基于双重光亲和标记策略的钩吻毒性成分作用靶标研究

Gelsemium elegans (Gardn. & Champ.) Benth., also known as "Duan-Chang-Cao", is a world-famous toxic plant. G. elegans poisoning accidents occurred frequently by eating or using by mistake and has become an urgent public health security problem now. Because of the unclear poisoning mechanisms, there are no specific and effective clinical treatments. More than 190 alkaloids were isolated from G. elegans and 14-hydroxygelsenicine was identified as the main poisonous component by our team, but the poisoning mechanisms and target remain unsolved. We developed Dual Photo-affinity labeling Strategy as a new method for improving the accuracy and efficiency of target-identification based on Photo-affinity labeling (PAL) in our previous study. Possessing high crosslinking yeild and efficiency of target-identification, this strategy has been successfully applied to target identification of natural products. As continuing research, this project intents to design probes of 14-hydroxygelsenicine based on Dual Photo-affinity labeling Strategy for toxicity target identification. This research will help to clarify the toxicity target and poisoning mechanisms of G. elegans and provide a valuable scientific basis for the development of antidotes and detoxification programs for G. elegans poisoning.

钩吻（又名“断肠草”）是一种剧毒的草药，民间因误食、误用所造成的钩吻中毒甚至死亡的事件时有发生，已成为亟需解决的公共卫生安全问题。钩吻的毒性机制不明确，尚无针对性的救治手段。本课题组前期从钩吻中分离鉴定了190余个生物碱类成分，并已明确14-羟基钩吻素己为钩吻的毒性成分，但其具体的作用靶标和毒理机制尚不清楚。本研究团队前期提出了基于经典光亲和标记技术（PAL）的双重光亲和标记策略，是一种可有效提高化合物靶标鉴定准确性的新方法，具有高标记效率及高靶标识别性，并已成功应用于天然产物的靶标识别研究中。本项目拟在前期研究基础上，通过采用双重光亲和标记策略构建14-羟基钩吻素己双重光亲和标记分子探针，并开展钩吻毒性靶标的识别与确证研究，以明确钩吻毒性成分的作用靶标，阐明其毒理机制，为钩吻中毒解毒剂的研发及解毒方案的制定提供科学依据。

钩吻（又名“断肠草”）是一种剧毒的草药，民间因误食、误用所造成的钩吻中毒甚至死亡的事件时有发生，已成为亟需解决的公共卫生安全问题。钩吻的毒性机制及作用靶标目前尚不明确，尚无针对性的救治手段。本项目在前期研究基础上，通过采用构建14-羟基钩吻素己双功能光亲和标记分子探针，开展钩吻毒性靶标的识别与确证研究，发现了一系列与呼吸作用相关的潜在作用靶标，并通过细胞标记、免疫荧光共定位、及蛋白质谱学、pulldown-western blot、CETSA等方法，对与NMDA关联密切的潜在靶标CAMKII进行了进一步确证，为钩吻中毒解毒剂的研发及解毒方案的制定提供了科学依据。