Sarcodonin类天然产物的结构优化及促神经轴突生长构效和作用机制研究

The enhancement of memory and learning by small molecules is of significant current interest to society. This approach could alleviate the course of neurodegenerative diseases such as Alzheimer's. Traditional medicinal resources constitute a valuable pool, in particular regarding the identification and synthesis of biologically active lead structures..1. In the previous study, we first demonstrated that both sarcodonin G and A, cyathane -type diterpene compounds isolated from the higher fungus Sarcodon scabrosus, possessed remarkable neuritogenic (neurite outgrowth-promoting) properties. In order to improve their bioactivities and to deeply elucidate mechanism of action, structural modifications of three positions C14, C15 and C19 on sarcodonin G and A are performed by using organic synthetic approaches to provide a chemical library of novel derivatives..2. Using PC12 cells and 1321N1 cells assays, evaluation of neuritogenic activities of the semisynthetic compounds will be conducted to find more active compounds..3. Structure–activity relationships of sarcodonin derivatives will be studied in detail to identify their active structural requirements..4. Effects of some potent active sarcodonin analogues on morphology of PC12 cells are carried out to examine..5. Effects of various specific kinases, such as the MAPK kinase (MEK) inhibitor PD98059, on neurite outgrowth from PC-12 cells induced by highly potent bioactive compound, will be tested by Western blotting analysis..6. Effects of bioactive compounds on intracellular cAMP levels in PC12 cells are determined..The aim of the present studies undertaken are to elucidate structure-activity relationships of this class of diterpenic compounds, mechanism of action of inducing significant neurite outgrowth in PC12 cells by the compounds with strong activity. These results are to lead to the discovery of potent neurotrophic compounds for blood brain barrier penetration and for the treatment of neurodegenerative conditions such as Alzheimer's.

申请人研究发现鸟巢二萜天然产物sarcodonin G 和A 具有显著增强神经生长因子（NGF）诱导神经轴突生长活性，而这些二萜化合物的分子结构和神经营养活性关系鲜见报道，以及它们对促NGF诱导神经轴突生长的作用机制尚不够清晰。为提高其生物活性，本项目通过大量分离sarcodonin、结合有机合成方法对其结构进行多位点改造和修饰以得到sarcodonin衍生物分子库；通过PC12细胞及1321N1细胞活性测定，筛选出具有高活性的促NGF介导的神经轴突生长活性的新化合物；探讨高活性化合物对NGF诱导PC12细胞轴突生长信号通路的影响。本研究目的在于探明sarcodonin类化合物增强NGF诱导细胞轴突生长的构效关系，从分子和细胞角度揭示高活性化合物调控NGF诱导细胞轴突生长的作用机制，为进一步研究神经发育与神经退行性疾病的生物学与生理学功能提供化学和药理学依据。

神经退行性疾病的发生与年龄密切相关，随着人口老龄化以及人口平均寿命的逐年增加，患有神经退行性疾病的人群越来越多。其中发病率最高的阿尔茨海默病(AD) 已被世界卫生组织列为21世纪严重危害人类健康的五大疾病之一。然而，目前的临床药物仅能缓解AD的症状。因此研发安全有效的AD防治药物，已成为关系国计民生的重大问题。.本研究半合成鸟巢烷型二萜sarcodonin系列衍生物60多个；从五种真菌发酵物中分离鉴定了65个化合物，包括40个新的sarcodonin类似物，首次发现含1,3二氧惡烷环的二萜和4,9-裂环二萜，并利用波谱技术、X－单晶衍射、化学反应以及园二色谱ECD方法确定了新化合物的分子结构和绝对构型。以PC12细胞为模型，筛选出40多个促NGF介导的神经突起生长活性化合物，探讨了活性化合物的构效关系；揭示了活性化合物erincine A通过TrkA/Erk1/2依赖途径促进NGF诱导PC12细胞突起生长；以PC12细胞和大鼠原代神经元为模型，首次发现了两个sarcodonin G含氟衍生物对神经突起表现相反的活性，促进突起生长（激动剂）和抑制突起生长（拮抗剂）作用，揭示了它们对NGF的受体Trek A具有双功能调节作用，它们可能结合于NGF与其Trk A受体组成的蛋白复合体上，对NGF激发的下游信号通路即PKC依赖的以及PKC不依赖的ERK/CREB产生不同的影响，使得PC12细胞的突起生长呈现不同的趋势；同时，LPS诱导的BV-2小胶质细胞炎症抑制活性显示，20多个化合物通过下调iNOS和COX酶显示抗神经炎作用。本研究为深入理解sarcodonin二萜的神经营养活性机制奠定基础，也为抗神经退行性疾病的药物设计提供了新思路。