β-咔啉类生物碱对果蝇雌性生殖干细胞分化Hedgehog信号通路的调控机制及分子优化

The regulation of small molecular compounds on the differentiation of insect germline stem cells has become a global research frontier, which brings novel targets in the research and development of new pesticides. In previous study, we found that natural product β-Carboline alkaloid and its modified chemicals could inhibit the differentiation of Drosophila female germline stem cell (fGSC) and the regulation of Hedgehog signaling pathway. However, the specific mechanism remains unclear, which results in the lack of the theoretical guidance for molecular structure optimization. In this project, the relationship among temperature, time and dosage in treatments is determined by Drosophila melanogaster as the model insect and α- spectrosome as a marker protein. In order to determine the functional target, the regulatory effect of β-Carboline alkaloids on the expression of Hh signaling pathway in Drosophila fGSC is further studied by hybridization, immunofluorescent histochemical staining, laser confocal scanning and CoMSIA techniques. Additionally, the chemical structure is modified using the 3D model of target protein, molecular docking and virtual screening of highly effective compounds while the structure-activity relationship between serial chemicals and target protein is studied by protein fluorescence quenching. Furthermore, highly effective compounds are synthesized after molecular structure optimization and their activities are verified by the inhibition of Drosophila fGSC differentiation and oviposition. Taken together, the prospective results would enrich our theoretical knowledge of insect toxicology and stem cells as well as provide novel insights and mechanical basic for developing β-carboline insect growth regulator in practice.

小分子化合物调控昆虫干细胞分化是昆虫毒理学研究新领域。前期研究发现天然产物β-咔啉类生物碱及改造物可抑制果蝇雌性生殖干细胞(fGSC)分化和调控Hedgehog信号通路，但机理不清，化合物合成改造缺乏靶标引导。本项目以α- spectrosome为标记蛋白，以黑腹果蝇为试虫，在确定化合物处理的适宜剂量与时间的基础上，运用遗传杂交、免疫组织化学、激光共聚焦、CoMSIA等技术，研究β-咔啉类生物碱对果蝇fGSC分化Hh通路成分表达的调控作用，调控Hh通路与其他主要信号通路的互作关系，明确作用靶标；建立靶标蛋白3D模型，虚拟筛选化合物，以分子对接研究指导合成系列化合物，研究与靶标蛋白结合、抑制fGSC分化和产卵抑制的构效关系；优化分子结构，合成验证活性。本项目理论上有助于丰富昆虫毒理学研究内容，促进开展农业昆虫干细胞研究，实践上有助于推动β-咔啉类新型昆虫生长发育调节剂创制率先取得突破。

小分子化合物调控干细胞分化是当前研究热点，具有巨大的应用潜力。本项目以α- spectrosome为标记蛋白，以黑腹果蝇、斜纹夜蛾和草地贪夜蛾离体培养细胞和活体昆虫作为试材，运用遗传杂交、免疫组织化学、激光共聚焦显微观察、CoMSIA等技术，在确定处理剂量与时间的基础上，明确了β-咔啉类生物碱抑制果蝇fGSC分化的时间-剂量-效率关系以及与产卵的相关性；研究了β-咔啉类生物碱、印楝素、喜树碱、姜黄素对果蝇fGSC分化Hh通路成分Hh、Smo等表达的调控作用，探讨了Hh通路与Dpp、Bam、EGFR等主要信号通路的互作关系，发现Harmine处理可引起Hh途径与EGFR途径协同增效互补的作用；明确Smo蛋白是β-咔啉类生物碱作用于Hh通路的主要靶标，建立靶标蛋白3D模型，虚拟筛选化合物，以分子对接优化分子结构，通过全新药物设计合成筛选优化了β-咔啉类生物碱先导化合物2个。本项目理论上有助于丰富昆虫毒理学研究内容，促进开展农业昆虫干细胞研究，实践上有助于推动β-咔啉类新型昆虫生长发育调节剂创制率先取得突破。项目获得化合物授权专利2件，发表SCI论文12篇，培养研究生5名。