吡喹酮衍生物DW-3-15靶向组蛋白乙酰转移酶（HAT）抗血吸虫的分子机制

Praziquantel is the only available effective anti-Schistosoma japonicum drug, and the long-term use of praziquantel as preventive chemotherapy increases the risk for the development of drug resistance. Therefore, the search for alternative chemotherapeutic agents is urgently needed. In our previous study, we have found a praziquantel derivative DW-3-15 exhibiting activities against both juvenile and adult Schistosoma japonicum. In the following studies, we have demonstrated that after treatment with PZQ and DW-3-15, there were significant difference in both morphology and ultrastructure of adult worms. In addition, DW-3-15 has no cross-resistance to PZQ-resistant worms induced by sub-lethal does of PZQ, and cytochalasin D which can completely block the anti-schistosomal effect of PZQ in vitro, cannot antagonize the effect of DW-3-15, indicating that the target of DW-3-15 is different from PZQ. Thereafter, tandem mass tags (TMT) for relative quantitation the protein profiles were used to assess the differentially expressed protein in the adult worm treated with DW-3-15, compared with the untreated normal adult worm. The results showed that the histone acetyltransferase (HAT) and ribosomal proteins of S. japonicum were significantly down-regulated after treatment with DW-3-15. It is noteworthy that the change of ribosomal protein expression profile can significantly affect the development and life process of S. japonicum. Moreover, HAT can remodel the structure of chromatin, and regulate the transcription and expression of the target genes. Hence, we hypothesize “DW-3-15 plays anti-schistosomal effect by targeting the HAT to alter the acetylation status of histone, then to regulate the transcription and expression of ribosomal protein related genes.” In this study, we will focus on the hypothesis and clarify the mechanism of DW-3-15 for anti-Schistosoma japonicum, which will provide scientific basis for development of new anti-schistosomal drug.

吡喹酮是抗日本血吸虫的惟一有效药物，长期使用吡喹酮，其抗药性已成为业内关注的热点问题，寻找抗血吸虫新药势在必行。本课题组研发的吡喹酮衍生物DW-3-15兼具抗日本血吸虫童虫和成虫效应。前期研究发现经吡喹酮和DW-3-15作用后，虫体形态及超微结构均有显著差异，且DW-3-15对经吡喹酮亚治疗剂量诱导的耐受虫体无交叉抗性，可完全拮抗吡喹酮杀虫效应的细胞松弛素亦不能完全拮抗DW-3-15的杀虫作用，提示二者靶点不一致。经TMT定量蛋白质组表达谱分析，DW-3-15处理后的虫体HAT及核糖体相关蛋白均显著下调。核糖体蛋白表达谱的改变可显著影响虫体的发育和生命进程，而HAT可重塑染色质结构，调控相关基因的转录和表达。据此提出假设：“DW-3-15通过靶向HAT影响组蛋白的乙酰化，调控核糖体蛋白相关基因的转录和表达，发挥抗血吸虫效应”，拟阐明DW-3-15的抗虫机制，为研制抗血吸虫新药提供科学依据。

寻找抗血吸虫新药一直是血吸虫病防治工作中的焦点问题。课题组前期研发的吡喹酮衍生物DW-3-15（ZL201110142538.2）兼具抗日本血吸虫童虫和成虫效应，前期研究发现经PZQ和DW-3-15作用后，虫体形态结构变化具有显著差异。且DW-3-15对经PZQ亚治疗剂量诱导的耐受虫体无交叉抗性，可完全拮抗PZQ杀虫效应的细胞松弛素亦不能完全拮抗DW-3-15的杀虫效应，提示二者作用靶点不一致。将PZQ与DW-3-15联合应用，对日本血吸虫成虫和童虫的杀虫效应具有协同增效作用，再次表明二者作用靶点不一致。应用TMT定量蛋白质组表达谱分析技术定量检测经DW-3-15作用后的成虫与正常成虫的差异表达蛋白，发现经DW-3-15处理后的虫体组蛋白乙酰转移酶(SjHAT)及核糖体相关蛋白均显著下调。核糖体蛋白表达谱的改变可显著影响虫体的生命进程，而SjHAT可重塑染色质结构，调控相关基因的转录和表达。因此提出假设：DW-3-15通过靶向HAT影响组蛋白的乙酰化，调控核糖体蛋白相关基因的转录和表达，发挥抗血吸虫效应。基于此，本项目首先通过体内外实验证明SjHAT的转录水平和蛋白表达水平随DW-3-15浓度增高而显著下降，显示SjHAT的下调与虫体死亡率有直接关系。其次，成功筛选到能够显著抑制SjHAT的抑制剂——姜黄素。姜黄素体外作用于不同发育阶段的虫体后，其SjHAT转录水平、虫体形态变化和虫体死亡率均显示出与DW-3-15相似的生物学效应。应用RNA干扰技术特异性的敲低SjHAT，虫体表型与DW-3-15作用后的部分表型相似，为SjHAT作为潜在药物靶点提供了直接证据。最后，通过体内外实验证实DW-3-15能够显著下调日本血吸虫成虫组蛋白H4乙酰化水平。结合前期研究结果，进一步推断经DW-3-15作用后，通过下调日本血吸虫SjHAT，抑制成虫组蛋白H4乙酰化，进而发挥杀虫作用。