抗肿瘤新颖安莎NA1的作用靶蛋白、结构优化和成药性研究

Ansamycins belong to type I polyketides. Most members of this family have broad bioactivities, which afford high ratios of new drugs. Previously, our group isolated nine new ansamycins from marine Actinomycetes. Among these compounds, NA1 is a new backbone naphthoquinone-type ansamycin. Our preliminary biological experiments indicated that NA1 induced G2/M cell cycle arrest and apoptosis in MDA-MB-435 cells through activating NOXs to generate reactive oxygen species (ROS). It is well known that the druggability of bioactive natural products is not desirable due to the limits of solubility, bioavailability and side effects. Therefore, this project is going to investigate the mechanism of action of NA1 inducing the production of ROS, and to design and synthesize potent derivatives of NA1 with good druggability. Firstly, it should be defined the main cellular target of NA1 in activating NOXs. Then, NA1 will be structurally modified and optimized to afford a focus chemical library containing more than 300 compounds. Thirdly, these compounds will be screened by various bioassays at the levels of molecule, cell and animal tests, which will afford 2-3 potent antitumor derivatives of NA1 with high specificity. Finally, the druggability of 2-3 potent antitumor derivatives will be studied, which shall put forward a base for the drug development of ansamycins, and be significant to prevention and chemotherapy of oncology.

安莎类化合物属于I型聚酮，其成员大多具有广泛的生理活性，成药率高。前期研究中，本课题组从海洋放线菌中分离得到了9个新安莎，其中NA1是新骨架萘醌类安莎。初步生物学实验表明，NA1可能激活NADPH氧化酶(NOXs)诱导MDA-MB-435细胞产生活性氧(ROS)，从而引起细胞G2/M期停滞和凋亡。众所周知，活性天然产物由于溶解性、生物利用度和毒副作用等局限，成药性不理想。本项目将力图阐明NA1诱导ROS产生的机制；并针对成药性进行NA1的结构优化。首先明确NA1激活NOXs的作用靶蛋白；其次合成8个系列约300个NA1衍生物；然后对目标分子进行细胞、分子和实验动物水平上的活性筛选，以期发现2-3个活性强、选择性高的衍生物；最后开展2-3个NA1衍生物的成药性初步评价，为研发新型、高效低毒的安莎类抗肿瘤候选药物奠定基础，对肿瘤的预防和治疗具有重要意义。

安莎类化合物属于I型聚酮，其成员大多具有广泛的生理活性，成药率高，其中最著名的有利福霉素、格尔德霉素和美登木素。前期研究本课题组从海洋放线菌中分离得到了新安莎NA1。初步生物学实验表明，NA1可能激活NADPH氧化酶(NOXs)诱导MDA-MB-435细胞产生活性氧(ROS)，从而引起细胞G2/M期停滞和凋亡。本项目研究结果证明NA1激活NOXs的作用靶蛋白是小GTPase Rac1，NA1通过结合Rac1激活NOXs诱导MDA-MB-435细胞产生ROS，从而引起细胞G2/M期停滞和凋亡；针对其溶解性较差、生物利用度较低和毒副作用，设计合成了7个系列约164个新安莎衍生物；细胞、分子和实验动物水平的活性筛选结果表明，F33和F36具有优秀的体内外抗肿瘤活性；体内肝毒性评价结果表明，F33注射组小鼠的AST和ALT水平均显著地低于GA组（P < 0.05）；作用机制研究结果表明，F33通过下调EMT转录因子Zeb1、Snail和Slug，同时上调黏附蛋白E-cadherin，从而对前列腺癌细胞的增值和转移表现出抑制活性；裸鼠体内抗LNCaP和DU-145细胞移植模型实验结果表明，化合物F33可以抑制肿瘤细胞LNCaP和DU-145的转移，可以作为潜在的抗前列腺癌候选新药；开展了NA1衍生物的成药性初步评价，为研发新型、高效低毒的安莎类抗肿瘤候选药物奠定基础；另外，系统开展了新安莎的深度挖掘，通过启动子置换、负调控基因的敲除和途径专一性正调控基因的过表达，从7株放线菌分别分离得到45个新安莎，其中包括3类新骨架，并且阐明新安莎NA1的生物合成机制，为安莎的萘环形成提供了的直接证据；还发现了鼠伤寒沙门氏菌III型分泌系统新天然产物抑制剂ansavaricin E、WG-15、WG-17和nocazine F。