基于靶向荧光探针的中药表观遗传调控物质发现方法研究

The aim of the present project is to resolve the scientific problem related to how to discover the epigenetic modulators from traditional Chinese medicines (TCMs). The applicant propose the design of fluorescent probes for targeting histone modifications by integrated use of multi-discipline techniques from analytical chemistry, material science and molecular biology. The synthesis of targeted fluorescent probes with ‘turn-on’ characteristics are achieved by conjunction of specific recognized peptide of histone modifications to the core molecules with aggregation-induced emission property. Those reversible histone modifications include acetylation and deacetylation of lysine, methylation and methyltransfer of arginine. The probes will be utilized to develop appropriate approaches to screening epigenetic modulators from traditional Chinese medicine. By the application of this novel technique, active compounds with regulatory effects on histone modifications will be identified from TCMs with tonifying Qi and Yin efficacy as well as clearing heat efficacy based on molecular, cellular and zebrafish models. To reveal the relationship between pharmacological mechanisms and traditional efficacy of those active compounds, regulatory patterns on histone modifications of different kinds of compounds from varied TCM will be compared and summarized. Thus, the mechanism of action of TCMs might be illustrated based on their effects on alleviating abnormal epigenetic modifications and regulating relevant gene transcription. It will contribute great scientific and scholar values for the development of basic theory of integrative Chinese medicine, as well as for the establishment of novel methodology for discovering modern Chinese medicine.

本申请围绕如何筛选发现中药表观遗传调控物质这一科学问题，以靶向组蛋白修饰的荧光探针设计为切入点，综合运用分析化学、材料学、分子生物学等多学科技术手段，从检测组蛋白乙酰/去乙酰化、甲基化/去甲基化等可逆修饰入手，以聚集诱导发光分子为母核，连接特异性识别肽段，设计合成具有“开—关”效应的靶向荧光探针，进而构建适用于中药药效物质筛选的表观遗传调控物质发现新方法。运用该方法在分子—细胞—模式生物三个层次，筛选益气养阴及清热活血类方药中具有组蛋白修饰调控活性的物质，并进一步研究不同类型药效物质的组蛋白修饰调控作用谱，从而研究发现不同功效与表观遗传修饰的相关性，从调节表观遗传修饰影响基因转录的角度阐释中药药效物质的作用机制。这对于中西医结合基础理论的创新发展及建立现代中药创制方法学均具有重大科学意义及学术价值。

表观遗传修饰异常被认为是心血管疾病、肿瘤等复杂性疾病发生发展的重要因素，已成为新药发现的一类重要靶点。本项目根据领域研究进展，围绕中药是否能有效调控表观遗传修饰、如何快速发现具有表观遗传调控作用的中药药效物质等科学问题开展研究，重点构建适合于中药复杂体系的表观遗传相关靶点筛选方法，从而快速、高效地寻找具有表观遗传调控作用的中药活性物质。本项目已按项目计划书完成研究任务。取得的主要成果为：①研究设计合成了组蛋白去乙酰化酶SIRT3、HDAC1特异性识别的底物肽段，分别构建了特异、灵敏的SIRT3、HDAC1活性荧光探针及体外快速筛选方法；②通过整合荧光探针和高内涵细胞成像技术，从益气养阴类中成药中筛选出来源于制何首乌的大黄素和来自于甘草的甘草异黄酮A可靶向激活SIRT3从而抗心肌肥大；此外，从中药化合物库中筛选发现了人参皂苷Rk3、原儿茶酸等具有抑制HDAC1活性的中药药效物质；③围绕表观遗传修饰相关的重要靶点干扰素刺激反应元件（ISRE），建立了一种知识挖掘与高通量筛选相结合的仲景方活性成分发现方法，筛选出显著激活ISRE的18个活性组分和3个活性化合物（芒柄花苷、黄芪皂苷I、亥茅酚苷），并验证了活性化合物调控p65磷酸化水平和核转位作用，为依据传统功效从方剂组分库中高效筛选发现中药活性物质提供了新思路；④构建心肌细胞肥大模型、主动脉弓狭窄和异丙肾上腺素诱导心力衰竭动物模型，从分子-细胞-动物水平多层次揭示大黄素、甘草异黄酮A通过激活SIRT3信号通路、抑制氧化应激等多途径抑制心肌肥大的作用机制。上述研究结果均证明了本项目的科学假说，即中药药效物质可通过调控表观遗传相关关键酶活性从而产生药效作用。项目研究成果发表SCI论文11篇，包括Pharmacol. Res.、Chem. Comm.等国际高水平期刊论文5篇，申请国家发明专利1项，相关研究论文已被Cir. Res.等杂志正面引用。相关研究成果获得中华中医药学会科学技术二等奖1项，列入中华中医药学会临床应用专家共识1项。