Rational Design of Bisubstrate-Type Analogues as Inhibitors of DNA Methyltransferases in Cancer Cells

Aberrant DNA hypermethylation of promoter of tumor suppressor genes is commonly observed in cancer, and its inhibition by small molecules is promising for their reactivation. Here we designed bisubstrate analogues-based inhibitors, by mimicking each substrate, the S-adenosyl-l-methionine and the deoxycytidine, and linking them together. This approach resulted in quinazoline–quinoline derivatives as potent inhibitors of DNMT3A and DNMT1, some showing certain isoform selectivity. We highlighted the importance of (i) the nature and rigidity of the linker between the two moieties for inhibition, as (ii) the presence of the nitrogen on the quinoline group, and (iii) of a hydrophobic group on the quinazoline. The most potent inhibitors induced demethylation of CDKN2A promoter in colon carcinoma HCT116 cells and its reactivation after 7 days of treatment. Furthermore, in a leukemia cell model system, we found a correlation between demethylation of the promoter induced by the treatment, chromatin opening at the promoter, and the reactivation of a reporter gene.

癌症中常可观察到肿瘤抑制基因启动子的异常DNA高甲基化现象，通过小分子抑制剂对该过程进行干预以重新激活此类抑癌基因，是颇具潜力的研究方向。本研究设计了基于双底物类似物的抑制剂：通过分别模拟两种底物——S-腺苷-L-甲硫氨酸（S-adenosyl-L-methionine）与脱氧胞苷（deoxycytidine），并将二者进行连接。基于该策略，我们获得了喹唑啉-喹啉类衍生物，其可作为DNA甲基转移酶3A（DNMT3A）与DNA甲基转移酶1（DNMT1）的强效抑制剂，部分化合物还表现出对特定同工型的选择性。我们重点阐明了三类影响抑制活性的关键因素：(i) 两个功能基团之间连接臂的性质与刚性；(ii) 喹啉环上氮原子的存在；(iii) 喹唑啉环上疏水取代基的引入。活性最优的抑制剂可在结肠癌HCT116细胞中诱导CDKN2A启动子去甲基化，并在给药7天后实现该基因的重新激活。此外，在白血病细胞模型体系中，我们发现药物处理诱导的启动子去甲基化、启动子区域染色质开放状态，与报告基因的重新激活三者之间存在显著相关性。