合成EZH2/LSD1双靶点抑制剂作为干预表观遗传的抗肿瘤新策略

Epigenetics regulates gene transcription without altering DNA sequences. Among epigenetic modifications, histone methylation plays a crucial role in malignant tumors. EZH2 and LSD1 are histone lysine methyltransferase and demethylase respectively, and they are crucial for the maintenance of histone methylation homeostasis. Aberrant expression of these two enzymes is closely associated with the tumorigenesis. Our recent study showed that the combination of an EZH2 inhibitor and a LSD1 inhibitor could achieve a highly synergistic cytotoxic effect against acute myeloid leukemia, but the underlying mechanisms remain unclear. Furthermore, due to the different chemical properties and pharmacokinetics of each drug, it is possible that the two compounds might not be able to simultaneously enter each of all tumor cells in vivo, leading to a decrease in synergy and therapeutic efficacy. Thus, we propose to (1) design and synthesize dual-target inhibitors of EZH2 and LSD1; (2) test the impact of these dual inhibitors on histone methylations, cellular metabolism, cell motility, and apoptosis, and investigate the underlying mechanisms; (3) use multiple animal models to validate the in vivo therapeutic effect of the dual-target inhibitors in term of inhibition of the tumor growth and metastasis, and further verify the synergistic mechanisms of these compounds in vivo. The successful accomplishment of our proposed project will provide crucial data for the development of novel inhibitors of epigenetic modifying enzymes as potential anticancer drugs, which has significant scientific and clinical implications.

表观遗传在不影响DNA序列情况下调节基因表达，其中组蛋白甲基化的改变在恶性肿瘤中起重要作用。EZH2和LSD1分别是组蛋白赖氨酸甲基酶和去甲基酶，是维持组蛋白甲基化平衡状态的关键因子，其异常表达与肿瘤密切相关。申请人近期发现联合使用EZH2和LSD1抑制剂可显著提高对急性髓性白血病细胞的体内外杀伤效果。然而，该协同作用的确切机制尚未明确；另外，由于这两种抑制剂的理化和药物代谢性质不同，在体内不一定能同时进入每一个肿瘤细胞，因此可能降低协同作用和疗效。据此，本项目拟: 1，设计与合成EZH2/LSD1双靶点抑制剂；2，研究共抑制EZH2和LSD1对组蛋白甲基化修饰的影响及其对肿瘤代谢、细胞迁移和凋亡的作用及机制；3，用多种动物模型验证双靶点抑制剂的体内抗肿瘤活性和抑制肿瘤转移的作用及机制。本项目将为表观遗传靶向抗肿瘤创新药物提供新颖实验数据和药物设计策略，具有重要科学研究价值和临床应用潜力。

本项目针对两个表观遗传修饰酶EZH2和LSD1单靶点抑制剂各自在有响应的肿瘤细胞种类不多以及治疗有效性不高的背景下设计和合成EZH2/LSD1双靶点抑制剂。项目实施过程中，我们首先对LSD1抑制剂进行了简要的构效关系研究，发现带有Boc官能团的化合物在急性髓性白血病细胞上具有更好的抗肿瘤活性。其次，我们也针对EZH2抑制剂不足以完全抑制淋巴瘤增殖的缺点发展了EZH2蛋白降解靶向嵌合体药物分子PROTAC，发现EZH2 PROTAC分子不仅对弥漫大B淋巴瘤有更好的抑制效果也对其它淋巴瘤有效。由于实体瘤对EZH2抑制剂和降解剂都不敏感，我们进一步设计和合成了EZH2/BRD4双靶点抑制剂，发现它们对实体瘤细胞生长抑制效果较好。最后，我们设计和合成了EZH2/LSD1双靶点抑制剂，发现化合物TXZ221具有针对前内列腺癌细胞增殖和迁移都具有较好的效果，酶活性测试和细胞实验进一步确证TXZ221能够同时干扰EZH2和LSD1，并且通过微小结构改造去除TXZ221一端的EZH2抑制或LSD1抑制证明两个靶点同时双抑制是TXZ221表现抗肿瘤效果更好的关键原因，为表观遗传抗肿瘤提供新药物骨架和新策略。项目实施过程中，我们发表了SCI论文13篇，申请了中国发明专利2项，培养了研究生3名。