Synthesis and Characterization of Transition-State Analogue Inhibitors against Human DNA Methyltransferase 1

Hypermethylation of CpG regions by
human DNA methyltransferase
1 (DNMT1) silences tumor-suppression genes, and inhibition of DNMT1
can reactivate silenced genes. The 5-azacytidines are approved inhibitors
of DNMT1, but their mutagenic mechanism limits their utility. A synthon
approach from the analogues of S-adenosylhomocysteine,
methionine, and deoxycytidine recapitulated the chemical features
of the DNMT1 transition state in the synthesis of 16 chemically stable
transition-state mimics. Inhibitors causing both full and partial
inhibition of purified DNMT1 were characterized. The inhibitors show
modest selectivity for DNMT1 versus DNMT3b. Active-site docking predicts
inhibitor interactions with S-adenosyl-l-methionine and deoxycytidine regions of the catalytic site, validated
by direct binding analysis. Inhibitor action with purified DNMT1 is
not reflected in cultured cells. A partial inhibitor activated cellular
DNA methylation, and a full inhibitor had no effect on cellular DNA
methylation. These compounds provide chemical access to a new family
of noncovalent DNMT chemical scaffolds for use in DNA methyltransferases.

人类DNA甲基转移酶1（DNMT1）介导的CpG区域高甲基化会沉默抑癌基因，而抑制DNMT1可重新激活已被沉默的基因。5-氮杂胞苷类（5-azacytidines）是获批的DNMT1抑制剂，但其诱变机制限制了其应用价值。本研究采用合成子（synthon）策略，以S-腺苷同型半胱氨酸（S-adenosylhomocysteine）、甲硫氨酸（methionine）以及脱氧胞苷（deoxycytidine）的类似物为原料，在合成16种化学稳定的过渡态模拟物的过程中，重现了DNMT1过渡态的化学特征。对纯化DNMT1具有完全抑制与部分抑制活性的抑制剂均得到了表征鉴定。相较于DNA甲基转移酶3b（DNMT3b），此类抑制剂对DNMT1仅表现出中等程度的选择性。活性位点对接实验预测，抑制剂可与催化位点中的S-腺苷-L-甲硫氨酸（S-adenosyl-L-methionine）区域以及脱氧胞苷区域发生相互作用，该预测通过直接结合分析得到了验证。针对纯化DNMT1的抑制剂活性无法在培养细胞中得到重现。部分抑制剂可激活细胞内的DNA甲基化过程，而完全抑制剂则对细胞内DNA甲基化无显著影响。此类化合物为研发一类可应用于DNA甲基转移酶的非共价结合型DNMT化学骨架提供了化学途径。