四种罗汉松科植物中STAT3抑制剂的发现及抗肿瘤活性研究

The constitutive activation of JAK2-STAT3 signaling pathway is crucial for the proliferation, anti-apoptosis, angiogenesis, metastasis, and immune escape of cancer cells. Inhibition of STAT3 is one of the effective strategies for targeted cancer therapy. In the preliminary study, we have obtained a series of diterpenoids from plants, established a novel three-tier screening strategy for STAT3 inhibitors, and discovered that K11, a derivative of the diterpenoid from Podocarpus macrophyllus, exhibited significant STAT3 inhibiting activity. Based on the above results, the aims of the present project are listed as follows: i) To screen the diterpenoids we have obtained in previous work and try to clarify their SAR and michanisms of action. ii) To separate the extracts of Podocarpus macrophyllus var. angustitolius, Nageia nagi, and N. wallichiana and rapidly obtain the novel bioactive metabolites and analogs under the guidance of STAT3-inhibiting assays. iii) To design and prepare the derivatives of the active compounds by computer-aided drug design, analyze the structure-activity relationship of STAT3 inhibition, and obtain the most potent one. iv) To test the anticancer, anti-angiogenesis, and/or anti-metastasis activity of the active compound, investigate its inhibitory activity on the JAK2-STAT3 signaling pathway, and clarify its michanism of action. v) To evaluate the in vivo anticancer activity of the active compound in tumor-bearing animals administered singly or in combination with other drugs. The results of this project will make basis for the discovery of new anticancer drugs.

JAK2-STAT3信号通路的持续激活对肿瘤细胞增殖、抗凋亡、新生血管生成、转移和免疫逃逸等功能至关重要，抑制STAT3是肿瘤靶向治疗的有效策略之一。前期研究完成了罗汉松成分研究，获得了一批二萜化合物，建立了STAT3抑制剂的筛选方法，并发现罗汉松二萜衍生物K11具有显著的STAT3抑制活性。本项目拟在前期的基础上，综合利用3种已建立的STAT3抑制剂筛选技术，首先加强已获得的二萜的筛选与活性研究；同时以活性筛选为指导，对筛选出有活性的狭叶罗汉松、竹柏和肉托竹柏等植物进行研究，主动发掘其中具有STAT3抑制活性的成分；以计算机辅助分子对接为指导，进行活性化合物的设计与结构修饰，开展重要代表化合物的抗肿瘤活性与构效关系研究；研究并阐明其通过抑制JAK2-STAT3通路发挥抗肿瘤作用的分子机制。为进一步认识JAK2-STAT3通路的调控特点，发现作用机制明确、具有自主知识产权的新药奠定基础。

本项目以活性为指导，采用各种分离技术对罗汉松(Podocarpus macrophyllus)、狭叶罗汉松(Podocarpus macrophyllus var. angustitolius)、竹柏(Nageia nagi)和肉托竹柏(Nageia wallichiana)、短叶罗汉松(Podocarpus chinensis)、长叶竹柏(Nageia fleuryi)、思茅松(Pinus kesiya var. langbianensis)和大穗野桐 (Mallotus macrostachyus)进行了活性成分研究与发掘，共分离获得各类化合物252个，采用NMR和HRMS等波谱学技术鉴定了其结构，其中包括二萜类化合物94个，新化合物29个。对前期发现有活性的罗汉松二萜化合物进行了富集，并对其进行了结构修饰，制备二萜衍生物41个。对分离获得的化合物及二萜衍生物进行了抗肿瘤活性测试，初步总结了其构效关系，筛选获得了强活性的化合物。对经筛选发现有强的肿瘤细胞增殖抑制活性的二萜衍生物3a和3d进行了抗肿瘤活性研究，发现化合物3a可使肿瘤细胞阻滞在G0/G1期，并能诱导肿瘤细胞凋亡，进一步研究发现该类化合物可抑制STAT3磷酸化和二聚体形成，进而抑制JAK2/STAT3信号通路的转录激活活性。上述研究工作的完成，丰富了对罗汉松科植物的认识，对阐明这几种植物的活性成分和新型抗肿瘤药物的发现具有重要意义。