基于血管正常化"时间窗"理论的多靶标抗血管生成先导物发现与评价

Pathological angiogenesis plays a critical role in numerous diseases. Vascular normalization induced by anti-angiogenesis drugs appears to play an essential role in the intervention for pathological angiogenesis. However, the compensatory activation of pro-angiogenesis factors shortens the vascular normalization "time window" and induces the occurrence of resistance. We have identified a novel biphenyl-aryl urea incorporated with salicylaldoxime (BPS-7) as potent and selective VEGFR-2 inhibitor through structural optimization of taspine. Fortunately, BPS-7 also displayed selective inhibitory activities against angiogenesis-related EphB4 and TIE-2. Moreover, it has been validated that the three angiogenesis-related RTKs could be compensatory activated by each other as alternative pro-angiogenic pathways. Accordingly, we propose that multiplex inhibition of VEGFR-2, EphB4, and TIE-2 would enhance anti-angiogenesis effect on the basis of vascular normalization concept. Encouraged by previous results, this project will develop multiplex inhibitors of VEGFR-2/EphB4/TIE-2 based on their highly conserved DFG-out conformation. We initiate a medicinal chemistry program to identify the key structural elements required for the kinase inhibition. The design strategies of "hybridization" and "mix-n-match" will be adopted with BPS-7 as lead compound. All the title compounds possess structural diversity with salicylaldoxime as hinge-binding moiety. It is a medicinal chemistry campaign aimed at improving the potency and selectivity profile of BPS-7. In order to evaluate the potency and selectivity of the title compounds, various biological assays will be carried out. This project will identify the first class of multiplex inhibitors of VEGFR-2/EphB4/TIE-2 with a "triplet" inhibition profile. They can not only stabilize the vascular normalization "time window", but also prevent the occurrence of resistance. The normalized vasculature can lead to an improvement in the delivery and efficacy of therapeutic drugs. This project may contribute to the discovery of novel anti-angiogenesis agents for the intervention of pathological angiogenesis-related diseases.

病理性血管生成会导致多种疾病，抗血管生成药物诱导的血管正常化有重要治疗意义；但促血管生成代偿性通路的激活，会缩短血管正常化的"时间窗"并导致耐药性。课题组前期发现的水杨醛肟类VEGFR-2抑制剂BPS-7，对促血管生成的受体酪氨酸激酶EphB4、TIE-2也有选择性抑制并有初步的血管正常化诱导效应。本课题基于血管正常化"时间窗"理论以及VEGFR-2/EphB4/TIE-2可代偿性激活的发现，以BPS-7为新型先导物，分析三种受体活性位点的保守构象并寻找共性结构域；在水杨醛肟基础上采用"Hybridization"和"Mix-n-match"的策略，构建满足共性结构域构象要求的化合物库，通过多水平活性筛选发现同时拮抗三条代偿性通路的多靶标抑制剂，以期实现①稳定血管正常化的"时间窗"；②降低耐药性发生。在抗血管生成的同时促进血管正常化，增强药物转运和敏感性，为新型抗血管生成药物发现奠定基础。

在国家自然科学基金(81573285)资助下，本项目以秦巴山区特有药用植物红毛七中的活性生物碱为先导化合物，通过结构优化发现了水杨醛肟类化合物BPS-7具有抗血管生成活性。基于此，提出BPS-7可作为血管生成调控功能分子研究的新型先导物。在此基础上，以水杨醛肟类BPS-7为先导物，开展了新一轮的结构优化研究。基于VEGFR-2、EphB4、TIE-2可代偿性激活的发现，分析三种受体活性位点的保守构象并寻找共性结构域；采用"Hybridization"和"Mix-n-match"策略，构建满足共性结构域构象要求的化合物库，通过多水平活性筛选发现同时拮抗三条代偿性通路的多靶标抑制剂。通过系统的生物活性评价，发现了一系列具有抗血管生成活性的化合物。特别是，在筛选过程中发现了有明确血管正常化诱导效应的喹唑啉酮化合物。深入研究发现：喹唑啉酮类活性分子QDAU5可显著提高血管内皮细胞和周细胞的覆盖率，降低血管渗透性，改善肿瘤组织缺氧状态，诱导肿瘤的异常血管趋于正常。利用转基因斑马鱼Tg(Flk-1:GFP)模型筛选发现：QDAU5能够促进低氧所致异常血管的正常化进程，逆转低氧引起的血管损伤。因此，喹唑啉酮类活性分子具有明确的血管正常化诱导效应，通过与EphrinB2的特异性亲和并抑制其磷酸化，发挥促血管正常化的活性，EphrinB2可能是喹唑啉酮活性分子的作用靶标。综上，本课题的研究结果，为血管正常化药物的发现提供了新靶标和新型先导物。. 课题组经过四年的深入研究，取得了一系列创新性成果，获陕西省科学技术二等奖和陕西高等学校科学技术一等奖各1项。发表SCI论文15篇（第一标注），其中11篇为JCR一区论文，影响因子大于5的论文有5篇，有8篇论文发表在药物化学权威期刊European Journal of Medicinal Chemistry上。申请国家发明专利16项，获得授权8项。课题组成员中1人晋升为教授，3人晋升为副教授，培养1名博士生、9名硕士生，有5名研究生获国家奖学金，4篇论文获评优秀硕士学位论文。在课题研究过程中，多次邀请国内外知名专家进行课题的指导与座谈交流，课题组成员也多次参加国内学术会议进行交流，促进了国内外学术交流。

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