Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China.
Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
Eur J Med Chem. 2024 Aug 5;274:116538. doi: 10.1016/j.ejmech.2024.116538. Epub 2024 May 27.
DNA methyltransferase 1 (DNMT1) is the primary enzyme responsible for maintaining DNA methylation patterns during cellular division, crucial for cancer development by suppressing tumor suppressor genes. In this study, we retained the phthalimide structure of N-phthaloyl-l-tryptophan (RG108) and substituted its indole ring with nitrogen-containing aromatic rings of varying sizes. We synthesized 3-(9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acids and confirmed them as DNMT1 inhibitors through protein affinity testing, radiometric method using tritium labeled SAM, and MTT assay. Preliminary structure-activity relationship analysis revealed that introducing substituents on the carbazole ring could enhance inhibitory activity, with S-configuration isomers showing greater activity than R-configuration ones. Notably, S-3-(3,6-di-tert-butyl-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7r-S) and S-3-(1,3,6-trichloro-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7t-S) exhibited significant DNMT1 enzyme inhibition activity, with IC values of 8.147 μM and 0.777 μM, respectively (compared to RG108 with an IC above 250 μM). Moreover, they demonstrated potential anti-proliferative activity on various tumor cell lines including A2780, HeLa, K562, and SiHa. Transcriptome analysis and KEGG pathway enrichment of K562 cells treated with 7r-S and 7t-S identified differentially expressed genes (DEGs) related to apoptosis and cell cycle pathways. Flow cytometry assays further indicated that 7r-S and 7t-S induce apoptosis in K562 cells and arrest them in the G0/G1 phase in a concentration-dependent manner. Molecular docking revealed that 7t-S may bind to the methyl donor S-adenosyl-l-methionine (SAM) site in DNMT1 with an orientation opposite to RG108, suggesting potential for deeper penetration into the DNMT1 pocket and laying the groundwork for further modifications.
DNA 甲基转移酶 1（DNMT1）是在细胞分裂过程中维持 DNA 甲基化模式的主要酶，通过抑制肿瘤抑制基因在癌症发展中起着至关重要的作用。在本研究中，我们保留了 N-邻苯二甲酰基-L-色氨酸（RG108）的酞亚胺结构，并将其吲哚环用不同大小的含氮芳环取代。我们合成了 3-（9H-咔唑-9-基）-2-（1,3-二氧代异吲哚啉-2-基）丙酸，并通过蛋白质亲和测试、氚标记 SAM 的放射性方法和 MTT 测定证实它们是 DNMT1 抑制剂。初步的构效关系分析表明，在咔唑环上引入取代基可以增强抑制活性，S-构型异构体的活性大于 R-构型异构体。值得注意的是，S-3-（3,6-二叔丁基-9H-咔唑-9-基）-2-（1,3-二氧代异吲哚啉-2-基）丙酸（7r-S）和 S-3-（1,3,6-三氯-9H-咔唑-9-基）-2-（1,3-二氧代异吲哚啉-2-基）丙酸（7t-S）对 DNMT1 酶具有显著的抑制活性，IC 值分别为 8.147 μM 和 0.777 μM（与 IC 值超过 250 μM 的 RG108 相比）。此外，它们在包括 A2780、HeLa、K562 和 SiHa 在内的各种肿瘤细胞系中表现出潜在的抗增殖活性。用 7r-S 和 7t-S 处理 K562 细胞的转录组分析和 KEGG 通路富集鉴定出与细胞凋亡和细胞周期途径相关的差异表达基因（DEGs）。流式细胞术检测进一步表明，7r-S 和 7t-S 以浓度依赖性方式诱导 K562 细胞凋亡并将其阻滞在 G0/G1 期。分子对接表明，7t-S 可能与 DNMT1 中的甲基供体 S-腺苷甲硫氨酸（SAM）结合位点结合，与 RG108 的取向相反，这表明它有可能更深地进入 DNMT1 口袋，并为进一步修饰奠定基础。
