UGT1A1抑制剂高效筛选与评价方法的建立及其在中药安全性评价中的应用

Inhibition on bilirubin metabolizing enzyme UGT1A1 is one of the most important reason for bilirubin metabolism imbalance and drug-drug interactions. Several Chinese herbs can induce hyperbilirubinemia, liver injury and herb-drug interactions via UGT1A1 inhibition. It is urgently needed to establish a fast and accurate method for rapid screening and characterization of UGT1A1 inhibitor from herbs in both academy and industry. Following our previous works, this project aimed to construct a screening platform for UGT1A1 inhibitors by using our self-developed fluorescence probe substrates for UGT1A1, which could be applied in complex biological systems. The platform will achieve a multi-system screening and precise evaluation of UGT1A1 inhibitors from tissue preparation to live cell and then to living organism. Meanwhile, the fingerprinting techniques combined with bioactivity-based assays will be used to construct a new system for screening and discovery of natural UGT1A1 inhibitors from herbal extract. All these newly developed platforms will be applied in high-throughput screening for UGT1A1 inhibitors from Chinese herbs, as well as screening and characterization for natural UGT1A1 inhibitors. This project is expected to enhance the overall levels of herb-drug interactions associated studies in China from both technical and theoretical aspects. Furthermore, this project will provide a scientific basis and research standard for selecting the essence and discard the dross from the Chinese medicine formula and guiding for rational application of herbs in clinic.

药物对胆红素代谢酶UGT1A1的抑制是引发胆红素代谢障碍和药药相互作用的一个重要原因。部分中草药可通过抑制UGT1A1引发高胆红素血症、肝功能异常及草药-药物相互作用等安全隐患，业界迫切需要构建适于中药等复杂研究对象的UGT1A1抑制剂的高效筛选与精准评价体系。本项目拟在前期工作基础上，借助自主研发的UGT1A1特异性荧光探针底物构建适于复杂生物体系的UGT1A1抑制剂筛选与评价方法，实现从组织制备物→活细胞→活体的多层次系统筛选和精准评价；同时采用中药化学指纹谱和抑制效应谱相结合的策略，构建谱效结合导向下的天然UGT1A1抑制剂的发现与评价体系，并将其用于中药对UGT1A1抑制作用的规模化筛选、天然UGT1A1抑制剂的高效发现及抑制作用的精细表征等研究。该项目有望从技术和理论两个层面提升我国中草药-药物相互作用研究的整体水平，同时为中药方剂的去粗取精及临床合理用药提供科学依据和研究示范。

项目背景：抑制UGT1A1是引发胆红素代谢障碍和药物相互作用的一个重要原因，但长期以来，业界一直缺少高效精准评价中草药对UGT1A1抑制作用的方法和风险预警体系。.主要研究内容：.1）.UGT1A1特异性荧光探针底物NHPN的设计研发.2）.适于复杂生物体系UGT1A1抑制剂高效筛选与评价方法的构建.3）.中药提取物/成分对UGT1A1抑制作用的规模化筛选.4）.谱效结合导向下发现天然UGT1A1抑制剂.5）.天然UGT1A1抑制剂的抑制机制研究和引发药物相互作用的风险预警.重要结果及关键数据：.1）设计研发了属性优良的UGT1A1荧光探针底物NHPN，实现了复杂生物体系（包括组织制备物、活细胞、活体）中UGT1A1活性的实时动态检测和精准分析，为UGT1A1相关的基础和应用研究提供了工具分子。.2）基于LC-FD构建了适于复杂生物体系的UGT1A1酶活检测新方法，其对UGT1A1的检测限达0.16 μg/mL。该方法被成功用于活细胞/活组织及组织制备物中UGT1A1的活性检测及UGT1A1抑制剂/诱导剂的高通量筛选。.3）完成了百余种中药提取物对UGT1A1抑制作用的规模化筛选。采用谱效结合的策略，揭示了银杏叶提取物、双黄连注射液、麦冬等中药抑制UGT1A1的效应物质和作用机制，并对其引发药物相互作用的风险进行了评估。.4）开展了百余种天然产物对UGT1A1酶的抑制作用和诱导作用研究，细致总结了黄酮类化合物-UGT1A1抑制的构效关系；发现部分黄酮类化合物还可显著诱导UGT1A1并揭示了其诱导机制。.5）设计研发了UGT酶广谱荧光底物4-HN-335，借助该广谱底物构建了基于微孔板酶标仪的荧光检测方法，可同步筛选和评价药物对十种人源UGT酶的抑制作用。.6）发表论文56篇，其中SCI论文54篇；申报国家发明专利2项。.科学意义：.1）通过研发属性优良的UGT1A1特异性荧光底物NHPN，实现了复杂生物体系中UGT1A1活性的高效检测和实时精准分析，为UGT1A1相关药理和毒理学研究提供了强有力的工具。.2）构建了谱效结合导向下的天然UGT1A1抑制剂的发现与评价体系，并用于中药对UGT1A1抑制作用的规模化筛选、天然UGT1A1抑制剂的高效发现及抑制作用的精细表征等。相关研究为UGT1A1介导的中药-药物相互作用提供了关键技术和研究示范。