多级靶向纳米粒在PAKs抑制剂中的应用及其抗肿瘤机制的研究

p21-Activated kinase 4 (PAK4) executes key functions in tumor invasion and metastasis，and thus emerges as a promising target of anti-tumor therapy. In our previous studies, the protein-ligand interactions, virtual library screening and biological assay were investigated for PAK4 inhibitors discovery. In this project, our aim is to design more potent and selective PAK4 inhibitors by means of techniques from areas of computational chemistry, molecular nanotechnology, cell biology. The studies will be focused on solving the following scientific problems: (1) modification of the lead compound for generating more diverse structures according to the PAK4 active site features, and prediction of the activities and drug-likeness through molecular docking and ADME/Tox evaluation; (2) The structures with good records of predictive activities and drug-likeness will be synthesized for further biological assay to validate the inhibitory effects; (3) 3D-QSAR model will be constructed to deeply investigate the structure-activity relationships of inhibitors; (4) The inhibitors with potent activities will be bonded to RGD modified polymer chain and prepared self-assembled nanoparticles for further research of the in vitro and in vivo anti-tumor activity to study the multistage targeted antitumor mechanism. Thus, a set of small-molecule PAK4 inhibitors and their polymer drug conjugates with novel chemical structures, potent activities should be obtained, which would lay a foundation for the new targeted anti-tumor drugs with independent intellectual property rights.

PAK4激酶在肿瘤细胞侵袭转移中具有重要作用，已成为肿瘤治疗的重要靶点。前期工作中，通过计算机模拟技术，针对PAK4进行了大量分子设计工作，发现具有PAK4抑制作用的小分子化合物。在此基础上，针对现有抑制剂结构无靶向性、活性无选择性等缺点，鉴于此靶点的突出作用，本研究拟解决如下几个科学问题：（1）分析靶酶活性位点特征，设计新的抑制剂结构，预测其活性及成药性；（2）对所合成的化合物进行抗肿瘤生物学活性测试；（3）建立3D-QSAR模型，深入探讨化合物的构效关系；（4）将活性突出的PAK4抑制剂通过化学键连接到RGD修饰的具有特定功能的高分子链上形成高分子键合物，自组装成纳米粒，进行体外和体内的抗肿瘤活性测试，以研究和探讨多级靶向抗肿瘤作用机制。通过本研究得到化学结构新颖和生物活性突出的小分子PAK4抑制剂及其高分子键合物，为创制拥有自主知识产权的新型靶向抗癌药奠定重要基础。

p21活化激酶4(p21-activated kinase 4，PAK4)是丝氨酸/苏氨酸蛋白激酶，参与体内多条信号通路，影响肿瘤细胞的增殖、存活、侵袭转移及凋亡，对肿瘤的发生发展起着重要作用。近年来，PAK4 成为抗肿瘤药物研发的新靶点，多种类型的PAK4抑制剂已被发现。本项目基于前期计算机模拟，明确了PAK4激酶活性位点的空间、电性、结构等特征，通过对虚拟筛选得到的苗头化合物分子和文献报道的高活性分子，进行结构改造、衍生化，设计并合成了3类50余个新型PAK4抑制剂，得到的衍生化合物中乙炔环戊醇类化合物对PAK4的抑制活性较强IC50=182.1nM，通过激酶选择性筛选实验发现，该化合物对II类PAKs具有很好的抑制活性，尤其对PAK4的抑制活性最高，具有明显的激酶选择性，通过细胞生物学机制研究和荷瘤nu/nu裸鼠体内药效学考察，明确了该化合物具有抑制非小细胞肺癌A549肿瘤增殖促进凋亡的作用，此类化合物具有进一步深入研究的价值。本项目还构建了cRGD和FA双靶向长循环脂质体，用于包载高活性PAK4抑制剂PF3758309，该递药系统具有明确的肿瘤靶向性，通过体外药效和荷瘤nu/nu裸鼠体内药效学考察，表示该递药系统有助于提高原药的药效并降低其毒副作用，该多级靶向纳米递药系统用于小分子靶向抑制剂的抗肿瘤作用研究具有研究意义和开发前景。