技术领域technical field
本发明属于药物化学领域，具体涉及一种多肽的筛选及其应用，尤其涉及一种能与人血管内皮生长因子受体-3(VEGFR-3)特异性结合的多肽及上述多肽的衍生产品在肿瘤靶向给药领域的应用。The present invention belongs to the field of medicinal chemistry, and in particular relates to the screening and application of a polypeptide, in particular to a polypeptide capable of specifically binding to human vascular endothelial growth factor receptor-3 (VEGFR-3) and derivatives of the above polypeptide. Applications in the field of tumor-targeted drug delivery.
背景技术Background technique
近年来，针对肿瘤细胞特有的分子信号通路的开发的分子靶向药物，层出不穷。但是随着研究的深入，其局限性开始突显，基因突变产生的耐药性问题的存在限制了其在临床的应用。靶向给药系统(targeteddrugdeliverysystem，TDDS)，是指载体将药物通过局部给药或全身血液循环而选择性地浓集定位于病变组织、器官、细胞或细胞内结构的新型给药系统，可使病变部位的药物浓度明显提高，从而减少用药量，增加用药安全性，是目前较有前景的研究方向。其中，靶向给药系统按作用方式不同，可分为被动靶向(passivetargeting)和主动靶向(activetargeting)；被动靶向是一种基于肿瘤组织的EPR(增强渗透和滞留)效应使递药系统靶向分布于肿瘤组织的策略，但是不均匀的粒径分布使得微粒进入血液循环后，易被网状内皮系统(RES)摄取而蓄积于肝、脾等器官产生毒性，而致密的肿瘤细胞外基质以及较高的肿瘤间质压极大地阻碍了药物进入肿瘤深层，造成肿瘤内药物的不均匀分布，为肿瘤的治愈又增加了难度。因而，借助肿瘤组织高表达的特异性受体的介导作用，增加递药系统在肿瘤组织、细胞或细胞器内分布的主动靶向给药系统的开发显得尤为重要。In recent years, molecular targeted drugs developed for the specific molecular signaling pathways of tumor cells have emerged in an endless stream. However, with the deepening of research, its limitations began to be highlighted, and the existence of drug resistance caused by gene mutations limited its clinical application. Targeted drug delivery system (TDDS) refers to a new type of drug delivery system in which the carrier selectively concentrates and localizes drugs in diseased tissues, organs, cells or intracellular structures through local administration or systemic blood circulation. The drug concentration in the lesion is significantly increased, thereby reducing the dosage of the drug and increasing the safety of the drug, which is a more promising research direction at present. Among them, the targeted drug delivery system can be divided into passive targeting and active targeting according to different modes of action; passive targeting is a kind of EPR (enhanced penetration and retention) effect based on tumor tissue to enable The system targets the strategy of distribution in tumor tissue, but the uneven particle size distribution makes the particles easy to be taken up by the reticuloendothelial system (RES) and accumulate in the liver, spleen and other organs to produce toxicity after entering the blood circulation, while dense tumor cells The extracellular matrix and high tumor interstitial pressure greatly hinder the drug from entering the deep layer of the tumor, resulting in the uneven distribution of the drug in the tumor, which makes it more difficult to cure the tumor. Therefore, it is particularly important to develop an active targeted drug delivery system that increases the distribution of drug delivery systems in tumor tissues, cells or organelles through the mediation of specific receptors highly expressed in tumor tissues.
小分子多肽具有相对分子质量小、活性高、免疫原性低、易于制备等特点，近年来广泛应用于肿瘤主动靶向治疗。利用肿瘤组织某些异常表达的蛋白，研究人员筛选出能特异性识别肿瘤组织的高活性小分子肽段，通过氨基酸侧链的功能基团连接于药物载体，能够显著提高肿瘤药物治疗的疗效。例如，靶向整合素受体的RGD多肽，靶向血管生成受体的K237多肽，对肿瘤转移和淋巴管生成影响较大的Lyp-1多肽等修饰的多肽靶向给药系统在癌症治疗领域研究较为深入。肿瘤靶向多肽不仅在靶向治疗中具有重要的应用价值，其还可以通过与分子成像试剂偶联，应用于肿瘤分子成像，提高肿瘤诊断的灵敏度和特异性。Small molecule peptides have the characteristics of small molecular weight, high activity, low immunogenicity, and easy preparation, and have been widely used in active targeted therapy of tumors in recent years. Using some abnormally expressed proteins in tumor tissue, researchers screen out highly active small molecular peptides that can specifically recognize tumor tissue, and link them to drug carriers through functional groups of amino acid side chains, which can significantly improve the efficacy of tumor drug therapy. For example, RGD polypeptides targeting integrin receptors, K237 polypeptides targeting angiogenesis receptors, and Lyp-1 polypeptides that have a greater impact on tumor metastasis and lymphangiogenesis and other modified polypeptide targeted drug delivery systems are widely used in the field of cancer treatment. The research is more in-depth. Tumor-targeting polypeptides not only have important application value in targeted therapy, but also can be applied to tumor molecular imaging by coupling with molecular imaging reagents to improve the sensitivity and specificity of tumor diagnosis.
目前，小分子多肽的发现主要有组合化学肽库，表面展示技术和基于结构的计算机辅助设计，其中，组合化学肽库的筛选合成周期长，效率较低，不易纯化，不适合大规模筛选；表面展示技术应用较多的有噬菌体展示，酵母展示，细菌展示等，以噬菌体展示技术为例，它是一种将编码多肽的外源基因或随机序列的DNA分子群与负责表达噬菌体外壳蛋白的基因融合后，使外源DNA所编码的蛋白质以融合蛋白形式表达在噬菌体外壳表面，导入各种外源基因的一组噬菌体集合构成一个展示各种外源多肽的噬菌体展示肽库，之后，将噬菌体集合与其各种靶分子(细胞表面受体、荷瘤小鼠等)进行多轮淘选(panning)，实现高通量亲和筛选活性多肽的方法。但构建库容量大、具有更多生物学意义的氨基酸序列仍需进一步的改进，且筛选过程繁琐，费时。计算机模拟筛选方法是一种计算机辅助筛选和化学合成筛选相结合的方法，基于肿瘤过度表达的蛋白的晶体结构，选取可能具有活性的氨基酸片段组成多肽库，用分子对接的方法进行评估虚拟筛选，缩小实验对象、提高实验筛选命中率。利用化学合成多肽，通过实验的手段进行验证，为寻找肿瘤靶向分子治疗药物或输送导向“弹头”开辟一条新颖、快捷、经济的途径。At present, the discovery of small molecule peptides mainly includes combinatorial chemical peptide library, surface display technology and structure-based computer-aided design. Among them, the screening and synthesis cycle of combinatorial chemical peptide library is long, the efficiency is low, it is not easy to purify, and it is not suitable for large-scale screening; Surface display technology is widely used in phage display, yeast display, bacterial display, etc. Taking phage display technology as an example, it is a method of combining foreign genes or random sequences of DNA molecules encoding polypeptides with the protein responsible for expressing phage coat proteins. After gene fusion, the protein encoded by the foreign DNA is expressed on the surface of the phage shell in the form of a fusion protein, and a set of phage collections that introduce various foreign genes constitute a phage display peptide library that displays various foreign polypeptides. After that, the The phage collection and its various target molecules (cell surface receptors, tumor-bearing mice, etc.) undergo multiple rounds of panning to achieve high-throughput affinity screening of active polypeptides. However, the construction of amino acid sequences with large library capacity and more biological significance still needs further improvement, and the screening process is cumbersome and time-consuming. The computer simulation screening method is a combination of computer-aided screening and chemical synthesis screening. Based on the crystal structure of proteins overexpressed in tumors, amino acid fragments that may be active are selected to form a polypeptide library, and molecular docking is used to evaluate virtual screening. Reduce the experimental objects and increase the hit rate of experimental screening. Using chemically synthesized peptides and verifying them through experiments will open up a novel, fast and economical way for finding tumor-targeted molecular therapeutic drugs or delivering guided "warheads".
血管内皮生长因子受体3(vascuLarendotheliagrowthfactorreceptor-3，VEGFR-3，又称FLT4)属于III型受体酪氨酸激酶(RTKs)亚家族，是血管内皮生长因子受体家族的一员，目前该家族已发现的血管内皮生长因子受体还有VEGFR-L、VEGFR-2，人们普通认为，VEGFR-1主要介导细胞骨架重排引起细胞迁移，并引起单核细胞趋化，调节VEGF与VEGFR-2的结合；VEGFR-2则主要介导与肿瘤有关的内皮细胞增殖和迁移，引起血管通透性升高，并有抗内皮细胞凋亡、维持内皮细胞存活的作用，在诱导肿瘤新血管的形成过程中发挥着最为重要的作用；而VEGFR-3主要介导淋巴内皮细胞的增殖和迁移，与淋巴血管的生成和和肿瘤细胞的扩散转移密切相关。与RTK家族其它成员类似，VEGFR-3结构上有如下特征：(1)胞外区由7个免疫球蛋白(Immunoglobulin)样区构成，其中第1～3个Ig样区之间为其与配体VEGF的结合位点，第4～7个Ig为受体二聚化位点，与激酶活性有关；(2)单次跨膜区；(3)胞内区分为近膜区，催化区酪氨酸激酶域，激酶插入域和C末端激酶自身磷酸化区。KariALitaLo等人在2013年解析了VEGFR-3胞外区(D1-D2，D4-D5)，胞外结构域中第5个免疫球蛋白样折叠在合成时被切割，切割后由一个二硫键连接。两个单体的VEGFR-3通过第四个免疫球蛋白域相互形成同源或异源二聚体，发挥生理活性。随着研究的深入，发现VEGFR-3在许多肿瘤中过度表达，如肺癌、乳腺癌、Kaposi′s肉瘤、肝癌和鼻咽癌、结肠癌和卵巢癌等。由于肿瘤细胞遗传的不稳定性，传统的细胞毒类抗肿瘤药物极易产生耐药性；已经证实，肿瘤的生长和转移必须依赖于新血管和淋巴管的形成，因此靶向抑制介导肿瘤的血管和淋巴管生成为抗肿瘤治疗和防止肿瘤转移提供了一个非细胞毒性的重要途径，同时血管内皮细胞在遗传学上是稳定的，不易产生变异而导致耐药性。目前已有许多公司正在这方面进行积极靶向治疗的尝试，并取得了积极的结果。BDD073抗体通过抑制VEGFR3的作用，成功抑制了HEL细胞的增殖。除了抗体之外，针对VEGFR-3信号转导通路设计的小分子激酶抑制剂的研究也在进行之中，比如AZD2171，BIBF1120等正在进行临床实验，但是无论是抗体还是小分子，均不是VEGFR-3特异性抑制剂，治疗效果不佳，临床进展缓慢。VascuLarendotheliagrowthfactor receptor-3 (VEGFR-3, also known as FLT4) belongs to the type III receptor tyrosine kinases (RTKs) subfamily and is a member of the vascular endothelial growth factor receptor family. The vascular endothelial growth factor receptors that have been discovered include VEGFR-L and VEGFR-2. It is generally believed that VEGFR-1 mainly mediates cytoskeleton rearrangement to cause cell migration, induce monocyte chemotaxis, and regulate VEGF and VEGFR-2. 2; VEGFR-2 mainly mediates the proliferation and migration of tumor-related endothelial cells, causing increased vascular permeability, anti-endothelial cell apoptosis, and maintaining the survival of endothelial cells. VEGFR-3 mainly mediates the proliferation and migration of lymphatic endothelial cells, and is closely related to the formation of lymphatic vessels and the spread and metastasis of tumor cells. Similar to other members of the RTK family, the structure of VEGFR-3 has the following characteristics: (1) The extracellular domain is composed of 7 immunoglobulin (Immunoglobulin)-like domains, among which the first to third Ig-like domains are between the Ig-like domains and the ligands. The 4th to 7th Ig is the receptor dimerization site, which is related to the kinase activity; (2) the single transmembrane region; (3) the intracellular region is divided into the near-membrane region and the catalytic region Amino acid kinase domain, kinase insertion domain and C-terminal kinase autophosphorylation region. KariALitaLo et al. analyzed the VEGFR-3 extracellular region (D1-D2, D4-D5) in 2013. The fifth immunoglobulin-like fold in the extracellular domain was cleaved during synthesis, and a disulfide bond formed after cleavage connect. The two monomers of VEGFR-3 form homologous or heterologous dimers with each other through the fourth immunoglobulin domain to exert physiological activity. With the deepening of research, it is found that VEGFR-3 is overexpressed in many tumors, such as lung cancer, breast cancer, Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, colon cancer and ovarian cancer. Due to the genetic instability of tumor cells, traditional cytotoxic antitumor drugs are prone to drug resistance; it has been confirmed that tumor growth and metastasis must depend on the formation of new blood vessels and lymphatic vessels, so targeted inhibition mediates tumor The angiogenesis and lymphangiogenesis of vascular endothelial cells provide an important non-cytotoxic way for anti-tumor therapy and prevention of tumor metastasis. At the same time, vascular endothelial cells are genetically stable and are not easy to mutate and cause drug resistance. At present, many companies are making active targeted therapy attempts in this area and have achieved positive results. BDD073 antibody successfully inhibited the proliferation of HEL cells by inhibiting the effect of VEGFR3. In addition to antibodies, research on small-molecule kinase inhibitors designed for the VEGFR-3 signal transduction pathway is also in progress, such as AZD2171, BIBF1120, etc. are undergoing clinical trials, but neither antibodies nor small molecules are VEGFR- 3 Specific inhibitors, the treatment effect is not good, and the clinical progress is slow.
针对VEGFR-3目前分子靶向药物研究的缺陷和局限性，可以对其研究思路进一步拓宽和改造，充分利用VEGFR-3胞外区晶体结构提供的三维信息，进行基于靶标结构的合理靶向多肽设计，将靶向多肽与生物相容性好，载药量高的聚合物胶束相结合，以多肽为靶头将活性药物输送至肿瘤细胞内部，提高药物的选择性和安全性。这一理念与近年来抗肿瘤新药研究的最新思路不谋而合，可谓是殊途同归。但是这一策略的研究关键依赖于多肽的特异性和高亲和力。目前已有针对VEGFR-3的多肽的出现，并在细胞水平上有效的靶向VEGFR-3信号通路，但并未有进一步的体内实验报道，因此，本领域迫切需要进一步的新型靶向多肽和多肽给药系统的开发和研究，也可能为肿瘤的成像和诊断提供新的手段。In view of the defects and limitations of the current research on molecularly targeted drugs for VEGFR-3, it is possible to further broaden and transform its research ideas, and make full use of the three-dimensional information provided by the crystal structure of the extracellular domain of VEGFR-3 to conduct reasonable targeting peptides based on the target structure. The design combines the targeting polypeptide with polymer micelles with good biocompatibility and high drug loading, and uses the polypeptide as the target to deliver the active drug into the tumor cells, improving the selectivity and safety of the drug. This concept coincides with the latest ideas in the research of new anti-tumor drugs in recent years, which can be said to lead to the same goal. However, the study of this strategy depends critically on the specificity and high affinity of the peptide. Peptides targeting VEGFR-3 have appeared and effectively target the VEGFR-3 signaling pathway at the cellular level, but no further in vivo experiments have been reported. Therefore, there is an urgent need for further novel targeting polypeptides and The development and research of peptide drug delivery systems may also provide new means for tumor imaging and diagnosis.
发明内容Contents of the invention
本发明的目的是针对现有技术的不足，利用计算机辅助设计和化学合成相结合的方法，合理筛选出与VEGFR-3高亲和力，分子量小，容易到达肿瘤组织部位的特异性多肽，并进行部分体外生物学实验验证。在肿瘤靶向给药、癌症诊断等方面彰显出较强的优越性，为以VEGFR-3为靶点的多肽抗肿瘤靶向药物系统的开发和设计提供有效的开发途径。The purpose of the present invention is to address the deficiencies of the prior art, using computer-aided design and chemical synthesis to rationally screen out specific polypeptides with high affinity to VEGFR-3, small molecular weight, and easy access to tumor tissue sites, and carry out partial Validation by in vitro biological experiments. It shows strong advantages in tumor targeted drug delivery, cancer diagnosis, etc., and provides an effective development path for the development and design of peptide anti-tumor targeted drug systems targeting VEGFR-3.
本发明的目的是通过以下技术方案来实现的：以VEGFR-3的晶体结构(4BSJ)为基础，全面分析其作用模式，利用FTmap软件寻找潜在的结合位点，并在远离VEGFR-3天然配体的潜在结合口袋附近，考虑氨基酸的理化性质和空间位置排序，构建倾向性多肽库，利用多种对接软件进行虚拟筛选，互为验证，同时综合考虑对接排名顺序、氢键成键能力、空间构象、静电互补、疏水作用和合成难易程度等因素，最终选出9条代表性的多肽进行合成并投入生物亲和力实验，其中有3条多肽表现出较强的细胞亲和活性。本发明氨基酸序列通式为CXXPFXP，X为20种天然氨基酸中的任一种或其D型氨基酸，优选的三条多肽的氨基酸序列分别为CIQPFYP、CVKPFYP、CVKTFDP。The purpose of the present invention is achieved through the following technical solutions: based on the crystal structure (4BSJ) of VEGFR-3, comprehensively analyze its mode of action, use FTmap software to find potential binding sites, and find the potential binding sites away from the natural ligand of VEGFR-3. In the vicinity of the potential binding pocket of the body, considering the physical and chemical properties of amino acids and the ordering of spatial positions, construct a preferential peptide library, use a variety of docking software for virtual screening, mutual verification, and comprehensively consider the docking ranking order, hydrogen bonding ability, space Based on factors such as conformation, electrostatic complementarity, hydrophobic interaction and synthesis difficulty, 9 representative peptides were finally selected for synthesis and put into bio-affinity experiments, among which 3 peptides showed strong cell-affinity activity. The general formula of the amino acid sequence of the present invention is CXXPFXP, X is any one of 20 kinds of natural amino acids or its D-type amino acid, and the amino acid sequences of the preferred three polypeptides are respectively CIQPFYP, CVKPFYP, and CVKTFDP.
上述的三种多肽经过生物信息学分析比对发现，与美国国立生物技术信息中心(NCBI)GenBankDNA序列数据库和Swiss-Prot蛋白数据库中的已知基因和蛋白无同源性，且国内外文献也均未见报道，由此可以得出利用本发明的方法获得的多肽结构新颖，具有潜在的应用价值。The above three polypeptides were found to have no homology with the known genes and proteins in the GenBank DNA sequence database and the Swiss-Prot protein database of the National Center for Biotechnology Information (NCBI) through bioinformatics analysis and comparison, and domestic and foreign literature also None of them have been reported, so it can be concluded that the polypeptide obtained by the method of the present invention has a novel structure and has potential application value.
第二方面，本发明的多肽可在C末端和或N或其他位置进行氨基酸替换，缺失或添加或连接其他基团。另外，本发明的多肽也涉及一种前述多肽的酰胺、酯或其盐，或该多肽形成的活性片段、活性衍生物或融合型多肽，或该多肽的二价体或多价体。In the second aspect, the polypeptide of the present invention may undergo amino acid substitution, deletion or addition or connection of other groups at the C-terminus and/or N or other positions. In addition, the polypeptide of the present invention also relates to an amide, ester or salt thereof of the aforementioned polypeptide, or an active fragment, active derivative or fusion polypeptide formed by the polypeptide, or a bivalent or multivalent body of the polypeptide.
第三方面，本发明所述的多肽或其衍生物与能杀伤癌细胞的制剂相缀合或混合，涉及一种多肽靶向连接的药物输送系统，该系统包括：纳米粒子、聚合物、脂质体、囊泡、固体脂质微粒、胶束、碳纳米管、细胞器、脂蛋白，量子点。优选地，所述的生物高分子材料为纳米材料。In the third aspect, the polypeptide or its derivative described in the present invention is conjugated or mixed with an agent capable of killing cancer cells, and relates to a drug delivery system for targeted linking of polypeptides. The system includes: nanoparticles, polymers, lipids Plastids, vesicles, solid lipid particles, micelles, carbon nanotubes, organelles, lipoproteins, quantum dots. Preferably, the biopolymer material is a nanomaterial.
第四方面，本发明提供了靶向多肽在制备肿瘤早期诊断和治疗试剂中的用途，涉及的多肽靶向药物输送系统至少包括一种活性物质。所述的显像制剂优选为放射性核素、放射性核素标记物或分子影像制剂中的任意一种。所述的药物优选为能杀伤癌细胞的化学药物、生物药物、纳米药物、光热治疗或光动力治疗药物。In the fourth aspect, the present invention provides the use of targeting polypeptides in the preparation of reagents for early diagnosis and treatment of tumors, and the polypeptide targeting drug delivery system involved at least one active substance. The imaging agent is preferably any one of radionuclide, radionuclide label or molecular imaging agent. The drug is preferably a chemical drug, a biological drug, a nano drug, a photothermal therapy or a photodynamic therapy drug capable of killing cancer cells.
第五方面，本发明的多肽及其衍生物用于预防和靶向治疗与VEGFR-3相关的肿瘤，所述的肿瘤包括但不限于肺癌、肝癌、结肠癌、乳腺癌、白血病等癌症疾病。In the fifth aspect, the polypeptide of the present invention and its derivatives are used for the prevention and targeted treatment of tumors related to VEGFR-3, including but not limited to lung cancer, liver cancer, colon cancer, breast cancer, leukemia and other cancer diseases.
与现有技术相比，本发明具有如下优势：Compared with the prior art, the present invention has the following advantages:
1.本发明利用计算机辅助设计和化学合成相结合的方法制备多肽，操作简单，成本低廉，能高效筛选出候选多肽。同时，优选多肽通过细胞摄取与定位，小鼠活体成像等体内外生物学验证，对肿瘤细胞的亲和力和特异性较强。本方法涉及多学科交叉和融合，开拓了“多肽设计-化学合成-生物筛选”研究模式，符合以药物化学为中心的现代创新药物研究的新趋势。1. The present invention uses computer-aided design and chemical synthesis to prepare polypeptides, which is simple to operate, low in cost, and can efficiently screen out candidate polypeptides. At the same time, it is preferred that the polypeptide has strong affinity and specificity for tumor cells through in vivo and in vitro biological verification such as cell uptake and localization, mouse live imaging, etc. This method involves multidisciplinary interdisciplinary and integration, and has opened up a research model of "peptide design-chemical synthesis-biological screening", which is in line with the new trend of modern innovative drug research centered on medicinal chemistry.
2.本发明筛选得到的优选多肽具有亲和力高，纯度高，毒性低，可以很好地结合在肿瘤细胞上，灵敏度高，可以应用于肿瘤的筛查、诊断。2. The preferred polypeptide screened by the present invention has high affinity, high purity, low toxicity, can be well bound to tumor cells, has high sensitivity, and can be applied to tumor screening and diagnosis.
3.本发明筛选得到的优选多肽具有良好的肿瘤靶向作用，可以作为靶向给药系统或基因药物载体的靶头分子，安全可靠，在肿瘤的治疗领域具有非常广阔的应用前景。3. The preferred polypeptide screened by the present invention has good tumor targeting effect, can be used as a target molecule of a targeted drug delivery system or a gene drug carrier, is safe and reliable, and has a very broad application prospect in the field of tumor treatment.
附图说明Description of drawings
图1为VEGFR-3靶向多肽的发现流程图Figure 1 is a flow chart for the discovery of VEGFR-3 targeting peptides
图2为VEGFR-3潜在的结合位点(PDB：4BSJ)，格点球形区域为VEGFR-3结合位点，黄色格点球体区域为我们选择用于对接的结合口袋Figure 2 shows the potential binding site of VEGFR-3 (PDB: 4BSJ), the gridded spherical area is the VEGFR-3 binding site, and the yellow gridded sphere area is the binding pocket we selected for docking
图3为VEGFR-3所选对接口袋的表面图Figure 3 is a surface view of the selected docking pocket for VEGFR-3
图4为VEGFR-3和优选多肽的对接结构图，多肽以球棒模式表示Figure 4 is a docking structure diagram of VEGFR-3 and a preferred polypeptide, and the polypeptide is represented in a ball-and-stick mode
图5为表面等离子共振方法验证不同浓度的多肽和蛋白VEGFR-3的动力学结合曲线Figure 5 is the kinetic binding curves of different concentrations of peptides and protein VEGFR-3 verified by surface plasmon resonance
图6为流式分析仪(FC)验证FITC标记优选多肽与高表达VEGFR-3的A549细胞和低表达VEGFR-3的HeLa、L-O2的细胞结合曲线图Figure 6 is a flow cytometer (FC) verification of the FITC-labeled preferred polypeptide and the A549 cells that highly express VEGFR-3 and the cell binding curves of HeLa and L-O2 that low express VEGFR-3
图7为激光共聚焦显微镜观察FITC优选多肽与高表达VEGFR-3的A549细胞和低表达VEGFR-3的HeLa、L-O2细胞的显微镜图片Figure 7 is a microscope picture of FITC preferred polypeptides and A549 cells with high expression of VEGFR-3 and HeLa and L-O2 cells with low expression of VEGFR-3 observed by laser confocal microscope
图8为MTT法检验优选多肽的安全性Fig. 8 is the safety of MTT method inspection preferred polypeptide
图9为PEG-PLL胶束的电子显微镜图片Figure 9 is an electron microscope picture of PEG-PLL micelles
具体实施例specific embodiment
下面结合具体实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。Below in conjunction with specific embodiment, further illustrate the present invention. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
1.VEGFR-3活性位点的寻找和倾向性多肽库的构建1. The search for active sites of VEGFR-3 and the construction of a preferential peptide library
在RCSBProteinDataBank(PDB)中，共有2个VEGFR-3的X光衍射晶体结构，我们选取其中VEGFR-3的D4-D5区域的晶体结构进行研究(PDBID：4BSJ)。在使用晶体结构前，首先利用2009的ProteinPreparationWizardWorkflow流程进行对晶体结构进行处理，进行加氢后利用OPLS2005力场对氢原子进行优化。随后，为了寻找合适的结合靶点，运用虚拟分子探针和2009的sitemap模块综合寻找VEGFR-3潜在的结合热点区，每个结合热点区具有各自不同的结构特征，我们选择远离天然配体VEGF与VEGFR-3的结合位点的site2热点区域作为多肽的结合口袋，以避免天然配体的竞争性干扰。考虑氨基酸的理化性质和空间位置排序，选取TYLRKIW作为有义肽，基于有义-反义多肽理论，构建倾向性多肽库。In RCSB Protein Data Bank (PDB), there are 2 X-ray diffraction crystal structures of VEGFR-3, and we selected the crystal structure of the D4-D5 region of VEGFR-3 for research (PDBID: 4BSJ). Before using the crystal structure, first use The ProteinPreparationWizardWorkflow process in 2009 processes the crystal structure, and optimizes the hydrogen atoms by using the OPLS2005 force field after hydrogenation. Subsequently, to find suitable binding targets, virtual molecular probes and The sitemap module in 2009 comprehensively searched for potential binding hotspots of VEGFR-3. Each binding hotspot has its own different structural characteristics. We selected the site2 hotspot far from the binding site of the natural ligand VEGF and VEGFR-3 as the binding peptide pocket to avoid competitive interference from natural ligands. Considering the physicochemical properties and spatial position ordering of amino acids, TYLRKIW was selected as the sense peptide, and based on the sense-antisense peptide theory, a preferential peptide library was constructed.
2.利用分子对接手段，筛选优选多肽2. Use molecular docking methods to screen for optimal peptides
分子对接利用锁钥原理，按照几何互补、能量互补以及化学环境互补的原则，计算多肽与蛋白结合的最佳构象和结合模式。该方法既计算了多肽的低能构象，又考虑了生物大分子的结构信息，因此在评估受体与小分子之间的相互作用时更加准确。我们采用Glide对接算法和GOLD对接算法等进行互为验证，综合考虑对接打分值、氢键成键能力、空间构象、静电互补、疏水作用和合成难易程度等因素，最终选出9条代表性的多肽进行合成并投入生物亲和力实验。Molecular docking uses the lock-and-key principle to calculate the optimal conformation and binding mode of polypeptides and proteins according to the principles of geometric complementarity, energy complementarity and chemical environment complementarity. This method not only calculates the low-energy conformation of peptides, but also considers the structural information of biomacromolecules, so it is more accurate in evaluating the interaction between receptors and small molecules. We used the Glide docking algorithm and the GOLD docking algorithm to verify each other, and finally selected 9 representative ones after comprehensive consideration of the docking scoring value, hydrogen bonding ability, spatial conformation, electrostatic complementarity, hydrophobic interaction, and synthesis difficulty. The peptides were synthesized and put into bioaffinity experiments.
3.固相合成法合成优选多肽3. Synthesis of preferred peptides by solid-phase synthesis
具体方法为通过混合裂分的方式将氨基酸随机地逐个偶联到固相树脂上，然后在强酸下将侧链保护基团去除，进而进行筛选。称取200mg的Tentage1-S-NH2树脂，按照固相多肽合成程序循环，加入200mg的等量氨基酸的HBTU，依次进行反应两个循环肽待偶联完毕后，脱保护，得到相应的多肽。The specific method is to randomly couple amino acids one by one to the solid-phase resin by means of mixed splitting, and then remove the side chain protecting group under strong acid, and then carry out screening. Weigh 200 mg of Tentage1-S-NH2 resin, cycle according to the solid-phase peptide synthesis procedure, add 200 mg of HBTU of the same amount of amino acid, and react in turn for two cycles. After the coupling is completed, the peptide is deprotected to obtain the corresponding peptide.
FITC标记的多肽是通过氨基己胺将多肽与FITC相连。FITC-labeled peptides are linked to FITC via aminohexylamine.
所得多肽均通过MS，HPLC确证。The obtained peptides were confirmed by MS and HPLC.
4.通过表面等离子共振成像(SPR)方法检测多肽与VEGFR-3的亲和作用4. Detection of the affinity between peptides and VEGFR-3 by surface plasmon resonance imaging (SPR)
以固定RecombinantHumanVEGFR-3的通道作为检测通道，未固定RecombinantHumanVEGFR-3的通道作为对照通道进行亲和力分析实验，过程如下：(1)表面平衡：HBS-EP缓冲液，以10μL/min的流速平衡芯片表面5min；(2)表面活化：注射‘NHS+EDC’1∶1混合液，以10μL/min的流速活化芯片表面7min；(3)耦联蛋白：注射RecombinantHumanVEGFR-3(稀释在10mM醋酸钠(pH5.0)缓冲液中)，以10μL/min的流速耦联约7min；对照通道省略此步骤；(4)表面封闭：注射乙醇胺，以10μL/min的流速封闭表面7min。经双扣减(对照通道及零浓度)后，在BiacoreT200evaLuationsoftware中进行“1∶1Binding”模型的拟合分析数据，Kd值达到1.6nM，说明该CIQPFYP多肽与VEGFR-3亲和力较好。The channel with immobilized RecombinantHumanVEGFR-3 was used as the detection channel, and the channel without immobilized RecombinantHumanVEGFR-3 was used as the control channel for affinity analysis experiments. The process is as follows: (1) Surface balance: HBS-EP buffer, balance the chip surface at a flow rate of 10 μL/min 5min; (2) Surface activation: inject 'NHS+EDC' 1:1 mixture, activate the surface of the chip at a flow rate of 10μL/min for 7min; (3) Coupling protein: inject RecombinantHumanVEGFR-3 (diluted in 10mM sodium acetate (pH5 .0) in buffer), coupled at a flow rate of 10 μL/min for about 7 minutes; this step was omitted for the control channel; (4) Surface blocking: inject ethanolamine, and block the surface at a flow rate of 10 μL/min for 7 minutes. After double subtraction (control channel and zero concentration), the fitting analysis data of the "1:1Binding" model was performed in BiacoreT200evaLuationsoftware, and the Kd value reached 1.6nM, indicating that the CIQPFYP polypeptide has a better affinity with VEGFR-3.
5.FITC标记多肽与表达VEGFR-3的A549细胞和低表达VEGFR-3的HeLaL-O2的细胞亲和力5. Cell affinity of FITC-labeled polypeptides to A549 cells expressing VEGFR-3 and HeLaL-O2 expressing VEGFR-3
细胞培养：A549、HeLa、L-O2细胞，均培养在含10％FBS的高糖DMEM培养基中，置37℃培养箱，5％CO2隔天传代一次，待细胞长满80％镜下视野时用0.25％胰蛋白酶消化传代，取对数生长期的细胞用于后续实验。Cell culture: A549, HeLa, and L-O2 cells were all cultured in high-glucose DMEM medium containing 10% FBS, placed in a 37°C incubator, and passaged every other day in 5% CO2 until the cells were 80% full. Digested and passaged with 0.25% trypsin at the visual field, and took the cells in the logarithmic growth phase for subsequent experiments.
为评估多肽与细胞的亲和能力，我们将多肽进行FITC荧光标记，并选择VEGFR-3表达水平不同的3种细胞株，分别进行亲和实验。取一定密度的细胞，接种于六孔板中，24小时后用PBS洗去未贴壁的细胞后，加入一定浓度的FITC-多肽孵育1小时后，然后弃掉培养基，用冷PBS清洗，消化，混悬。采用MACSQuantAnalyzer10细胞流式仪检测FITC的信号，并利用Flowjo软件处理数据。In order to evaluate the affinity between the polypeptide and the cells, we labeled the polypeptide with FITC fluorescence, and selected three cell lines with different expression levels of VEGFR-3 to conduct affinity experiments respectively. Take a certain density of cells and inoculate them in a six-well plate. After 24 hours, wash off the unattached cells with PBS, add a certain concentration of FITC-polypeptide and incubate for 1 hour, then discard the medium and wash with cold PBS. Digestion, suspension. The signal of FITC was detected by MACSQuantAnalyzer10 cytometer, and the data was processed by Flowjo software.
6.FITC标记多肽对A549、HeLa、L-O2细胞的摄取能力6. The uptake ability of FITC-labeled polypeptide to A549, HeLa, L-O2 cells
将细胞按以上方法培养，按一定密度接种于35mm单孔皿，24小时用PBS洗去未贴壁的细胞后，加入一定浓度的FITC-多肽孵育1小时后，然后弃掉培养基，用冷PBS清洗两次细胞。用4％多聚甲醛溶液室温下固定细胞10分钟后用冷PBS清洗细胞两次，然后用DAPI对细胞核染色，用激光共聚焦显微镜观察多肽在细胞内的分布情况。Culture the cells according to the above method, inoculate them in a 35mm single-well dish at a certain density, wash off the unattached cells with PBS for 24 hours, add a certain concentration of FITC-polypeptide and incubate for 1 hour, then discard the medium, and use cold Cells were washed twice with PBS. Fix the cells with 4% paraformaldehyde solution at room temperature for 10 minutes, wash the cells twice with cold PBS, then stain the nuclei with DAPI, and observe the distribution of polypeptides in the cells with a laser confocal microscope.
7.MTT方法来检测优选多肽的细胞毒性7. MTT method to detect cytotoxicity of preferred polypeptides
将细胞按以上方法培养，按一定密度接种于96孔板。一天后，用含不同材料的200μL有血清培养基DMEM给细胞换液，培养24小时。24小时用PBS洗去未贴壁的细胞后，加入一定浓度的FITC-多肽孵育过夜。每孔中加入25μLMTT溶液，继续在37℃培养箱中培养4小时，用Bio-Rad96微孔读数仪在490nm读吸光值。细胞存活率用PBS组均一化。实验重复3次。The cells were cultured according to the above method, and seeded in a 96-well plate at a certain density. One day later, the cells were replaced with 200 μL serum-containing medium DMEM containing different materials, and cultured for 24 hours. After 24 hours, unattached cells were washed away with PBS, and a certain concentration of FITC-polypeptide was added to incubate overnight. Add 25 μL of MTT solution to each well, continue to incubate in a 37° C. incubator for 4 hours, and read the absorbance at 490 nm with a Bio-Rad96 microwell reader. Cell viability was normalized with PBS group. The experiment was repeated three times.
8.N-苄氧羰基-L-赖氨酸酸酐(Lys(Z)-NCA)的制备8. Preparation of N-benzyloxycarbonyl-L-lysine anhydride (Lys(Z)-NCA)
称取Lys(z)(苄氧羰基-赖氨酸)0.175g(0.591mmol)混悬于25mL无水THF，通入氮气除氧约10min后，加入适量三光气，50℃反应30min后，若反应液浑浊，则缓慢补加少量三光气，直至溶液澄清，将反应液倒入无水正己烷中，析出白色固体，抽滤后得到粗品，用无水THF/正己烷体系重结晶，得到纯品，产率98％。Weigh 0.175 g (0.591 mmol) of Lys(z) (benzyloxycarbonyl-lysine) and suspend it in 25 mL of anhydrous THF. After passing through nitrogen gas to remove oxygen for about 10 min, add an appropriate amount of triphosgene and react at 50 ° C for 30 min. If the reaction solution is turbid, slowly add a small amount of triphosgene until the solution is clear. Pour the reaction solution into anhydrous n-hexane to precipitate a white solid. After suction filtration, the crude product is obtained, which is recrystallized with anhydrous THF/n-hexane system to obtain pure Product, yield 98%.
9.Acetal-PEG-NH2(乙缩醛基-聚乙二醇氨基)的制备9. Preparation of Acetal-PEG-NH2 (acetal-polyethylene glycol amino)
将耐压管抽真空后，氮气保护，3，3-二乙氧基丙醇160μL(1mmol)和适量萘钾溶液溶于25mL无水THF后加入到耐药管中，然后将其放入-78℃冷却后，加入环氧乙烷溶液，氮气保护后转入室温下反应两天后，加入甲醇和冰醋酸终止反应，并用乙醚析出得Acetal-PEG-OH。称取AcetaL-PEG-OH1g溶于适量无水二氯甲烷中，随后向该溶液中加入三乙胺少量和对甲苯磺酰氯0.03g(0.25mmol)，室温搅拌24小时后，反应液体用1moL/LHCL溶液萃取三次，取有机相，并用无水硫酸钠干燥有机相，抽滤后加入乙醚析出，得到白色固体对乙缩醛基-聚乙二醇-对甲苯磺酸酯基(Acetal-PEG-OTs)。将乙醇上述产物溶于20mL无水DMF，加入少量邻苯二甲酰亚胺钾盐120℃条件反应6小时，减压蒸除DMF后，得到乙缩醛基-聚乙二醇-邻苯二甲亚氨基(Acetal-PEG-PI)。将所得产物溶于4mL和少量水合肼中，70℃条件下反应12小时，最后用乙醚析出沉淀，过滤得AcetaL-PEG-NH2。After evacuating the pressure tube, nitrogen protection, 160 μL (1 mmol) of 3,3-diethoxypropanol and an appropriate amount of potassium naphthalene solution dissolved in 25 mL of anhydrous THF were added to the drug-resistant tube, and then put into- After cooling at 78°C, add ethylene oxide solution, nitrogen protection, then transfer to room temperature for two days, add methanol and glacial acetic acid to terminate the reaction, and precipitate with ether to obtain Acetal-PEG-OH. Weigh 1g of AcetaL-PEG-OH and dissolve it in an appropriate amount of anhydrous dichloromethane, then add a small amount of triethylamine and 0.03g (0.25mmol) of p-toluenesulfonyl chloride to the solution, stir at room temperature for 24 hours, and then dissolve the reaction liquid with 1moL/ The LHCL solution was extracted three times, the organic phase was taken, and the organic phase was dried with anhydrous sodium sulfate. After suction filtration, ether was added to precipitate, and a white solid p-acetal-polyethylene glycol-p-toluenesulfonate (Acetal-PEG-p-toluenesulfonate) was obtained. OTs). Dissolve the above product in ethanol in 20 mL of anhydrous DMF, add a small amount of phthalimide potassium salt and react at 120°C for 6 hours, and distill off the DMF under reduced pressure to obtain acetal-polyethylene glycol-phthalate Acetal-PEG-PI. The obtained product was dissolved in 4 mL and a small amount of hydrazine hydrate, reacted at 70°C for 12 hours, and finally precipitated with diethyl ether, and filtered to obtain AcetaL-PEG-NH2 .
10.Acetal-PEG-PLL-NH2(乙缩醛基-聚乙二醇-聚赖氨酸)的制备10. Preparation of Acetal-PEG-PLL-NH2 (acetal-polyethylene glycol-polylysine)
以Acetal-PEG-NH2为大分子引发剂，将Lys(Z)-NCA和Acetal-PEG-NH2按一定比例溶于25mL无水DMF中，搅拌溶解，氮气反应72小时，反应液浑浊。加入大量乙醚，析出得到Acetal-PEG-PLL白色固体。将该固体溶于冰醋酸中，冰浴搅拌下加入大量的33％HBr/AcOH溶液，反应2小时后在无水乙醚中沉淀离心得到粗产物。再把粗产物加入到DMSO中溶解，直接装入到透析袋中透析，在去离子水中透析24小时后，转入到氨水中透析24小时，然后在pH5.0的HCL溶液中透析24小时，最后在去离子水中透析24小时，冷冻干燥处理得到Acetal-PEG-PLL-NH2固体。Using Acetal-PEG-NH2 as a macroinitiator, Lys(Z)-NCA and Acetal-PEG-NH2 were dissolved in 25mL of anhydrous DMF in a certain proportion, stirred and dissolved, and reacted with nitrogen for 72 hours, and the reaction solution was turbid. A large amount of diethyl ether was added, and Acetal-PEG-PLL was precipitated as a white solid. The solid was dissolved in glacial acetic acid, and a large amount of 33% HBr/AcOH solution was added with stirring in an ice bath. After 2 hours of reaction, the crude product was precipitated and centrifuged in anhydrous ether. Then add the crude product to DMSO to dissolve, put it directly into the dialysis bag for dialysis, after dialysis in deionized water for 24 hours, transfer to ammonia water for dialysis for 24 hours, and then dialyze in HCL solution with pH 5.0 for 24 hours, Finally, it was dialyzed in deionized water for 24 hours, and freeze-dried to obtain Acetal-PEG-PLL-NH2 solid.
11.Acetal-PEG-PLL-多肽胶束的制备11. Preparation of Acetal-PEG-PLL-polypeptide micelles
称取Acetal-PEG-PLL-NH220mg溶解在HAC缓冲溶液(pH4.0，HAC，8mL)，加入优选多肽15mg，反应5天后，将反应液装入到透析袋中透析，在PBS缓冲溶液(pH7.4)中透析24小时后，转入去离子水中透析24小时(上述透析过程中，每隔8h换一次透析外液)，冷冻干燥处理得到Acetal-PEG-PLL-多肽固体。将上述产品完全溶解于10mL的DMF后，在搅拌的条件下，缓慢滴加少量去离子水，继续搅拌1-2小时后，使聚合物与混合溶液达到溶解平衡，然后直接加入到透析袋中(截留分子量为3500Da)，在2L去离子水中透析48小时，每隔12小时换一次水除去DMF。然后使用水相滤头过滤(0.22μM)透析袋内液，过滤掉大分子沉淀和聚集的胶束颗粒，得到Acetal-PEG-PLL-多肽胶束。Weigh 20 mg of Acetal-PEG-PLL-NH2 and dissolve it in HAC buffer solution (pH 4.0, HAC, 8 mL), add 15 mg of the preferred polypeptide, react for 5 days, put the reaction solution into a dialysis bag for dialysis, in PBS buffer solution (pH7.4) after 24 hours of dialysis, transfer to deionized water for dialysis for 24 hours (during the above dialysis process, the dialysis fluid was changed every 8 hours), freeze-dry to obtain Acetal-PEG-PLL-polypeptide solid. After the above product is completely dissolved in 10mL of DMF, slowly add a small amount of deionized water dropwise under stirring conditions, and continue stirring for 1-2 hours to make the polymer and the mixed solution reach a dissolution equilibrium, and then directly add it to the dialysis bag (Molecular weight cut-off is 3500Da), dialyze in 2L deionized water for 48 hours, change water once every 12 hours and remove DMF. Then use the water phase filter head to filter (0.22μM) the liquid in the dialysis bag to filter out macromolecular precipitates and aggregated micelle particles to obtain Acetal-PEG-PLL-polypeptide micelles.
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