Pauzaite Tekle, Tollitt James, Sopaci Betul, Caprani Louise, Iwanowytsch Olivia, Thacker Urvi, Hardy John G, Allinson Sarah L, Copeland Nikki A
Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, UK.
Materials Science Institute, Lancaster University, Lancaster LA1 4YW, UK.
Biomedicines. 2022 Aug 19;10(8):2012. doi: 10.3390/biomedicines10082012.
Precise regulation of DNA replication complex assembly requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) activities to activate the replicative helicase complex and initiate DNA replication. Chemical probes have been essential in the molecular analysis of DDK-mediated regulation of MCM2-7 activation and the initiation phase of DNA replication. Here, the inhibitory activity of two distinct DDK inhibitor chemotypes, PHA-767491 and XL-413, were assessed in cell-free and cell-based proliferation assays. PHA-767491 and XL-413 show distinct effects at the level of cellular proliferation, initiation of DNA replication and replisome activity. XL-413 and PHA-767491 both reduce DDK-specific phosphorylation of MCM2 but show differential potency in prevention of S-phase entry. DNA combing and DNA replication assays show that PHA-767491 is a potent inhibitor of the initiation phase of DNA replication but XL413 has weak activity. Importantly, PHA-767491 decreased E2F-mediated transcription of the G1/S regulators cyclin A2, cyclin E1 and cyclin E2, and this effect was independent of CDK9 inhibition. Significantly, the enhanced inhibitory profile of PHA-767491 is mediated by potent inhibition of both DDK and the CDK2-Rb-E2F transcriptional network, that provides the molecular basis for its increased anti-proliferative effects in RB cancer cell lines.
DNA复制复合物组装的精确调控需要细胞周期蛋白依赖性激酶（CDK）和Dbf4依赖性激酶（DDK）的活性来激活复制解旋酶复合物并启动DNA复制。化学探针对于DDK介导的MCM2-7激活调控及DNA复制起始阶段的分子分析至关重要。在此，在无细胞和基于细胞的增殖试验中评估了两种不同的DDK抑制剂化学类型PHA-767491和XL-413的抑制活性。PHA-767491和XL-413在细胞增殖、DNA复制起始和复制体活性水平上表现出不同的作用。XL-413和PHA-767491均降低MCM2的DDK特异性磷酸化，但在阻止S期进入方面表现出不同的效力。DNA梳理和DNA复制试验表明，PHA-767491是DNA复制起始阶段的有效抑制剂，但XL413活性较弱。重要的是，PHA-767491降低了E2F介导的G1/S调节因子细胞周期蛋白A2、细胞周期蛋白E1和细胞周期蛋白E2的转录，且这种作用独立于对CDK9的抑制。值得注意的是，PHA-767491增强的抑制作用是通过对DDK和CDK2-Rb-E2F转录网络的有效抑制介导的，这为其在RB癌细胞系中增强的抗增殖作用提供了分子基础。
