌药物，其特征在于，包含如权利要求1～3任一项所述的mTOR抑制剂，以及医学上可接受的载体。A tumor suppressor drug, characterized by comprising the mTOR inhibitor according to any one of claims 1 to 3, and a medically acceptable carrier. 一种抗衰老药物，其特征在于，包含如权利要求1～3任一项所述的mTOR抑制剂，以及医学上可接受的载体。An anti-aging drug, characterized by comprising the mTOR inhibitor according to any one of claims 1 to 3, and a medically acceptable carrier. 如权利要求1所述的mTOR抑制剂在制备提高mTOR信号通路中物质磷酸化水平的药物/试剂中的应用，其中，所述mTOR信号通路中物质为底物S6K及其下游S6。The application of the mTOR inhibitor according to claim 1 in the preparation of drugs/reagents that increase the phosphorylation level of substances in the mTOR signaling pathway, wherein the substances in the mTOR signaling pathway are substrate S6K and its downstream S6. 如权利要求1所述的mTOR抑制剂在制备通过抑制mTORC1信号通路的活性预防和/或治疗癌症、以及抗衰老药物中的应用，其特征在于，所述癌症为乳腺癌或胰腺癌。The application of the mTOR inhibitor according to claim 1 in the preparation of drugs for preventing and/or treating cancer and anti-aging by inhibiting the activity of the mTORC1 signaling pathway, wherein the cancer is breast cancer or pancreatic cancer.
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